Decreased IL-12 production and Th1 cell development by acetyl salicylic acid-mediated inhibition of NF-kappaB. 
IL-12 is a 75-kDa heterodimeric cytokine composed of two covalently linked p35 and p40 chains. This pro-inflammatory cytokine plays a prominent role in the development of Th1 cell-mediated immune responses. Th1 cell-mediated immune responses have been implicated in the pathogenesis of chronic inflammatory autoimmune diseases. Thus, IL-12 appears to be a critical factor in the generation and maintenance of chronic inflammatory conditions. In this study, we investigated the effects of a commonly prescribed anti-inflammatory drug, acetyl salicylic acid (ASA), on IL-12 production and Th1 cell development. ASA was found to inhibit secretion of the IL-12 heterodimer as well as p40 monomer by human monocytic cells. This was associated with the down-regulation of IL-12p40 mRNA expression. Analysis of the regulation of the p40 gene promoter revealed that ASA inhibited NF-kappaB activation and binding to the p40-kappaB site in the p40 promoter, leading to transcriptional repression of the p40 gene. Addition of ASA to an in vitro T helper cell differentiation system, at concentrations compatible with plasma levels reached during anti-inflammatory therapy, resulted in reduced development of Th1 cells. These results suggest that the inhibition of NF-kappaB activation by ASA leads to down-regulation of IL-12 production and inhibition of Th1 cell development. 


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NF-kappa B controls expression of inhibitor I kappa B alpha: evidence for an inducible autoregulatory pathway. 
The eukaryotic transcription factor nuclear factor-kappa B (NF-kappa B) participates in many parts of the genetic program mediating T lymphocyte activation and growth. Nuclear expression of NF-kappa B occurs after its induced dissociation from its cytoplasmic inhibitor I kappa B alpha. Phorbol ester and tumor necrosis factor-alpha induction of nuclear NF-kappa B is associated with both the degradation of performed I kappa B alpha and the activation of I kappa B alpha gene expression. Transfection studies indicate that the I kappa B alpha gene is specifically induced by the 65-kilodalton transactivating subunit of NF-kappa B. Association of the newly synthesized I kappa B alpha with p65 restores intracellular inhibition of NF-kappa B DNA binding activity and prolongs the survival of this labile inhibitor. Together, these results show that NF-kappa B controls the expression of I kappa B alpha by means of an inducible autoregulatory pathway. 


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Activation of the NF-kappaB transcription factor in a T-lymphocytic cell line by hypochlorous acid. 
Reactive oxygen species (ROS) such as hydrogen peroxide serve as second messengers in the induction of the transcription factor NF-kappaB, and hence in the activation and replication of human immunodeficiency virus type 1 (HIV-1) in human cells. During inflammatory reactions, many oxidative species are produced, one of which is hypochlorous acid (HOCl), which is responsible for the microbicidal effects of activated human polymorphonuclear leukocytes. Treatment of a T-lymphocytic cell line with micromolar concentrations of HOCl promoted the appearance of transcription factor NF-kappaB (the heterodimer p50/p65) in the nucleus of the cells, even in the absence of de novo protein synthesis. Western blot analysis of the NF-kappaB inhibitory subunits (IkappaB) demonstrated that both IkappaB-alpha proteolysis and p105 processing were induced by the treatment. NF-kappaB activation was very effective when cells were subjected to hyperthermia before being treated with HOCl. Various antioxidants, such as pyrrolidine dithiocarbamate, p-bromophenacyl-bromide and nordihydroguaiaretic acid could strongly reduce NF-kappaB translocation, demonstrating the importance of oxidative species in the transduction mechanism. Moreover, ACH-2 cells treated with HOCl or H2O2 released tumour necrosis factor-alpha (TNF-alpha) in the supernatants. The importance of TNF-alpha release in NF-kappaB induction by HOCl or H2O2 was demonstrated by the fact that: (1) the nuclear appearance of NF-kappaB was promoted in untreated cells; and (2) synergism between TNF-alpha and HOCl was detected. Collectively, these results suggest that HOCl should be considered as an oxidative species capable of inducing NF-kappaB in a T-lymphocytic cell line through a transduction mechanism involving ROS, and having a long-distance effect through subsequent TNF-alpha release. 


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Human interferon regulatory factor 2 gene. Intron-exon organization and functional analysis of 5'-flanking region. 
Interferon regulatory factor 2 (IRF-2) is a transcriptional regulatory protein that terminates interferon beta expression initiated by interferon regulatory factor 1. In this study, we isolated the genomic DNA for human IRF-2 gene, determined the intron-exon structure of the human IRF-2 gene, mapped the major transcription initiation site, identified a number of potential regulatory elements in the 5'-flanking region, and localized the IRF-2 gene on human chromosome 4. The IRF-2 promoter region contains a CpG island, with several GC boxes, a putative NF-kappa B-binding site, and a CAAT box, but no TATA box. When the promoter region was linked with a heterologous reporter gene, we found that the promoter region is inducible by both interferons (interferon-alpha and -gamma) and interferon regulatory factor 1. The region which induced these inductions was identified as being confined to 40 nucleotides 5' to the major transcriptional initiation site by testing a series of clones with truncated promoter of IRF-2. This region contains elements which are shared with the transcriptional enhancers of other genes including interferon regulatory factor 1, interferon beta, and interferon-inducible genes. These data suggest that interferon regulatory factor 1 not only triggers the activation of the interferon signal transduction pathway, but also may play a role in limiting the duration of this response by activating the transcription of IRF-2. 


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IL-10 inhibits nuclear factor-kappa B/Rel nuclear activity in CD3-stimulated human peripheral T lymphocytes. 
IL-10 markedly reduces nuclear factor (NF)-kappa B/Rel nuclear activity induced in PBMC by stimulation with the anti-CD3 mAb OKT3. The inhibition is exerted specifically on the NF-kappa B/Rel activation induced by mAb OKT3, and not that produced by PMA. As judged by supershifting the DNA-protein complexes with Abs recognizing specific components of the NF-kappa B/Rel protein family, the p50/p65 (Rel A) heterodimeric form of NF-kappa B is primarily affected. The maximal effect is observed at the IL-10 concentration of 20 U/ml. IL-10 inhibitory activity is exerted on T lymphocytes and is mediated by monocytes. Indeed, monocytes pretreated with IL-10 are able so inhibit NF-kappa B nuclear activity in purified T lymphocytes stimulated with OKT3. Soluble factors do not appear to be involved in the mechanism of inhibition. On the other hand, the up-regulation of CD80 Ag, found on monocytes obtained from PBMC incubated with OKT3, is not detected after addition of IL-10, and the anti-CD28 mAb CLB-CD28/1 restores the NF-kappa B/Rel nuclear activity in IL-10-inhibited lymphocytes. Therefore, the NF-kappa B/Rel inhibition might be ascribed to a lack of cooperation between accessory cells and T lymphocytes, resulting from down-regulation of a costimulatory molecule, such as CD80, produced by IL-10 on activated monocytes. Our results demonstrate that IL-10 can inhibit the induction of NF-kappa B/Rel nuclear activity in CD3-stimulated T lymphocytes. Since inappropriate activation of kappa B-driven genes has a physiopathologic role in a number of diseases, such as HIV infection, our findings support the possibility of using this cytokine to suppress an undesirable activation of these transcription factors. 


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TNFalpha cooperates with the protein kinase A pathway to synergistically increase HIV-1 LTR transcription via downstream TRE-like cAMP response elements. 
Activating protein-1 (AP-1) binding TPA responsive elements (TRE) are located downstream of the transcription initiation site in the U5 region of the HIV-1 long terminal repeat (LTR). These downstream sequence elements, termed DSE, can bind both AP-1 and CREB/ATF transcription factors. Recently, we demonstrated that the DSE are also cAMP-responsive elements (CRE), since they mediated activation signals elicited by cholera toxin (Ctx), a potent activator of the cAMP-dependent protein kinase A (PKA) signal transduction pathway. In the present study, we demonstrate that the HIV-1 DSE can mediate the transcriptional synergy elicited by the combination of Ctx and TNFalpha. Ctx combined with TNFalpha or IL-1beta to produce a synergistic increase in p24 antigen production in U1 promonocytic cells. Transfection studies of LTR reporter constructs indicated that mutation of the DSE sites abrogated the LTR-mediated synergy induced by Ctx and TNFalpha, whereas the synergy induced by Ctx and IL-1beta was unaffected, suggesting TNFalpha and IL-1beta cooperate differently with the cAMP/PKA activation pathway to induce HIV-1 expression in U1 cells. Because the DSE are also TRE sites, we assessed the effect of the agonist combinations on AP-1-dependent transcription. TNFalpha as well as IL-1beta cooperated with Ctx to produce a synergistic activation of AP-1-mediated transcription. These data indicate that the TRE-like cAMP-responsive DSE sites within the 5'-untranslated leader can mediate the transcriptional cooperativity between TNFalpha and the cAMP/PKA pathway. Since the DSE and TRE sites cannot bind CREB/ATF homodimers, we propose a mechanism in which the HIV-1 DSE bind heterodimers composed of both AP-1 and CREB/ATF proteins. Copyright 1997 Academic Press. 


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Signaling events induced by lipopolysaccharide-activated toll-like receptor 2. 
Human Toll-like receptor 2 (TLR2) is a signaling receptor that responds to LPS and activates NF-kappaB. Here, we investigate further the events triggered by TLR2 in response to LPS. We show that TLR2 associates with the high-affinity LPS binding protein membrane CD14 to serve as an LPS receptor complex, and that LPS treatment enhances the oligomerization of TLR2. Concomitant with receptor oligomerization, the IL-1R-associated kinase (IRAK) is recruited to the TLR2 complex. Intracellular deletion variants of TLR2 lacking C-terminal 13 or 141 aa fail to recruit IRAK, which is consistent with the inability of these mutants to transmit LPS cellular signaling. Moreover, both deletion mutants could still form complexes with wild-type TLR2 and act in a dominant-negative (DN) fashion to block TLR2-mediated signal transduction. DN constructs of myeloid differentiation protein, IRAK, TNF receptor-associated factor 6, and NF-kappaB-inducing kinase, when coexpressed with TLR2, abrogate TLR2-mediated NF-kappaB activation. These results reveal a conserved signaling pathway for TLR2 and IL-1Rs and suggest a molecular mechanism for the inhibition of TLR2 by DN variants. 


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The role of p16 in the E2F-dependent thymidine kinase regulation. 
The role of alterations of the MTS1 tumor suppressor gene on chromosome 9p21, which encodes p16, the inhibitor of cyclin-dependent-kinase-4 and 6, in tumorigenesis is not yet clear. Phosphorylation of the retinoblastoma protein by cyclin-dependent kinases 4 and 6 prevents its interaction with the transcription factor E2F, which subsequently promotes the expression of S phase regulated genes, such as thymidine kinase. Although a role of p16 in this regulation has been presumed, there is no proof so far that loss of this tumor suppressor gene really affects E2F-mediated regulations. We investigated the regulation of thymidine kinase in phytohemagglutinin-stimulated normal human lymphocytes and in the p16-negative human acute lymphoblastic leukemia cell lines, MOLT-4 and CEM. Compared to normal lymphocytes, MOLT-4 and CEM cells exhibited an altered cell cycle regulation of thymidine kinase, a much higher intracellular activity of this enzyme, and higher thymidine kinase mRNA expression. Transient expression of p16 in normal human lymphocytes caused arrest in G1, but was without effect on the cell growth of MOLT-4 and CEM cells, although all of them express functional retinoblastoma protein. Nevertheless, in the two leukemia cell lines transient overexpression of p16 reestablished the normal regulation of thymidine kinase, paralleled by an increase of the underphosphorylated form of retinoblastoma protein and decrease of free E2F bound to its motif in the thymidine kinase promoter. We demonstrate that loss of p16 causes upregulation of this DNA precursor pathway enzyme via activation of E2F by a mechanism involving retinoblastoma protein. 


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Modulation of endogenous IL-1 beta and IL-1 receptor antagonist results in opposing effects on HIV expression in chronically infected monocytic cells. 
A proportion of HIV-infected individuals experience episodes of localized or systemic bacterial infections caused by Gram-negative bacteria. Many of the clinical side effects of these infections are associated with the production of proinflammatory cytokines, which are induced primarily by LPS, a constituent of the bacterial cell wall of Gram-negative bacteria. The present study examines the mechanisms involved in LPS-mediated induction of HIV expression in U1 cells, a promonocytic cell line chronically infected with HIV. Stimulation of U1 cells by LPS alone induced minimal levels of HIV expression, which was significantly enhanced by granulocyte-macrophage colony-stimulating factor (GM-CSF). Costimulation of U1 cells with LPS plus GM-CSF resulted in the accumulation of steady-state levels of HIV RNA; however, only a weak induction of HIV long terminal repeat-driven transcription, which was not associated with the activation of the cellular transcription factor nuclear factor-kappa B, was noted. Costimulation of cells with LPS plus GM-CSF induced the production of proinflammatory cytokines, IL-8, IL-1 beta and IL-6, but not TNF-alpha. IL-1 receptor antagonist (ra) inhibited LPS enhancement of HIV expression in GM-CSF-stimulated cells, suggesting that endogenous IL-1 was involved in LPS-mediated viral production. In this regard, anti-inflammatory cytokines inhibited LPS plus GM-CSF-stimulated HIV expression, and this effect closely correlated with inhibition of IL-1 beta release and, in particular, with up-regulation of endogenous IL-1ra production. Thus, the balance between an endogenously produced viral inducer (IL-1 beta ) and an inhibitor (IL-1ra) may represent an important pathway leading to modulation of HIV expression from monocytic cells. 


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Activation of the Janus kinase 3-STAT5a pathway after CD40 triggering of human monocytes but not of resting B cells. 
CD40/CD40 ligand interactions play a key role in the immune responses of B lymphocytes, monocytes, and dendritic cells. The signal transduction events triggered by cross-linking of the CD40 receptor have been widely studied in B cell lines, but little is known about signaling following CD40 stimulation of monocytes and resting tonsillar B cells. Therefore, we studied the CD40 pathway in highly purified human monocytes and resting B cells. After CD40 triggering, a similar activation of the NF-kappaB (but not of the AP-1) transcription factor complex occurred in both cell preparations. However, the components of the NF-kappaB complexes were different in monocytes and B cells, because p50 is part of the NF-kappaB complex induced by CD40 triggering in both monocytes and B cells, whereas p65 was only induced in B cells. In contrast, although the Janus kinase 3 tyrosine kinase was associated with CD40 molecules in both monocytes and resting B cells, Janus kinase 3 phosphorylation induction was observed only in CD40-activated monocytes, with subsequent induction of STAT5a DNA binding activity in the nucleus. These results suggest that the activation signals in human B cells and monocytes differ following CD40 stimulation. This observation is consistent with the detection of normal CD40-induced monocyte activation in patients with CD40 ligand+ hyper IgM syndrome in whom a defect in CD40-induced B cell activation has been reported. 


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Identification of an I kappa B alpha-associated protein kinase in a human monocytic cell line and determination of its phosphorylation sites on I kappa B alpha. 
Nuclear factor kappa B (NF-kappa B) is stored in the cytoplasm as an inactive form through interaction with I kappa B. Stimulation of cells leads to a rapid phosphorylation of I kappa B alpha, which is presumed to be important for the subsequent degradation. We have recently reported the establishment of a lipopolysaccharide (LPS)-dependent cell-free activation system of NF-kappa B in association with the induction of I kappa B alpha phosphorylation. In this study, we have identified a kinase in cell extracts from the LPS-stimulated human monocytic cell line, THP-1, that specifically binds and phosphorylates I kappa B alpha. LPS stimulation transiently enhanced the I kappa B alpha-bound kinase activity in THP-1 cells. Mutational analyses of I kappa B alpha and competition experiments with the synthetic peptides identified major phosphorylation sites by the bound kinase as Ser and Thr residues in the C-terminal acidic domain of I kappa B alpha. Moreover, we show that the peptide, corresponding to the C-terminal acidic domain of I kappa B alpha, blocked the LPS-induced NF-kappa B activation as well as inducible phosphorylation of endogenous I kappa B alpha in a cell-free system using THP-1 cells. These results suggested that the bound kinase is involved in the signaling pathway of LPS by inducing the phosphorylation of the C-terminal region of I kappa B alpha and subsequent dissociation of the NF-kappa B.I kappa B alpha complex. 


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Regulation of interleukin-1beta transcription by Epstein-Barr virus involves a number of latent proteins via their interaction with RBP. 
Epstein-Barr virus (EBV) infects B cells, resulting in the outgrowth of immortalised lymphoblastoid cell lines (LCLs). Here, we demonstrate through the use of intracellular staining that interleukin-1beta (IL-1beta) is expressed in LCLs and investigate the influence of the individual latent proteins on the expression of IL-1beta. Using RT-PCR, IL-1beta was shown to be up-regulated in EBV-transformed LCLs as well as in group III Burkitt's lymphoma (BL) cell lines, compared with group I BL cell lines. The up-regulation of IL-1beta message could be mediated by the latent membrane protein-1, EBV nuclear proteins 2, 3, 4, and 6 genes. Electrophoretic mobility shift assays (EMSAs) demonstrated that the -300 region of the IL-1beta promoter, which contains a nuclear factor-kappaB (NF-kappaB) binding site, contained a functional RBP binding site. Binding of RBP to this site could be inhibited by addition of EBV nuclear proteins 3 and 6, suggesting that these proteins displace RBP from its recognition sequence, removing transcriptional repression and allowing gene transcription to occur. In group I BL cells, containing low levels of NF-kappaB, only RBP binding was observed in EMSAs, whereas NF-kappaB binding could be demonstrated in EBV-transformed B cell lines containing high levels of activated NF-kappaB. In addition, the expression of latent membrane protein-1 led to activation of NF-kappaB that was capable of binding the IL-1beta promoter. The study demonstrates that EBV can up-regulate IL-1beta expression, possibly by using RBP, NF-kappaB, or both. Copyright 1998 Academic Press. 


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Uncoupling activation-dependent HS1 phosphorylation from nuclear factor of activated T cells transcriptional activation in Jurkat T cells: differential signaling through CD3 and the costimulatory receptors CD2 and CD28. 
CD3, CD2, and CD28 are functionally distinct receptors on T lymphocytes. Engagement of any of these receptors induces the rapid tyrosine phosphorylation of a shared group of intracellular signaling proteins, including Vav, Cbl, p85 phosphoinositide 3-kinase, and the Src family kinases Lck and Fyn. Ligation of CD3 also induces the tyrosine phosphorylation of HS1, a 75-kDa hematopoietic cell-specific intracellular signaling protein of unknown function. We have examined changes in HS1 phosphorylation after differential stimulation of CD3, CD2, and CD28 to elucidate its role in T cells and to further delineate the signaling pathways recruited by these receptors. Unlike ligation of CD3, stimulation with anti-CD28 mAb or CHO cells expressing the CD28 ligands CD80 or CD86 did not lead to tyrosine phosphorylation of HS1 in Jurkat T cells. Additionally, no tyrosine phosphorylation of HS1 was induced by mitogenic pairs of anti-CD2 mAbs capable of activating the transcription factor NFAT (nuclear factor of activated T cells). Costimulation through CD28 and/or CD2 did not modulate the CD3-dependent phosphorylation of HS1. In vivo studies indicated that CD3-induced HSI phosphorylation was dependent upon both the Src family tyrosine kinase Lck and the tyrosine phosphatase CD45, did not require MEK1 kinase activity, and was regulated by protein kinase C activation. Thus, although CD3, CD28, and CD2 activate many of the same signaling molecules, they differed in their capacity to induce the tyrosine phosphorylation of HSI. Furthermore, activation-dependent tyrosine phosphorylation of HS1 was not required for NFAT transcriptional activation. 


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Cytokine rescue from glucocorticoid induced apoptosis in T cells is mediated through inhibition of IkappaBalpha. 
We previously reported that dexamethasone (DEX), a synthetic glucocorticoid, causes apoptosis in mature Th cell lines, and that this induction of cell death is prevented by specific cytokines, namely, by IL-2 in Th1 cells and by IL-4 in Th2 cells. We now show that this differential rescue by specific cytokines in Th cells correlates with the level of IkappaBalpha that is regulated by DEX and cytokines. In both cell types the cellular levels of IkappaBalpha mRNA and protein were evaluated by DEX treatment. Interestingly, the DEX-mediated IkappaBalpha induction was completely inhibited by IL-2, but not IL-4, in Th1 cells, while the reverse profile was seen in Th2 cells. In both cell types, the cytokine that inhibits the induction of IkappaBalpha by DEX, also rescues these cells from DEX-induced apoptosis, although the rescue cytokine is different in Th1 and Th2 cells. Our results imply that T cells need to maintain a certain level of NF-kappaB transcriptional activity in order to survive; up- or down-regulation of nuclear NF kappaB through modulation of IkappaBalpha expression by cytokines or DEX may lead to cell survival or cell death, respectively. 


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Regulation of Id3 cell cycle function by Cdk-2-dependent phosphorylation. 
The functions of basic helix-loop-helix (bHLH) transcription factors in activating differentiation-linked gene expression and in inducing G1 cell cycle arrest are negatively regulated by members of the Id family of HLH proteins. These bHLH antagonists are induced during a mitogenic signalling response, and they function by sequestering their bHLH targets in inactive heterodimers that are unable to bind to specific gene regulatory (E box) sequences. Recently, cyclin E-Cdk2- and cyclin A-Cdk2-dependent phosphorylation of a single conserved serine residue (Ser5) in Id2 has been shown to occur during late G1-to-S phase transition of the cell cycle, and this neutralizes the function of Id2 in abrogating E-box-dependent bHLH homo- or heterodimer complex formation in vitro (E.Hara, M.Hall, and G.Peters, EMBO J.16:332-342, 1997). We now show that an analogous cell-cycle-regulated phosphorylation of Id3 alters the specificity of Id3 for abrogating both E-box-dependent bHLH homo- or heterodimer complex formation in vitro and E-box-dependent reporter gene function in vivo. Furthermore, compared with wild-type Id3, an Id3 Asp5 mutant (mimicking phosphorylation) is unable to promote cell cycle S phase entry in transfected fibroblasts, whereas an Id3 Ala5 mutant (ablating phosphorylation) displays an activity significantly greater than that of wild-type Id3 protein. Cdk2-dependent phosphorylation therefore provides a switch during late G1-to-S phase that both nullifies an early G1 cell cycle regulatory function of Id3 and modulates its target bHLH specificity. These data also demonstrate that the ability of Id3 to promote cell cycle S phase entry is not simply a function of its ability to modulate bHLH heterodimer-dependent gene expression and establish a biologically important mechanism through which Cdk2 and Id-bHLH functions are integrated in the coordination of cell proliferation and differentiation. 


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Induction of cytokine expression in leukocytes by binding of thrombin-stimulated platelets. 
BACKGROUND: Activated platelets tether and activate myeloid leukocytes. To investigate the potential relevance of this mechanism in acute myocardial infarction (AMI), we examined cytokine induction by leukocyte-platelet adhesion and the occurrence of leukocyte-platelet conjugates in patients with AMI. METHODS AND RESULTS: We obtained peripheral venous blood samples in 20 patients with AMI before and daily for 5 days after direct percutaneous transluminal coronary angioplasty (PTCA) and in 20 patients undergoing elective PTCA. Throughout the study period, CD41 immunofluorescence of leukocytes (flow cytometry) revealed increased leukocyte-platelet adhesion in patients with AMI compared with control patients (mean +/- SE of fluorescence [channels] before PTCA: 77 +/- 16 versus 35 +/- 9; P = .003). In vitro, thrombin-stimulated fixed platelets bound to neutrophils and monocytes. Within 2 hours, this resulted in increased mRNA for interleukin (IL),1 beta, IL-8, and monocyte chemoattractant protein (MCP)-1 in unfractionated leukocytes. After 4 hours, IL-1 beta and IL-8 concentration of the cell-free supernatant had increased by 268 +/- 36% and 210 +/- 7%, respectively, and cellular MCP-1 content had increased by 170 +/- 8%. Addition of activated platelets to adherent monocytes had a similar effect and was associated with nuclear factor-kappa B activation. Inhibition of binding by anti-P selectin antibodies reduced the effect of activated platelets on cytokine production. CONCLUSIONS: In patients with AMI, leukocyte-platelet adhesion is increased. Binding of activated platelets induces IL-1 beta, IL-8, and MCP-1 in leukocytes. Our findings suggest that leukocyte-platelet adhesion contributes to the regulation of inflammatory responses in AMI. 


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Paternal expression of WT1 in human fibroblasts and lymphocytes. 
The Wilms' tumor suppressor gene ( WT1 ) was previously identified as being imprinted, with frequent maternal expression in human placentae and fetal brains. We examined the allele-specific expression of WT1 in cultured human fibroblasts from 15 individuals. Seven of 15 fibroblast lines were heterozygous for polymorphic alleles, and the expression patterns were variable, i.e., equal, unequal or monoallelic paternal expression in three, two and two cases, respectively. Exclusive paternal expression of WT1 was also shown in non-cultured peripheral lymphocytes from the latter two individuals. The allele-specific expression profiles of other imprinted genes, IGF2 and H19, on human chromosome 11 were constant and consistent with those in other tissues. Our unexpected observations of paternal or biallelic expression of WT1 in fibroblasts and lymphocytes, together with the previous findings of maternal or biallelic expression in placentae and brains, suggest that the allele-specific regulatory system of WT1 is unique and may be controlled by a putative tissue- and individual-specific modifier. 


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Overproduction of NFKB2 (lyt-10) and c-Rel: a mechanism for HTLV-I Tax-mediated trans-activation via the NF-kappa B signalling pathway. 
Molecular, biochemical and epidemiological evidence implicate HTLV-I as an etiologic agent of adult T cell leukemia (ATL). The Tax protein of HTLV-I, a positive transcriptional activator of HTLV-I gene expression, is a viral oncogene that also increases transcription of cellular genes including GM-CSF, IL-2R alpha and IL-2. One of the cellular targets of the trans-activating effects of Tax is the NF-kappa B/Rel family of transcription factors, pleiotropic regulators of immunoregulatory, cytokine and viral gene expression. In this report, we demonstrate that NFKB2 (lyt-10) and c-Rel are overexpressed in HTLV-I infected and Tax-expressing cells and, together, account for the majority of the constitutive NF-kappa B binding activity in these cells before and after PMA stimulation. Most importantly, we show a Tax-dependent correlation between expression of NFKB2(p100) and processing to the DNA binding NFKB2(p52) form, induction of c-Rel, and trans-activation of NF-kappa B-mediated gene expression. Furthermore, the NFKB2 precursor is physically associated with c-Rel and with Tax in HTLV-I infected cells. We propose that NFKB2 synthesis and processing allows continuous nuclear expression of an otherwise cytoplasmic protein and, in conjunction with overexpression of c-Rel, NFKB2 alters the NF-kappa B signalling pathway and contributes to leukemic transformation of T cells by HTLV-I. 


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Abnormal regulation of the IL-2 promoter in lpr CD4-CD8- T lymphocytes results in constitutive expression of a novel nuclear factor of activated T cells-binding factor. 
The inert quality of MRL-Ipr/Ipr (Ipr) peripheral CD4-CD8- (CD4-8-) T cells manifests primarily as an inability to proliferate or produce IL-2 in response to TCR or mitogenic stimulation. Yet these same cells do initiate early TCR-mediated signaling events, such as generation of inositol phosphates and increased intracellular calcium. They also display constitutively high levels of p59fyn and CD3 zeta tyrosine phosphorylation. The generation of second messengers in T cells normally leads to downstream signaling that results in transcriptional activation of the IL-2 gene. We, therefore, compared the activation state of the IL-2 gene promoter region in freshly isolated and stimulated Ipr CD4-8- T cells with that of normal T lymphocytes. Levels of the octamer, NF-kappa B (p50-p65 heterodimer), and AP-1 transcriptional factors are constitutively elevated in freshly isolated Ipr CD4-8- T cells, consistent with the activated phenotype of these cells. Upon stimulation with mitogens, formation of the transactivating complex, nuclear factor of activated T cells (NF-AT), occurs with normal kinetics in Ipr CD4-8- T cells. Yet, the levels of the activating NF-AT complex never reach those observed in similarly stimulated normal T cells. Furthermore, nuclear extracts from Ipr CD4-8- T cells display high levels of a novel specific binding activity at the NF-AT site, which is present at much lower levels in freshly isolated normal T lymphocytes. Upon mitogenic stimulation, the binding activity of the novel NF-AT-binding factor is rapidly down-regulated in normal T cells, but persists at high levels in Ipr CD4-8- T cells. These two abnormalities at the NF-AT site provide a potential mechanism to account for the defect in IL-2 production from Ipr CD4-8- T cells. 


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Impaired fetal thymocyte development after efficient adenovirus-mediated inhibition of NF-kappa B activation. 
We introduce a new experimental system combining adenovirus-mediated gene transfer and fetal thymic organ culture (FTOC). This system allowed us to efficiently express in developing thymocytes a mutant form of the NF-kappa B inhibitor I kappa B alpha (mut-I kappa B) and to study the maturation defects occurring when NF-kappa B activation is inhibited during fetal development. Fetal thymocytes infected with adenovirus containing mut-I kappa B were found to develop normally until the CD44-CD25+, CD4-CD8- double-negative stage, while production of more mature double-positive and single-positive populations was strongly decreased. Proliferation, as measured by the percentage of cells in cycle appeared normal, as did rearrangement and expression of the TCR beta-chain. However, apoptosis was much higher in FTOC infected with adenovirus containing mut-I kappa B than in FTOC infected with a control virus. Taken together, these results suggest that NF-kappa B plays a crucial role in ensuring the differentiation and survival of thymocytes in the early stages of their development. 


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An IL-2 response element in the human IL-2 receptor alpha chain promoter is a composite element that binds Stat5, Elf-1, HMG-I(Y) and a GATA family protein. 
Expression of the human interleukin-2 (IL-2) receptor alpha chain gene is potently upregulated by its own ligand, IL-2. In this study, we characterize an essential upstream IL-2 response element that contains both consensus and non-consensus GAS motifs, two putative Ets binding sites (EBS), one of which overlaps the consensus GAS motif, and a GATA motif, which overlaps the non-consensus GAS motif. We demonstrate that although the individual components of this element do not respond to IL-2, together they form a composite element capable of conferring IL-2 responsiveness to a heterologous promoter. Multiple factors including Stat5, Elf-1, HMG-I(Y) and GATA family proteins bind to the IL-2 response element and mutation of any one of these binding sites diminishes the activity of this element. An unidentified Ets family protein binds to the EBS overlapping the consensus GAS motif and appears to negatively regulate the human IL-2R alpha promoter. Thus, IL-2-induced IL-2R alpha promoter activity requires a complex upstream element, which appears to contain binding sites for both positive and negative regulatory factors. 


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Genomic organization, sequence, and transcriptional regulation of the human eotaxin gene. 
Eotaxin is an eosinophil specific beta-chemokine assumed to be involved in eosinophilic inflammatory diseases such as atopic dermatitis, allergic rhinitis, asthma and parasitic infections. Its expression is stimulus- and cell-specific. We here describe the genomic organisation (3 exons of 132, 112 and 542 bp and 2 introns of 1211 and 378 bp) and sequence including 3 kb of DNA from the immediate 5' upstream region of the human eotaxin gene. Among the regulatory promoter elements potentially regulating eotaxin gene expression and/or mediating the effects of anti-inflammatory drugs we identified consensus sequences known to interact with nuclear factors like NF-IL6, AP-1, a NF-kappa-B like consensus sequence and gamma-interferon- as well as glucocorticoid response elements. 


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Calcium-dependent immediate-early gene induction in lymphocytes is negatively regulated by p21Ha-ras. 
The induction of immediate-early (IE) response genes, such as egr-1, c-fos, and c-jun, occurs rapidly after the activation of T lymphocytes. The process of activation involves calcium mobilization, activation of protein kinase C (PKC), and phosphorylation of tyrosine kinases. p21(ras), a guanine nucleotide binding factor, mediates T-cell signal transduction through PKC-dependent and PKC-independent pathways. The involvement of p21(ras) in the regulation of calcium-dependent signals has been suggested through analysis of its role in the activation of NF-AT. We have investigated the inductions of the IE genes in response to calcium signals in Jurkat cells (in the presence of activated p21(ras)) and their correlated consequences. The expression of activated p21(ras) negatively regulated the induction of IE genes by calcium ionophore. This inhibition of calcium-activated IE gene induction was reversed by treatment with cyclosporin A, suggesting the involvement of calcineurin in this regulation. A later result of inhibition of this activation pathway by p21(ras) was down-regulation of the activity of the transcription factor AP-1 and subsequent coordinate reductions in IL-2 gene expression and protein production. These results suggest that p2l(ras) is an essential mediator in generating not only positive but also negative modulatory mechanisms controlling the competence of T cells in response to inductive stimulations. 


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The lymphotoxin promoter is stimulated by HTLV-I tax activation of NF-kappa B in human T-cell lines. 
The HTLV-I transcriptional activator tax was used to gain insight into the mechanism of lymphotoxin (LT; TNF-beta) gene induction. Tax-expressing cell lines produce LT biologic activity. An LT promoter (LT-293) CAT construct that contained an NF-kappa B site was active in the LT-producing C81-66-45 cell line, which contains defective HTLV-I but expresses tax. The observation that a mutated LT-kappa B construct (M1-CAT) was inactive in C81-66-45, confirmed the importance of NF-kappa B in LT gene expression. Tax was transfected into HTLV-I-negative human T-cell lines. Jurkat T cells stably expressing tax contained elevated levels of NF-kappa B that directly bound to the LT-kappa B site. Tax co-transfected with reporter constructs into Jurkat cells maximally activated HTLV-I-LTR-CAT and kappa B-fos-CAT and also activated LT-293 to a lesser extent. In JM T cells, tax induced LT-293 activity by two- to four-fold, though there was no induction of M1-CAT. The increase in LT-293 CAT activity mirrored the increase in LT biologic activity seen under these conditions. These studies, the first to demonstrate induction of LT promoter activity over basal levels, indicate that HTLV-I tax causes low-level activation of both endogenous LT and the LT promoter, at least in part through activation of NF-kappa B. 


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Inhibition of NF-kappa B activity in human T lymphocytes induces caspase-dependent apoptosis without detectable activation of caspase-1 and -3. 
NF-kappa B is involved in the transcriptional control of various genes that act as extrinsic and intrinsic survival factors for T cells. Our findings show that suppression of NF-kappa B activity with cell-permeable SN50 peptide, which masks the nuclear localization sequence of NF-kappa B1 dimers and prevents their nuclear localization, induces apoptosis in resting normal human PBL. Inhibition of NF-kappa B resulted in the externalization of phosphatidylserine, induction of DNA breaks, and morphological changes consistent with apoptosis. DNA fragmentation was efficiently blocked by the caspase inhibitor Z-VAD-fmk and partially blocked by Ac-DEVD-fmk, suggesting that SN50-mediated apoptosis is caspase-dependent. Interestingly, apoptosis induced by NF-kappa B suppression, in contrast to that induced by TPEN (N,N,N',N'-tetrakis [2-pyridylmethyl]ethylenediamine) or soluble Fas ligand (CD95), was observed in the absence of active death effector proteases caspase-1-like (IL-1 converting enzyme), caspase-3-like (CPP32/Yama/apopain), and caspase-6-like and without cleavage of caspase-3 substrates poly(ADP-ribose) polymerase and DNA fragmentation factor-45. These findings suggest either low level of activation is required or that different caspases are involved. Preactivation of T cells resulting in NF-kappa B nuclear translocation protected cells from SN50-induced apoptosis. Our findings demonstrate an essential role of NF-kappa B in survival of naive PBL. 


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Dysregulation of monocytic nuclear factor-kappa B by oxidized low-density lipoprotein. 
Nuclear factor-kappa B (NF-kappa B)/Rel transcription factors may be involved in atherosclerosis, as is suggested by the presence of activated NF-kappa B in human atherosclerotic lesions. The aim of the present study was to investigate the effects of oxidized LDL (oxLDL) on the NF-kappa B system in human THP-1 monocytic cells as well as adherent monocytes. Our results demonstrate that short-term incubation of these cells with oxLDL activated p50/p65 containing NF-kappa B dimers and induced the expression of the target gene IL-8. This activation of NF-kappa B was inhibited by the antioxidant and H2O2 scavenger pyrrolidine dithiocarbamate and the proteasome inhibitor PSI. The oxLDL-induced NF-kappa B activation was accompanied by an initial depletion of I kappa B-alpha followed by a slight transient increase in the level of this inhibitor protein. In contrast, long-term treatment with oxLDL prevented the lipopolysaccharide-induced depletion of I kappa B-alpha, accompanied by an inhibition of both NF-kappa B activation and the expression of tumor necrosis factor-alpha and interleukin-1 beta genes. These observations provide additional evidence that oxLDL is a potent modulator of gene expression and suggest that (dys)regulation of NF-kappa B/Rel is likely to play an important role in atherogenesis. 


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The interleukin-8 AP-1 and kappa B-like sites are genetic end targets of FK506-sensitive pathway accompanied by calcium mobilization. 
FK506, an immunosuppressant, inhibits the production of several cytokines in T lymphocytes. We observed that FK506 suppressed the transcription of a chemotactic cytokine, interleukin-8 (IL-8) in a human T cell line, Jurkat cells, activated by phorbol 12-myristate 13-acetate (PMA) and calcium (Ca2+) ionophore (ionomycin). By deleted and mutated analysis of the IL-8 promoters, the AP-1 and kappa B-like sites were identified as the responsive elements for PMA and ionomycin. FK506 suppressed the transcriptions through the AP-1 or kappa B-like sites induced by PMA plus Ca(2+)-mobilizing agents, but not those induced by Ca(2+)-independent stimuli. In gel retardation analysis, FK506 had little effect on the binding to the AP-1 site of PMA/ionomycin-induced nuclear factors, which were recognized with anti-JunD or c-Fos antibody. In contrast, FK506 or EGTA (Ca2+ chelator) similarly affected the formation of kappa B-like site binding complexes, which were not recognized by any antibodies against the human Rel family proteins (c-Rel, p65, p50, and p49). Furthermore, we confirmed the previous report that FK506 suppressed the PMA/ionomycin-induced activation through authentic kappa B site of immunoglobulin (Ig) gene, to which NF-kappa B binding was also decreased by FK506, indicating that both IL-8 kappa B-like site and Ig kappa B site are FK506-sensitive in spite of the difference of binding factors. Our results indicate that not only the reported IL-2 NF-AT and NFIL-2A sites and Ig kappa B site, but also the IL-8 AP-1 and kappa B-like sites are terminals of FK506-sensitive pathway involving Ca2+ mobilization. 


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Interactions of a transcriptional activator in the env gene of the mouse mammary tumor virus with activation-dependent, T cell-specific transacting factors. 
The mouse mammary tumor virus env gene contains a transcriptional activator (META) that can control transcription of the adjacent long terminal repeat region. Transcriptional control by META parallels that of several lymphokine genes, being specific to T cells, dependent on their activation, and inhibited by the immunosuppressive drug cyclosporine (CsA). DNase I footprinting indicated that nuclear factors from activated T lymphocytes bound a promoter-proximal site, META(P), and a promoter-distal site, META(D+), within the 400-base pair META region. Nuclear factors from unstimulated, but not from activated cells, bound a site, META(D-), adjacent to META(D+). META(D+) directed transcription of a linked luciferase gene, and gel shift analysis revealed binding of inducible, CsA-sensitive T cell factors, in parallel with transfection results. Authentic NFAT and NF-kappaB targets did not compete for the META(D+) binding factor(s). The SV40 core sequence competed for META(D+) binding factors, but META(D+) failed to compete for the complexes obtained with the SV40 probe. Our results, taken together, indicate that META(D+) is a novel transcriptional enhancer element that is similar in its cell-type specificity, activation dependence, and CsA sensitivity to the NFAT element. It may be relevant to the role of MMTV in expression of Mls antigens or the induction of T cell lymphomas. 


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Cytomegalovirus immediate early genes upregulate interleukin-6 gene expression. 
BACKGROUND: The immediate early genes (IE) of human cytomegalovirus (CMV) can be expressed in monocytic cells and are known to regulate viral and cellular genes. Interleukin-6 (IL-6) plays a central role in numerous inflammatory and immune processes. Interleukin-6 levels are increased in lung transplant patients clinically diagnosed with CMV pneumonitis. The regulation of IL-6 is dependent on various stimuli that include lipopolysaccharide (LPS), viruses, and other cytokines. These studies examined the ability of CMV IE gene products to modulate IL-6 production. METHODS: THP-1 cells, a monocytic cell line, were transfected with the CMV IE genes. Interleukin-6 protein and IL-6 mRNA were measured in control and CMV immediate early transfected cells. Cotransfection of CMV IE genes and IL-6 chloramphenicol acetyl transferase (CAT) or IL-6 luciferase constructs were used to study IL-6 promoter activity. RESULTS: Interleukin-6 protein and mRNA production were significantly increased in cells transfected with the CMV IE genes and stimulated with LPS compared to LPS-stimulated control cells. Cytomegalovirus IE gene products significantly enhanced LPS stimulation of IL-6 promoter activity in both IL-6 CAT and IL-6 luciferase assays. A deletion construct that contains a NF-kappa B site but is missing the multiple response region demonstrated a continued increase in IL-6 luciferase activity in LPS-stimulated CMV transfected cells. CONCLUSION: Cytomegalovirus immediate early gene products significantly enhanced expression of IL-6 in LPS-stimulated cells. The increase in IL-6 luciferase activity occurs in the absence of the multiple response region, the area of the IL-6 promoter responsive to IL-1, TNF alpha, cyclic amp, and phorbol 12-myristate 13-acetate. The ability of CMV IE gene products to enhance IL-6 production may play an important role in immune inflammatory states associated with CMV infection. 


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Costimulation requirement for AP-1 and NF-kappa B transcription factor activation in T cells. 
The transcriptional activity of the IL-2 promoter requires T-cell costimulation delivered by the TCR and the auxiliary receptor CD28. Several transcription factors participate in IL-2 promoter activation, among which are AP-1-like factors and NF-kappa B. Protein phosphorylation has an important role in the regulation of these two factors: (1) it induces the transactivating capacity of the AP-1 protein c-Jun; and (2) it is involved in the release of the cytoplasmic inhibitor, I kappa B, from NF-kappa B, allowing translocation of the latter into the nucleus. We have recently shown that both phosphorylation processes require T-cell costimulation. Furthermore, in activated T cells, the kinetics of the two phosphorylation events are essentially similar. According to our results, however, the kinases responsible for the two processes are distinct entities. Whereas TPCK inhibits phosphorylation of I kappa B and, consequently, activation of NF-kappa B, it markedly enhances the activity of JNK, the MAP kinase-related kinase that phosphorylates the transactivation domain of c-Jun. We, therefore, propose the activation scheme presented in FIGURE 3 for T-cell costimulation. Costimulation results in the activation of a signaling pathway that leads to the simultaneous induction of the two transcription factors, AP-1 and NF-kappa B. Integration of the signals generated by TCR and CD28 engagement occurs along this pathway, which then bifurcates to induce I kappa B phosphorylation and NF-kappa B activation on the one hand, and JNK activation and c-Jun phosphorylation on the other. We are currently engaged in defining where the two signals integrate along the AP-1/NF-kappa B pathway. 


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The Epstein-Barr virus oncogene product latent membrane protein 1 engages the tumor necrosis factor receptor-associated death domain protein to mediate B lymphocyte growth transformation and activate NF-kappaB. 
The Epstein-Barr virus latent membrane protein 1 (LMP1) is essential for the transformation of B lymphocytes into lymphoblastoid cell lines. Previous data are consistent with a model that LMP1 is a constitutively activated receptor that transduces signals for transformation through its carboxyl-terminal cytoplasmic tail. One transformation effector site (TES1), located within the membrane proximal 45 residues of the cytoplasmic tail, constitutively engages tumor necrosis factor receptor-associated factors. Signals from TES1 are sufficient to drive initial proliferation of infected resting B lymphocytes, but most lymphoblastoid cells infected with a virus that does not express the 155 residues beyond TES1 fail to grow as long-term cell lines. We now find that mutating two tyrosines to an isoleucine at the carboxyl end of the cytoplasmic tail cripples the ability of EBV to cause lymphoblastoid cell outgrowth, thereby marking a second transformation effector site, TES2. A yeast two-hybrid screen identified TES2 interacting proteins, including the tumor necrosis factor receptor-associated death domain protein (TRADD). TRADD was the only protein that interacted with wild-type TES2 and not with isoleucine-mutated TES2. TRADD associated with wild-type LMP1 but not with isoleucine-mutated LMP1 in mammalian cells, and TRADD constitutively associated with LMP1 in EBV-transformed cells. In transfection assays, TRADD and TES2 synergistically mediated high-level NF-kappaB activation. These results indicate that LMP1 appropriates TRADD to enable efficient long-term lymphoblastoid cell outgrowth. High-level NF-kappaB activation also appears to be a critical component of long-term outgrowth. 


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ETS transcription factors regulate an enhancer activity in the third intron of TNF-alpha. 
We describe an enhancer site in the third intron of tumor necrosis factor alpha (TNF-alpha). A reporter construct containing the 5'-flanking region of the mouse TNF-alpha gene displayed weak activity when transfected into RAW264.7 macrophage-like cells. The addition of the third intron of TNF-alpha to this construct resulted in an enhancement of CAT protein. This enhancement was eliminated if a conserved 20-bp sequence was removed from the intron or if a dominant-negative ets-binding factor was co-transfected with the reporter gene. Mutations of this site that destroyed potential ets transcription factor binding sites had reduced transcriptional activity. The major transcription factor that bound to the oligonucleotide was confirmed to be GABP by supershift and competition analysis. In RAW264.7 cells, the binding was constitutive, however, in bone marrow-derived macrophages binding activity was shown to be interferon-gamma inducible. This may imply a role for ets transcription factors in the production of TNF-alpha. 


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Xenogeneic human serum promotes leukocyte adhesion to porcine endothelium under flow conditions, possibly through the activation of the transcription factor NF-kappa B. 
Endothelial cell activation and leukocyte infiltration are a consistent feature of discordant xenograft rejection. Here we evaluated whether xenogeneic serum, as a source of xenoreactive natural antibodies and complement, induced endothelial cell activation with consequent leukocyte adhesion under flow conditions. Porcine aortic endothelial cells (PAEC) were incubated for 1 hr 30 min or 5 hr with 10% homologous porcine serum (control) or 10% xenogeneic human serum and then perfused with total human leukocytes in a parallel plate flow chamber under laminar flow (1.5 dynes/cm2). Adherent cells were counted by digital image analysis. Xenogeneic human serum significantly (P < 0.01) increased the number of adherent leukocytes as compared with porcine serum. A similar adhesive response was elicited by TNF alpha (100 U/ml), one of the most potent inducers of endothelial cell adhesive properties, here used as positive control. In order to elucidate possible mechanisms underlying endothelial cell activation by xenogeneic serum, we focussed on transcription factor NF-kappa B, a central regulator for the induction of different genes, including adhesive molecules and chemoattractants. By confocal fluorescence microscopy, we observed a positive staining for NF-kappa B (p65 subunit) in the nuclei of PAEC exposed for 1 hr 30 min to human serum, which indicated NF-kappa B activation in this setting. At variance, in PAEC incubated with the homologous serum, NF-kappa B was strictly localized in the cell cytoplasm. Treatment of PAEC exposed to xenogeneic serum with the NF-kappa B inhibitors pyrrolidinedithiocarbamate (PDTC, 25 microM) and tosyl-phechloromethylketone (TPCK, 25 microM) significantly (P < 0.01) reduced leukocyte adhesion in respect to PAEC treated with human serum alone. Findings that xenogeneic serum promotes leukocyte-endothelium interaction possibly through NF-kappa B activation might be relevant for designing future therapeutic strategies aimed at prolonging xenograft survival. 


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Epstein-Barr virus replicative gene transcription during de novo infection of human thymocytes: simultaneous early expression of BZLF-1 and its repressor RAZ. 
Epstein-Barr virus (EBV) is known to infect B cells and epithelial cells. We and others have shown that EBV can also infect a subset of thymocytes. Infection of thymocytes was accompanied by the appearance of linear EBV genome within 8 hr of infection. Circularization of the EBV genome was not detected. This is in contrast to the infection in B cells where the genome can circularize within 24 hr of infection. The appearance of the BamHI ZLF-1 gene product, ZEBRA, by RT-PCR, was observed within 8 hr of infection. The appearance of a novel fusion transcript (RAZ), which comprised regions of the BZLF-1 locus and the adjacent BRLF-1 locus, was detected by RT-PCR. ZEBRA protein was also identified in infected thymocytes by immunoprecipitation. In addition, we demonstrated that the EBNA-1 gene in infected thymocytes was transcribed from the Fp promoter, rather than from the Cp/Wp promoter which is used in latently infected B cells. Transcripts encoding gp350/220, the major coat protein of EBV, were identified, but we did not find any evidence of transcription from the LMP-2A or EBER-1 loci in infected thymocytes. These observations suggest that de novo EBV infection of thymocytes differs from infection of B cells. The main difference is that with thymocytes, no evidence could be found that the virus ever circularizes. Rather, EBV remains in a linear configuration from which replicative genes are transcribed. 


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Second messenger up-regulation of androgen receptor gene transcription is absent in androgen insensitive human prostatic carcinoma cell lines, PC-3 and DU-145. 
A theoretical pathway of transcriptional regulation of the androgen receptor (AR) gene is via a cAMP response element (CRE) present in its promoter region (-508 to -501). After 20 h of stimulation with 8-bromo-cAMP, AR mRNA was upregulated in LNCaP but not in either PC-3 or DU-145 cell lines. We have demonstrated that the level of CRE binding protein (CREB) was the same in all cell lines and that the putative AR-CRE forms specific and compatible protein interactions with CREB. The ability to regulate AR gene transcription via the second messenger pathway is lost in the PC-3 and DU-145 cell lines. This may be an important primary mechanism of androgen insensitivity in prostate cancer. 


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Induction of tyrosine phosphorylation and T-cell activation by vanadate peroxide, an inhibitor of protein tyrosine phosphatases. 
Rapid tyrosine phosphorylation of key cellular proteins is a crucial event in the transduction of activation signals to T-lymphocytes. The regulatory role of protein tyrosine phosphatases (PTPases) in this process was explored by studying the effects of a powerful PTPase inhibitor, vanadate peroxide (pervanadate), on the activation cascade of Jurkat human leukaemic T-cells. Pervanadate induced activation of the tyrosine kinases lck and fyn (4- and 3-fold respectively) and a dramatic increase in tyrosine phosphorylation of cellular proteins, notably phospholipase C gamma 1. After this event, we observed a rise in intracellular Ca2+ concentration, corresponding to an influx. This effect required surface expression of the CD45 PTPase and was not observed in CD45-deficient variants of Jurkat cells. In the CD45-negative variant, the effect of pervanadate on tyrosine phosphorylation was globally decreased and some phosphorylated substrates were specifically missing. Pervanadate also stimulated transcription of the c-fos gene and accumulation of its mRNA as well as several other hallmarks of T-lymphocyte activation such as surface expression of the CD69 antigen and the interleukin 2 receptor alpha-chain (CD25). Pervanadate synergized with signals delivered by T-cell antigen receptor engagement or by a phorbol ester to induce interleukin 2 production. Pervanadate activated NF-kappa B, as shown by an increase in DNA-binding activity of this transcription factor. We thus conclude that PTPases play a crucial role in the negative regulation of signal transduction culminating in T-lymphocyte activation. Moreover, induction of tyrosine phosphorylation appears sufficient per se to initiate a complete activation programme. 


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Relationship between IkappaBalpha constitutive expression, TNFalpha synthesis, and apoptosis in EBV-infected lymphoblastoid cells. 
In order to understand the role of NF-kappaB in EBV transformation we have established stably transfected IkappaBalpha into lymphoblastoid cells. Two clones were obtained in which the loss of NF-kappaB binding activity correlated with the constitutive expression of the transgenic IkappaBalpha. Protein latency expression was determined by immunocytochemistry. Expression of surface markers, intracytoplasmic content of cytokines cell cycle analysis after BrdU incorporation and DNA staining with propidium iodide were studied by flow cytometry. Percentage of apoptotic cells was determined by in-situ labelling of DNA strand breaks. No significative changes in EBV latency nor in cell surface marker expression was found. In contrast, intracytoplasmic TNFalpha levels were strongly reduced in transfected clones. Furthermore, 30% of IkappaBalpha transfected cells were apoptotic after 8 h of TNFalpha treatment. This correlated with a strong reduction of BrdU incorporation after 24 h of TNFalpha treatment. No effect was seen with non transfected cells or with cells transfected with a control plasmid. Our results suggest that the TNFalpha gene could be one of the targets of NF-kappaB in EBV infected cells and that NF-kappaB protects EBV-infected cells from apoptosis induced by TNFalpha, which may favour the proliferative effect of this cytokine. 


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High molecular weight dextran sulfate increases the activity of NF-kappaB-regulated promoter in monocyte-derived macrophages. 
It is known that sulfated polysaccharides can mimic the action of common T-cell mitogens. To investigate the molecular basis of the mitogenic effect of high molecular weight dextran sulfate (HMDS), monocyte-derived macrophages were transfected with recombinant plasmid containing chloramphenicol acetyl transferase (CAT) reporter gene under the control of the HIV-1 long terminal repeat (LTR) promoter, which is regulated by transcription factor NF-kappaB. We observed that HMDS, similar to bacterial lipopolysaccharide (LPS), increases the expression of CAT reporter gene suggesting increased activity of NF-kappaB. The activation of NF-kappaB correlated with the increased expression of B7.1 molecules. It was postulated that this NF-kappaB-regulated promoter might play a role in the activation of the accessory cells as well as the rate of replication of HIV-1 in monocyte-derived macrophages. 


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Monoclonal anti-endothelial cell antibodies from a patient with Takayasu arteritis activate endothelial cells from large vessels. 
OBJECTIVE: To create monoclonal anti-endothelial cell antibodies (mAECA) from a patient with Takayasu arteritis to evaluate their ability to activate human umbilical vein endothelial cells (HUVEC), and to characterize the mechanism of EC activation. METHODS: A panel of mAECA was generated from peripheral blood lymphocytes of a patient with Takayasu arteritis, using Epstein-Barr virus transformation. Activity against macrovascular EC (HUVEC) and microvascular EC (human bone marrow EC immortalized by SV40) antigens was detected by enzyme-linked immunosorbent assay. Inhibition studies were used to select the monoclonal antibodies (mAECA) which share the same EC epitope binding specificity as the total IgG-AECA from the Takayasu arteritis patient. The binding of the mAECA to human aortic EC was studied by immunohistochemistry. The secretion levels of interleukin-6 (IL-6) and von Willebrand factor (vWF) were determined, to serve as markers for EC activation. The activated EC were examined for the adherence of a monocytic cell line (U937), as well as for expression of vascular cell adhesion molecule 1, intercellular adhesion molecule 1, and E-selectin. In addition, nuclear extracts of the mAECA-treated EC were analyzed for the induction of translocation of nuclear factor kappaB (NF-kappaB), using a specific NF-kappaB oligoprobe in an electrophoretic mobility shift assay. RESULTS: Six mAECA were selected, the mixture of which produced 100% inhibition of binding of the original IgG (from the patient with Takayasu arteritis) to HUVEC. All mAECA possessed high activity against macrovascular EC, but none had significant antimicrovascular EC activity. The mAECA, but not normal human IgG, had anti-human aortic EC activity. Four of the 6 mAECA activated EC, manifested by increased IL-6 and vWF secretion. The 4 mAECA induced EC expression of adhesion molecules and increased adhesion of U937 monocytic cells to EC. In addition, these mAECA stimulated the nuclear translocation of the NF-kappaB transcription factor. CONCLUSION: Our findings suggest that AECA may directly stimulate EC in Takayasu arteritis through elevation of adhesion molecule expression associated with NF-kappaB activation and adhesion of monocytes, and may therefore play a pathogenic role in the development of the vasculopathy in Takayasu arteritis. 


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Possible differences in the mechanism(s) of action of different glucocorticoid hormone compounds. 
Different glucocorticoid hormones (GCH) show differences in the intensity and in the kinetics of their immunomodulating activity. The mechanism(s) of action of GCH is under investigation, but is has been noted that they exert immune activity via the genomic pathway. We have studied the effects of prednisone (PDN), deflazacort (DFC), and dexamethasone (DXM) on the production of cytokines (IL-2, IL-6, TNF-alpha, IL-10) by peripheral T lymphocytes, and the effects on the inhibition of NF-kB DNA binding activity by activated Jurkat cell line. The data obtained show that the three GCH molecules exert an immunosuppression on cytokine production by T lymphocytes and a strong decrease in the nuclear translocation of NF-kB in Jurkat cells; moreover, (a) not all the cytokines investigated were affected, and not with the same intensity, by the three GCH and (b) DXM inhibited the binding activity of NF-kB less than that of DFC and PDN. These data are in agreement with the concept that different GCH compounds might differ in their binding and affinity properties, tissue-specific metabolism, and interaction with transcription factor. 


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Abnormal NF-kappa B activity in T lymphocytes from patients with systemic lupus erythematosus is associated with decreased p65-RelA protein expression. 
Numerous cellular and biochemical abnormalities in immune regulation have been described in patients with systemic lupus erythematosus (SLE), including surface Ag receptor-initiated signaling events and lymphokine production. Because NF-kappa B contributes to the transcription of numerous inflammatory genes and has been shown to be a molecular target of antiinflammatory drugs, we sought to characterize the functional role of the NF-kappa B protein complex in lupus T cells. Freshly isolated T cells from lupus patients, rheumatoid arthritis (RA) patients, and normal individuals were activated physiologically via the TCR with anti-CD3 and anti-CD28 Abs to assess proximal membrane signaling, and with PMA and a calcium ionophore (A23187) to bypass membrane-mediated signaling events. We measured the NF-kappa B binding activity in nuclear extracts by gel shift analysis. When compared with normal cells, the activation of NF-kappa B activity in SLE patients was significantly decreased in SLE, but not in RA, patients. NF-kappa B binding activity was absent in several SLE patients who were not receiving any medication, including corticosteroids. Also, NF-kappa B activity remained absent in follow-up studies. In supershift experiments using specific Abs, we showed that, in the group of SLE patients who displayed undetectable NF-kappa B activity, p65 complexes were not formed. Finally, immunoblot analysis of nuclear extracts showed decreased or absent p65 protein levels. As p65 complexes are transcriptionally active in comparison to the p50 homodimer, this novel finding may provide insight on the origin of abnormal cytokine or other gene transcription in SLE patients. 


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cAMP-dependent regulation of proenkephalin by JunD and JunB: positive and negative effects of AP-1 proteins. 
We demonstrate that JunD, a component of the AP-1 transcription factor complex, activates transcription of the human proenkephalin gene in a fashion that is completely dependent upon the cAMP-dependent protein kinase, protein kinase A. Activation of proenkephalin transcription by JunD is dependent upon a previously characterized cAMP-, phorbol ester-, and Ca(2+)-inducible enhancer, and JunD is shown to bind the enhancer as a homodimer. Another component of the AP-1 transcription complex, JunB, is shown to inhibit activation mediated by JunD. As a homodimer JunB is unable to bind the enhancer; however in the presence of c-Fos, high-affinity binding is observed. Furthermore, JunD is shown to activate transcription of genes linked to both cAMP and phorbol ester response elements in a protein kinase A-dependent fashion, further blurring the distinction between these response elements. These results demonstrate that the transcriptional activity of an AP-1-related protein is regulated by the cAMP-dependent second-messenger pathway and suggest that JunD and other AP-1-related proteins may play an important role in the regulation of gene expression by cAMP-dependent intracellular signaling pathways. 


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Triggering of the human interleukin-6 gene by interferon-gamma and tumor necrosis factor-alpha in monocytic cells involves cooperation between interferon regulatory factor-1, NF kappa B, and Sp1 transcription factors. 
We investigated the molecular basis of the synergistic induction by interferon-gamma (IFN-gamma)/tumor necrosis factor-alpha (TNF-alpha) of human interleukin-6 (IL-6) gene in THP-1 monocytic cells, and compared it with the basis of this induction by lipopolysaccharide (LPS). Functional studies with IL-6 promoter demonstrated that three regions are the targets of the IFN-gamma and/or TNF-alpha action, whereas only one of these regions seemed to be implicated in LPS activation. The three regions concerned are: 1) a region between -73 and -36, which is the minimal element inducible by LPS or TNF-alpha; 2) an element located between -181 and -73, which appeared to regulate the response to IFN-gamma and TNF-alpha negatively; and 3) a distal element upstream of -224, which was inducible by IFN-gamma alone. LPS signaling was found to involve NF kappa B activation by the p50/p65 heterodimers. Synergistic induction of the IL-6 gene by IFN-gamma and TNF-alpha, in monocytic cells, involved cooperation between the IRF-1 and NF kappa B p65 homodimers with concomitant removal of the negative effect of the retinoblastoma control element present in the IL-6 promoter. This removal occurred by activation of the constitutive Sp1 factor, whose increased binding activity and phosphorylation were mediated by IFN-gamma. 


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The involvement of multiple tumor necrosis factor receptor (TNFR)-associated factors in the signaling mechanisms of receptor activator of NF-kappaB, a member of the TNFR superfamily. 
Receptor activator of NF-kappaB (RANK) is a recently identified member of the tumor necrosis factor receptor superfamily and is expressed on activated T cells and dendritic cells. Its cognate ligand (RANKL) plays significant roles in the activation of dendritic cell function and osteoclast differentiation. We demonstrate here the interaction of RANK with tumor necrosis factor receptor-associated factors (TRAFs) 1, 2, 3, 5, and 6 both in vitro and in cells. Mapping of the structural requirements for TRAF/RANK interaction revealed multiple TRAF binding sites clustered in two distinct domains in the RANK cytoplasmic tail. These TRAF binding domains were shown to be functionally important for the RANK-dependent induction of NF-kappaB and c-Jun NH2-terminal kinase activities. Site-directed mutagenesis demonstrated that these TRAF binding sites exhibited selective binding for different TRAF proteins. In particular, TRAF6 interacted with membrane-proximal determinants distinct from those binding TRAFs 1, 2, 3, and 5. When this membrane-proximal TRAF6 interaction domain was deleted, RANK-mediated NF-kappaB signaling was completely inhibited while c-Jun NH2-terminal kinase activation was partially inhibited. An NH2-terminal truncation mutant of TRAF6 inhibited RANKL-mediated NF-kappaB activation, but failed to affect constitutive signaling induced by receptor overexpression, revealing a selective role for TRAF6 in ligand-induced activation events. 


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Monocytic cell type-specific transcriptional induction of collagenase. 
Interstitial collagenase (MMP-1), a metalloproteinase produced by resident and inflammatory cells during connective tissue turnover, cleaves type I collagen fibrils. This catalytic event is rate limiting in remodeling of tissues rich in fibrillar collagen such as the skin and lungs. The regulation of collagenase expression is cell-type specific; bacterial LPS and zymosan, a yeast cell wall derivative, are potent inducers of collagenase expression in macrophages, but do not alter fibroblast collagenase expression. Since promoter elements controlling collagenase transcription in monocytic cells have not been previously defined, we sought to delineate responsive cis-acting elements of the collagenase promoter in transiently transfected human (U937) and murine (J774) monocytic cell lines. Deletion constructs containing as little as 72 bp of 5' -flanking sequence of the collagenase promoter were sufficient for LPS- or zymosan-mediated transcriptional induction, whereas phorbol inducibility exhibited an absolute requirement for upstream elements including the polyoma enhancer A-binding protein-3 site (-83 to -91) and TTCA sequence (-102 to -105) in both monocytic cells and fibroblasts. Mutagenesis of the activator protein-1 [AP-1] site at -72 abolished basal promoter activity and LPS/zymosan inducibility, while mutagenesis of an NF-kappaB-like site at -20 to -10 had no effect. Nuclear extracts from LPS- and zymosan-treated cells showed strong AP-1 activity by gel-shift analysis, and supershift analysis showed the AP-1 complexes contained specific members of both the jun and fos gene families. These data indicate that, in contrast to most LPS effects, AP-1, but not nuclear factor-kappaB, mediates LPS induction of collagenase transcription in macrophagelike cells. Furthermore, as compared to regulation by phorbol ester, collagenase induction in monocytic cells by cell wall derivatives of bacteria or yeast is largely independent of upstream promoter sequences. 


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CD30-dependent degradation of TRAF2: implications for negative regulation of TRAF signaling and the control of cell survival. 
CD30 is a cell-surface receptor that can augment lymphocyte activation and survival through its ability to induce the transcription factor NF-kappaB. CD30, however, has also been implicated in the induction of apoptotic cell death of lymphocytes. Here we show that one of the effects of CD30 signal transduction is to render cells sensitive to apoptosis induced by the type 1 tumor necrosis factor receptor (TNFR1). This sensitization is dependent on the TRAF-binding sites within the CD30 cytoplasmic domain. One of the proteins that binds to these sites is TRAF2, a signal transduction molecule that is also utilized by TNFR1 to mediate the activation of several downstream kinases and transcription factors. During CD30 signal transduction, we found that binding of TRAF2 to the cytoplasmic domain of CD30 results in the rapid depletion of TRAF2 and the associated protein TRAF1 by proteolysis. These data suggest a model in which CD30 limits its own ability to transduce cell survival signals through signal-coupled depletion of TRAF2. Depletion of intracellular TRAF2 and its coassociated proteins also increased the sensitivity of the cell to undergoing apoptosis during activation of death-inducing receptors such as TNFR1. Consistent with this hypothesis, expression of a dominant-negative form of TRAF2 was found to potentiate TNFR1-mediated death. These studies provide a potential mechanism through which CD30, as well as other TRAF-binding members of the TNFR superfamily, can negatively regulate cell survival. 


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Active suppression of the class II transactivator-encoding AIR-1 locus is responsible for the lack of major histocompatibility complex class II gene expression observed during differentiation from B cells to plasma cells. 
In this study the genetic control of major histocompatibility complex (MHC) class II gene expression during the transition from B cell to plasma cell has been analyzed. Class II molecules are not expressed in plasma cells because of an active suppression resulting in the abrogation of class II gene transcription. We show here that the plasma cell-specific repressor function, designated SIR (suppressor of immune response genes), does not act directly on the transcription of class II genes, but instead on the transcription of the AIR-1 gene, whose product, the class II transactivator (CIITA), is fundamental for the regulation of the constitutive and inducible expression of MHC class II genes. This was unambiguously demonstrated by the fact that plasmacytoma x B cell hybrids carrying an AIR-1 locus derived from CIITA-expressing cells do not express CIITA-specific transcripts. Transfection of a cDNA containing the human CIITA coding sequence under the control of an heterologous promoter restores expression of human MHC class II genes in the hybrids and is responsible for de novo expression of mouse MHC class II genes in both the mouse plasmacytoma cell line and the hybrids. These results confirm and extend the notion of the functional conservation of the AIR-1 gene product across species barriers. Interestingly, in CIITA-transfected cell hybrids, cell surface expression of the human HLA-DQ heterodimer was not observed. This result was not attributable to lack of HLA-DQ alpha or -DQ beta transcription, because both transcripts were present in the CIITA-transfected hybrids, although at reduced levels. These findings further support our previous observations on the distinct regulation of expression of the human HLA-DQ class II subset, which may be thus controlled at the posttranscriptional level by a CIITA-independent mechanism. 


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An isotype-specific activator of major histocompatibility complex (MHC) class II genes that is independent of class II transactivator. 
Patients with one type of major histocompatibility complex class II combined immunodeficiency have mutations in a gene termed class II transactivator (CIITA), which coordinately controls the transcription of the three major human class II genes, HLA-DR, -DQ, and -DP. However, the experimentally derived B-lymphoblastoid cell line, clone 13, expresses high levels of HLADQ in the absence of HLA-DR and HLA-DP, despite its mapping by complementation analysis to this group. It was possible that one of the clone 13 CIITA alleles bore a mutation that allowed HLA-DQ, but not HLA-DR or -DP transcription. Alternatively, another factor, distinct from CIITA, might control HLA-DQ expression. We report here that ectopic expression of CIITA cDNAs derived by reverse transcriptase polymerase chain reaction from clone 13 do not restore expression of HLA-DQ in another CIITA-deficient cell line, RJ2.2.5. In addition, no CIITA protein is detectable in clone 13 nuclear extracts. In contrast, somatic cell fusion between clone 13 and RJ2.2.5 restored expression of the HLA-DQ haplotype encoded by the RJ2.2.5 DQB gene. Taken together, these data demonstrate the existence of an HLA-DQ isotype-specific trans-acting factor, which functions independently of CIITA. 


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CIITA activates the expression of MHC class II genes in mouse T cells. 
It has long been a puzzle that MHC class II molecules are expressed in human T cells after activation but not in mouse T cells; this expression is believed to play a role in the cell mediated immune response. Recently the MHC class II transactivator (CIITA) has been reported to be a major regulatory factor for both the constitutive and IFN inducible expression of MHC class II genes. Here we show that human T cells expressing MHC class II have CIITA transcripts while MHC class II-negative human T cells and mouse T cells do not. The expression of MHC class II genes in mouse T cells can be reconstituted upon transfection with the human CIITA cDNA. These data indicate that the expression of CIITA explains the expression or lack of expression of MHC class II in human and mouse T cells respectively. 


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Membrane-associated lymphotoxin on natural killer cells activates endothelial cells via an NF-kappaB-dependent pathway. 
BACKGROUND: Inhibition of complement in small animal models of xenotransplantation has demonstrated graft infiltration with natural killer (NK) cells and monocytes associated with endothelial cell (EC) activation. We have previously demonstrated that human NK cells activate porcine EC in vitro, which results in adhesion molecule expression and cytokine secretion. In this study, we used the NK cell line NK92 to define the molecular and cellular basis of NK cell-mediated EC activation. METHODS: EC were transfected with either reporter constructs containing the luciferase gene driven either by E-selectin or interleukin (IL)-8 promoters or a synthetic NF-kappaB-dependent promoter. In addition, a dominant-negative mutant tumor necrosis factor receptor I (TNFRI) expression vector was co-transfected in inhibition studies. Forty-eight hours after transfection, EC were stimulated with NK cells or NK cell membrane extracts for 7 hr and activation was measured by a luciferase assay. RESULTS: Co-culture of NK cells with transfected EC enhanced E-selectin, IL-8, and NF-kappaB-dependent promoter activity. NK cell membrane extracts retained the capacity to activate EC and induced nuclear translocation of NF-kappaB (p50 and p65). Western blotting of NK cell and membrane extracts detected the presence of Lymphotoxin-alpha (LTalpha) but not tumor necrosis factor-alpha. Furthermore, LTalpha was secreted in NK:EC co-cultures. Co-transfection with dominant-negative mutant TNFRI inhibited EC activation by NK cell membrane extracts and by NK cells by 80% and 47%, respectively. The same pattern of inhibition was observed using anti-human LT sera. CONCLUSIONS: Human NK cell membrane-bound LT signals across species via TNFRI, leading to NF-kappaB nuclear translocation and transcription of E-selectin and IL-8, which results in EC activation. The discrepancy in the degree of inhibition by membrane extracts and NK cells with mutant TNFRI suggests that additional pathways are utilized by NK cells to activate EC. 


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NF-kappaB only partially mediates Epstein-Barr virus latent membrane protein 1 activation of B cells. 
The latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) is required for EBV-induced immortalization of human B cells and causes tumorigenic transformation of cell lines. LMP1 expression induces phenotypic changes resembling B cell activation, such as cell size increase and up-regulation of cell surface activation markers. LMP1 contains two domains that activate the transcription factor NF-kappaB, one through interactions with TRAF proteins and the other with the TRADD protein. The purpose of the present study was to investigate the importance of NF-kappaB induction in the up-regulation of the B cell activation markers ICAM-1 and CD71 by LMP1. This study shows that expression of LMP1 activates transcription from p50/p65- and c-Rel- responsive promoters, and that this activity can be completely inhibited by expression of a dominant inhibitory IkappaB mutant. ICAM-1 and CD71 are nevertheless up-regulated by LMP1 in primary B cells and cell lines expressing the dominant IkappaB. Furthermore, LMP1-induced cell size increase of primary B cells was unaffected by IkappaB expression. It was concluded that even when LMP1 is unable to activate NF-kappaB, it is still capable of inducing certain characteristics of activated B cells, strongly suggesting that LMP1 can also activate cells independently of NF-kappaB. 


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Cell-specific bifunctional role of Jun oncogene family members on glucocorticoid receptor-dependent transcription. 
Interaction between protein kinase C (PKC)- and glucocorticoid receptor (GR)-mediated signaling is suggested by the ability of the PKC activating phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) to inhibit GR-dependent transcription of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR). Here we report that this interference is cell specific, as TPA augmented dexamethasone-induced transcriptional activation of the MMTV LTR in several T cell lines but was inhibitory in NIH-3T3 fibroblasts. TPA-GR synergism was determined to have occurred at the GR-responsive element (GRE) level by functional analysis of deletion mutants or synthetic GRE oligonucleotides driving chloramphenicol acetyl-transferase expression. Synergism required an intact GR DNA-binding domain, whereas amino- or carboxyl-terminal domains were dispensable. The effect was abrogated by the PKC inhibitor staurosporine, suggesting a role for PKC. Increased c-jun, jun-B, and jun-D expression above basal levels and increased transcriptional activity of AP-1/TPA responsive elements fused to chloramphenicol acetyl-transferase vectors were observed in T cells treated with TPA alone or in combination with dexamethasone. The ability of Jun proteins to cooperate with GR in T cells has been investigated after transfection of c-jun, jun-B, or jun-D expression vectors, which augmented GR-dependent transcription from either MMTV LTR or GRE. Conversely, c-jun and jun-B transfection blunted GR-dependent transcription in HeLa cells. The presence of c-fos had a negative influence on GR function and correlated with the cell-specific synergistic or antagonistic activity of Jun with respect to GR; high basal expression of c-fos as well as AP-1 DNA binding and transcriptional activity were observed in HeLa cells, but not in T cells. Furthermore overexpression of exogenous c-fos has an inhibitory effect on GR-dependent transcription from GRE in T cells. We propose that Jun plays a bifunctional role on GR-dependent transcriptional activation of GRE, selecting either synergistic or antagonistic activity depending on the cell-specific microenvironment. In this regard, intracellular levels of c-fos appear to be influential. 


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Effects of overexpression of IL-1 receptor-associated kinase on NFkappaB activation, IL-2 production and stress-activated protein kinases in the murine T cell line EL4. 
The association and activation of the IL-1 receptor-associated protein kinase (IRAK) to the IL-1 receptor complex is one of the earliest events detectable in IL-1 signal transduction. We generated permanent clones of the murine T cell line EL4 6.1 overexpressing human (h)IRAK to evaluate the role of this kinase in IL-1 signaling. Overexpression of hIRAK enhanced IL-1-stimulated activation of the transcription factor NFkappaB, whereas a truncated form (N-IRAK) specifically inhibited IL-1-dependent NFkappaB activity. In clones stably overexpressing hIRAK a weak constitutive activation of NFkappaB correlated with a low basal IL-2 production which was enhanced in an IL-1-dependent manner. Compared to the parental cell line the dose-response curve of IL-1-induced IL-2 production was shifted in both potency and efficacy. These results demonstrate that IRAK directly triggers NFkappaB-mediated gene expression in EL4 cells. Qualitatively different effects were observed for the IL-1-induced activation of stress-activated protein (SAP) kinases: permanent overexpression of IRAK did not affect the dose dependence but prolonged the kinetics of IL-1-induced activation of SAP kinases, suggesting that this signaling branch may be regulated by distinct mechanisms. 


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Inhibition of p105 processing by NF-kappaB proteins in transiently transfected cells. 
Regulation of the transcription factor NF-kappaB involves proteasome-mediated processing of the NF-kappaB1 p105 precursor protein, which generates the p50 subunit of NF-kappaB. The processing of p105 occurs constitutively in vivo but can be markedly enhanced by various cellular activation agents, although the underlying regulatory mechanism is not yet clear. In the present study, we demonstrate that signal-mediated induction of p105 processing in human T cells is associated with de novo synthesis of this precursor protein. Transient transfection studies performed in COS7 cells revealed that the newly synthesized p105 protein appears to be more rapidly processed compared to its accumulated form that is already associated with the processed product p50. Interestingly, the processing rate of p105 is markedly inhibited in cells co-transfected with p50 or other NF-kappaB subunits, including RelA and c-Rel, that physically interact with p105. These findings suggest that the processing of p105 is subject to negative regulation by the various NF-kappaB subunits. We further demonstrate that p105 undergoes degradation in lipopolysaccharide-stimulated human monocytic cells. However, the inducible degradation of p105 is not coupled with the generation of p50. Together, these studies demonstrate that the processing and inducible degradation of p105 are differentially regulated. 


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The role of jun and fos gene family members in 12-O-tetradecanoylphorbol-13-acetate induced hemopoietic differentiation. 
Terminal differentiation of the leukemic cell lines U-937 and HL-60 by 12-O-tetradecanoylphorbol-13-acetate is accompanied by marked changes in gene expression. In this study, we demonstrate that the expression of jun and fos gene family members is induced with variable kinetics during 12-O-tetradecanoylphorbol-13-acetate induced differentiation, with c-jun expression best paralleling differentiation. The generation of AP-1 complexes, as measured by DNA binding activity, closely parallels morphological differentiation. Furthermore, the ability of these complexes to regulate gene expression is demonstrated by increased transcription from an AP-1 driven reporter construct and marked increases in the expression of endogenous AP-1 regulated genes. Differentiation assays using water soluble phorbol esters reveal that differentiation becomes irreversible soon after AP-1 appears. This tight correlation between c-jun expression, the generation of AP-1 activity, and differentiation suggests a critical role for this gene and transcriptional complex during this process. 


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Calcineurin activates transcription from the GM-CSF promoter in synergy with either protein kinase C or NF-kappa B/AP-1 in T cells. 
Two cis-acting elements GM-kappa B/GC-box and CLE0, of the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene are required for maximal induction in Jurkat T cells by costimulation with phorbol-12-myristate acetate (PMA) and Ca2+ ionophore (A23187). The GM-kappa B sequence is recognized by NF-kappa B, which is mainly induced by PMA. The CLE0 sequence interacts with factors, related to a PMA-induced AP-1 and a PMA/A23187-induced NF-AT. We examined whether signal transducing components in T cells can activate transcription of the GM-CSF gene. Cotransfection of NF-kappa B (p50/p65)- or AP-1 (c-Jun/c-Fos)- expression vectors into Jurkat cells with a luciferase reporter containing the GM-CSF promoter did not stimulate transcription from the GM-CSF promoter. In contrast, cotransfection with a combination of NF-kappa B and AP-1 significantly augmented transcription from the GM-CSF promoter containing the GM-kappa B/GC-box and the CLE0 (AP-1/NF-AT). Expression of a constitutively active calcineurin (CN), a Ca2+/calmodulin-dependent protein phosphatase, potentiated by two fold the transcriptional activation by NF-kappa B/AP-1. Both constitutively active forms of CN and protein kinase C (PKC) synergistically activated transcription from the GM-CSF promoter. These results suggest that cooperation among NF-kappa B-, AP-1- and NF-AT-binding sequences is required for induction of the GM-CSF gene through PKC- and Ca2+- signaling pathways downstream of T cell activation. 


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Stimulation of CD40 on immunogenic human malignant melanomas augments their cytotoxic T lymphocyte-mediated lysis and induces apoptosis. 
Here, we report the functional expression of CD40 on human malignant melanomas (MMs). Comparison of tumor specimen from MM precursor lesions, primary tumors, and metastases revealed that CD40 surface expression is down-regulated during tumor progression. CD40 expression was confirmed in 7 human MM cell lines established from immunogenic primary tumors or metastases, whereas 11 cell lines established from advanced stages were CD40 negative. CD40 expression could be enhanced in CD40-positive MM by stimulation with IFN-gamma and tumor necrosis factor-alpha but not by interleukin (IL)-1beta or CD40 triggering. CD40 ligation on MM by CD40L-transfected murine L-cells or by a soluble CD40L fusion protein up-regulated their expression of intercellular adhesion molecule-1 and MHC class I and class II molecules and their secretion of IL-6, IL-8, tumor necrosis factor-a, and granulocyte macrophage colony-stimulating factor and also induced a rapid activation of the transcription factor nuclear factor kappaB. Furthermore, CD40 ligation of a HLA-A2+, MelanA/MART1+ MM cell line enhanced its susceptibility to specific lysis by a HLA-A2-restricted, MelanA/MART-1-specific CTL clone. Finally, CD40 ligation induced growth inhibition and apoptosis in MM. These results indicate that CD40-CD40L interactions may play an important role in augmenting antitumor immunity and inducing apoptosis in some CD40-positive immunogenic human MMs. 


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Involvement of adenylate cyclase and p70(S6)-kinase activation in IL-10 up-regulation in human monocytes by gp41 envelope protein of human immunodeficiency virus type 1. 
Our previous results show that recombinant gp41 (aa565-647), the extracellular domain of HIV-1 transmembrane glycoprotein, stimulates interleukin-10 (IL-10) production in human monocytes. The signal cascade transducing this effect is not yet clear. In this study, we examined whether gp41-induced IL-10 up-regulation is mediated by the previously described synergistic activation of cAMP and NF-kappaB pathways. gp41 induced cAMP accumulation in monocytes in a time- and concentration-dependent manner and the adenylate cyclase inhibitor SQ 22536 suppressed gp41-induced IL-10 production in monocytes. In contrast, gp41 failed to stimulate NF-kappaB binding activity in as much as no NF-kappaB bound to the main NF-kappaB-binding site 2 of the IL-10 promoter after addition of gp41. We also examined the involvement of other signal transduction pathways. Specific inhibitors of p70(S6)-kinase (rapamycin), and Gi protein (pertussis toxin), prevented induction of IL-10 production by gp41 in monocytes, while inhibitors of the phosphatidylinositol 3-kinase (PI 3-kinase) (wortmannin) and mitogen-activated protein kinase (MAPK) pathway (PD 98059) did not. Thus HIV-1 gp41-induced IL-10 up-regulation in monocytes may not involve NF-kappaB, MAPK, or PI 3-kinase activation, but rather may operate through activation of adenylate cyclase and pertussis-toxin-sensitive Gi/Go protein to effect p70(S6)-kinase activation. 


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Characterization of a cofactor that regulates dimerization of a mammalian homeodomain protein. 
Dimerization among transcription factors has become a recurrent theme in the regulation of eukaryotic gene expression. Hepatocyte nuclear factor-1 alpha (HNF-1 alpha) is a homeodomain-containing protein that functions as a dimer. A dimerization cofactor of HNF-1 alpha (DCoH) was identified that displayed a restricted tissue distribution and did not bind to DNA, but, rather, selectively stabilized HNF-1 alpha dimers. The formation of a stable tetrameric DCoH-HNF-1 alpha complex, which required the dimerization domain of HNF-1 alpha, did not change the DNA binding characteristics of HNF-1 alpha, but enhanced its transcriptional activity. However, DCoH did not confer transcriptional activation to the GAL4 DNA binding domain. These results indicate that DCoH regulates formation of transcriptionally active tetrameric complexes and may contribute to the developmental specificity of the complex. 


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Inhibitory effect of E3330, a novel quinone derivative able to suppress tumor necrosis factor-alpha generation, on activation of nuclear factor-kappa B. 
(2E)-3-[5-(2,3-Dimethoxy-6-methyl-1,4-benzoquinoyl)]-2-nonyl-2- propenoic acid (E3330), is a novel agent with hepatoprotective activity. We report the effect of E3330 on transcriptional activation of tumor necrosis factor (TNF)-alpha gene and on nuclear factor (NF)-kappa B activation. Nuclear run-on experiments showed that E3330 decreases transcriptional activation of TNF-alpha gene induced by lipopolysaccharide (LPS) stimulation in human peripheral monocytes. To investigate the inhibitory mechanisms, we constructed a secreted-type placental alkaline phosphatase (PLAP) reporter gene whose transcription is controlled by a 1.4-kb human TNF-alpha promoter. A stable transformant of the PLAP reporter gene derived from human monocytic cell line showed very little activity on the promoter before stimulation, whereas LPS stimulation led to a dramatic increase in PLAP activity. E3330 inhibited this induced promoter activity in a dose-dependent manner. There are four putative NF-kappa B binding sites (kappa B-1, kappa B-2, kappa B-3, kappa B-4) in human TNF-alpha promoter. By using mutated promoter-PLAP plasmids, we established that these NF-kappa B sites were necessary for induction of TNF-alpha transcription on stimulation with LPS. A gel retardation experiment with synthetic double-stranded oligonucleotides showed that activated NF-kappa B consisting of p50/p65 heterodimer bound to all four putative NF-kappa B DNA probes, suggesting that all four putative NF-kappa B recognition sites play an important role in inducible TNF-alpha expression. E3330 decreased activated NF-kappa B in nuclei, suggesting that E3330 inhibits NF-kappa B activation and/or translocation of the nuclei. Western blotting analysis with anti-I kappa B-alpha antibody indicated that E3330 inhibited degradation of I kappa B-alpha, which is an inhibitory protein of NF-kappa B, in LPS-stimulated monocytes. E3330 may suppress the production of active oxygen species serving as common messengers to activate NF-kappa B. 


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Activation of the interleukin 6 gene by Mycobacterium tuberculosis or lipopolysaccharide is mediated by nuclear factors NF-IL6 and NF-kappa B [published erratum appears in Proc Natl Acad Sci U S A 1995 Apr 11;92(8):3632] 
The host response to Mycobacterium tuberculosis includes granuloma formation at sites of infection and systemic symptoms. Cytokines have been identified by immunohistochemistry in granulomas in animal models of bacillus Calmette-Guerin (BCG) infection and are released by mononuclear phagocytes upon stimulation by mycobacterial proteins. In this regard, the cytokine interleukin 6 (IL-6) may play a role in the clinical manifestations and pathological events of tuberculosis infection. We have demonstrated that lipoarabinomannan (LAM) from the mycobacterial cell wall, which was virtually devoid of lipopolysaccharide (LPS), stimulated mononuclear phagocytes to release IL-6 in a dose-response manner. LAM and LPS were potent inducers of IL-6 gene expression in peripheral blood monocytes. Both LAM- and LPS-inducible IL-6 promoter activity was localized to a DNA fragment, positions -158 to -49, by deletion analysis and chloramphenicol acetyltransferase assay. Two nuclear factor NF-IL6 (positions -153 to -145 and -83 to -75) and one nuclear factor NF-kappa B (positions -72 to -63) motifs are present within this fragment. Site-directed mutagenesis of one or more of these motifs within the IL-6 promoter demonstrated that each has positive regulatory activity and that they could act in a function- and orientation-independent manner. Deletion of all three elements abolished inducibility of IL-6 promoter activity by both LAM and LPS. We conclude that the NF-IL6 and NF-kappa B sites mediate IL-6 induction in response to both LPS and LAM, acting as bacterial or mycobacterial response elements. 


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The class II trans-activator CIITA interacts with the TBP-associated factor TAFII32. 
The class II trans- activator (CIITA) is the main transcriptional co-activator for the expression of MHC class II proteins. Its N-terminal 125 amino acids function as an independent transcriptional activation domain. Analyses of the primary amino acid sequence of the activation domain predict the presence of three alpha-helices, each with a high proportion of acidic residues. Using site-directed mutagenesis, we found that two of these predicted alpha-helices are required for full transcriptional activation by CIITA. Moreover, a CIITA protein in which both functional alpha-helices have been deleted displays a dominant negative phenotype. This activation domain of CIITA interacts with the 32 kDa subunit of the general transcription complex TFIID, TAFII32. Decreased transcriptional activation by N-terminal deletions of CIITA is correlated directly with their reduced binding to TAFII32. We conclude that interactions between TAFII32 and CIITA are responsible for activation of class II genes. 


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Transcriptional regulation of the interleukin-2 gene in normal human peripheral blood T cells. Convergence of costimulatory signals and differences from transformed T cells. 
To study transcriptional regulation in normal human T cells, we have optimized conditions for transient transfection. Interleukin-2 (IL-2) promoter-reporter gene behavior closely parallels the endogenous gene in response to T cell receptor and costimulatory signals. As assessed with mutagenized promoters, the most important IL-2 cis-regulatory elements in normal T cells are the proximal AP-1 site and the NF- kappaB site. Both primary activation, with phytohemagglutinin or antibodies to CD3, and costimulation, provided by pairs of CD2 antibodies or B7-positive (B cells) or B7-negative (endothelial) accessory cells, are mediated through the same cis-elements. Interestingly, the nuclear factor of activated T cell sites are much less important in normal T cells than in Jurkat T cells. We conclude that IL-2 transcriptional regulation differs in tumor cell lines compared with normal T cells and that different costimulatory signals converge on the same cis-elements in the IL-2 promoter. 


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Interleukin 10 induced c-fos expression in human B cells by activation of divergent protein kinases. 
IL-10 is a potent mediator of human B cell growth and plasma cell formation. However, signal transduction of IL-10 in B cells is poorly understood. In this study the effect of IL-10 on the expression of the protooncogene c-fos was investigated, because Fos plays a potential role in the regulation of B cell proliferation and differentiation. B cells were purified from buffy coat preparations of healthy blood donors by positive selection using an anti CD20 monoclonal antibody and a MiniMACS separation unit. B cells were prestimulated with SAC for 48 hrs. Then, cells were incubated with medium or IL-10 (100 ng/ml) for 10 to 120 min. RNA was extracted by phenol/chloroform and c-fos expression was analyzed by PCR assisted mRNA assay. A significant 2-4 fold increase of c-fos expression was observed within 30 min of stimulation with IL-10 (p < 0.01). After 2 hrs c-fos expression declined to basal levels. The effect of IL-10 was dose-dependent with a maximum stimulation using 100 ng/ml of IL-10. The IL-10 effect on c-fos expression was not blocked by polymyxin B. Using the tyrosine kinase inhibitor genistein (10 microM) a complete inhibition of IL-10 induced c-fos expression was observed. In addition, H-7 (10 microM), a specific inhibitor of serine/threonine kinases, significantly blocked IL-10 mediated c-fos expression (p < 0.05). In conclusion, these data show that IL-10 induces c-fos expression in human B-cells by activation of tyrosine and serine/threonine kinases. Since this is the first report on IL-10 induced signal transduction, these data may help to identify the intracellular mechanisms by which IL-10 stimulates human B-cells. 


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GM-CSF and IL-2 share common control mechanisms in response to costimulatory signals in T cells. 
Antigen complexed with major histocompatibility complex class I or II molecules on the surface of antigen presenting cells interacts with the T cell receptor (TCR) on the surface of T cells and initiates an activation cascade. So called costimulatory signals, mediated by other cell surface interactions or soluble cytokines produced by antigen presenting cells, are also required for complete T cell activation. High levels of cytokine gene expression in T cells also required both TCR and costimulatory signals. The granulocyte-macrophage colony-stimulating factor requires sequences in the promoter as well as a powerful enhancer located 3kb upstream to respond to TCR-like signals. These promoter and enhancer regions are mainly activated by the transcription factor nuclear factor of activated T cells (NFAT). The activation of NFAT by TCR signals has been well described for interleukin-2 (IL-2) and IL-4 gene transcription in T cells. Costimulatory signals, such as activation of the CD28 cell surface molecule on T cells, lead to activation through a distinct region of the granulocyte-macrophage colony-stimulating factor (GM-CSF) promoter. This region is termed the CK-1 or CD28RE and appears to bind specific members of the NF-kappa B family of transcription factors. Human T leukemia virus type 1 (HTLV-1) infects T cells and can lead to increase GM-CSF expression. We have found that the HTLV-1 transactivator protein, tax, acts as a costimulatory signal for GM-CSF and IL-2 gene transcription, in that it can cooperate with TCR signals to mediate high level gene expression. Tax activates the GM-CSF promoter through the CK-1/CD28RE region and also activates nuclear factor-kappa B binding to this region. However, other transcription factors or coactivators of NF-kappa B are required for tax activation but these remain to be identified. The CK-1/CD28RE of GM-CSF shows a high degree of similarity to the IL-2 CD28RE and the IL-3 gene also contains a related region. This observation, together with the fact that both GM-CSF and IL-2 respond to TCR signals via NFAT, implies a high degree of conservation in the regulation of cytokine gene expression in T cells. 


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IL-7 reconstitutes multiple aspects of v-Abl-mediated signaling. 
The mechanism by which early lymphoid cells are selectively transformed by v-Abl is currently unknown. Previous studies have shown constitutive activation of IL-4 and IL-7 signaling pathways, as measured by activation of Janus protein kinase (JAK)1, JAK3, STAT5, and STAT6, in pre-B cells transformed by v-Abl. To determine whether activation of these cytokine signaling pathways by v-Abl is important in the cellular events induced by the Abelson murine leukemia virus, the effects of IL-4 and IL-7 on pre-B cells transformed with a temperature-sensitive v-Abl mutant were examined. Whereas IL-4 had little or no effect, IL-7 delayed both the apoptosis and cell cycle arrest that occur upon v-Abl kinase inactivation. IL-7 also delayed the decreases in the levels of c-Myc, Bcl-2, and Bcl-xL that occur upon loss of v-Abl kinase activity. IL-7 did not maintain v-Abl-mediated differentiation arrest of the pre-B cells, as activation of NF-kappaB and RAG gene transcription was unaffected by IL-7. These results identify a potential role for IL-7 signaling pathways in transformation by v-Abl while demonstrating that a combination of IL-4 and IL-7 signaling cannot substitute for an active v-Abl kinase in transformed pre-B cells. 


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Constitutive activation of NF-kappaB in primary adult T-cell leukemia cells. 
Human T-cell leukemia virus type I (HTLV-I) is an etiologic agent of adult T-cell leukemia (ATL). The viral protein Tax induces the activation and nuclear translocalization of transcription factor NF-kappaB, which is proposed to play a crucial role in the transformation of T cells by HTLV-I. However, the HTLV-I genes including Tax are not expressed significantly in primary leukemic cells from ATL patients. In this study, we examined the basis for NF-kappaB activation in freshly isolated leukemic cells from ATL patients. We found that leukemic cells from ATL patients, like HTLV-I-infected T-cell lines, display constitutive NF-kappaB DNA binding activity and increased degradation of IkappaBalpha (an inhibitor of NF-kappaB). Whereas the NF-kappaB binding activity in Tax-expressing T-cell lines consisted mostly of p50/c-Rel, fresh ATL samples contained p50/p50 and p50/p65 heterodimers. One T-cell line derived from ATL leukemic cells, TL-Om1, displayed constitutive NF-kappaB activity, as well as enhanced degradation of IkappaBalpha, despite the lack of detectable Tax expression. Interestingly, the NF-kappaB in TL-Om1 consists of p50/p50 and p50/p65 like that in fresh primary leukemic cells. Our results suggest that activation of NF-kappaB occurs through a Tax-independent mechanism in leukemic cells of ATL patients, possibly due to differential NF-kappaB subunit activation. 


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Ligand-dependent repression of the erythroid transcription factor GATA-1 by the estrogen receptor. 
High-dose estrogen administration induces anemia in mammals. In chickens, estrogens stimulate outgrowth of bone marrow-derived erythroid progenitor cells and delay their maturation. This delay is associated with down-regulation of many erythroid cell-specific genes, including alpha- and beta-globin, band 3, band 4.1, and the erythroid cell-specific histone H5. We show here that estrogens also reduce the number of erythroid progenitor cells in primary human bone marrow cultures. To address potential mechanisms by which estrogens suppress erythropoiesis, we have examined their effects on GATA-1, an erythroid transcription factor that participates in the regulation of the majority of erythroid cell-specific genes and is necessary for full maturation of erythrocytes. We demonstrate that the transcriptional activity of GATA-1 is strongly repressed by the estrogen receptor (ER) in a ligand-dependent manner and that this repression is reversible in the presence of 4-hydroxytamoxifen. ER-mediated repression of GATA-1 activity occurs on an artificial promoter containing a single GATA-binding site, as well as in the context of an intact promoter which is normally regulated by GATA-1. GATA-1 and ER bind to each other in vitro in the absence of DNA. In coimmunoprecipitation experiments using transfected COS cells, GATA-1 and ER associate in a ligand-dependent manner. Mapping experiments indicate that GATA-1 and the ER form at least two contacts, which involve the finger region and the N-terminal activation domain of GATA-1. We speculate that estrogens exert effects on erythropoiesis by modulating GATA-1 activity through protein-protein interaction with the ER. (ABSTRACT TRUNCATED AT 250 WORDS) 


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HMG-I binds to GATA motifs: implications for an HPFH syndrome. 
We have examined binding of the nuclear protein HMG-I to the human gamma-globin promoter. We find that HMG-I binds preferentially to the more 3' of a pair of GATA motifs in the gamma-globin promoter; this paired motif is bound by the erythroid factor GATA-1. A naturally occurring mutation (-175 T-C) in the area bound by HMG-I results in overexpression of gamma-globin in adult red blood cells (HPFH) and up-regulation of the gamma-globin promoter in in vitro expression assays; HMG-I does not bind to this mutant sequence. A survey of GATA motifs from other globin cis-elements demonstrates HMG-I binding to most of them. These findings implicate HMG-I in the HPFH phenotype; we speculate that it may participate in the formation of multiprotein complexes that regulate globin gene expression. 


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A nongenomic mechanism for progesterone-mediated immunosuppression: inhibition of K+ channels, Ca2+ signaling, and gene expression in T lymphocytes. 
The mechanism by which progesterone causes localized suppression of the immune response during pregnancy has remained elusive. Using human T lymphocytes and T cell lines, we show that progesterone, at concentrations found in the placenta, rapidly and reversibly blocks voltage-gated and calcium-activated K+ channels (KV and KCa, respectively), resulting in depolarization of the membrane potential. As a result, Ca2+ signaling and nuclear factor of activated T cells (NF-AT)-driven gene expression are inhibited. Progesterone acts distally to the initial steps of T cell receptor (TCR)-mediated signal transduction, since it blocks sustained Ca2+ signals after thapsigargin stimulation, as well as oscillatory Ca2+ signals, but not the Ca2+ transient after TCR stimulation. K+ channel blockade by progesterone is specific; other steroid hormones had little or no effect, although the progesterone antagonist RU 486 also blocked KV and KCa channels. Progesterone effectively blocked a broad spectrum of K+ channels, reducing both Kv1.3 and charybdotoxin-resistant components of KV current and KCa current in T cells, as well as blocking several cloned KV channels expressed in cell lines. Progesterone had little or no effect on a cloned voltage-gated Na+ channel, an inward rectifier K+ channel, or on lymphocyte Ca2+ and Cl- channels. We propose that direct inhibition of K+ channels in T cells by progesterone contributes to progesterone-induced immunosuppression. 


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Involvement of Rel, Fos, and Jun proteins in binding activity to the IL-2 promoter CD28 response element/AP-1 sequence in human T cells. 
CD28 is an important costimulatory molecule in the activation of human T cells. Costimulation of T cells through both the Ag receptor and CD28 leads to high level IL-2 production, which is vital to the development of an immune response in vivo. Previous reports have suggested the CD28 stimulation contributes to the activation of the IL-2 promoter by up-regulating the activity of several transcription factors, including AP-1 and nuclear factor-kappaB (NF-kappaB)/Rel family members as well as an uncharacterized transcription factor called CD28 response complex. While several lines of investigation have suggested that NF-kappaB/Rel family members make up the CD28 response complex transcription factor, other work has not supported this conclusion. Recent studies suggest that the CD28 response element (CD28RE) does not function independently but works instead in conjunction with the adjacent promoter proximal AP-1-binding site and this hypothesis is confirmed here. Also in the current study, binding activity to the CD28RE/AP-1 sequence of the IL-2 promoter is evaluated. Although four specific complexes can be detected binding to this sequence, only one of these complexes is specific for both the CD28RE and the adjacent AP-1 site. Of the NF-kappaB/Rel family members tested, this CD28RE/AP-1-specific complex contains predominantly c-Rel, despite the fact that both p50 and RelA can efficiently bind to the CD28RE. c-Fos and c-Jun are also found in this CD28RE/AP-1-specific complex. These data indicate that functional complexes encompassing both the CD28RE and the AP-1-binding sites influence IL-2 promoter activity in CD28-costimulated T cells. 


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Monocyte tethering by P-selectin regulates monocyte chemotactic protein-1 and tumor necrosis factor-alpha secretion. Signal integration and NF-kappa B translocation [see comments] 
Adhesion molecules that tether circulating leukocytes to endothelial cells may also transduce or modulate outside-in signals for cellular activation, providing an initial regulatory point in the inflammatory response. Adhesion of human monocytes to P-selectin, the most rapidly expressed endothelial tethering factor, increased the secretion of monocyte chemotactic protein-1 (MCP-1) and tumor necrosis factor-alpha (TNF-alpha) by the leukocytes when they were stimulated with platelet-activating factor. Increased cytokine secretion was specifically inhibited by G1, an anti-P-selectin mAb that prevents P-selectin from binding to its ligand (P-selectin glycoprotein ligand-1) on myeloid cells. Moreover, tethering by P-selectin specifically enhanced nuclear translocation of nuclear factor-kappa B (NF-kappa B), a transcription factor required for expression of MCP-1, TNF-alpha, and other immediate-early genes. These results demonstrate that P-selectin, through its ligands on monocytes, may locally regulate cytokine secretion in inflamed tissues. 


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Signal transduction abnormalities in T lymphocytes from patients with advanced renal carcinoma: clinical relevance and effects of cytokine therapy. 
Studies have demonstrated abnormalities of the CD3/T-cell antigen receptor (TCR) and pathways of signal transduction in T lymphocytes from animals and patients with advanced malignancy. Diminished expression of TCRzeta and p56(lck) that are associated with the TCR and reduced nuclear localization of RelA containing nuclear factor kappaB (NFkappaB) complexes have been noted. These defects have been described in T cells from patients with malignant melanoma, renal cell carcinoma (RCC), ovarian cancer, and colorectal cancer. Preliminary observations also indicate possible correlation with clinical variables such as stage in selected instances. To further characterize altered expression of TCRzeta, p56(lck), and impaired activation of NFkappaB, T lymphocytes were obtained from 65 patients with RCC, the majority of whom were receiving combination cytokine therapy [interleukin (IL)-2, IFN alpha-containing regimens] and 37 control individuals. In 29 of these patients, levels of TCRzeta and p56(lck) were determined by Western blots of T-cell lysates and semiquantitated using densitometry. Relative levels were then correlated with a series of clinical variables including response to therapy, performance status, survival, disease sites, age, and others. In another group of 28 patients (three individuals from the first group), the frequency of abnormal NFkappaB activation was studied using electrophoretic mobility shift assays after activation of T cells with phorbol myristate acetate/ionomycin or anti-CD3 monoclonal antibody. Changes in these signaling molecules during cytokine treatment were also investigated. TCRzeta and p56(lck) were detected in the peripheral blood T cells in 27 of 29 patients, and overall, reduced levels were noted visually in 12 of 29 (41%) and 13 of 29 (45%) individuals, respectively. When levels were semiquantitated using densitometry, significant decreases of TCRzeta (P = 0.029) and p56(lck) (P = 0.029) but not CD3epsilon (P = 0.131), compared with control levels, were found. In patients treated with IL-2/IFN alpha-based therapy, relative levels of TCRzeta increased significantly (P = 0.002) on day 15 of cycle one compared with the baseline. Correlations of TCRzeta or p56(lck) levels with response or disease variables, except for lower TCRzeta levels (P < 0.001) in the presence of bone metastases, were not found. Abnormal NFkappaB activation after stimulation with phorbol myristate acetate/ionomycin and/or anti-CD3 monoclonal antibody was found in 59% of patients (17 of 28) and was not accounted for by the advanced age of the study cohort. Activation of NFkappaB in peripheral blood T cells was inducible during cytokine therapy in four of six individuals who displayed impaired NFkappaB activity prior to therapy. Moreover, impaired activation of NFkappaB does not appear linked to a reduction of TCRzeta expression, because in five patients, normal TCRzeta levels were present although kappaB binding was not inducible. In the majority of patients with advanced RCC, peripheral blood T cells express TCRzeta and p56(lck), and in a subset, reduced levels of these TCRzeta associated molecules are seen that may increase during cytokine-based therapy. Abnormal activation of NFkappaB is also present in >50% of patients and may also revert to normal during IL-2/IFN alpha-based treatment. This alteration in NFkappaB activation occurred in the presence of normal expression of TCRzeta-associated signaling elements. The clinical significance of these findings remains unclear. 


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Activation of protein kinase C and elevation of cAMP interact synergistically to raise c-Fos and AP-1 activity in Jurkat cells. 
We have earlier found that in Jurkat cells activation of protein kinase C (PKC) enhances the cyclic adenosine monophosphate (cAMP) accumulation induced by adenosine receptor stimulation or activation of Gs. Here we have therefore examined the effect of the phorbol ester PMA (phorbol 12-myristate 13-acetate) which stimulates PKC and a combination of the adenosine receptor agonist NECA (5'-(N-ethyl)-carboxamido adenosine) and forskolin to raise cAMP, on the levels of c-Fos and Jun and on the binding and transcriptional activity of the transcription factor, activator protein-1 (AP-1). PMA treatment caused a concentration- and time-dependent increase in both c-Fos and Jun immunoreactivity in contrast to cAMP elevation that had only a slight effect. Both PMA and the combination of NECA and forskolin acted together either to increase (c-Fos) or decrease (Jun) protein levels as well as increasing AP-1 binding, as judged by gel-shift assay, and AP-1 transcriptional activity. Furthermore there was a clear-cut synergy between the PKC stimulator and the cAMP elevating agents. The results demonstrate that the simultaneous activation of PKC and elevation of cAMP leads to an enhanced AP-1 transcriptional activity in a T-leukemia cell line, suggesting that the previously observed interaction between the parallel signal transduction pathways may have functional consequences at the level of gene transcription. 


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Differential effects of lipopolysaccharide and tumor necrosis factor on monocytic IkappaB kinase signalsome activation and IkappaB proteolysis. 
The inflammatory mediators lipopolysaccharide (LPS) and tumor necrosis factor (TNF) are potent activators of NF-kappaB. This study compared the effect of these stimuli on endogenous IkappaB kinase (IKK) signalsome activation and IkappaB phosphorylation/proteolysis in human monocytic cells and investigated the role of the signalsome proteins IKK-alpha, IKK-beta, NF-kappaB-inducing kinase (NIK), IKK-gamma (NF-kappaB essential modulator), and IKK complex-associated protein. Kinase assays showed that TNF elicited a rapid but short-lived induction of IKK activity with a 3-fold greater effect on IKK-alpha than on IKK-beta, peaking at 5 min. In contrast, LPS predominantly stimulated IKK-beta activity, which slowly increased, peaking at 30 min. A second peak was observed at a later time point following LPS stimulation, which consisted of both IKK-alpha and -beta activity. The endogenous levels of the signalsome components were unaffected by stimulation. Furthermore, our studies showed association of the IKK-alpha/beta heterodimer with NIK, IkappaB-alpha and -epsilon in unstimulated cells. Exposure to LPS or TNF led to differential patterns of IkappaB-alpha and IkappaB-epsilon disappearance from and reassembly with the signalsome, whereas IKK-alpha, IKK-beta, and NIK remained complex-associated. NIK cannot phosphorylate IkappaB-alpha directly, but it appears to be a functionally important subunit, because mutated NIK inhibited stimulus-induced kappaB-dependent transcription more effectively than mutated IKK-alpha or -beta. Overexpression of IKK complex-associated protein inhibited stimulus-mediated transcription, whereas NF-kappaB essential modulator enhanced it. The understanding of LPS- and TNF-induced signaling may allow the development of specific strategies to treat sepsis-associated disease. 


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Extinction of immunoglobulin gene expression in B cells upon fusion with HeLa cells is preceded by rapid nuclear depletion of essential transcription factors and is accompanied by widespread inactivation of genes expressed in a B cell-specific manner. 
When immunoglobulin (Ig) expressing B cells are fused with non-B cells, Ig expression is rapidly suppressed at the level of transcription, a phenomenon termed extinction. Here we demonstrate that fusion of HeLa cells with either diploid or tetraploid B cells (Daudi) results in widespread extinction of several other B cell-encoded genes that are expressed in a B cell-specific manner. In contrast, expression of B cell-expressed genes that are not dependent on cell-specific controls is unaffected. We show that the molecular mechanism(s) underlying Ig gene extinction can be explained, at least in part, by a lack of transcription factors that are essential for Ig gene transcription. These transcription factors are either not produced due to block of transcription of their respective genes (Oct-2, OBF-1, PU.1), or are rendered inactive posttranslationally (NF-kappa B, E47). By isolating Daudi x HeLa heterokaryons a few hours after fusion, we have studied the initial fate of two B cell-specific transcription factors involved in Ig gene transcription, Oct-2 and NF-kappa B. This report provides the first demonstration that upon fusion with HeLa cells, the nuclear contents of B cell-expressed transcription factors are depleted within a few hours with kinetics that are as fast or faster than that of Ig gene extinction. Thus, the extinguishing mechanism is effective very early after fusion. We suggest that extinction of Ig genes is part of a global mechanism that suppresses the differentiation program foreign to the HeLa phenotype. 


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Sequential development of structural and functional alterations in T cells from tumor-bearing mice. 
The TCR alpha beta or -gamma delta chains bind the peptide ligand, whereas the associated CD3 deltaepsilongamma and TCR zeta subunits couple the TCR to intracellular signal transduction components. Recently, several groups have described marked alterations in signal transduction elements in T cells from cancer patients or in mice bearing tumor for a few weeks (>26 days). The sequence in which these alterations develop is unknown. The aim of this study was to explore the kinetics of the development of alterations in signal transduction molecules (TCR zeta chain, NF kappaB family proteins, and tyrosine kinase p56(lck)) in mice bearing MC38 colon adenocarcinoma. The results demonstrate that alterations in NF kappaB family proteins, specifically the failure of p65 translocation to the nucleus, occur earlier and more frequently than the decrease in zeta-chain. These defects are paralleled by an impaired ability to produce Th1 cytokines (IL-2 and IFN-gamma). These initial changes are followed by the eventual loss of TCR zeta chain and p56(lck) and a marked decrease in cytotoxic function. An increased rate of lysosomal degradation is one of the mechanisms responsible for the loss of zeta-chain. 


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Thymocytes control the CD4 gene differently from mature T lymphocytes. 
We analyzed the activity of the enhancer, the promoter and the silencer of the human CD4 gene during T cell development using transgenic mice. Immunofluorescence studies on thymic populations of mice carrying transgenes in various combinations of these regulatory DNA elements revealed that thymocytes control the CD4 gene in a different manner than mature peripheral T lymphocytes. The 5'-positive regulatory unit, consisting of the promoter and the 5' enhancer, is already active at the CD4-CD8-double-negative (DN) stage of development. However, its activity becomes lower in the double-positive and a fraction of the CD4+ CD8int/- cell population, indicating that an additional enhancer, located in either the first or the third intron of the CD4 gene, is required for CD4 gene expression in this population. The other studied regulatory element is the minimal CD4 silencer which inhibits CD4 gene expression in peripheral CD8 T lymphocytes. This silencer is inactive in the most immature DN thymocytes, which probably use a distinct silencer mechanism to down-regulate CD4 gene expression. Unexpectedly, the CD4 silencer is also active in CD4+ CD8int/- cells of the thymus, implying that an anti-silencer may be required to resume CD4 expression in this cell population. Altogether, the CD4 gene is regulated by several positive and negative regulatory mechanisms which come into play in a developmentally coordinated manner. 


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Differential regulation of 4E-BP1 and 4E-BP2, two repressors of translation initiation, during human myeloid cell differentiation. 
Human myeloid differentiation is accompanied by a decrease in cell proliferation. Because the translation rate is an important determinant of cell proliferation, we have investigated translation initiation during human myeloid cell differentiation using the HL-60 promyelocytic leukemia cell line and the U-937 monoblastic cell line. A decrease in the translation rate is observed when the cells are induced to differentiate along the monocytic/macrophage pathway or along the granulocytic pathway. The inhibition in protein synthesis correlates with specific regulation of two repressors of translation initiation, 4E-BP1 and 4E-BP2. Induction of HL-60 and U-937 cell differentiation into monocytes/macrophages by IFN-gamma or PMA results in a dephosphorylation and consequent activation of 4E-BP1. Dephosphorylation of 4E-BP1 was also observed when U-937 cells were induced to differentiate into monocytes/macrophages following treatment with retinoic acid or DMSO. In contrast, treatment of HL-60 cells with retinoic acid or DMSO, which results in a granulocytic differentiation of these cells, decreases 4E-BP1 amount without affecting its phosphorylation and strongly increases 4E-BP2 amount. Taken together, these data provide evidence for differential regulation of the translational machinery during human myeloid differentiation, specific to the monocytic/macrophage pathway or to the granulocytic pathway. 


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Apoptosis signaling pathways in normal T cells: differential activity of Bcl-2 and IL-1beta-converting enzyme family protease inhibitors on glucocorticoid- and Fas-mediated cytotoxicity. 
Fas-mediated apoptosis plays an important role in regulating the immune response in peripheral T cells. Restimulation of T cell blasts up-regulates Fas and Fas ligand expression, with subsequent interaction leading to cell death. Overexpression of Bcl-2 in tumor cells blocks apoptosis induced by many stimuli, but inhibition of Fas-mediated killing has not been consistently observed. To examine the behavior of Bcl-2 in normal cells, T cell blasts were transiently transfected with Bcl-2 and related gene products to determine the effect on apoptotic signaling. Transient overexpression of Bcl-2 in mouse and human T cell blasts did not block Fas-mediated apoptosis, whereas etoposide- and glucocorticoid-induced cytotoxicity was potently inhibited. Expression of Bcl-xL and adenovirus E1B 19K did not interfere with anti-Fas killing. In contrast, interleukin-1beta-converting enzyme family protease inhibitors Ac-DEVD-CHO and CrmA blocked Fas-mediated apoptosis. These results suggest that peripheral T cells use distinct apoptosis signaling pathways with differential sensitivity to Bcl-2 and interleukin-1beta-converting enzyme family protease inhibitors. Since T cells normally express Bcl-2 and Bcl-xL following activation, their inability to block Fas-mediated apoptosis may allow for the elimination of self-reactive cells and the appropriate regulation of immune responses. 


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Activation of pp90rsk and early growth response-1 gene expression by pokeweed mitogen in human B cells. 
The present studies have examined the effects of pokeweed mitogen (PWM) on the induction of early growth response-1 gene (EGR-1) in normal human B cells. PWM regulates EGR-1 gene expression by both transcriptional and post-transcriptional mechanisms. Transient transfection assays with EGR-1 promoter fragments linked to the chloramphenicol acetyltransferase (CAT) gene demonstrated that PWM induced EGR-1 transcription is conferred by the CArG motif (C C[AT]6GG) in the EGR-1 promoter. The results further demonstrated the activation of S6 kinase (pp90rsk), evidenced by phosphorylation of S6 and serum response factor (SRF) peptides, in PWM treated B cells. Taken together, these findings suggest that PWM is able to initiate an intracytoplasmic signalling cascade and EGR-1 induction in normal human B cells. 


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Inhibition of transcription factors belonging to the rel/NF-kappa B family by a transdominant negative mutant. 
The KBF1 factor, which binds to the enhancer A located in the promoter of the mouse MHC class I gene H-2Kb, is indistinguishable from the p50 DNA binding subunit of the transcription factor NF-kappa B, which regulates a series of genes involved in immune and inflammatory responses. The KBF1/p50 factor binds as a homodimer but can also form heterodimers with the products of other members of the same family, like the c-rel and v-rel (proto)oncogenes. The dimerization domain of KBF1/p50 is contained between amino acids 201 and 367. A mutant of KBF1/p50 (delta SP), unable to bind to DNA but able to form homo- or heterodimers, has been constructed. This protein reduces or abolishes in vitro the DNA binding activity of wild-type proteins of the same family (KBF1/p50, c- and v-rel). This mutant also functions in vivo as a trans-acting dominant negative regulator: the transcriptional inducibility of the HIV long terminal repeat (which contains two potential NF-kappa B binding sites) by phorbol ester (PMA) is inhibited when it is co-transfected into CD4+ T cells with the delta SP mutant. Similarly the basal as well as TNF or IL1-induced activity of the MHC class I H-2Kb promoter can be inhibited by this mutant in two different cell lines. These results constitute the first formal demonstration that these genes are regulated by members of the rel/NF-kappa B family. 


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Functional association of Nmi with Stat5 and Stat1 in IL-2- and IFNgamma-mediated signaling. 
Using the coiled-coil region of Stat5b as the bait in a yeast two-hybrid screen, we identified the association of Nmi, a protein of unknown function previously reported as an N-Myc interactor. We further show that Nmi interacts with all STATs except Stat2. We evaluated two cytokine systems, IL-2 and IFNgamma, and demonstrate that Nmi augments STAT-mediated transcription in response to these cytokines. Interestingly, Nmi lacks an intrinsic transcriptional activation domain; instead, Nmi enhances the association of CBP/p300 coactivator proteins with Stat1 and Stat5, and together with CBP/p300 can augment IL-2- and IFNgamma-dependent transcription. Therefore, our data not only reveal that Nmi can potentiate STAT-dependent transcription, but also suggest that it can augment coactivator protein recruitment to at least some members of a group of sequence-specific transcription factors. 


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Four P-like elements are required for optimal transcription of the mouse IL-4 gene: involvement of a distinct set of nuclear factor of activated T cells and activator protein-1 family proteins. 
We previously identified the P sequence as a critical regulatory element of the human IL-4 promoter. In the mouse IL-4 promoter, there are five elements homologous to the human P sequence designated conserved lymphokine element 0 (CLE0), P, P2, P3 and P4. To characterize the role of these P-like elements and their binding factors in the native promoter, we did transient transfection and electrophoretic mobility shift assays (EMSA). Transfection of EL-4 cells with the IL-4 promoter-reporter constructs carrying mutated P-like elements showed that four P-like elements, CLE0, P, P2 and P4, but not P3, were required for optimal activation of the IL-4 promoter. EMSA showed that both constitutive and inducible complexes bound to CLE0, P, P2 and P4, whereas only a constitutive complex bound to P3. In competition and antibody supershift assays in EMSA, complexes formed with P or P2 proved to contain nuclear factor of activated T cells (NFAT) family proteins as major components. Activator protein (AP)-1 family proteins interacted with CLE0, P, P2 and P4. NFAT/AP-1 complex formed only with P and P2. Cross-competition assays among the P-like elements revealed element-specific and common complexes. Six tandem repeats of the P element linked to the SV40 promoter responded to phorbol 12-myristate 13-acetate, while that of other elements did not. It would thus appear that components of each P-like element-binding complexes are not identical and may coordinately contribute to transcriptional activity. 


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HIV enhancer activity perpetuated by NF-kappa B induction on infection of monocytes [see comments] 
Permissiveness to replication of human immunodeficiency virus (HIV) differs in T lymphocytes and macrophages. In T cells, HIV transcription is poorly detected in vivo. Cloned, normal T lymphocytes show very little, if any, basal activity of the HIV enhancer and low nuclear expression of NF-kappa B, a potent transcriptional activator of the HIV enhancer. In contrast, fixed tissue macrophages express detectable HIV proteins, indicating permanent virus transcription. One explanation for the perpetuation of virus infection in macrophages could be sustained nuclear NF-kappa B expression. However, the U937 monocytic cell line, which is fully permissive to HIV replication, is known to express only low levels of nuclear NF-kappa B. We show here that chronic HIV infection results in both induction of a nuclear factor with antigenic properties indistinguishable from those of NF-kappa B and permanently increased HIV enhancer activity. This phenomenon, which is independent of tumour necrosis factor, is associated with HIV replication, and is thus likely to explain at least in part the perpetuation of HIV infection in monocytes. 


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Functional roles of the transcription factor Oct-2A and the high mobility group protein I/Y in HLA-DRA gene expression. 
The class II major histocompatibility complex gene HLA-DRA is expressed in B cells, activated T lymphocytes, and in antigen-presenting cells. In addition, HLA-DRA gene expression is inducible in a variety of cell types by interferon-gamma (IFN-gamma). Here we show that the lymphoid-specific transcription factor Oct-2A plays a critical role in HLA-DRA gene expression in class II-positive B cell lines, and that the high mobility group protein (HMG) I/Y binds to multiple sites within the DRA promoter, including the Oct-2A binding site. Coexpression of HMG I/Y and Oct-2 in cell lines lacking Oct-2 results in high levels of HLA-DRA gene expression, and in vitro DNA-binding studies reveal that HMG I/Y stimulates Oct-2A binding to the HLA-DRA promoter. Thus, Oct-2A and HMG I/Y may synergize to activate HLA-DRA expression in B cells. By contrast, Oct-2A is not involved in the IFN-gamma induction of the HLA-DRA gene in HeLa cells, but antisense HMG I/Y dramatically decreases the level of induction. We conclude that distinct sets of transcription factors are involved in the two modes of HLA-DRA expression, and that HMG I/Y may be important for B cell-specific expression, and is essential for IFN-gamma induction. 


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Activation of E2F-mediated transcription by human T-cell leukemia virus type I Tax protein in a p16(INK4A)-negative T-cell line. 
The human T-cell leukemia virus type I (HTLV-I) is a causative agent of adult T-cell leukemia. Although the exact mechanism by which HTLV-I contributes to leukemogenesis is still unclear, the Tax protein is thought to play a major role in this process. This 40-kDa polypeptide is able to interact with the tumor suppressor p16(INK4A). Consequently, Tax can activate the signaling pathway that lead to the release of E2F that in turn induces expression of factors required for cell cycle progression. In this paper, we demonstrate that Tax can also activate E2F-mediated transcription independently of p16(INK4A). Indeed, when Tax is coexpressed with the E2F-1 transcription factor in CEM T-cells, which lack expression of p16(INK4A), it strongly potentiates the E2F-dependent activation of a reporter construct driven by a promoter containing E2F binding sites. This stimulation is abrogated by mutations affecting the E2F-binding sites. In addition, Tax also stimulates the transcription of the E2F-1 gene itself. Using Tax mutants that fail to activate either ATF- or NF-kappaB-dependent promoters and different 5' truncation mutants of the E2F-1 promoter, we show that the Tax-dependent transcriptional control of the E2F1 gene involves, at least in part, the ATF binding site located in the E2F-1 promoter. 


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Activation-dependent transcriptional regulation of the human Fas promoter requires NF-kappaB p50-p65 recruitment. 
Fas (CD95) and Fas ligand (CD95L) are an interacting receptor-ligand pair required for immune homeostasis. Lymphocyte activation results in the upregulation of Fas expression and the acquisition of sensitivity to FasL-mediated apoptosis. Although Fas upregulation is central to the preservation of immunologic tolerance, little is known about the molecular machinery underlying this process. To investigate the events involved in activation-induced Fas upregulation, we have examined mRNA accumulation, fas promoter activity, and protein expression in the Jurkat T-cell line treated with phorbol myristate acetate and ionomycin (P/I), pharmacological mimics of T-cell receptor activation. Although resting Jurkat cells express Fas, Fas mRNA was induced approximately 10-fold in 2 h upon P/I stimulation. Using sequential deletion mutants of the human fas promoter in transient transfection assays, we identified a 47-bp sequence (positions -306 to -260 relative to the ATG) required for activation-driven fas upregulation. Sequence analysis revealed the presence of a previously unrecognized composite binding site for both the Sp1 and NF-kappaB transcription factors at positions -295 to -286. Electrophoretic mobility shift assay (EMSA) and supershift analyses of this region documented constitutive binding of Sp1 in unactivated nuclear extracts and inducible binding of p50-p65 NF-kappaB heterodimers after P/I activation. Sp1 and NF-kappaB transcription factor binding was shown to be mutually exclusive by EMSA displacement studies with purified recombinant Sp1 and recombinant p50. The functional contribution of the kappaB-Sp1 composite site in P/I-inducible fas promoter activation was verified by using kappaB-Sp1 concatamers (-295 to -286) in a thymidine kinase promoter-driven reporter construct and native promoter constructs in Jurkat cells overexpressing IkappaB-alpha. Site-directed mutagenesis of the critical guanine nucleotides in the kappaB-Sp1 element documented the essential role of this site in activation-dependent fas promoter induction. 


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HIV type 1 protease activation of NF-kappa B within T lymphoid cells. 
NF-kappa B is a nuclear protein of the rel oncogene family capable of enhancing transcription of several cellular genes, including IL-2 and the IL-2 receptor, and viral genes transcribed from the HIV-1 LTR. It has been reported that HIV-1 protease may cleave the NF-kappa B precursor to its active form in vitro. In this study the effects of HIV protease on NF-kappa B precursor activation were examined in Jurkat T cells by introducing a protease expression vector into the cells. Increased NF-kappa B activity was observed and this increased activity was blocked by a specific inhibitor of the viral protease. Viral transcription, as measured using LTR-CAT assays, was only slightly enhanced in the HIV-protease expressing cells, while secretion of IL-2 and expression of the IL-2 receptor were not affected. The limited activation of NF-kappa B by HIV protease appears unlikely to have a significant effect on virus expression or T cell function. 


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The regulation of HIV by retinoic acid correlates with cellular expression of the retinoic acid receptors. 
OBJECTIVES: To analyze the effect of retinoic acids (RA) on HIV-1 expression and correlate this effect with expression levels of RA receptors (RARs) in T-lymphoid and monocytoid cell lines. DESIGN AND METHODS: The effect of all-trans and 9-cis RA on HIV-1 production in T-lymphoid (H9, CEM) and monocytoid (U937,THP-1) cell lines was measured during acute and chronic infection. The expression levels of human RAR alpha (hRAR alpha, receptor for all-trans RA) and the human retinoid-X receptor alpha (hRXR alpha receptor for 9-cis RA) were determined by Northern blot analysis. RESULTS: Both all-trans and 9-cis RA inhibited virus replication in HIV-1 IIIB-infected monocytoid cells, in the presence and absence of the co-stimulatory agent phorbol myristate acetate (PMA). The retinoids had weak or no stimulatory effects on HIV production by T-cell lines. HIV production by PMA-stimulated T-cell lines was inhibited by these retinoids. The 9-cis RA was generally more effective than all-trans RA in inhibiting HIV production and in combination generally more effective than the single agents alone. Human RAR alpha was expressed in H9, U937 and THP-1 cells, but almost undetectable in CEM cells. Human RXR alpha was significantly expressed in U937 and THP-1 cells, weakly expressed in H9 cells and not detectable in CEM cells. After stimulation by PMA, RXR alpha expression increased in H9 and U937 cells but not in CEM cells. Human RAR alpha expression was unchanged in H9 and CEM cells, and elevated in U937 cells, after PMA stimulation. CONCLUSION: The effect of RA on HIV-1 expression was cell-type-dependent and partially correlated with cellular expression of RARs. Endogenous or exogenously administered RA may have a significant role in HIV regulation. 


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Mycobacterium tuberculosis mannose-capped lipoarabinomannan can induce NF-kappaB-dependent activation of human immunodeficiency virus type 1 long terminal repeat in T cells. 
Tuberculosis has emerged as an epidemic, extended by the large number of individuals infected with human immunodeficiency virus type 1 (HIV-1). The major goal of this study was to determine whether the mycobacterial cell wall component mannose-capped lipoarabinomannan (ManLAM) of Mycobacterium tuberculosis (M. tuberculosis) could activate transcription of HIV-1 in T cells with the use of an in vitro cell culture system. These experiments are of prime importance considering that CD4-expressing T lymphocytes represent the major virus reservoir in the peripheral blood of infected individuals. Using the 1G5 cell line harbouring the luciferase reporter gene under the control of the HIV-1 LTR, it was first found that culture protein filtrates (CFP) from M. tuberculosis or purified ManLAM could activate HIV-1 LTR-dependent gene expression unlike similarly prepared CFP extracts devoid of ManLAM. The implication of protein tyrosine kinase(s), protein kinase A and/or protein kinase C was highlighted by the abrogation of the ManLAM-mediated activation of HIV-1 LTR-driven gene expression using herbimycin A and H7. It was also determined, using electrophoresis mobility shift assays, that M. tuberculosis ManLAM led to the nuclear translocation of the transcription factor NF-kappaB. M. tuberculosis ManLAM resulted in clear induction of the luciferase gene placed under the control of the wild-type, but not the kappaB-mutated, HIV-1 LTR region. Finally, the ManLAM-mediated activation of HIV-1 LTR transcription was found to be independent of the autocrine or paracrine action of endogenous TNF-alpha. The results suggest that M. tuberculosis can upregulate HIV-1 expression in T cells and could thus have the potential to influence the pathogenesis of HIV-1 infection. 


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Effects of glucocorticoids on transcription factor activation in human peripheral blood mononuclear cells. 
Glucocorticoids have an inhibitory effect on inflammatory and immune responses, and this may be through the modulation of transcription factor binding to DNA. The interaction of the transcription factors, activator protein-1 (AP-1), nuclear factor kappa B (NF kappa B), and cAMP-responsive element binding protein (CREB) with DNA and glucocorticoid receptors (GR) was analyzed in human peripheral blood mononuclear cells by gel mobility shift assays. TNF-alpha, IL-1 beta and phorbol myristate acetate (PMA) treatment increased AP-1 and NF kappa B DNA binding by up to 200% but decreased CREB binding (38%) over a 60-min time course. Dexamethasone produced a rapid and sustained increase in glucocorticoid response element binding and a concomitant 40-50% decrease in AP-1, NF kappa B, and CREB DNA binding that was blocked by combined dexamethasone and cytokine or PMA treatment. These latter effects were due to increases in the nuclear localization of GR, not to reduced amounts of the other transcription factors. This suggests that in these cells GR within the nucleus interacts with cytokine-stimulated transcription factors by the process of cross coupling. This may be an important molecular site of steroid action. 


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Competent transcription initiation by RNA polymerase II in cell-free extracts from xeroderma pigmentosum groups B and D in an optimized RNA transcription assay. 
The human autosomal recessive disease, xeroderma pigmentosum (XP), can result from mutations in any one of seven genes, designated XPA through XPG. Of these, the XPB and XPD genes encode proteins that are subunits of a general transcription factor, TFIIH, involved in both nucleotide excision repair (NER) and initiation of mRNA transcription by RNA polymerase II. In humans, mutation of the XPB or XPD gene impairs NER, resulting in hyper-sensitivity to sunlight and greatly increased skin tumor formation. However, no transcription deficiency has been demonstrated in either XP-B or XP-D. We have employed an optimized cell-free RNA transcription assay to analyze transcription activity of XP-B and XP-D. Although the growth rate was normal, the XP-B and XP-D cells contained reduced amounts of TFIIH. Extracts prepared from XP-B and XP-D lymphoblastoid cells exhibited similar transcription activity from the adenovirus major late promoter when compared to that in extracts from normal cells. Thus, we conclude that the XP-B and XP-D lymphoblastoid cells do not have impaired RNA transcription activity. We consider the possible consequences of the reduced cellular content of TFIIH for the clinical symptoms in XP-B or XP-D patients, and discuss a 'conditional phenotype' that may involve an impairment of cellular function only under certain growth conditions. 


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Targeted remodeling of human beta-globin promoter chromatin structure produces increased expression and decreased silencing. 
The chromatin structure of the human beta-globin gene locus assumes a transcriptionally-active conformation in erythroid cells. One feature of this chromatin reorganization is the formation of DNase 1 hypersensitive sites in the regions of active globin gene promoters. This reorganization requires the globin locus control region and is associated with normal expression of the beta-like globin genes. To determine whether it is possible to artificially enhance the opening of the chromatin structure of a minimal beta-globin promoter, we placed a 101bp, erythroid-specific DNase 1 hypersensitive site-forming element (HSFE) immediately upstream of the beta-globin promoter and gene. This element includes binding sites for NF-E2, AP-1, GATA-1 and Sp-1. Constructs were stably transfected into murine erythroleukemia cells and promoter chromatin structure and gene expression were analyzed. The HSFE induced an area of enhanced DNase 1 hypersensitivity extending from the transcriptional start site to -300bp of the artificial promoter and significantly increased the proportion of beta-globin promoters in an open chromatin configuration. This remodeling of promoter chromatin structure resulted in 3-fold increases in beta-globin gene transcription and induction, and inhibited long-term beta-globin gene silencing. These results indicate that a relatively small cis-acting element is able to enhance remodeling of promoter chromatin structure resulting in increased beta-globin gene expression. 


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Transcriptional control of steroid-regulated apoptosis in murine thymoma cells. 
Early studies in murine T cell lines indicated that transcriptional transactivation functions encoded in the glucocorticoid receptor (GR) N-terminal domain are required for glucocorticoid-mediated apoptosis. However, more recent studies in human T cell lines have suggested that the N-terminal domain is not necessary for steroid-regulated apoptosis and that GR-mediated transrepression may be the more critical mechanism. To better understand the contribution of the GR N-terminal transactivation domain in mediating murine thymocyte apoptosis, we stably transfected GR, GR variants, and the androgen receptor (AR) into receptor-negative S49 murine thymoma cells. GR expression levels were shown to be rate-limiting for initiating the apoptotic pathway, and a positive correlation between steroid sensitivity and GR-mediated induction of an integrated mouse mammary tumor virus (MMTV) LTR reporter gene was observed. Analysis of GR chimeric receptors containing the potent VP16 and E1A viral transactivation domains in place of the GR N terminus revealed that even low level expression of these receptors resulted in both enhanced steroid sensitivity and MMTV induction, thus supporting a role for transactivation in apoptosis. In contrast, we found that AR can initiate apoptosis in S49 cells after treatment with 5 alpha-dihydrotestosterone, despite its relative inability to induce high level expression of MMTV. To investigate this further, we examined the steroid-regulated expression of an endogenous thymocyte-specific gene called GIG18. We found that GIG18 was rapidly induced to comparable levels by both AR and GR, demonstrating that AR can indeed function as a transcriptional activator in S49 cells and, moreover, that GIG18 induction may be a marker of early apoptotic events in steroid-treated cells. Taken together, these results support our conclusion that transcriptional transactivation is a necessary signaling component of S49 cell apoptosis, although an additional role for GR-mediated transrepression cannot be excluded. 


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Infection and replication of Tat- human immunodeficiency viruses: genetic analyses of LTR and tat mutations in primary and long-term human lymphoid cells. 
Tat is an essential regulatory protein for the replication of human immunodeficiency virus (HIV). Mutations in the tat gene have been shown to block HIV replication in human T cells. Several studies have established that Tat releases an elongation block to the transcription of HIV long terminal repeat (LTR); however, it is not known whether this mechanism alone is sufficient to explain the block to HIV replication in human T cells when Tat is absent. It is possible that Tat is also needed for other functions during HIV replication. To test these hypotheses, we studied several tat mutants, including two stop codon mutants and one deletion mutant using replication-competent HIV-1 constructs carrying wild-type or mutant LTRs with modifications in the NF-kappa B and/or Sp1 binding sites. In this study, we show that Tat- HIV-1 with wild-type LTRs can replicate in HeLa cells, and the virus produced from HeLa cells can infect primary peripheral blood lymphocytes and macrophages. It was found that the propagation of the Tat mutants containing wild-type LTRs was less efficient than that of the LTR-modified Tat mutants. Large amounts of viral RNA and particles were synthesized in infections established using the tat mutants that contain modified LTRs. However, this efficient propagation of the LTR-modified tat mutants was restricted to some lymphoid cell lines that have been transformed with other viruses. Thus, despite its essential role for releasing an elongation block, Tat is not otherwise absolutely required for synthesis of full-length HIV transcripts and assembly of virus particles. Direct sequencing of the viral genomes and reinfection kinetics showed no evidence of wild-type reversion even after prolonged infection with the Tat- virus. The implications for in vivo HIV-1 replication and potential application of this system to the study of alternative Tat function are discussed. 


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The human prointerleukin 1 beta gene requires DNA sequences both proximal and distal to the transcription start site for tissue-specific induction. 
In these studies, we have identified DNA sequences and specific protein interactions necessary for transcriptional regulation of the human prointerleukin 1 beta (proIL-1 beta) gene. A cell-type-independent lipopolysaccharide (LPS)-responsive enhancer element located between -3757 and -2729 bp upstream from the transcription start site (cap site) consisted of at least six discrete subregions which were essential to the maximal induction by LPS in transfected monocytes. The enhancer also appeared to mediate phorbol myristate acetate induction in monocytes and IL-1 responsiveness in fibroblasts. Deletion and base substitution mutations along with DNA binding studies demonstrated that the enhancer contained a minimum of three functional protein binding sequences, two of which appeared to be important for gene induction. One of the essential proteins which bound to the enhancer was similar or identical to members of the C/EBP family of transcription factors required for both IL-1- and LPS-specific induction of the IL-6 gene (i.e., the NF-IL6 proteins). When ligated to the proIL-1 beta cap site-proximal region (located between -131 to +12), both the proIL-1 beta and the simian virus 40 enhancer elements functioned more efficiently in monocytes than in HeLa cells, which are not normally competent for IL-1 beta expression. When ligated to the murine c-fos promoter, however, the proIL-1 beta enhancer was inducible in phorbol myristate acetate-stimulated HeLa cells, suggesting the existence of a proIL-1 beta promoter-proximal requirement for tissue specificity. 


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Interaction of HTLV-I Tax with the human proteasome: implications for NF-kappa B induction. 
The human T-cell leukemia virus type I (HTLV-I) has been etiologically associated with the development of the adult T-cell leukemia (ATL) as well as degenerative neurologic syndrome termed tropical spastic paraparesis (TSP). HTLV-I encodes a potent transactivator protein termed Tax that appears to play an important role in the process of T-cell immortalization. Even though the mechanisms by which Tax induces transformation are still unknown, it seems likely that the ability of Tax to alter the expression of many cellular genes plays an important part in this process. Tax does not bind directly to DNA but rather deregulates the activity of cellular transcription factors. One family of host transcription factors whose activity is altered by Tax includes NF-kappa B/Rel. These transcription factors are post-transcriptionally regulated by their assembly with a second family of inhibitory proteins termed I kappa B that serve to sequester the NF-kappa B/Rel complexes in the cytoplasm. Upon cellular activation, I kappa B alpha is phosphorylated, polyubiquitinated, and degraded in the proteasome. This proteolytic event liberates NF-kappa B, permitting its rapid translocation into the nucleus where it binds to its cognate enhancer elements. Similarly, the p105 precursor of the NF-kappa B p50 subunit is also post-translationally processed in the proteasome. The mechanisms by which Tax activates NF-kappa B remain unclear, and findings presented in the literature are often controversial. We identified a physical interaction between Tax and the HsN3 subunit of the human proteasome. This raises the intriguing possibility that physical association of the HsN3 proteasome subunit with HTLV-I Tax coupled with the independent interaction of Tax with either p100 or p65-I kappa B alpha targets these cytoplasmic NF-kappa B/Rel complexes to the proteasome for processing. 


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X-rays-induced secretion of cellular factor(s) that enhance(s) HIV-1 promoter transcription in various non-irradiated transfected cell lines. 
Various cellular stress agents like ionizing radiation exposure could activate human immunodeficiency virus type 1 (HIV- 1) replication or reporter gene expression. In addition, extracellular factor(s) released by X-ray-treated human colonic carcinoma cell line (HT29) might activate the long terminal repeat (LTR) of HIV-1 in non-irradiated HT29 cells. In the present report we show that in various transiently or stably transfected cell lines, X-ray irradiation up-regulates HIV-1 LTR transcription through the kappaB regulatory elements. A factor(s), which is processed by and acts upon a variety of cell types, was detected by addition to non-irradiated cells of either X-ray-treated cells or a conditioned medium taken from irradiated cultures. The magnitude of responsiveness is cell type dependent. In addition, X-ray activation of HIV-1 LTR in transiently or stably transfected cell lines is inhibited by a potent antioxidant drug, pyrrolidine dithiocarbamate and by another drug, known for its role in the trapping of growth factors, suramin. The importance of these observations in the pathophysiology of patients with AIDS-related cancers treated by radiotherapy remains to be established. 


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TRAMP, a novel apoptosis-mediating receptor with sequence homology to tumor necrosis factor receptor 1 and Fas(Apo-1/CD95). 
A novel member of the tumor necrosis factor (TNF) receptor family, designated TRAMP, has been identified. The structural organization of the 393 amino acid long human TRAMP is most homologous to TNF receptor 1. TRAMP is abundantly expressed on thymocytes and lymphocytes. Its extracellular domain is composed of four cysteine-rich domains, and the cytoplasmic region contains a death domain known to signal apoptosis. Overexpression of TRAMP leads to two major responses, NF-kappaB activation and apoptosis. TRAMP-induced cell death is inhibited by an inhibitor of ICE-like proteases, but not by Bcl-2. In addition, TRAMP does not appear to interact with any of the known apoptosis-inducing ligands of the TNF family. 


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Interactions between the class II transactivator and CREB binding protein increase transcription of major histocompatibility complex class II genes. 
Class II major histocompatibility (class II) genes are regulated in a B-cell-specific and gamma interferon-inducible fashion. The master switch for the expression of these genes is the class II transactivator (CIITA). In this report, we demonstrate that one of the functions of CIITA is to recruit the CREB binding protein (CBP) to class II promoters. Not only functional but also specific binding interactions between CIITA and CBP were demonstrated. Moreover, a dominant negative form of CBP decreased the activity of class II promoters and levels of class II determinants on the surface of cells. Finally, the inhibition of class II gene expression by the glucocorticoid hormone could be attributed to the squelching of CBP by the glucocorticoid receptor. We conclude that CBP, a histone acetyltransferase, plays an important role in the transcription of class II genes. 


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A mammalian histone deacetylase related to the yeast transcriptional regulator Rpd3p [see comments] 
Trapoxin is a microbially derived cyclotetrapeptide that inhibits histone deacetylation in vivo and causes mammalian cells to arrest in the cell cycle. A trapoxin affinity matrix was used to isolate two nuclear proteins that copurified with histone deacetylase activity. Both proteins were identified by peptide microsequencing, and a complementary DNA encoding the histone deacetylase catalytic subunit (HD1) was cloned from a human Jurkat T cell library. As the predicted protein is very similar to the yeast transcriptional regulator Rpd3p, these results support a role for histone deacetylase as a key regulator of eukaryotic transcription. 


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BCL-6 expression during B-cell activation. 
Translocations involving the BCL-6 gene are common in the diffuse large cell subtype of non-Hodgkin's lymphoma. Invariably, the BCL-6 coding region is intact, but its 5' untranslated region is replaced with sequences from the translocation partner. The present study shows that BCL-6 expression is regulated in lymphocytes during mitogenic stimulation. Resting B and T lymphocytes contain high levels of BCL-6 mRNA. Stimulation of mouse B cells with anti-IgM or IgD antibodies, bacterial lipopolysaccharide, phorbol 12-myristate 13-acetate plus ionomycin, or CD40 ligand led to a five-fold to 35-fold decrease in BCL-6 mRNA levels. Similar downregulation of BCL-6 mRNA was seen in human B cells stimulated with Staphylococcus aureus plus interleukin-2 or anti-IgM antibodies and in human T lymphocytes stimulated with phytohemagglutinin. BCL-6 mRNA levels began to decrease 8 to 16 hours after stimulation, before cells entered S phase. Although polyclonal activation of B cells in vitro invariably decreased BCL-6 MRNA expression, activated B cells from human germinal centers expressed BCL-6 mRNA at levels comparable to the levels in resting B cells. Despite these similar mRNA levels, BCL-6 protein expression was threefold to 34-fold higher in germinal center B cells than in resting B cells, suggesting that BCL-6 protein levels are controlled by translational or posttranslational mechanisms. These observations suggest that the germinal center reaction provides unique activation signals to B cells that allow for continued, high-level BCL-6 expression. 


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Involvement of an SAF-like transcription factor in the activation of serum amyloid A gene in monocyte/macrophage cells by lipopolysaccharide. 
Serum amyloid A (SAA) has been linked to atherosclerosis because of its ability to remodel high-density lipoprotein by the depletion of apolipoprotein A1, its ability to bind cholesterol, and its presence in the atherosclerotic plaques of coronary and carotid arteries. In the present study, we investigated the induction mechanism of SAA gene in THP-1 monocyte/macrophage cells which play a critical role in the development of atherosclerotic fatty streak and plaque formation. We and others have shown that SAA gene is induced in monocyte/macrophage cells by lipopolysaccharide (LPS). By promoter function analysis, we show that the SAA promoter sequence between -280 and -226 can confer LPS responsiveness. Gel electrophoretic mobility shift assay detected an induced DNA-binding activity in these cells in response to LPS. Characterization of the DNA-binding protein by UV cross-linking, Southwestern blot, and antibody ablation/supershift assays revealed that it is similar to a recently reported nuclear factor designated SAF. These results demonstrated that LPS-mediated SAA gene induction in monocyte/macrophage cells is primarily due to the induction of SAF activity. 


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The spatial distribution of human immunoglobulin genes within the nucleus: evidence for gene topography independent of cell type and transcriptional activity. 
The three-dimensional positioning of immunoglobulin (Ig) genes within the nucleus of human cells was investigated using in situ hybridization and confocal microscopy. The visualization of heavy and light chain genes in B-lymphoid cells showed that the three Ig genes are differentially and nonrandomly distributed in different nuclear subvolumes: the kappa genes were found to be preferentially confined to an outer nuclear volume, whereas the gamma and lambda genes consistently occupied more central positions within the nucleus, the lambda genes being more interior when compared with the gamma genes. The data further show that these overall topographical distributions are independent of gene transcriptional activity and are conserved in different cell types. Although subtle gene movements within those defined topographical regions cannot be excluded by this study, the results indicate that tissue specificity of gene expression is not accompanied by drastic changes in gene nuclear topography, rather suggesting that gene organization within the nucleus may be primarily dependent on structural constraints imposed on the respective chromosomes. 


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V3 loop of human immunodeficiency virus type 1 suppresses interleukin 2-induced T cell growth [published erratum appears in AIDS Res Hum Retroviruses 1997 May 1;13(7):633] 
We tested the effect of three linear or two loop peptides derived from the V3 region of the HTLV-III BH10 clone or the SF2 strain of human immunodeficiency virus type 1 on IL-2-driven T cell proliferation. V3-BH10, which consists of 42 amino acids and has a loop structure, suppressed IL-2-driven proliferation of all IL-2-dependent cells [Kit225, ED-40515(+), KT-3, 7-day PHA-blasts, and fresh peripheral blood mononuclear cells] tested, whereas it did not suppress the cell growth of IL-2-independent cell lines (Hut102, Molt-4, and Jurkat). This suppressive effect was also seen in IL-2-driven cell growth of CD8-positive lymphocytes purified from 7-day PHA-blasts, indicating that CD4 molecules were not required for the suppression. The treatment with anti-V3 loop monoclonal antibody (902 antibody) completely abolished the suppressive effect of V3-BH10. In addition, V3-BH10 generated the arrest of Kit225 cells and also purified CD8-positive lymphocytes in G1 phase in the presence of IL-2. Neither chromatin condensation nor DNA fragmentation was detected in Kit225 cells cultured with V3-BH10 and IL-2. V3-BH10 neither blocked radiolabeled IL-2 binding to IL-2 receptors nor affected tyrosyl phosphorylation of several cellular proteins (p120, p98, p96, p54, and p38), which is immediately induced by IL-2 stimulation. However, V3-BH10 enhanced IL-2-induced mRNA expression of c-fos but not c-myc or junB. Thus, the binding of V3 loop of gp120 to the cell surface molecule(s) appears to affect intracellular IL-2 signaling, which leads to the suppression of IL-2-induced T cell growth. 


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Interleukin 6-induced differentiation of a human B cell line into IgM-secreting plasma cells is mediated by c-fos. 
The role of the protooncogene c-fos in interleukin (IL) 6-induced B cell differentiation was assessed. Treatment of SKW 6.4 cells with IL 6 induced a transient and early stimulation of c-fos sense mRNA expression. The effect appeared within 30 min and returned to basal levels after 2 h. The addition of antisense oligonucleotides to c-fos significantly inhibited IL 6-induced IgM production by SKW 6.4 cells (p less than 0.001), whereas control oligonucleotides had no inhibitory effect. These results indicate that activation of c-fos is involved in IL 6-induced differentiation of SKW 6.4 cells into IgM-secreting cells. 


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Tissue-specific expression of the platelet GPIIb gene. 
One of the major objectives in the study of thrombogenesis is to determine the mechanisms by which a hematopoietic progenitor is activated and committed to the megakaryocytic lineage. Recent development of primary cultures of human megakaryocytes and the molecular cloning of genes that are specific to this lineage offer the possibility of getting some insights into the genetic mechanisms that control megakaryocytopoiesis. One gene of interest is the glycoprotein IIb (GPIIb) gene; GPIIb, the alpha subunit of the platelet cytoadhesin GPIIb-IIIa, is produced in megakaryocytes at an early stage of the differentiation, whereas the other subunit of this complex, GPIIIa, is expressed in other cells. For these reasons, the 5'-flanking region of the GPIIb gene was used to identify the regions that interact with DNA-binding nuclear factors. A fragment extending from -643 to +33 is capable of controlling the tissue-specific expression of the CAT gene in transfection experiments. Within this region, we have identified several sequences that are implicated in DNA protein interactions as shown in DNAse I footprints and gel mobility shift assays. One region, centered at -54, is similar to a nuclear factor E1-binding site, and a region located at position -233 contains a CCAAT motif. Two domains centered at positions -345 and -540, respectively, bind proteins that are present in megakaryocytic cells and nonrelated cells as well. Finally, two other domains, located at positions -460 and -510, interact with proteins that are only present in megakaryocytic cells. In addition, deletion of the region containing these two domains results in a significant decrease of the promoter activity. It is very likely that these domains bind megakaryocyte-specific nuclear proteins acting as positive transcription factors. 


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HIV-1 Nef protein inhibits the recruitment of AP-1 DNA-binding activity in human T-cells. 
The human immunodeficiency virus type 1 long terminal repeat, HIV-1-LTR, contains binding sites for several cellular transcription factors which contribute to HIV-1 gene expression. Our previous studies on the function of the HIV-1-encoded Nef protein suggested that Nef may be an inhibitor HIV-1 transcription. To determine whether Nef affects the binding of cellular factors implicated in HIV-1 regulation, 32P-labeled oligonucleotides corresponding to the binding sites were incubated with nuclear extracts prepared from Nef-expressing T-cell lines that were not stimulated or were stimulated with T-cell mitogens. We found that Nef inhibited the recruitment of AP-1 DNA-binding activity in mitogen-stimulated human T-cells. Additionally, Nef expressing cells were transiently transfected with a plasmid in which HIV-1 AP-1 DNA recognition sequences were cloned downstream of the chloramphenicol acetyltransferase (CAT) gene. Mitogen-mediated transcriptional activation of the CAT gene in this construct was inhibited in Nef-expressing cells but not in control cells. These studies suggest that, by inhibiting AP-1 activation, Nef may play a role in regulating HIV-1 gene expression in infected T-cells. 


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Synergism between two distinct elements of the HTLV-I enhancer during activation by the trans-activator of HTLV-I. 
We have conducted functional studies of the enhancer elements of human T-cell leukemia virus type I (HTLV-I) using the human T-cell lines Jurkat and MOLT 4, which are negative for HTLV-I, and MT-2 and TL-Mor, which carry the proviral genome of HTLV-I. Two distinct elements have been implicated in function of the HTLV-I enhancer. One is the 21-base-pair (bp) core element that is responsible for trans-activation by the HTLV-I trans-activator p40tax and that has the ability to bind to cyclic-AMP responsive element binding factor (CREB)-like factor(s). The other is a region interposed between the 21-bp elements. In this study we demonstrate that a subfragment (C26) in the region between the 21-bp elements is involved in trans-activation by p40tax, possibly through binding to an NF-kappa B-like nuclear factor or factors. Formation of the protein-DNA complex with the C26 subfragment was positively affected by p40tax. The C26 element conferred partial responsiveness to p40tax when linked to one copy of the 21-bp element that, by itself, showed little activation in response to p40tax. However, the C26 element alone, even when repeated, could not be activated by p40tax, unlike other NF-kappa B-binding elements. In contrast, the C26 element itself was profoundly activated upon stimulation with 12-O-tetradecanoylphorbol-13-acetate. These findings therefore suggest that the HTLV-I enhancer contains multiple functional elements, including binding sites for at least CREB- and NF-kappa B-like factors, which synergistically cooperate in activation of the HTLV-I enhancer in response to p40tax. Our results also demonstrate that TPA-dependent activation of the HTLV-I enhancer may be mediated through the C26 element. 


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Regulation of sialoadhesin expression on rat macrophages. Induction by glucocorticoids and enhancement by IFN-beta, IFN-gamma, IL-4, and lipopolysaccharide. 
Sialoadhesin is a macrophage-restricted member of the Ig superfamily that mediates adhesion with lymphoid and myeloid cells. It is expressed on a subpopulation of macrophages in lymphoid tissues and in chronic inflammation (e.g., during autoimmune diseases). We have studied the regulation of sialoadhesin expression in vitro and show that glucocorticoids (GC) induce sialoadhesin expression on freshly isolated rat macrophages and the rat macrophage cell line R2. The cytokines IFN-beta, IFN-gamma, IL-4, and LPS, although unable to induce sialoadhesin expression by themselves, were able to enhance GC-mediated induction of sialoadhesin. Sialoadhesin expression was functional as shown by cell adhesion assays with human RBCs. Northern blotting experiments indicated that regulation predominantly occurred at the mRNA level. Comparison of the different combinations of GC and cytokines/LPS revealed differences in the level of GC-dependent enhancement of sialoadhesin expression, with IFN-beta and IL-4 being more potent than IFN-gamma and LPS. Moreover, the effects of IFN-gamma and LPS could be reproduced by priming, whereas IFN-beta and IL-4 were required simultaneously with GC. The regulation of sialoadhesin expression was mediated by the GC receptor, and not by mineralocorticoid receptor, as shown by inhibition experiments with specific antagonists. Finally, it is demonstrated that macrophages in the adrenal gland, the major site of endogenous GC production, express sialoadhesin. This study demonstrates that GC act as a primary inducer of sialoadhesin expression on rat macrophages, and that the response can be enhanced by IFN-beta, T cell-derived cytokines, or LPS. 


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Transcription factor activation in lymphokine activated killer cells and lymphocytes from patients receiving IL-2 immunotherapy. 
Administration of the cytokine interleukin-2 (IL-2) can result in therapeutic benefits for individuals with renal cell carcinoma and melanoma. Here we report an analysis of the transcription factor families AP-1, Sp1, NF-kappaB, and signal transducers and activators of transcription (STAT) in cancer patients' lymphocytes before and after IL-2 immunotherapy, as assessed by a gel-shift assay. An in vitro surrogate of IL-2 immunotherapy is the incubation of fresh peripheral blood mononuclear cells (PBMC) from healthy individuals in IL-2 for several days, resulting in the production of lymphokine-activated killer (LAK) activity in these cultures. One purpose of this study was to describe the profile of transcription factor activation in these different populations, and assess whether the patterns observed correlated with functional differences in these cells. Prior to in vivo IL-2 administration, the typical binding pattern of transcription factors in PBMC from patients resembled that seen in fresh PBMC from healthy individuals. Over a 3-week course of IL-2 therapy, in most patients the binding patterns of AP-1 , Sp1, and NF-kappaB proteins changed to resemble those seen in PBMC activated by IL-2 in vitro. However, the cells obtained from IL-2-treated patients did not have low-level constitutive expression of STAT binding factors as did LAK cells. When these patient cells were further stimulated by IL-2 in vitro, additional differences in STAT induction patterns were noted. These data provide further information on the molecular events occurring in immune cells generated through in vivo and in vitro administration of IL-2, and further document that there is not a precise congruence between PBMC activated in vivo and in vitro by IL-2. 


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A family of serine proteases expressed exclusively in myelo-monocytic cells specifically processes the nuclear factor-kappa B subunit p65 in vitro and may impair human immunodeficiency virus replication in these cells. 
Two groups of U937 promonocytic cells were obtained by limiting dilution cloning which differed strikingly in their ability to support human immunodeficiency virus 1 (HIV-1) replication. "Plus" clones replicated the virus efficiently, whereas "minus" clones did not. We examined these clones for differences in nuclear factor (NF)-kappa B activity which might account for the observed phenomenon. Stimulation of plus clones liberated the classical p50-p65 complex from cytoplasmic pools, whereas minus clones produced an apparently novel, faster-migrating complex, as judged by electrophoretic mobility shift assays. It is surprising that the faster-migrating complex was composed also of p50 and p65. However, the p65 subunit was COOH-terminally truncated, as shown by immunoprecipitation. The truncation resulted from limited proteolysis of p65 during cellular extraction which released particular lysosomal serine proteases, such as elastase, cathepsin G, and proteinase 3. These specific proteases are coordinately expressed and were present exclusively in the minus U937 clones, but not in the plus clones, as demonstrated in the case of cathepsin G. In addition, these proteases were detected in certain subclones of THP-1 and HL-60 cells and in primary monocytes, in each case correlating with the truncated from of p65. We demonstrate in vitro cleavage of p65 by purified elastase and cathepsin G. It is possible that particular serine proteases may have inhibiting effects on the replication of HIV-1 in myelo-monocytic cells. The data also demonstrate that special precautions must be taken when making extracts from myelo-monocytic cells. 


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Nuclear factor-kappaB activation in human monocytes stimulated with lipopolysaccharide is inhibited by fibroblast conditioned medium and exogenous PGE2. 
The nuclear factor kappaB (NF-kappaB) is thought to be crucially involved in the gene activation of several cytokines, including tumor necrosis factor alpha (TNF). Previously, we showed that fibroblast conditioned medium (FCM) is able to inhibit both TNF mRNA accumulation and protein release in peripheral blood-derived human monocytes (PBM) stimulated with lipopolysaccharide (LPS). In this study we have investigated the effect of FCM on the LPS-induced DNA-binding activity of NF-kappaB, by means of electrophoretic shift assay (EMSA). We provide evidence that FCM strongly inhibits the LPS-induced NF-kappaB activation in PBM. Furthermore, we show that exogenous PGE2 mimics the NF-kappaB inhibitory effect of FCM. On the other hand, FCM produced in the presence of indomethacin does not inhibit NF-kappaB activation by LPS. Our results lend further support to the hypothesis that inflammatory and immune responses of monocytes/macrophages may be modulated at the molecular level by signals originating from tissue structural cells such as fibroblasts. 


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Interferon-gamma potentiates the antiviral activity and the expression of interferon-stimulated genes induced by interferon-alpha in U937 cells. 
Binding of type I interferon (IFN-alpha/beta) to specific receptors results in the rapid transcriptional activation, independent of protein synthesis, of IFN-alpha-stimulated genes (ISGs) in human fibroblasts and HeLa and Daudi cell lines. The binding of ISGF3 (IFN-stimulated gene factor 3) to the conserved IFN-stimulated response element (ISRE) results in transcriptional activation. This factor is composed of a DNA-binding protein (ISGF3 gamma), which normally is present in the cytoplasm, and other IFN-alpha-activated proteins which preexist as latent cytoplasmic precursors (ISGF3 alpha). We have found that ISG expression in the monocytic U937 cell line differs from most cell lines previously examined. U937 cells express both type I and type II IFN receptors, but only IFN-alpha is capable of inducing antiviral protection in these cells. Pretreatment with IFN-gamma potentiates the IFN-alpha-induced protection, but IFN-gamma alone does not have any antiviral activity. ISG15 mRNA accumulation in U937 cells is not detectable before 6 h of IFN-alpha treatment, peaks at 24 h, and requires protein synthesis. Although IFN-gamma alone does not induce ISG expression, IFN-gamma pretreatment markedly increases and hastens ISG expression and transcriptional induction. Nuclear extracts assayed for the presence of ISRE binding factors by electrophoretic mobility shift assays show that ISGF3 is induced by IFN-alpha within 6 h from undetectable basal levels in untreated U937 cells. Activation of ISGF3 alpha, the latent component of ISGF3, occurs rapidly. However, the increase in ISGF3 activity ultimately correlates with the accumulation of ISGF3 gamma induced by IFN-alpha or IFN-gamma. (ABSTRACT TRUNCATED AT 250 WORDS) 


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Interferon-alpha activates multiple STAT proteins and upregulates proliferation-associated IL-2Ralpha, c-myc, and pim-1 genes in human T cells. 
Interferon-alpha (IFN-alpha) is a pleiotropic cytokine that has antiviral, antiproliferative, and immunoregulatory functions. There is increasing evidence that IFN-alpha has an important role in T-cell biology. We have analyzed the expression of IL-2Ralpha, c-myc, and pim-1 genes in anti-CD3-activated human T lymphocytes. The induction of these genes is associated with interleukin-2 (IL-2)-induced T-cell proliferation. Treatment of T lymphocytes with IFN-alpha, IL-2, IL-12, and IL-15 upregulated IL-2Ralpha, c-myc, and pim-1 gene expression. IFN-alpha also sensitized T cells to IL-2-induced proliferation, further suggesting that IFN-alpha may be involved in the regulation of T-cell mitogenesis. When we analyzed the nature of STAT proteins capable of binding to IL-2Ralpha, pim-1, and IRF-1 GAS elements after cytokine stimulation, we observed IFN-alpha-induced binding of STAT1, STAT3, and STAT4, but not STAT5 to all of these elements. Yet, IFN-alpha was able to activate binding of STAT5 to the high-affinity IFP53 GAS site. IFN-alpha enhanced tyrosine phosphorylation of STAT1, STAT3, STAT4, STAT5a, and STAT5b. IL-12 induced STAT4 and IL-2 and IL-15 induced STAT5 binding to the GAS elements. Taken together, our results suggest that IFN-alpha, IL-2, IL-12, and IL-15 have overlapping activities on human T cells. These findings thus emphasize the importance of IFN-alpha as a T-cell regulatory cytokine. 


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An alternatively spliced isoform of the Spi-B transcription factor. 
Spi-B is an Ets transcription factor related to the oncoprotein Spi-1/PU.1 and highly expressed in B lymphoid cells. The Ets proteins share a conserved Ets domain that mediates specific DNA binding. Spi-B binds DNA sequences containing a core 5'-GGAA-3' and activates transcription through this motif. Up to date, the biological function of Spi-B remains unknown. Here, we describe the characterization of an alternatively spliced variant of Spi-B, named deltaSpi-B, which has lost the Ets domain. In B lymphoid cells, deltaspi-B and spi-B mRNAs were present simultaneously in a ratio of around 10%. DeltaSpi-B product was not able to bind DNA and was recovered in cytoplasmic cellular extracts. We raise the hypothesis that delta Spi-B might affect Spi-B function by recruiting factors involved in Spi-B activity. 


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ZAP-70 tyrosine kinase, CD45, and T cell receptor involvement in UV- and H2O2-induced T cell signal transduction. 
Several mammalian responses to UV irradiation, including the activation of NF-kappa B, are believed to involve tyrosine phosphorylation. UV irradiation and H2O2 treatment of T lymphocytes induce protein tyrosine phosphorylation and Ca2+ signals similar to those observed following biological stimulation. We have examined the role of cell surface molecules in these responses. Normal T lymphocytes whose surface expression of CD3 was depleted showed impaired UV-induced tyrosine phosphorylation and Ca2+ signals. Similarly, Jurkat T cell lines deficient in CD3 or CD45 expression also gave impaired UV responses. However, all these cell types still gave strong Ca2+ and tyrosine phosphorylation responses to H2O2. The T cell tyrosine kinase ZAP-70 was found to be highly responsive to UV and H2O2 treatment. ZAP-70 responsiveness to UV required expression of both CD3 and CD45, whereas only CD3 was required for the response to H2O2. UV-induced activation of NF-kappa B was blocked by CD3 depletion, indicating the importance of such cell surface molecules in biological responses to UV. In nonlymphoid cells, the epidermal growth factor receptor displayed increased tyrosine phosphorylation within seconds of UV irradiation. These results suggest that UV-induced signal transduction is mediated via cell surface receptors that normally respond to biological stimulation, whereas H2O2 is able to partially bypass this requirement. 


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Bcl-2 protein inhibits bufalin-induced apoptosis through inhibition of mitogen-activated protein kinase activation in human leukemia U937 cells. 
In a previous study, we demonstrated that bufalin, which is an active principle of Chinese medicine, chan'su, caused apoptosis in human leukemia U937 cells by anomalous activation of mitogen-activated protein kinase (MAPK) via the signaling pathway of Ras, Raf-1, and MAPK kinase-1. Here, we report the effect of overexpression of bcl-2 in U937 cells on the signaling pathway of apoptosis that is induced by bufalin. The results indicated that the apoptosis induced by bufalin in U937 cells was significantly inhibited by overexpression of the Bcl-2 protein. No significant difference was detected in the activation of MAPK kinase-1 that is induced by bufalin in wild-type or Bcl-2-overexpressed U937 cells; however, the activation of MAPK by bufalin was significantly attenuated in the cells overexpressing Bcl-2. Bufalin treatment activated activator protein-1 transcriptional activity; however, this activation was decreased to 40% in bcl-2-overexpressed U937 cells. These results indicate that Bcl-2 acts downstream of MAPK kinase-1 but upstream of MAPK and suggest that, in the signaling pathway of the apoptotic process induced by bufalin, the transcriptional activity of activator protein-1 may be down-regulated through the inhibition of MAPK activity by Bcl-2. 


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STAT1 activation during monocyte to macrophage maturation: role of adhesion molecules. 
Human monocytes isolated from peripheral blood of healthy donors show a time-dependent differentiation into macrophages upon in vitro cultivation, closely mimicking their in vivo migration and maturation into extravascular tissues. The mediator(s) of this maturation process has not been yet defined. We investigated the involvement of signal transducers and activators of transcription (STAT) factors in this phenomenon and reported the specific, time-dependent, activation of STAT1 protein starting at day 0/1 of cultivation and maximally expressed at day 5. STAT1 activity was evident on the STAT binding sequences (SBE) present in the promoters of genes which are up-regulated during monocyte to macrophage maturation such as FcgammaRI and ICAM-1, and in the promoter of the transcription factor IFN regulatory factor-1. Moreover, the effect of cell adhesion to fibronectin or laminin was studied to investigate mechanisms involved in STAT1 activation. Compared with monocytes adherent on plastic surfaces, freshly isolated cells allowed to adhere either to fibronectin- or laminin-coated flasks exhibited an increased STAT1 binding activity both in control and in IFN-gamma-treated cells. The molecular events leading to enhanced STAT1 activation and cytokine responsiveness concerned both Y701 and S727 STAT1 phosphorylation. Exogenous addition of transforming growth factor-beta, which exerts an inhibitory effect on some monocytic differentiation markers, inhibited macrophage maturation, integrin expression and STAT1 binding activity. Taken together these results indicate that STAT1 plays a pivotal role in the differentiation/maturation process of monocytes as an early transcription factor initially activated by adherence and then able to modulate the expression of functional genes, such as ICAM-1 and FcgammaRI. 


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Nuclear localization and formation of beta-catenin-lymphoid enhancer factor 1 complexes are not sufficient for activation of gene expression. 
In response to activation of the Wnt signaling pathway, beta-catenin accumulates in the nucleus, where it cooperates with LEF/TCF (for lymphoid enhancer factor and T-cell factor) transcription factors to activate gene expression. The mechanisms by which beta-catenin undergoes this shift in location and participates in activation of gene transcription are unknown. We demonstrate here that beta-catenin can be imported into the nucleus independently of LEF/TCF binding, and it may also be exported from nuclei. We have introduced a small deletion within beta-catenin (Delta19) that disrupts binding to LEF-1, E-cadherin, and APC but not axin. This Delta19 beta-catenin mutant localizes to the nucleus because it may not be efficiently sequestered in the cytoplasm. The nuclear localization of Delta19 definitively demonstrates that the mechanisms by which beta-catenin localizes in the nucleus are completely independent of LEF/TCF factors. beta-Catenin and LEF-1 complexes can activate reporter gene expression in a transformed T-lymphocyte cell line (Jurkat) but not in normal T lymphocytes, even though both factors are nuclear. Thus, localization of both factors to the nucleus is not sufficient for activation of gene expression. Excess beta-catenin can squelch reporter gene activation by LEF-1-beta-catenin complexes but not activation by the transcription factor VP16. Taken together, these data suggest that a third component is necessary for gene activation and that this third component may vary with cell type. 


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One gene, two transcripts: isolation of an alternative transcript encoding for the autoantigen La/SS-B from a cDNA library of a patient with primary Sjogrens' syndrome. 
A cDNA library was prepared from peripheral blood lymphocytes of an autoimmune patient with primary Sjogrens' syndrome. The cDNA library was screened with the patients own autoimmune serum being monospecific for the nuclear autoantigen La/SS-B. Thereby an alternative type of La mRNA was identified that differed from the known La mRNA due to an exchange of the exon 1. Sequencing of the genomic region between the exons 1 and 2 showed that the alternative 5'-end is a part of the intron. In addition, the presence of an alternative promoter site, which exists within the intron downstream of the exon 1, became evident. In consequence, the alternative La mRNA is the result of a promoter switching combined with an alternative splicing mechanism. In the intron, further transcription factor binding sites, including a NF-kappa B element, were identified leading to the suggestion that the expression of the gene encoding for the nuclear autoantigen La/SS-B alters in dependence on disease conditions. 


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Regulation of the tissue factor gene in human monocytic cells. Role of AP-1, NF-kappa B/Rel, and Sp1 proteins in uninduced and lipopolysaccharide-induced expression. 
Tissue factor (TF) expression by peripheral blood monocytes during sepsis initiates intravascular thrombosis. Bacterial lipopolysaccharide (LPS) rapidly induces TF gene transcription in monocytes. The human TF promoter contains binding sites for the transcription factors AP-1, c-Rel/p65, Egr-1, and Sp1. NF-kappa B/Rel proteins have been shown to physically interact with both AP-1 and Sp1 proteins. In this study, we investigated the role of these transcription factors in uninduced and LPS-induced TF gene expression in human monocytic THP-1 cells. Deletional analysis indicated that five Sp1 sites mediated basal expression in uninduced cells. The two AP-1 sites bound c-Fos/c-Jun heterodimers in both unstimulated and LPS-stimulated cells. Maximal LPS induction of the TF promoter required the two AP-1 sites and the kappa B site within the LPS response element. Disruption of the conserved spacing between the proximal AP-1 site and the kappa B site abolished LPS induction. Replacement of the two AP-1 sites with intrinsically bent DNA partially restored LPS induction, suggesting an additional structural role for the AP-1 sites. Synergistic transactivation of the LPS response element in Drosophila Schneider cells by coexpression of c-Fos, c-Jun, c-Rel, and p65 or c-Jun and p65 required the transactivation domains of c-Jun and p65. These data indicated that c-Fos/c-Jun, c-Rel/p65, and Sp1 regulate TF gene expression in human monocytic cells. 


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Nuclear factor-IL6 activates the human IL-4 promoter in T cells. 
Positive regulatory element I (PRE-I) is a strong enhancer element essential for expression of the human IL-4 gene. To identify transcription factors binding to PRE-I, we screened a cDNA expression library from Jurkat T cells and isolated a cDNA encoding nuclear factor (NF)-IL6 (also known as C/EBP beta). NF-IL6 mRNA was found in human Jurkat T cells and in the mouse Th2 clone D10, but not in Th1 clone 29. rNF-IL6 expressed in bacteria was shown to specifically bind to PRE-I. PRE-I forms multiple DNA-protein complexes with nuclear extracts from Jurkat cells. Some of these complexes were demonstrated to contain NF-IL6 by using anti-C/EBP beta Abs. Overexpression of NF-IL6 enhanced expression of the chloramphenicol acetyl transferase reporter gene linked to the PRE-I-thymidine kinase or the human IL-4 promoter more than 10-fold in Jurkat cells. Promoter deletion studies revealed two additional NF-IL6 binding sites located at positions -44 to -36 (C/EBP proximal) and -87 to -79 (C/EBP medial), respectively. Our results demonstrate that NF-IL6 is involved in transcriptional activation of the human IL-4 promoter in T cells. 


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An 11-base-pair DNA sequence motif apparently unique to the human interleukin 4 gene confers responsiveness to T-cell activation signals. 
We have identified a DNA segment that confers responsiveness to antigen stimulation signals on the human interleukin (IL) 4 gene in Jurkat cells. The human IL-4 gene, of 10 kilobases, is composed of four exons and three introns. A cis-acting element (P sequence) resides in the 5' upstream region; no additional DNA segments with enhancer activity were identified in the human IL-4 gene. For further mapping purposes, a fusion promoter was constructed with the granulocyte/macrophage colony-stimulating factor basic promoter containing 60 base pairs of sequence upstream from the cap site of the mouse granulocyte/macrophage colony-stimulating factor gene and various lengths of the 5' upstream sequence of the IL-4 gene. The P sequence was located between positions -79 and -69 relative to the transcription start site of the human IL-4 gene, and this location was confirmed by base-substitution mutations. The plasmids carrying multiple copies of the P sequence showed higher responsiveness to the stimulation. The binding protein(s) that recognize the P sequence of the IL-4 gene were identified by DNA-mobility-shift assays. The binding of NF(P) (a DNA binding protein that specifically recognizes the P sequence) to the P sequence was abolished when oligonucleotides carrying base substitutions were used, indicating that the NF(P) interaction is sequence-specific and that binding specificity of the protein paralleled the sequence requirements for IL-4 expression in vivo. The P sequence does not share homology with the 5' upstream sequence of the IL-2 gene, even though surrounding sequences of the IL-4 gene share high homology with the IL-2 gene. We conclude that a different set of proteins recognize IL-2 and IL-4 genes. 


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PPARalpha activators inhibit cytokine-induced vascular cell adhesion molecule-1 expression in human endothelial cells. 
BACKGROUND: Adhesion molecule expression on the endothelial cell (EC) surface is critical for leukocyte recruitment to atherosclerotic lesions. Better understanding of transcriptional regulation of adhesion molecules in ECs may provide important insight into plaque formation. Peroxisome proliferator-activated receptor-alpha (PPARalpha), a member of the nuclear receptor family, regulates gene expression in response to certain fatty acids and fibric acid derivatives. The present study investigated PPARalpha expression in human ECs and their regulation of vascular cell adhesion molecule-1 (VCAM-1). METHODS AND RESULTS: Immunohistochemistry revealed that human carotid artery ECs express PPARalpha. Pretreatment of cultured human ECs with the PPARalpha activators fenofibrate or WY14643 inhibited TNF-alpha-induced VCAM-1 in a time- and concentration-dependent manner, an effect not seen with PPARgamma activators. Both PPARalpha activators decreased cytokine-induced VCAM-1 mRNA expression without altering its mRNA half-life. Transient transfection of deletional VCAM-1 promoter constructs and electrophoretic mobility shift assays suggest that fenofibrate inhibits VCAM-1 transcription in part by inhibiting NF-kappaB. Finally, PPARalpha activators significantly reduced adhesion of U937 cells to cultured human ECs. CONCLUSIONS: Human ECs express PPARalpha, a potentially important regulator of atherogenesis through its transcriptional control of VCAM-1 gene expression. Such findings also have implications regarding the clinical use of lipid-lowering agents, like fibric acids, which can activate PPARalpha. 


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Inducible expression and phosphorylation of coactivator BOB.1/OBF.1 in T cells [see comments] 
BOB.1/OBF.1 is a transcriptional coactivator that is constitutively expressed in B cells and interacts with the Oct1 and Oct2 transcription factors. Upon activation of Jurkat T cells and primary murine thymocytes with phorbol esters and ionomycin, BOB.1/OBF.1 expression and transactivation function were induced. BOB.1/OBF.1 was phosphorylated at Ser184 both in vivo and in vitro, and this modification was required for inducible activation. Mutation of Ser184 also diminished transactivation function in B cells, suggesting that the activating phosphorylation that is inducible in T cells is constitutively present in B cells. Thus, BOB.1/OBF.1 is a transcriptional coactivator that is critically regulated by posttranslational modifications to mediate cell type-specific gene expression. 


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Oncogenic forms of NOTCH1 lacking either the primary binding site for RBP-Jkappa or nuclear localization sequences retain the ability to associate with RBP-Jkappa and activate transcription. 
Truncated forms of the NOTCH1 transmembrane receptor engineered to resemble mutant forms of NOTCH1 found in certain cases of human T cell leukemia/lymphoma (T-ALL) efficiently induce T-ALL when expressed in the bone marrow of mice. Unlike full-sized NOTCH1, two such truncated forms of the protein either lacking a major portion of the extracellular domain (DeltaE) or consisting only of the intracellular domain (ICN) were found to activate transcription in cultured cells, presumably through RBP-Jkappa response elements within DNA. Both truncated forms also bound to the transcription factor RBP-Jkappa in extracts prepared from human and murine T-ALL cell lines. Transcriptional activation required the presence of a weak RBP-Jkappa-binding site within the NOTCH1 ankyrin repeat region of the intracellular domain. Unexpectedly, a second, stronger RBP-Jkappa-binding site, which lies within the intracellular domain close to the transmembrane region and significantly augments association with RBP-Jkappa, was not needed for oncogenesis or for transcriptional activation. While ICN appeared primarily in the nucleus, DeltaE localized to cytoplasmic and nuclear membranes, suggesting that intranuclear localization is not essential for oncogenesis or transcriptional activation. In support of this interpretation, mutation of putative nuclear localization sequences decreased nuclear localization and increased transcriptional activation by membrane-bound DeltaE. Transcriptional activation by this mutant form of membrane-bound DeltaE was approximately equivalent to that produced by intranuclear ICN. These data are most consistent with NOTCH1 oncogenesis and transcriptional activation being independent of association with RBP-Jkappa at promoter sites. 


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Impaired induction of c-fos/c-jun genes and of transcriptional regulatory proteins binding distinct c-fos/c-jun promoter elements in activated human T cells during aging. 
The activation of transcriptional factor c-Fos/c-Jun AP-1 is essential for normal T cell responsiveness and is often impaired in T cells during aging. In the present study, we investigated whether aberrancies in the regulation of c-fos/c-jun at the mRNA or protein level might underlie the age-associated impairments of AP-1 in human T cells. Whereas T cells from young subjects stimulated with cross-linked anti-CD3epsilon mAb OKT3 plus PMA or with the lectin PHA plus PMA demonstrated considerable increases in c-Fos protein expression, the expression of c-Fos but not c-Jun was markedly reduced in stimulated T cells from certain elderly subjects. In addition, RNase protection assays revealed that anti-CD3/PMA-stimulated T cells from a substantial proportion of elderly subjects exhibited decreased levels of c-fos and/or c-jun mRNA compared to T cells from young subjects. Using electrophoretic mobility shift assays, the levels of nuclear regulatory proteins recognizing the AP-1 consensus TRE motif, the proximal c-jun TRE-like promoter element, and the c-fos serum response element (SRE) were determined in resting and stimulated T cells. Although the stimulation of T cells from young subjects resulted in coordinated increases of nuclear protein complexes binding the AP-1 TRE, c-jun TRE, and c-fos SRE DNA sequence motifs, age-related reductions in the activation of AP-1 were accompanied by decreased levels of c-jun TRE and c-fos SRE binding complexes. Furthermore, the nuclear protein complexes binding the SRE motif induced in activated T cells of young and elderly subjects contained serum response factor and Elk-1 pointing toward age-related defects in the activation of transcriptional regulatory proteins distinct from c-jun/AP-1. These results suggest that underlying aberrancies in the induction of c-fos/c-jun as well as their nuclear regulatory proteins may contribute to the age-related impairments of AP-1 activation in human T cells. 


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SLP-76 and Vav function in separate, but overlapping pathways to augment interleukin-2 promoter activity. 
SLP-76 and Vav, two hematopoietic cell specific molecules, are critical for T cell development and activation. Following T cell antigen receptor stimulation, SLP-76 and Vav both undergo tyrosine phosphorylation and associate with each other via the SH2 domain of Vav and phosphorylated tyrosines of SLP-76. Furthermore, SLP-76 and Vav have a synergistic effect on interleukin (IL)-2 promoter activity in T cells. In this report, we show that two tyrosines, Tyr-113 and Tyr-128, of SLP-76 are required for its binding to Vav, both in vitro and in intact cells. Surprisingly, we find also that the interaction between SLP-76 and Vav is not required for their cooperation in augmenting IL-2 promoter activity, as the two molecules appear to function in different signaling pathways upstream of IL-2 gene expression. Overexpression of SLP-76 in the Jurkat T cell line potentiates the activities of both nuclear factor of activated T cells and AP-1 transcription factors. In contrast, overexpression of Vav leads to enhanced nuclear factor of activated T cells activity without affecting AP-1. Additionally, overexpression of Vav, but not SLP-76, augments CD28-induced IL-2 promoter activity. These findings suggest that the synergy between SLP-76 and Vav in regulating IL-2 gene expression reflects the cooperation between different signaling pathways. 


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Human immunodeficiency virus type-2 gene expression: two enhancers and their activation by T-cell activators. 
The human immunodeficiency viruses (HIVs) may include a spectrum of retroviruses with varying potential to infect their host, undergo long periods of latent infection, and induce pathology. Since expression of the viruses is in large part regulated by the sequence elements in their long terminal repeats (LTRs), this study was directed to an analysis of the regulatory elements in the HIV-2 LTR. The HIV-2 LTR was found to contain two enhancers. One of these enhancers is, in part, identical to the HIV-1 enhancer. This enhancer in HIV-1 is the T-cell activation response element; in HIV-2, however, it is the second enhancer that is mainly responsible for activation in response to T-cell activators. The second enhancer interacts with two nuclear binding proteins (85 kD and 27 kD mobility) that appear to be required for optimal enhancer function and activation. Observations such as these encourage the speculation that there may be subtle differences in the regulation of HIV-1 and HIV-2 expression that may be relevant to the possible longer latency and reduced pathogenicity of HIV-2. 


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Vitamin E therapy of acute CCl4-induced hepatic injury in mice is associated with inhibition of nuclear factor kappa B binding. 
Oxidative stress, with reactive oxygen intermediate formation, may represent a common mechanism by which liver injury is induced by diverse etiologies. Oxidative stress enhances nuclear factor kappa B (NF-kappa B) activity, and NF-kappa B activity has been shown to enhance the expression of cytotoxic cytokines. Acute hepatic injury caused by reactive oxygen intermediate production was induced by an intraperitoneal injection of CCl4 in mice. This injury was significantly inhibited by intravenous pretreatment of the mice with a water-soluble emulsion of alpha-tocopherol. Alpha-tocopherol treatment of the mice given the CCl4 also reduced the NF-kappa B binding to levels approaching those found in normal mice. In vitro treatment of a monocyte/macrophage cell line with CCl4 led to enhanced NF-kappa B binding and an increase in tumor necrosis factor-alpha (TNF-alpha) messenger RNA levels. Liver specimens taken from patients with acute fulminant hepatitis had markedly increased NF-kappa B binding activity in comparison with the binding of normal livers. These data demonstrate that abolishing acute hepatic injury with alpha-tocopherol, a free radical scavenger, also eliminated increased NF-kappa B binding. It is tempting to speculate that enhanced NF-kappa B expression caused by free radical production/oxidative stress may modulate liver injury, perhaps through an effect on cytotoxic cytokine synthesis. 


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Limited proteolysis for assaying ligand binding affinities of nuclear receptors. 
The binding of natural or synthetic ligands to nuclear receptors is the triggering event leading to gene transcription activation or repression. Ligand binding to the ligand binding domain of these receptors induces conformational changes that are evidenced by an increased resistance of this domain to proteases. In vitro labeled receptors were incubated with various synthetic or natural agonists or antagonists and submitted to trypsin digestion. Proteolysis products were separated by SDS-PAGE and quantified. The amount of trypsin-resistant fragments was proportional to receptor occupancy by the ligand, and allowed the determination of dissociation constants (kDa). Using the wild-type or mutated human retinoic acid receptor alpha as a model, kDa values determined by classical competition binding assays using tritiated ligands are in agreement with those measured by the proteolytic assay. This method was successfully extended to human retinoic X receptor alpha, glucocorticoid receptor, and progesterone receptor, thus providing a basis for a new, faster assay to determine simultaneously the affinity and conformation of receptors when bound to a given ligand. 


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Isolation of a candidate repressor/activator, NF-E1 (YY-1, delta), that binds to the immunoglobulin kappa 3' enhancer and the immunoglobulin heavy-chain mu E1 site. 
We have determined that the developmental control of immunoglobulin kappa 3' enhancer (kappa E3') activity is the result of the combined influence of positive- and negative-acting elements. We show that a central core in the kappa E3' enhancer is active at the pre-B-cell stage but is repressed by flanking negative-acting elements. The negative-acting sequences repress enhancer activity in a position- and orientation-independent manner at the pre-B-cell stage. We have isolated a human cDNA clone encoding a zinc finger protein (NF-E1) that binds to the negative-acting segment of the kappa E3' enhancer. This protein also binds to the immunoglobulin heavy-chain enhancer mu E1 site. NF-E1 is encoded by the same gene as the YY-1 protein, which binds to the adeno-associated virus P5 promoter. NF-E1 is also the human homologue of the mouse delta protein, which binds to ribosomal protein gene promoters. The predicted amino acid sequence of this protein contains features characteristic of transcriptional activators as well as transcriptional repressors. Cotransfection studies with this cDNA indicate that it can repress basal promoter activity. The apparent dual function of this protein is discussed. 


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Cyclosporin A interferes with the inducible degradation of NF-kappa B inhibitors, but not with the processing of p105/NF-kappa B1 in T cells. 
The transcription factor NF-kappa B controls the induction of numerous cytokine promoters during the activation of T lymphocytes. Inhibition of T cell activation by the immunosuppressants cyclosporin A (CsA) and FK506 exerts a suppressive effect on the induction of these NF-kappa B-controlled cytokine promoters. We show for human Jurkat T leukemia cells, as well as human and mouse primary T lymphocytes, that this inhibitory effect is accompanied by an impaired nuclear translocation of the Rel proteins c-Rel, RelA/p65 and NF-kappa B1/p50, whereas the nuclear appearance of RelB remains unaffected. CsA does not interfere with the synthesis of Rel proteins, but prevents the inducible degradation of cytosolic NF-kappa B inhibitors I kappa B alpha and I kappa B beta upon T cell activation. CsA neither inhibits the processing of the NF-kappa B1 precursor p105 to p50, nor does it "stabilize" the C-terminal portion of p105, I kappa B gamma, which is degraded during p105 processing to mature p50. These results indicate that CsA interferes with a specific event in the signal-induced degradation of I kappa B alpha and I kappa B beta, but does not affect the processing of NF-kappa B1/p105 to p50. 


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Structural and functional characterization of the human CD36 gene promoter: identification of a proximal PEBP2/CBF site. 
CD36 is a cell surface glycoprotein composed of a single polypeptide chain, which interacts with thrombospondin, collagens type I and IV, oxidized low density lipoprotein, fatty acids, anionic phospholipids, and erythrocytes parasitized with Plasmodium falciparum. Its expression is restricted to a few cell types, including monocyte/macrophages. In these cells, CD36 is involved in phagocytosis of apoptotic cells, and foam cell formation by uptake of oxidized low density lipoprotein. To study the molecular mechanisms that control the transcription of the CD36 gene in monocytic cells we have isolated and analyzed the CD36 promoter. Transient expression experiments of 5'-deletion fragments of the CD36 promoter coupled to luciferase demonstrated that as few as 158 base pairs upstream from the transcription initiation site were sufficient to direct the monocyte-specific transcription of the reporter gene. Within the above region, the fragment spanning nucleotides -158 to -90 was required for optimal transcription in monocytic cells. Biochemical analysis of the region -158/-90 revealed a binding site for transcription factors of the polyomavirus enhancer-binding protein 2/core-binding factor (PEBP2/CBF) family at position -103. Disruption of the PEBP2/CBF site markedly diminished the role of the PEBP2/CBF factors in the constitutive transcription of the CD36 gene. The involvement of members of the PEBP2/CBF family in chromosome translocations associated with acute myeloid leukemia, and in the transcriptional regulation of the myeloid-specific genes encoding for myeloperoxidase, elastase, and the colony-stimulating factor receptor, highlights the relevance of the regulation of the CD36 gene promoter in monocytic cells by members of the PEBP2/CBF family. 


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Transcription factor activation and functional stimulation of human monocytes. 
Activation of expression of genes encoding transcription factors: c-fos and c-jun and formation of AP1 transcriptional complex in human monocytes was investigated. It was found that lipopolysaccharide induced strongly both c-fos and c-jun expression as well as AP1 formation. Interferon gamma activated strongly c-fos and weakly c-jun and AP1. Tumor necrosis factor induced slightly c-fos and had almost no effect on c-jun and AP1. The data suggest that differences in functional responses elicited in monocytes by all three factors may be dependent on different routes on nuclear signalling employed by the factors. 


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Potent and stable attenuation of live-HIV-1 by gain of a proteolysis-resistant inhibitor of NF-kappaB (IkappaB-alphaS32/36A) and the implications for vaccine development. 
Live-attenuated human immunodeficiency viruses (HIVs) are candidates for Acquired Immunodeficiency Syndrome (AIDS) vaccine. Based on the simian immunodeficiency virus (SIV) model for AIDS, loss-of-function (e.g. deletion of accessory genes such as nef) has been forwarded as a primary approach for creating enfeebled, but replication-competent, HIV-1/SIV. Regrettably, recent evidence suggests that loss-of-function alone is not always sufficient to prevent the emergence of virulent mutants. New strategies that attenuate via mechanisms distinct from loss-of-function are needed for enhancing the safety phenotype of viral genome. Here, we propose gain-of-function to be used simultaneously with loss-of-function as a novel approach for attenuating HIV-1. We have constructed an HIV-1 genome carrying the cDNA of a proteolysis-resistant nuclear factor-kappaB inhibitor (IkappaB-alphaS32/36A) in the nef region. HIV-1 expressing IkappaB-alphaS32/36A down-regulates viral expression and is highly attenuated in both Jurkat and peripheral blood mononuclear cells. We provide formal proof that the phenotypic and attenuating characteristics of IkappaB-alphaS32/36A permit its stable maintenance in a live, replicating HIV-1 despite 180 days of forced ex vivo passaging in tissue culture. As compared with other open-reading frames embedded into HIV/SIV genome, this degree of stability is unprecedented. Thus, IkappaB-alphaS32/36A offers proof-of-principle that artifactually gained functions, when used to attenuate the replication of live HIV-1, can be stable. These findings illustrate gain-of-function as a feasible strategy for developing safer live-attenuated HIVs to be tested as candidates for AIDS vaccine. 


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Control of I kappa B-alpha proteolysis by site-specific, signal-induced phosphorylation. 
I kappa B-alpha inhibits transcription factor NF-kappa B by retaining it in the cytoplasm. Various stimuli, typically those associated with stress or pathogens, rapidly inactivate I kappa B-alpha. This liberates NF-kappa B to translocate to the nucleus and initiate transcription of genes important for the defense of the organism. Activation of NF-kappa B correlates with phosphorylation of I kappa B-alpha and requires the proteolysis of this inhibitor. When either serine-32 or serine-36 of I kappa B-alpha was mutated, the protein did not undergo signal-induced phosphorylation or degradation, and NF-kappa B could not be activated. These results suggest that phosphorylation at one or both of these residues is critical for activation of NF-kappa B. 


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TAR-independent transactivation by Tat in cells derived from the CNS: a novel mechanism of HIV-1 gene regulation. 
The Tat protein of human immunodeficiency virus type 1 (HIV-1) is essential for productive infection and is a potential target for antiviral therapy. Tat, a potent activator of HIV-1 gene expression, serves to greatly increase the rate of transcription directed by the viral promoter. This induction, which seems to be an important component in the progression of acquired immune deficiency syndrome (AIDS), may be due to increased transcriptional initiation, increased transcriptional elongation, or a combination of these processes. Much attention has been focused on the interaction of Tat with a specific RNA target termed TAR (transactivation responsive) which is present in the leader sequence of all HIV-1 mRNAs. This interaction is believed to be an important component of the mechanism of transactivation. In this report we demonstrate that in certain CNS-derived cells Tat is capable of activating HIV-1 through a TAR-independent pathway. A Tat-responsive element is found upstream within the viral promoter that in glial-derived cell lines allows transactivation in the absence of TAR. Deletion mapping and hybrid promoter constructs demonstrate that the newly identified Tat-responsive element corresponds to a sequence within the viral long terminal repeat (LTR) previously identified as the HIV-1 enhancer, or NF-kappa B domain. DNA band-shift analysis reveals NF-kappa B binding activity in glial cells that differs from that present in T lymphoid cells. Further, we observe that TAR-deleted mutants of HIV-1 demonstrate normal late gene expression in glial cells as evidenced by syncytia formation and production of viral p24 antigen. (ABSTRACT TRUNCATED AT 250 WORDS) 


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Biphasic control of nuclear factor-kappa B activation by the T cell receptor complex: role of tumor necrosis factor alpha. 
The regulation of nuclear factor (NF)-kappa B activation by the T cell receptor (TcR)/CD3 complex in primary human T cells has been studied at various times after activation. Only p50 NF-kappa B protein bound the kappa B element of interleukin-2 receptor (IL-2R) alpha chain promoter on resting T cells. However, immediately after TcR/CD3 cross-linking (after approximately 1 h; immediate) binding of p50.p65 heterodimers was observed. p50.c-rel heterodimers were also detected bound to this sequence at early time points (7-16 h; early), and both remained active at later time points (40 h; late) after activation. This regulation takes place mainly at the level of nuclear translocation of p65 and c-rel, at immediate and early time points. Activation also induced c-rel and p105/p50 mRNA synthesis, but not p65 mRNA whose expression was constitutive. Interestingly, all those early and late events, but not the immediate ones, were inhibited by a neutralizing anti-tumor necrosis factor alpha (TNF-alpha) monoclonal antibody. Similarly, cycloheximide prevented the p65 and c-rel translocation and consequent formation of active binding heterodimers, at early and late times. Cyclosporin A impaired not only early and late, but also immediate events; however, addition of TNF-alpha prevented all inhibition. These results indicate that the regulation of NF-kappa B activation during T cell activation by TcR/CD3 signals is biphasic: TcR/CD3 triggers its immediate translocation, which is transient if no TNF-alpha is present. TNF-alpha, therefore, emerges as the main factor responsible for a second phase of NF-kappa B regulation, controlling both translocation of p65 and c-rel, and new mRNA synthesis for c-rel and p105/p50. 


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Transcriptional and post-transcriptional regulation of c-jun expression during monocytic differentiation of human myeloid leukemic cells. 
AP-1, the polypeptide product of c-jun, recognizes and binds to specific DNA sequences and stimulates transcription of genes responsive to certain growth factors and phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate (TPA). We studied the effects of TPA on the regulation of c-jun gene expression in HL-60 cells during monocytic differentiation. Low levels of c-jun transcripts were detectable in untreated HL-60 leukemic cells, increased significantly by 6 h, and reached near maximal levels by 24 h of exposure to 32 nM TPA. Similar kinetics of c-jun induction by TPA were observed in human U-937 and THP-1 monocytic leukemia cells. Similar findings were obtained with bryostatin 1 (10 nM), another activator of protein kinase C and inducer of monocytic differentiation. Furthermore, 1,25-dihydroxyvitamin D3 (0.5 microM), a structurally distinct agent which also induces HL-60 monocytic differentiation, increased c-jun expression. TPA treatment of HL-60 cells in the presence of cycloheximide was associated with superinduction of c-jun transcripts. Run-on analysis demonstrated detectable levels of c-jun gene transcription in untreated HL-60 cells, and that exposure to TPA increases this rate 3.3-fold. Treatment of HL-60 cells with both TPA and cycloheximide had no effect on the rates of c-jun transcription. The half-life of c-jun RNA as determined by treating HL-60 cells with TPA and actinomycin D was 30 min. In contrast, the half-life of c-jun RNA in TPA-treated HL-60 cells exposed to cycloheximide and actinomycin D was greater than 2 h. These findings suggested that the increase in c-jun RNA observed during TPA-induced monocytic differentiation is mediated by both transcriptional and post-transcriptional mechanisms. 


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Differential regulation of proto-oncogenes c-jun and c-fos in T lymphocytes activated through CD28. 
The T cell surface molecule CD28 binds to ligands on accessory cells and APCs, playing an important costimulatory role in the response of T cells to Ags. Our knowledge of the intracellular signaling pathways coupled to this receptor is incomplete. In addition to activation of phospholipase C gamma 1, ligation of this receptor also seems to activate a calcium-independent, CD28-specific pathway. In this paper, we report that cross-linking of CD28 (but not CD2, CD5, LFA-1, or CD7) leads to an elevation of c-jun mRNA, with only minimal activation of c-fos expression. CD28-dependent induction of c-jun expression requires protein tyrosine kinase activity, but does not depend on activation of a phorbol ester-responsive protein kinase C or elevation of cytosolic calcium. Furthermore, CD28-dependent elevation of c-jun mRNA does not appear to be mediated at the level of mRNA stability. A mechanism is suggested whereby expression of c-jun and junB, in the absence of members of the fos family, can prevent inappropriate activation of T cells caused by ligation of CD28 in the absence of a specific antigenic stimulus. 


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Antioxidants inhibit monocyte adhesion by suppressing nuclear factor-kappa B mobilization and induction of vascular cell adhesion molecule-1 in endothelial cells stimulated to generate radicals. 
Cell adhesion to endothelial cells stimulated by tumor necrosis factor-alpha (TNF) is due to induction of surface receptors, such as vascular cell adhesion molecule-1 (VCAM-1). The antioxidant pyrrolidine dithiocarbamate (PDTC) specifically inhibits activation of nuclear factor-kappa B (NF-kappa B). Since kappa B motifs are present in VCAM-1 and intercellular adhesion molecule-1 (ICAM-1) promoters, we used PDTC to study the regulatory mechanisms of VCAM-1 and ICAM-1 induction and subsequent monocyte adhesion in TNF-treated human umbilical vein endothelial cells (HUVECs). PDTC or N-acetylcysteine dose dependently reduced TNF-induced VCAM-1 but not ICAM-1 surface protein (also in human umbilical arterial endothelial cells) and mRNA expression (by 70% at 100 mumol/L PDTC) in HUVECs as assessed by flow cytometry and polymerase chain reaction. Gel-shift analysis in HUVECs demonstrated that PDTC prevented NF-kappa B mobilization by TNF, suggesting that only VCAM-1 induction was controlled by NF-kappa B. Since HUVECs released superoxide anions in response to TNF, and H2O2 induces VCAM-1, PDTC may act as a radical scavenger. Although ICAM-1 induction was unaffected, inhibitors of NADPH oxidase (apocynin) or cytochrome P-450 (SKF525a) suppressed VCAM-1 induction by TNF, revealing that several radical-generating systems are involved in its regulation. PDTC, apocynin, or SKF525a decreased adhesion of monocytic U937 cells to TNF-treated HUVECs (by 75% at 100 mumol/L PDTC). Inhibition by anti-VCAM-1 monoclonal antibody 1G11 indicated that U937 adhesion was VCAM-1 dependent and suppression by antioxidants was due to reduced VCAM-1 induction. (ABSTRACT TRUNCATED AT 250 WORDS) 


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Reduction of tumour necrosis factor alpha expression and signalling in peripheral blood mononuclear cells from patients with thalassaemia or sickle cell anaemia upon treatment with desferrioxamine. 
Recent evidence indicates that the rate of progression of the HIV-1 disease is significantly reduced in thalassaemia major patients upon treatment with high doses of desferrioxamine (DFX). The authors have previously demonstrated that in vitro exposure of mononuclear cells to DFX decreases the bioavailability of tumour necrosis factor alpha (TNF-alpha) which has a stimulatory effect on HIV-1 replication. In this study, therefore, TNF-alpha bioavailability from mononuclear cells isolated from 10 patients with thalassaemia or sickle cell anaemia given DFX as compared to 10 untreated subjects has been evaluated. Evidence is presented showing that DFX treatment reduces TNF-alpha bioavailability (P<0.05) by inhibiting its steady state (P<0.05) and by enhancing its inactivation through binding to soluble TNF-alpha receptor type II (P<0.05). We also show that DFX treatment limits the in vivo activation of NF-kappaB, a transcription factor involved in both TNF-alpha gene transcription and TNF-alpha signalling (P<0.005). We conclude that TNF-alpha bioavailability and signalling are impaired in patients upon DFX treatment. This mechanism may contribute to delayed progression of the HIV-1 infection in vivo. Copyright 1999 Academic Press. 


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Heterodimerization and transcriptional activation in vitro by NF-kappa B proteins. 
The NF-kappa B family of transcription proteins represents multiple DNA binding, rel related polypeptides that contribute to regulation of genes involved in immune responsiveness and inflammation, as well as activation of the HIV long terminal repeat. In this study multiple NF-kappa B related polypeptides ranging from 85 to 45 kDa were examined for their capacity to interact with the PRDII regulatory element of interferon beta and were shown to possess distinct intrinsic DNA binding affinities for this NF-kappa B site and form multiple DNA binding homo- and heterodimer complexes in co-renaturation experiments. Furthermore, using DNA templates containing two copies of the PRDII domain linked to the rabbit beta globin gene, the purified polypeptides specifically stimulated NF-kappa B dependent transcription in an in vitro reconstitution assay as heterodimers but not as p50 homodimers. These experiments emphasize the role of NF-kappa B dimerization as a distinct level of transcriptional control that may permit functional diversification of a limited number of regulatory proteins. 


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A nuclear factor NF-GM2 that interacts with a regulatory region of the GM-CSF gene essential for its induction in responses to T-cell activation: purification from human T-cell leukemia line Jurkat cells and similarity to NF-kappa B. 
Activation of T cells by antigen, lectin, or a combination of phorbol-12-myristate acetate (PMA) and calcium ionophore (A23187) leads to the induction of genes for a set of lymphokines, including granulocyte-macrophage colony-stimulating factor (GM-CSF). We demonstrated in earlier studies that the upstream region of the mouse GM-CSF promoter at positions between -95 and -73 is essential for transcriptional activation in response to PMA/A23187. This region contains two DNA-binding motifs, GM2 and GC-box. The GM2 sequence (GGTAGTTCCC) is recognized by an inducible factor NF-GM2; the other (CCGCCC) by constitutive factors A1, A2, and B. To elucidate the mechanism of GM-CSF gene activation, we have purified the inducible factor NF-GM2 from the nuclear extract of stimulated Jurkat cells on the basis of specific DNA-binding activity. The purified NF-GM2 consists of 50 (p50) and 65 kDa (p65) polypeptides and has a binding activity specific for both the GM-CSF and immunoglobulin kappa (GGAAAGTCCC) enhancers. Electrophoretically purified p50 alone can form a protein-DNA complex, but in the mixture, p50 associates preferentially with p65 to form the NF-GM2 complex. In addition, p65 gave per se, with low affinity, a protein-DNA complex that migrated more slowly than native NF-GM2 complex. Furthermore, an antiserum against KBF1 (identical to 50 kDa NF-kappa B protein) reacted with the p50 of NF-GM2, indicating that the NF-GM2 polypeptide cannot be immunologically differentiated from the 50 kDa subunit of NF-kappa B. The purified NF-GM2 activated in vitro transcription from the kappa B enhancer, while it failed to stimulate transcription from the GM-CSF promoter harboring the GM2 sequence. This suggests that the activation mechanism of the GM-CSF gene through the GM2/GC-box sequence is different from that of genes carrying the kappa B enhancer alone. 


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Retinoic acid-induced modulation of IL-2 mRNA production and IL-2 receptor expression on T cells. 
BACKGROUND: Retinoic acid (RA) has important immune-modulating effects on both T and B cell function. Our laboratory has shown that RA can enhance in vitro polyclonal B cell immunoglobulin (Ig) response. Investigating cytokines known to affect B cell differentiation, we have recently shown that IL-6 production is augmented by RA. In the present study we have examined the immune modulating effects of RA on IL-2 mRNA, another important cytokine for B cell immunoglobulin production, the expression of IL-2 receptors on T cells, and the RA nuclear receptors. METHODS: Purified T cells were obtained from adenoidal tissues, and incubated with RA (10(-7) M) or DMSO solvent/media control for 0, 6-8, and 24 h. Total mRNA was extracted from T cells, and using RT-PCR, changes in the production of IL-2 and RA receptors (RAR)-alpha,beta,gamma mRNA were determined. The effects of RA on IL-2-alpha receptor expression was determined by flow cytometry on T cells. CONCLUSION: These studies suggest that RA can augment IL-2 mRNA production by T cells with a possible paracrine effect on IL-2R-alpha expression. These changes appear to be mediated by RAR-alpha. Thus, IL-2 may be another important cytokine modulated by RA in the immune response. 


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IL-1 receptor and TCR signals synergize to activate NF-kappa B-mediated gene transcription. 
Previous studies have demonstrated that IL-1 receptor (IL-1R)- and TCR-initiated signals can interact synergistically to increase the rate of transcription of several lymphokine and lymphokine receptor genes during the competence phase of the activation program in T helper lymphocytes. In this report we describe how signals initiated through the type I IL-1R interact with signals from the antigen receptor to synergistically augment the transactivating properties of NF-kappa B. The synergistic antigen receptor initiated signals are mediated through protein kinase C because they can be mimicked by the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, but not with calcium ionophores; and are staurosporine sensitive but cyclosporine resistant. Gel shift analyses demonstrate that NF-kappa B nuclear translocation is stimulated primarily by IL-1 rather than by antigen receptor signals. Western blot and phosphorylation analyses demonstrate that the synergistic effect on NF-kappa B functional activity is independent of I kappa B alpha (MAD3)-NF-kappa B dissociation in the cytosol and is not associated with I kappa B nuclear translocation. The IL-1-induced NF-kappa B DNA nuclear localization is transient and can be prolonged either by an antigen receptor-initiated signal or by inhibiting protein synthesis. These results suggest that IL-1 induces both NF-kappa B nuclear translocation and the synthesis of a protein(s) responsible for terminating NF-kappa B-DNA interaction in the nucleus. Antigen receptor signals prolong NF-kappa B-DNA interaction, probably by functionally antagonizing the IL-1-induced synthesis of a protein(s) responsible for the transient NF-kappa B-DNA interaction and consequently synergistically enhance IL-1-induced NF-kappa B-dependent gene transcription. 


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Inhibition of NF-AT-dependent transcription by NF-kappa B: implications for differential gene expression in T helper cell subsets. 
Activation of individual CD4+ T cells results in differential lymphokine expression: interleukin 2 (IL-2) is preferentially produced by T helper type 1 (TH1) cells, which are involved in cell-mediated immune responses, whereas IL-4 is synthesized by TH2 cells, which are essential for humoral immunity. The Ca(2+)-dependent factor NF-ATp plays a key role in the inducible transcription of both these lymphokine genes. However, while IL2 expression requires the contribution of Ca(2+)- and protein kinase C-dependent signals, we report that activation of human IL4 transcription through the Ca(2+)-dependent pathway is diminished by protein kinase C stimulation in Jurkat T cells. This phenomenon is due to mutually exclusive binding of NF-ATp and NF-kappa B to the P sequence, an element located 69 bp upstream of the IL4 transcription initiation site. Human IL4 promoter-mediated transcription is downregulated in Jurkat cells stimulated with the NF-kappa B-activating cytokine tumor necrosis factor alpha and suppressed in RelA-overexpressing cells. In contrast, protein kinase C stimulation or RelA overexpression does not affect the activity of a human IL4 promoter containing a mouse P sequence, which is a higher-affinity site for NF-ATp and a lower-affinity site for RelA. Thus, competition between two general transcriptional activators, RelA and NF-ATp, mediates the inhibitory effect of protein kinase C stimulation on IL4 expression and may contribute to differential gene expression in TH cells. 


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Inhibition of NF-kappa B activation in vitro and in vivo: role of 26S proteasome. 
It is becoming increasingly apparent that NF-kappa B plays a critical role in regulating the inflammatory response. Data obtained from studies in our laboratories demonstrate that the proteasome plays an important role in the inflammatory cascade by regulating the activation of NF-kappa B. Indeed, the availability of selective and orally active proteasome inhibitors should prove useful in delineating the roles of the proteasome and NF-kappa B in other pathophysiological conditions such as cancer and heart disease. 


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HIV1 infection of human monocytes and macrophages promotes induction or translocation of NF-KB-related factors. 
In 1991, we demonstrated, using electrophoretic mobility shift assays, that 3 different factors (termed B1, B2 and B3) with affinity for the KB-enhancer target sequence were specifically detected in nuclear extracts from HIV1-infected monocytes and macrophages. The B2 factor was induced in the nuclei of these cells only upon HIV1 infection. The B3 factor was only slightly evident in nuclei of uninfected cells but was readily detectable in nuclei of infected monocytes. Its expression remained very low in nuclei of HIV1-infected macrophages. In this paper, we demonstrate that the B2 factor is expressed in the cytosol of monocytes and macrophages as a DNA-binding protein, indicating that it is not associated with an inhibitor (IKB). This factor remained clustered in the cytosol and was translocated to the nuclei only after HIV1 infection. The B3 factor is detected in the cytosol only when cells are HIV1-infected. The role of HIV1 infection in the expression and the translocation of these factors is discussed. 


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The intracellular parasite Theileria parva protects infected T cells from apoptosis. 
Parasites have evolved a plethora of strategies to ensure their survival. The intracellular parasite Theileria parva secures its propagation and spreads through the infected animal by infecting and transforming T cells, inducing their continuous proliferation and rendering them metastatic. In previous work, we have shown that the parasite induces constitutive activation of the transcription factor NF-kappaB, by inducing the constitutive degradation of its cytoplasmic inhibitors. The biological significance of NF-kappaB activation in T. parva-infected cells, however, has not yet been defined. Cells that have been transformed by viruses or oncogenes can persist only if they manage to avoid destruction by the apoptotic mechanisms that are activated on transformation and that contribute to maintain cellular homeostasis. We now demonstrate that parasite-induced NF-kappaB activation plays a crucial role in the survival of T. parva-transformed T cells by conveying protection against an apoptotic signal that accompanies parasite-mediated transformation. Consequently, inhibition of NF-kappaB nuclear translocation and the expression of dominant negative mutant forms of components of the NF-kappaB activation pathway, such as IkappaBalpha or p65, prompt rapid apoptosis of T. parva-transformed T cells. Our findings offer important insights into parasite survival strategies and demonstrate that parasite-induced constitutive NF-kappaB activation is an essential step in maintaining the transformed phenotype of the infected cells. 


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Posttranscriptional regulation of macrophage tissue factor expression by antioxidants. 
Tissue factor (TF) expression by cells of monocyte/macrophage lineage represents an important mechanism underlying the initiation of fibrin deposition at sites of extravascular inflammation. Recent evidence suggests a role for oxidant stress in the signalling pathway of various cell types by virtue of its ability to induce DNA binding of various transcription factors, including nuclear factor kappa B and AP-1. The effect of antioxidant treatment on lipopolysaccharide (LPS)-induced TF expression was examined in murine peritoneal macrophages and human monocytes. Both pyrrolidine dithiocarbamate, an oxidant scavenger, and N-acetyl-cysteine, a precursor of the endogenous antioxidant glutathione, inhibited stimulation of macrophage procoagulant activity by LPS. Northern blot analysis showed that neither of these agents reduced LPS-stimulated TF mRNA accumulation, thereby suggesting a posttranscriptional mechanism for the effect. Immunofluorescence studies of human monocytes using polyclonal anti-TF antibody showed that N-acetyl-cysteine treatment prevented the characteristic plasmalemmal localization of TF antigen that occurs in response to LPS. Western blot analysis showed that N-acetyl-cysteine reduced the accumulation of the 47-kD mature glycoprotein in LPS-treated cells, a finding consistent with the results of the immunofluorescence studies. Furthermore, these conditions did not result in an accumulation of the less mature forms of TF. When considered together, these data suggest that antioxidants exert their effects by impairing translation and/or by causing degradation of newly translated protein. The effect of antioxidants on tumor necrosis factor appeared to be species specific, with no effect on LPS-induced tumor necrosis factor in murine cells, but with inhibition in human monocytes. The posttranscriptional effect of antioxidants on TF expression data suggests a novel mechanism whereby these agents might modulate monocyte/macrophage activation. 


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Natural variants of the HIV-1 long terminal repeat: analysis of promoters with duplicated DNA regulatory motifs. 
Sequence variation in the long terminal repeat (LTR) region of HIV-1 was analyzed in viral isolates of 17 infected individuals. Two classes of LTR size variants were found. One HIV-1 variant was detected containing an additional binding site for the transcription factor Sp1. Another LTR size variation was observed in four patients in a region just upstream of the NF-kappa B enhancer. This variation was the result of a duplication of a short DNA sequence (CTG-motif). Cell culture experiments demonstrated that the natural variant with four Sp1 sites had a slightly higher promoter activity and viral replication rate than the isogenic control LTR with three Sp1 sites. No positive effect of the duplicated CTG-motif could be detected. In order to measure small differences in virus production more accurately, equal amounts of a size variant and the wild-type plasmid were cotransfected into T-cells. The virus with four Sp1 sites did outgrow the three Sp1 virus in 35 days of culture and CTG-monomer virus outcompeted the CTG-dimer virus in 42 days. Based on these results we estimate a 5-10% difference in virus production of the LTR variants when compared to that of wild-type. 


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Human immunodeficiency virus type 1 Nef protein down-regulates transcription factors NF-kappa B and AP-1 in human T cells in vitro after T-cell receptor stimulation. 
Human immunodeficiency virus type 1 (HIV-1) negative factor (Nef) has been shown to down-regulate the transcription factors NF-kappa B and AP-1 in vitro. To define the mechanism of action of the Nef protein, the signal transduction pathways which may be affected in T cells by constitutive expression of the nef gene were examined. Stimulation of T cells with tumor necrosis factor, interleukin-1, or lipopolysaccharide resulted in the recruitment of transcriptional factors to a similar level whether or not the cells expressed the nef gene. On the other hand, stimulation of T cells by mitogens or antibodies to the T-cell receptor (TCR)-CD3 complex resulted in the down-regulation of transcriptional factors NF-kappa B and AP-1 in cells expressing the nef gene compared with cells not expressing the nef gene. Because the Nef protein does not affect the surface expression of the CD3-TCR complex, we conclude that the Nef protein down-regulates the transcriptional factors NF-kappa B and AP-1 in T cells in vitro through an effect on the TCR-dependent signal transduction pathway. 


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Induction of relA(p65) and I kappa B alpha subunit expression during differentiation of human peripheral blood monocytes to macrophages. 
We evaluated the expression and DNA binding activity of nuclear factor (NF)-kappa B subunits in human peripheral blood monocytes and in monocyte-derived macrophages (MDMs). Constitutive DNA binding activity consisting of p50 homodimers was detected in nuclear extracts from both cell types. An additional complex composed of p50/RelA(p65) heterodimers appeared only in nuclear extracts from 7-day MDMs. Immunoblot analysis showed that the p50 subunit was constitutively expressed in monocytes and MDMs. In contrast, the RelA(p65) subunit was barely detectable in monocytes, but its level increased markedly in MDMs. Analysis of RelA(p65) mRNA revealed that the stability of RelA(p65) mRNA was significantly higher in MDMs, compared with monocytes. In MDMs, an upregulation of I kappa B alpha synthesis as well as the appearance of a novel M(r) 40,000 form of I kappa B alpha were also observed. These results suggest that macrophage differentiation results in the expression of active p50/RelA(p65) heterodimers with the capacity to activate target gene expression. The parallel induction of I kappa B alpha synthesis may allow for the continuous presence of a cytoplasmic reservoir of p50/RelA(p65) complexes that are readily available for inducer-mediated stimulation. 


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Phosphatidylinositides bind to plasma membrane CD14 and can prevent monocyte activation by bacterial lipopolysaccharide. 
Although bacterial lipopolysaccharides (LPS) and several other microbial agonists can bind to mCD14 (membrane CD14), a cell-surface receptor found principally on monocytes and neutrophils, host-derived mCD14 ligands are poorly defined. We report here that phosphatidylinositol (PtdIns), phosphatidylinositol-4-phosphate, and other phosphatidylinositides can bind to mCD14. Phosphatidylserine (PS), another anionic glycerophospholipid, binds to mCD14 with lower apparent affinity than does PtdIns. LPS-binding protein, a lipid transfer protein found in serum, facilitates both PS- and PtdIns-mCD14 binding. PtdIns binding to mCD14 can be blocked by anti-CD14 monoclonal antibodies that inhibit LPS-mCD14 binding, and PtdIns can inhibit both LPS-mCD14 binding and LPS-induced responses in monocytes. Serum-equilibrated PtdIns also binds to mCD14-expressing cells, raising the possibility that endogenous PtdIns may modulate cellular responses to LPS and other mCD14 ligands in vivo. 


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The peri-kappa B site mediates human immunodeficiency virus type 2 enhancer activation in monocytes but not in T cells. 
Human immunodeficiency virus type 2 (HIV-2), like HIV-1, causes AIDS and is associated with AIDS cases primarily in West Africa. HIV-1 and HIV-2 display significant differences in nucleic acid sequence and in the natural history of clinical disease. Consistent with these differences, we have previously demonstrated that the enhancer/promoter region of HIV-2 functions quite differently from that of HIV-1. Whereas activation of the HIV-1 enhancer following T-cell stimulation is mediated largely through binding of the transcription factor NF-kappa B to two adjacent kappa B sites in the HIV-1 long terminal repeat, activation of the HIV-2 enhancer in monocytes and T cells is dependent on four cis-acting elements: a single kappa B site, two purine-rich binding sites, PuB1 and PuB2, and a pets site. We have now identified a novel cis-acting element within the HIV-2 enhancer, immediately upstream of the kappa B site, designated peri-kappa B. This site is conserved among isolates of HIV-2 and the closely related simian immunodeficiency virus, and transfection assays show this site to mediate HIV-2 enhancer activation following stimulation of monocytic but not T-cell lines. This is the first description of an HIV-2 enhancer element which displays such monocyte specificity, and no comparable enhancer element has been clearly defined for HIV-1. While a nuclear factor(s) from both peripheral blood monocytes and T cells binds the peri-kappa B site, electrophoretic mobility shift assays suggest that either a different protein binds to this site in monocytes versus T cells or that the protein recognizing this enhancer element undergoes differential modification in monocytes and T cells, thus supporting the transfection data. Further, while specific constitutive binding to the peri-kappa B site is seen in monocytes, stimulation with phorbol esters induces additional, specific binding. Understanding the monocyte-specific function of the peri-kappa B factor may ultimately provide insight into the different role monocytes and T cells play in HIV pathogenesis. 


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Suppressive effects of anti-inflammatory agents on human endothelial cell activation and induction of heat shock proteins. 
BACKGROUND: Studies from our laboratory have shown that the earliest stages of atherosclerosis may be mediated by an autoimmune reaction against heat shock protein 60 (Hsp60). The interactions of Hsp60-specific T cells with arterial endothelial cells (EC) require expression of both Hsp60 and certain adhesion molecules shown to be induced simultaneously in EC by mechanical and other types of stress. Recently, it was shown that suppression of T cell-mediated immune responses by cyclosporin A (CyA) enhanced atherosclerotic lesion formation in mice. In contrast, aspirin was found to lower the risk of myocardial infarction in men. These conflicting observations may be due to different effects of anti-inflammatory agents on adhesion molecule and Hsp expression in EC, respectively. MATERIAL AND METHODS: In the present study, we analyzed the effects of CyA, aspirin, and indomethacin on T cell proliferation using a proliferation assay. To explore the expression of adhesion molecules, monocyte chemoattractant protein-1 (MCP-1), and Hsp60 in human umbilical vein endothelial cells (HUVECs), Northern blot analyses were used. To examine the activation status of the transcription factors nuclear factor kappaB (NF-kappaB) and heat shock factor-1 (HSF-1), electrophoretic mobility shift assays were performed. RESULTS: With the exception of indomethacin, the used immunosuppressive and anti-inflammatory agents significantly inhibited T cell proliferation in response to influenza virus antigen in a dose-dependent manner. Interestingly, CyA and indomethacin did not suppress tumor necrosis factor-alpha (TNF-alpha)-induced adhesion molecule expression on HUVECs, whereas aspirin had an inhibitory effect. These observations correlated with the modulation of NF-kappaB activity in EC. All agents tested induced expression of Hsp60 6 hr after application. In addition, aspirin and indomethacin, but not CyA, induced Hsp70 expression in HUVECs that correlated with induction of HSF-1 activity. CONCLUSION: Our results show that the tested agents (except indomethacin) are inhibitors of the T cell-mediated immune response, as expected, that aspirin is an effective suppressor of adhesion molecule expression, and that all three agents can induce Hsp60 in HUVECs. These data provide the molecular basis for the notion that (1) part of the anti-atherogenic effect of aspirin may be due to the prevention of the adhesion of sensitized T cells to stressed EC; (2) that part of the atherosclerosis-promoting effect of CyA may be due to its potential as an inducer of Hsp60 expression and its inability to down-regulate adhesion molecule expression on EC; and (3) that down-regulation of MCP-1 expression by aspirin may result in decreased recruitment of monocytes into the arterial intima beneath stressed EC. 


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Distinct roles of the molecular chaperone hsp90 in modulating dioxin receptor function via the basic helix-loop-helix and PAS domains. 
The intracellular dioxin receptor mediates signal transduction by dioxin and functions as a ligand-activated transcription factor. It contains a basic helix-loop-helix (bHLH) motif contiguous with a Per-Arnt-Sim (PAS) homology region. In extracts from nonstimulated cells the receptor is recovered in an inducible cytoplasmic form associated with the 90-kDa heat shock protein (hsp90), a molecular chaperone. We have reconstituted ligand-dependent activation of the receptor to a DNA-binding form by using the dioxin receptor and its bHLH-PAS partner factor Arnt expressed by in vitro translation in reticulocyte lysate. Deletion of the PAS domain of the receptor resulted in constitutive dimerization with Arnt. In contrast, this receptor mutant showed low levels of xenobiotic response element-binding activity, indicating that the PAS domain may be important for DNA-binding affinity and/or specificity of the receptor. It was not possible to reconstitute dioxin receptor function with proteins expressed in wheat germ lysate. In line with these observations, reticulocyte lysate but not wheat germ lysate promoted the association of de novo synthesized dioxin receptor with hsp90. At least two distinct domains of the receptor mediated interaction with hsp90: the ligand-binding domain located within the PAS region and, surprisingly, the bHLH domain. Whereas ligand-binding activity correlated with association with hsp90, bHLH-hsp90 interaction appeared to be important for DNA-binding activity but not for dimerization of the receptor. Several distinct roles for hsp90 in modulating dioxin receptor function are therefore likely: correct folding of the ligand-binding domain, interference with Arnt heterodimerization, and folding of a DNA-binding conformation of the bHLH domain. Thus, the dioxin receptor system provides a complex and interesting model of the regulation of transcription factors by hsp90. 


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Danazol decreases transcription of estrogen receptor gene in human monocytes. 
1. Administration of danazol for over one month reduced the levels of estrogen receptor (ER) and its mRNA to approximately 50 and 20%, respectively in monocytes. 2. Danazol did not alter the degradation rate of ER mRNA in monocytes. 3. Danazol decreased the transcription rate of ER gene to approximately 50% in monocytes in a run-on assay. 4. Danazol may release estrogen predominance via the reduction of transcription for ER gene, which leads to the reduction of ER mRNA and ER expressions in monocytes. 


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An intricate arrangement of binding sites for the Ets family of transcription factors regulates activity of the alpha 4 integrin gene promoter. 
alpha 4 integrins mediate cell-cell and cell-extracellular matrix interactions that are critical for maturation and function of the immune system as well as differentiation of skeletal muscle. Here we examine molecular mechanisms controlling the pattern of alpha 4 expression. The activity of constructs containing 5' deletion mutants of the alpha 4 gene promoter was compared in transfection assays into cell lines that express alpha 4 and cell lines that do not. The sequence between position -42 and -76 base pairs (bp) was required for efficient transcription in cells that express alpha 4, but it showed no activity in HeLa cells, which do not express alpha 4. Three binding sites for the Ets family of transcription factors are found in this region: two adjacent sites at positions -50 and -54 bp and a more 5' site at position -67 bp. Using a series of constructs containing deletions and mutations in this region, we found that the 3'-most site alone was sufficient for binding GA-binding protein alpha (GABP alpha)/GABP beta and for a low level of transcriptional activation. When all three sites were present, a second complex "a" was detected, which contains an unknown member of the Ets family. Formation of complex a was cell-type specific and correlated with a high level of transcription. Deletion of the 5'-most Ets site had no effect on binding to GABP alpha/GABP beta, but it eliminated a. Concomitant with this loss of a, a new Ets-1-containing complex "c" appeared. Complex c substituted efficiently for complex a in transcriptional activation. We conclude that although neither of the two 5'-most Ets sites alone binds nuclear protein, they appear to act as modulators which control the pattern of Ets proteins that bind the alpha 4 gene promoter. This arrangement of Ets sites, coupled with the tissue- and developmental-specific expression of Ets members, likely play a key role in defining the pattern of alpha 4 integrin. 


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Kappa B-specific DNA binding proteins are differentially inhibited by enhancer mutations and biological oxidation. 
Kappa B (kappa B) enhancer binding proteins isolated from the nuclei of activated human T cells produce two distinct nucleoprotein complexes when incubated with the kappa B element from the interleukin-2 receptor-alpha (IL-2R alpha) gene. These two DNA-protein complexes are composed of at least four host proteins (p50, p55, p75, p85), each of which shares structural similarity with the v-rel oncogene product. Nuclear expression of these proteins is induced with distinctly biphasic kinetics following phorbol ester activation of T cells (p55/p75 early and p50/p85 late). DNA-protein crosslinking studies have revealed that the more rapidly migrating B2 complex contains both p50 and p55 while the more slowly migrating B1 complex is composed of p50, p55, p75, and p85. Site-directed mutagenesis of the wild-type IL-2R alpha kappa B enhancer (GGGGAATCTCCC) has revealed that the binding of p50 and p55 (B2 complex) is particularly sensitive to alteration of the 5' triplet of deoxyguanosine residues. In contrast, formation of the B1 complex, reflecting the binding of p75 and p85, critically depends upon the more 3' sequences of this enhancer element. DNA binding by all four of these Rel-related factors is blocked by selective chemical modification of lysine and arginine residues, suggesting that both of these basic amino acids are required for binding to the kappa B element. Similarly, covalent modification of free sulfhydryl groups with diamide (reversible) or N-ethylmaleimide (irreversible) results in a complete loss of DNA binding activity. In contrast, mild oxidation with glucose oxidase selectively inhibits p75 and p85 binding while not blocking p50 and p55 interactions. These findings suggest that reduced cysteine thiols play an important role in the DNA binding activity of this family of Rel-related transcription factors. 


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Involvement of tyrosine phosphorylation in endothelial adhesion molecule induction. 
Induction of endothelial adhesion molecules by the cytokine tumor necrosis factor-alpha (TNF) can occur independently of protein kinase C and activation of a protein tyrosine kinase (PTK) has recently been implicated in the upregulation of vascular cell adhesion molecule 1 (VCAM-1) by interleukin-4 (IL-4) on endothelial cells. We demonstrate that the PTK inhibitors herbimycin A or genistein suppress induction of endothelial VCAM-1 and E-selectin, as well as subsequent monocytic cell adhesion to endothelial cells stimulated by TNF. Inhibition studies indicate that specific tyrosine phosphorylation following PTK activation is involved in the mobilization of the transcription factor, nuclear factor kappa B, and VCAM-1 mRNA expression. This may have implications for pathophysiological conditions that involve the upregulation of these molecules (e.g. inflammation and atherosclerosis). 


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Glycation-dependent, reactive oxygen species-mediated suppression of the insulin gene promoter activity in HIT cells. 
Prolonged poor glycemic control in non-insulin-dependent diabetes mellitus patients often leads to a decline in insulin secretion from pancreatic beta cells, accompanied by a decrease in the insulin content of the cells. As a step toward elucidating the pathophysiological background of the so-called glucose toxicity to pancreatic beta cells, we induced glycation in HIT-T15 cells using a sugar with strong deoxidizing activity, D-ribose, and examined the effects on insulin gene transcription. The results of reporter gene analyses revealed that the insulin gene promoter is more sensitive to glycation than the control beta-actin gene promoter; approximately 50 and 80% of the insulin gene promoter activity was lost when the cells were kept for 3 d in the presence of 40 and 60 mM D-ribose, respectively. In agreement with this, decrease in the insulin mRNA and insulin content was observed in the glycation-induced cells. Also, gel mobility shift analyses using specific antiserum revealed decrease in the DNA-binding activity of an insulin gene transcription factor, PDX-1/IPF1/STF-1. These effects of D-ribose seemed almost irreversible but could be prevented by addition of 1 mM aminoguanidine or 10 mM N-acetylcysteine, thus suggesting that glycation and reactive oxygen species, generated through the glycation reaction, serve as mediators of the phenomena. These observations suggest that protein glycation in pancreatic beta cells, which occurs in vivo under chronic hyperglycemia, suppresses insulin gene transcription and thus can explain part of the beta cell glucose toxicity. 


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Lactobacilli and vaginal host defense: activation of the human immunodeficiency virus type 1 long terminal repeat, cytokine production, and NF-kappaB. 
Lactobacilli, a component of the normal vaginal flora, can activate the human immunodeficiency virus (HIV)-1 long terminal repeat (LTR) in the Jurkat T lymphocyte and THP-1 macrophage cell lines. Activation of the LTR in Jurkat cells was strongly enhanced by vanadate and inhibited by catalase, implicating H2O2. In contrast, activation in THP-1 cells occurred in the absence of vanadate and was unaffected by catalase. The active material partitioned into the phenol layer on hot aqueous phenol extraction. Lactobacilli also increased tumor necrosis factor-alphaand interleukin-1betaproduction and activated NF-kappaB in THP-1 cells and increased tumor necrosis factor-alphaproduction by human monocytes. Human vaginal fluid specimens had comparable properties, which correlated with their bacterial content. These findings suggest the presence in vaginal fluid of agent(s) derived from indigenous bacteria that can activate the HIV-1 LTR, cytokine production, and NF-kappaB in cells of macrophage lineage, with possible influence on vaginal physiology and host defense. 


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Human T-cell leukemia virus type 1 Tax induction of NF-kappaB involves activation of the IkappaB kinase alpha (IKKalpha) and IKKbeta cellular kinases. 
Tax corresponds to a 40-kDa transforming protein from the pathogenic retrovirus human T-cell leukemia virus type 1 (HTLV-1) that activates nuclear expression of the NF-kappaB/Rel family of transcription factors by an unknown mechanism. Tax expression promotes N-terminal phosphorylation and degradation of IkappaB alpha, a principal cytoplasmic inhibitor of NF-kappaB. Our studies now demonstrate that HTLV-1 Tax activates the recently identified cellular kinases IkappaB kinase alpha (IKKalpha) and IKKbeta, which normally phosphorylate IkappaB alpha on both of its N-terminal regulatory serines in response to tumor necrosis factor alpha (TNF-alpha) and interleukin-1 (IL-1) stimulation. In contrast, a mutant of Tax termed M22, which does not induce NF-kappaB, fails to activate either IKKalpha or IKKbeta. Furthermore, endogenous IKK enzymatic activity was significantly elevated in HTLV-1-infected and Tax-expressing T-cell lines. Transfection of kinase-deficient mutants of IKKalpha and IKKbeta into either human Jurkat T or 293 cells also inhibits NF-kappaB-dependent reporter gene expression induced by Tax. Similarly, a kinase-deficient mutant of NIK (NF-kappaB-inducing kinase), which represents an upstream kinase in the TNF-alpha and IL-1 signaling pathways leading to IKKalpha and IKKbeta activation, blocks Tax induction of NF-kappaB. However, plasma membrane-proximal elements in these proinflammatory cytokine pathways are apparently not involved since dominant negative mutants of the TRAF2 and TRAF6 adaptors, which effectively block signaling through the cytoplasmic tails of the TNF-alpha and IL-1 receptors, respectively, do not inhibit Tax induction of NF-kappaB. Together, these studies demonstrate that HTLV-1 Tax exploits a distal part of the proinflammatory cytokine signaling cascade leading to induction of NF-kappaB. The pathological alteration of this cytokine pathway leading to NF-kappaB activation by Tax may play a central role in HTLV-1-mediated transformation of human T cells, clinically manifested as the adult T-cell leukemia. 


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Transcriptional analysis of Epstein-Barr virus gene expression in EBV-positive gastric carcinoma: unique viral latency in the tumour cells. 
Although case-oriented evidence for an association of Epstein-Barr virus (EBV) with gastric carcinoma has been accumulating recently, the interaction(s) between EBV and gastric epithelial cells is/are largely unknown. In this study, we examined seven EBV-positive gastric carcinoma tissues for viral gene expression at the mRNA level, from which studies on the EBV oncogenicity in human epithelial cells will benefit. Reverse transcription-PCR analysis showed that all seven EBV-positive tumour tissues constitutively expressed EBV nuclear antigen (EBNA) 1 mRNA, but not EBNA2 mRNA. The EBNA transcription was initiated from one of three EBNA promoters, Qp: by contrast, both Cp and Wp were silent, thus resulting in the lack of EBNA2 mRNA. Latent membrane protein (LMP) 2A mRNA was detected in three of seven cases; however, neither LMP1 nor LMP2B mRNA was detected in any of the tumours tested. Transcripts from the BamHI-A region of the viral genome were detectable in all cases. BZLF1 mRNA and the product, an immediate-early gene for EBV replication, was not expressed in any of them, thereby suggesting that the tumour cells carried EBV genomes in a tightly latent form. These findings further extended our previous data regarding EBV latency in gastric carcinoma cells at the protein level, and have affirmed that the programme of viral gene expression in the tumour more closely resembles 'latency I' represented by Burkitt's lymphoma than 'latency II' represented by the majority of nasopharyngeal carcinomas. 


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Differential interaction of nuclear factors with the PRE-I enhancer element of the human IL-4 promoter in different T cell subsets. 
The immunomodulatory cytokine IL-4 affects cells of most hemopoietic lineages. IL-4 is secreted by activated Th2 but not Th1 cells and plays a major role in the immune response by modulating the differentiation of naive Th cells toward the Th2 phenotype. We have previously identified an enhancer element, PRE-I, that is essential for the function of the human IL-4 promoter. To investigate the mechanisms responsible for tissue-specific expression of the IL-4 gene, we analyzed nuclear factors binding to the PRE-I site and compared the binding activities of these factors to the IL-4 promoter of Th1 and Th2 cells. We show that PRE-I interacts with PMA- and PMA/ionomycin-inducible, cyclosporin A-sensitive nuclear factors. Using anti-C/EBPbeta (NF-IL6), anti-C/EBPdelta (NF-IL6beta), anti-NF-ATc, anti-NF-ATp, anti-Fos, and anti-Jun Abs we demonstrate that the previously identified PRE-I binding factor POS-1 is composed of different transcription factors in different Th cell subsets. In the IL-4-producing Th0-like human Jurkat and mouse EL-4 cells, POS-1 (designated POS-1a) contains NF-IL6beta and Jun. In the mouse Th2 D10 cells and in the human Th2 clones, POS-1 (designated POS-1b) contains NF-IL6beta, Jun, and NF-ATc/p. In contrast, POS-1 was not found in nuclear extracts of human Th1 clones. These findings suggest that PRE-I may play a role in the differential regulation of IL-4 gene expression levels. 


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Inhibition of protein phosphatases by okadaic acid induces AP1 in human T cells. 
To examine the role of protein phosphatases in T cell activation, Jurkat cells were treated with okadaic acid, an inhibitor of type 1 and 2A phosphatases, and nuclear extracts were examined for the presence of AP1 as a measure of early T cell activation. Okadaic acid was found to be a potent inducer of AP1. In contrast to phorbol esters such as phorbol myristate acetate (PMA), the induction of AP1 by okadaic acid occurs predominantly by transcriptional activation of the jun and fos family of proto-oncogenes. Surprisingly, while the addition of phytohemagglutinin further enhanced the induction of AP1, the addition of PMA inhibited it. Okadaic acid treatment was found to dramatically increase mRNA transcripts of the jun family of proto-oncogenes including c-jun, junD, and junB and to a lesser extent the fos family including c-fos and fra-1. By comparison, PMA is a very inefficient inducer of the jun gene family in Jurkat cells. Similar to its effect on the induction of AP1 by okadaic acid, PMA inhibits the induction of c-jun mRNA by okadaic acid. Transfection of c-jun promoter constructs confirmed the marked difference between PMA and okadaic acid in inducing c-jun transcription. The induction of AP1 by okadaic acid suggests that protein phosphatases 1 and 2A (PP1 and PP2A) may be involved in T cell activation as important negative regulators of the transcription factor AP1. 


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Molecular mechanisms of anoxia/reoxygenation-induced neutrophil adherence to cultured endothelial cells. 
The objectives of this study were to (1) determine the time course of neutrophil adhesion to monolayers of human umbilical vein endothelial cells (HUVECs) that were exposed to 60 minutes of anoxia followed by 30 to 600 minutes of reoxygenation and (2) define the mechanisms responsible for both the early (minutes) and late (hours) hyperadhesivity of postanoxic HUVECs to human neutrophils. The results clearly demonstrate that anoxia/reoxygenation (A/R) leads to a biphasic increase in neutrophil adhesion to HUVECs, with peak responses occurring at 30 minutes (phase 1) and 240 minutes (phase 2) after reoxygenation. Oxypurinol and catalase inhibited phase-1 adhesion, suggesting a role for xanthine oxidase and H2O2. In comparison, platelet activating factor (PAF) contributed to both phases of neutrophil adhesion. Anti-intercellular adhesion molecule-1 (ICAM-1) and anti-P-selectin antibodies (monoclonal antibodies [mAbs]) attenuated phase-1 neutrophil adhesion, consistent with roles for constitutively expressed ICAM-1 and enhanced surface expression of preformed P-selectin. Phase-2 neutrophil adhesion was attenuated by an anti-E-selectin mAb, indicating a dominant role of this adhesion molecule in the late phase response. Pretreatment with actinomycin D and cycloheximide or with competing ds-oligonucleotides containing the nuclear factor-kappa B or activator protein-1 cognate DNA sequences significantly attenuated phase-2 response, suggesting a role for de novo macromolecule synthesis. Surface expression of ICAM-1, P-selectin, and E-selectin on HUVECs correlated with the phase-1 and -2 neutrophil adhesion responses. Collectively, these findings indicate that A/R elicits a two-phase neutrophil-endothelial cell adhesion response that involves transcription-independent and transcription-dependent surface expression of different endothelial cell adhesion molecules. 


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Costimulation of human CD4+ T cells with LFA-3 and B7 induce distinct effects on AP-1 and NF-kappa B transcription factors. 
We have earlier shown that stimulation of human CD4+ T cells with SEA presented on Chinese hamster ovary (CHO)-DR transfectants coexpressing either B7 or LFA-3 resulted in distinct cytokine profiles. We now demonstrate that B7, but not LFA-3, strongly costimulated IL-2 transcription and mRNA expression in CD4+ T cells. Maximal increase in IL-2 transcription was recorded with CHO-DR/B7/LFA-3, suggesting a cooperative effect of B7 and LFA-3 at the transcriptional level. Gel-shift analysis demonstrated that stimulation of CD4+ T cells with CHO-DR and staphylococcal enterotoxin A was sufficient to induce significant amounts of NF-kappa B binding proteins, whereas induction of AP-1 binding proteins required costimulation. LFA-3 induced moderate levels of AP-1, but did not influence the levels of NF-kappa B, while B7 costimulation strongly induced both AP-1 and substantially enhanced NF-kappa B binding proteins. The CHO-DR/B7/LFA-3 triple transfectant induced a further increase in AP-1 and NF-kappa B binding proteins compared with the double transfectants. The level of Oct-1 binding proteins remained similar in all samples. Super-shift analysis revealed that the NF-kappa B complex of costimulated CD4+ T cells contained large amounts of p50, substantial amounts of p65, and marginal levels of c-Rel proteins. The AP-1 binding proteins contained c-Jun, Jun-D, and Fra-1, but marginal amounts of Jun-B and c-Fos. Our results indicate distinct effects of B7 and LFA-3 costimulation on the activity of AP-1 and NF-kappa B. These may partly account for the differential effects of B7 and LFA-3 costimulation on IL-2 expression. 


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Blockade of T-cell activation by dithiocarbamates involves novel mechanisms of inhibition of nuclear factor of activated T cells. 
Dithiocarbamates (DTCs) have recently been reported as powerful inhibitors of NF-kappaB activation in a number of cell types. Given the role of this transcription factor in the regulation of gene expression in the inflammatory response, NF-kappaB inhibitors have been suggested as potential therapeutic drugs for inflammatory diseases. We show here that DTCs inhibited both interleukin 2 (IL-2) synthesis and membrane expression of antigens which are induced during T-cell activation. This inhibition, which occurred with a parallel activation of c-Jun transactivating functions and expression, was reflected by transfection experiments at the IL-2 promoter level, and involved not only the inhibition of NF-kappaB-driven reporter activation but also that of nuclear factor of activated T cells (NFAT). Accordingly, electrophoretic mobility shift assays (EMSAs) indicated that pyrrolidine DTC (PDTC) prevented NF-kappaB, and NFAT DNA-binding activity in T cells stimulated with either phorbol myristate acetate plus ionophore or antibodies against the CD3-T-cell receptor complex and simultaneously activated the binding of AP-1. Furthermore, PDTC differentially targeted both NFATp and NFATc family members, inhibiting the transactivation functions of NFATp and mRNA induction of NFATc. Strikingly, Western blotting and immunocytochemical experiments indicated that PDTC promoted a transient and rapid shuttling of NFATp and NFATc, leading to their accelerated export from the nucleus of activated T cells. We propose that the activation of an NFAT kinase by PDTC could be responsible for the rapid shuttling of the NFAT, therefore transiently converting the sustained transactivation of this transcription factor that occurs during lymphocyte activation, and show that c-Jun NH2-terminal kinase (JNK) can act by directly phosphorylating NFATp. In addition, the combined inhibitory effects on NFAT and NF-KB support a potential use of DTCs as immunosuppressants. 


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Activation of human macrophages by mechanical ventilation in vitro. 
Positive-pressure mechanical ventilation supports gas exchange in patients with respiratory failure but is also responsible for significant lung injury. In this study, we have developed an in vitro model in which isolated lung cells can be submitted to a prolonged cyclic pressure-stretching strain resembling that of conventional mechanical ventilation. In this model, cells cultured on a Silastic membrane were elongated up to 7% of their initial diameter, corresponding to a 12% increase in cell surface. The lung macrophage was identified as the main cellular source for critical inflammatory mediators such as tumor necrosis factor-alpha, the chemokines interleukin (IL)-8 and -6, and matrix metalloproteinase-9 in this model system of mechanical ventilation. These mediators were measured in supernatants from ventilated alveolar macrophages, monocyte-derived macrophages, and promonocytic THP-1 cells. Nuclear factor-kappaB was found to be activated in ventilated macrophages. Synergistic proinflammatory effects of mechanical stress and molecules such as bacterial endotoxin were observed, suggesting that mechanical ventilation might be particularly deleterious in preinjured or infected lungs. Dexamethasone prevented IL-8 and tumor necrosis factor-alpha secretion in ventilated macrophages. Mechanical ventilation induced low levels of IL-8 secretion by alveolar type II-like cells. Other lung cell types such as endothelial cells, bronchial cells, and fibroblasts failed to produce IL-8 in response to a prolonged cyclic pressure-stretching load. This model is of particular value for exploring physical stress-induced signaling pathways, as well as for testing the effects of novel ventilatory strategies or adjunctive substances aimed at modulating cell activation induced by mechanical ventilation. 


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Temporal control of IgH gene expression in developing B cells by the 3' locus control region. 
The suggested roles of the downstream 3' regions acting as a Locus Control Region (LCR), have allowed comparisons to be made between the regulation of the IgH locus with other model systems whose gene expression is governed by LCR activity. Here we summarize the importance of the IgH 3'LCR and its putative functional role in IgH gene expression and compare it with the 5'LCR regulatory region of the human beta-globin locus. 


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Transcriptional regulation of interleukin 3 (IL3) in primary human T lymphocytes. Role of AP-1- and octamer-binding proteins in control of IL3 gene expression. 
We have investigated the molecular and biochemical basis for activation of interleukin 3 (IL3) gene expression in primary human T lymphocytes following CD3 and CD2 receptor stimulation or activation by phytohemagglutinin plus phorbol 12-myristate 13-acetate. Using transfection and reporter gene assays specifically designed for primary T lymphocytes in conjunction with gel retardation assays, Western blot analyses and UV cross-linking studies, we found that c-Jun, c-Fos, and octamer-binding proteins play a major role in transcriptional activation of the IL3 gene via their interaction with two specific regions contained within the IL3 5'-flanking sequence. Additionally, the region between bases -107 and -59 of the IL3 promoter containing putative AP-2 and Sp1 binding motifs appears necessary for basal level expression of the IL3 gene. The data also indicate that CD2 receptor activation and phytohemagglutinin plus phorbol 12-myristate 13-acetate stimulation augment T cell IL3 gene expression through the same cis- and trans-activating signals. These results should contribute to a better understanding of the regulation of IL3 gene expression in human T lymphocytes. 


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Tcf-1-mediated transcription in T lymphocytes: differential role for glycogen synthase kinase-3 in fibroblasts and T cells. 
Beta-catenin is the vertebrate homolog of the Drosophila segment polarity gene Armadillo and plays roles in both cell-cell adhesion and transduction of the Wnt signaling cascade. Recently, members of the Lef/Tcf transcription factor family have been identified as protein partners of beta-catenin, explaining how beta-catenin alters gene expression. Here we report that in T cells, Tcf-1 also becomes transcriptionally active through interaction with beta-catenin, suggesting that the Wnt signal transduction pathway is operational in T lymphocytes as well. However, although Wnt signals are known to inhibit the activity of the negative regulatory protein kinase glycogen synthase kinase-3beta (GSK-3beta), resulting in increased levels of beta-catenin, we find no evidence for involvement of GSK-3beta in Tcf-mediated transcription in T cells. That is, a dominant negative GSK-3beta does not specifically activate Tcf transcription and stimuli (lithium or phytohemagglutinin) that inhibit GSK-3beta activity also do not activate Tcf reporter genes. Thus, inhibition of GSK-3beta is insufficient to activate Tcf-dependent transcription in T lymphocytes. In contrast, in C57MG fibroblast cells, lithium inactivates GSK-3beta and induces Tcf-controlled transcription. This is the first demonstration that lithium can alter gene expression of Tcf-responsive genes, and points to a difference in regulation of Wnt signaling between fibroblasts and lymphocytes. 


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Identification of a novel factor that interacts with an immunoglobulin heavy-chain promoter and stimulates transcription in conjunction with the lymphoid cell-specific factor OTF2. 
The tissue-specific expression of the MOPC 141 immunoglobulin heavy-chain gene was studied by using in vitro transcription. B-cell-specific transcription of this gene was dependent on the octamer element 5'-ATGCAAAG-3', located in the upstream region of this promoter and in the promoters of all other immunoglobulin heavy- and light-chain genes. The interaction of purified octamer transcription factors 1 and 2 (OTF1 and OTF2) with the MOPC 141 promoter was studied by using electrophoretic mobility shift assays and DNase I footprinting. Purified OTF1 from HeLa cells and OTF1 and OTF2 from B cells bound to identical sequences within the heavy-chain promoter. The OTF interactions we observed extended over the heptamer element 5'-CTCAGGA-3', and it seems likely that the binding of the purified factors involves cooperation between octamer and heptamer sites in this promoter. In addition to these elements, we identified a second regulatory element, the N element with the sequence 5'-GGAACCTCCCCC-3'. The N element could independently mediate low levels of transcription in both B-cell and HeLa-cell extracts, and, in conjunction with the octamer element, it can promote high levels of transcription in B-cell extracts. The N element bound a transcription factor, NTF, that is ubiquitous in cell-type distribution, and NTF was distinct from any of the previously described proteins that bind to similar sequences. Based on these results, we propose that NTF and OTF2 interactions (both with their cognate DNA elements and possibly at the protein-protein level) may be critical to B-cell-specific expression and that these interactions provide additional pathways for regulating gene expression. 


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Fibroblast growth factor-1 (FGF-1) enhances IL-2 production and nuclear translocation of NF-kappaB in FGF receptor-bearing Jurkat T cells. 
Fibroblast growth factors (FGFs) are heparin-binding proteins crucial to embryogenesis, angiogenesis, and wound healing. FGF-1 is abundantly expressed in the synovium in rheumatoid arthritis and in rejecting allografts, sites of chronic immune-mediated inflammation. The frequency of FGF-1-responsive T cells is increased in the peripheral blood of these disorders, and a high percentage of infiltrating T cells in rheumatoid arthritis synovium express receptors for FGF-1. To understand the action of FGF-1 in T cells, studies were initiated in Jurkat T cells that express the signaling isoform of FGF receptor-1. These experiments show that FGF-1 stimulation of Jurkat T cells provides a second signal that augments TCR-mediated IL-2 production. Analogous to costimulation via CD28, this activity is mediated through activation of Rel/kappaB, a family of transcription factors known to regulate IL-2 and other activation-inducible proteins. FGF-1 alone induces modest nuclear translocation of kappaB-binding proteins, and this translocation is enhanced by the combination of anti-CD3 and FGF-1. This NF-kappaB binding complex is composed of transcriptionally active p65(RelA)/p50 heterodimers and results primarily from the targeted degradation of IkappaB-alpha, an inhibitor that sequesters Rel/kappaB in the cytoplasm. These data are the first to show a connection between FGF-1 signaling and NF-kappaB activation outside of embryonic development. The signaling events that link FGF receptor-1 engagement and NF-kappaB activation in Jurkat are probably distinct from the CD28 costimulation pathway, since FGF-1-induced Rel/kappaB binding proteins do not contain significant levels of c-Rel and are not identical with the CD28 response complex. 


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Activation of NF-kappa B by interleukin 2 in human blood monocytes. 
We report here that interleukin 2 (IL-2) acts on human blood monocytes by enhancing binding activity of the transcription factor NF-kappa B to its consensus sequence in the 5' regulatory enhancer region of the IL-2 receptor alpha chain (p55). Similarly, IL-2 activates NF-kappa B in the human monocytic cell line U 937, but not in resting human T-cells. This effect is detectable within 15 min and peaks 1 h after exposure to IL-2. Enhanced NF-kappa B binding activity is followed by functional activation in that inducibility of the IL-2 receptor alpha chain is mediated by enhanced NF-kappa B binding and that a heterologous promoter containing the NF-kappa B consensus sequence (-291 to -245) of the IL-2 receptor alpha chain gene is activated. In addition, IL-2 is capable of increasing transcript levels of the p50 gene coding for the p50 subunit of the NF-kappa B transcription factor, whereas mRNA levels of the p65 NF-kappa B gene remained unchanged. 


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A transcriptional regulatory element is associated with a nuclease-hypersensitive site in the pol gene of human immunodeficiency virus type 1. 
Analysis of the chromatin organization of the integrated human immunodeficiency virus type 1 (HIV-1) genome has previously revealed a major constitutive DNase I-hypersensitive site associated with the pol gene (E. Verdin, J. Virol. 65:6790-6799, 1991). In the present report, high-resolution mapping of this site with DNase I and micrococcal nuclease identified a nucleosome-free region centered around nucleotides (nt) 4490 to 4766. A 500-bp fragment encompassing this hypersensitive site (nt 4481 to 4982) exhibited transcription-enhancing activity (two- to threefold) when it was cloned in its natural position with respect to the HIV-1 promoter after transient transfection in U937 and CEM cells. Using in vitro footprinting and gel shift assays, we have identified four distinct binding sites for nuclear proteins within this positive regulatory element. Site B (nt 4519 to 4545) specifically bound four distinct nuclear protein complexes: a ubiquitous factor, a T-cell-specific factor, a B-cell-specific factor, and the monocyte/macrophage- and B-cell-specific transcription factor PU.1/Spi-1. In most HIV-1 isolates in which this PU box was not conserved, it was replaced by a binding site for the related factor Ets1. Factors binding to site C (nt 4681 to 4701) had a DNA-binding specificity similar to that of factors binding to site B, except for PU.1/Spi-1. A GC box containing a binding site for Sp1 was identified (nt 4623 to 4631). Site D (nt 4816 to 4851) specifically bound a ubiquitously expressed factor. These results identify a transcriptional regulatory element associated with a nuclease-hypersensitive site in the pol gene of HIV-1 and suggest that its activity may be controlled by a complex interplay of cis-regulatory elements. 


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Induction of endothelial cell surface adhesion molecules by tumor necrosis factor is blocked by protein tyrosine phosphatase inhibitors: role of the nuclear transcription factor NF-kappa B. 
Recent studies from our laboratory have indicated that protein tyrosine phosphatase (PTPase) inhibitors can down-modulate the tumor necrosis factor (TNF)-mediated activation of the nuclear transcription factor NF-kappa B in ML-1a, a monocytic cell line (Singh and Aggarwal, J. Biol. Chem. 1995: 270: 10631). Since TNF is one of the major inducers of various adhesion molecules in human endothelial cells and their expression is known to require the activation of NF-kappa B, we examined the effect of PTPase inhibitors on the TNF-mediated induction of intracellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1 and endothelial leukocyte adhesion molecule (ELAM)-1. Like ML-1a, human dermal microvessel endothelial cells (MVEC) treated with TNF rapidly activated (within 30 min) NF-kappa B; this effect was completely abolished by co-treatment with phenylarsine oxide (PAO), a specific inhibitor of PTPase. The induction of ICAM-1, VCAM-1, and ELAM-1 by TNF in MVEC occurred within 6 h and was also completely down-regulated by PAO in a dose-dependent manner. PAO was found to be effective even when added 3 h after TNF, suggesting a rapid mode of action of this inhibitor. Besides PAO, other inhibitors of PTPase, including pervanadate and diamide, also blocked TNF-dependent NF-kappa B activation and induction of all the three adhesion proteins. Consistent with these results, the attachment of monocytes to MVEC was also blocked by the PTPase inhibitors. Thus, overall, our results demonstrate that a PTPase is involved either directly or indirectly in the pathway leading to the induction of endothelial cell adhesion molecules by TNF. Because of their role in cell adhesion, PTPase may provide a novel target of drug development for treatment of inflammation, atherogenesis, and tumor metastasis. 


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Inhibition of activation of transcription factor AP-1 by CD28 signalling in human T-cells. 
Co-stimulation of T-lymphocytes by T-cell receptor (TcR) occupancy and activation of the CD28 surface molecule results in enhanced proliferation and interleukin 2 (IL-2) production. The increase in IL-2 gene expression triggered by CD28 involves a kappa B-like sequence in the 5'-regulatory region of the IL-2 promoter, called CD28-responsive element. Stimulation of T-cells by agonistic anti-CD28 antibodies in conjunction with phorbol 12-myristate 13-acetate (PMA)- or TcR-derived signals induces the enhanced activation of the transcription factor NF-kappa B. Here we report that CD28 engagement, however, exerts opposite effects on the transcription factor AP-1. Whereas anti-CD28 together with PMA increased the DNA binding and trans-activation activity of NF-kappa B, PMA-induced activation of AP-1 was significantly suppressed. The inhibitory effect exerted by anti-CD28 was observed at the level of DNA binding as well as in functional reporter-gene assays. These results suggest that the two transcription factors are independently regulated and may perform different functions during T-cell activation. 


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Integrin-mediated tyrosine phosphorylation and cytokine message induction in monocytic cells. A possible signaling role for the Syk tyrosine kinase. 
Activation of cytoplasmic tyrosine kinases is an important aspect of signal transduction mediated by integrins. In the human monocytic cell line THP-1, either integrin-dependent cell adhesion to fibronectin or ligation of beta 1 integrins with antibodies causes a rapid and intense tyrosine phosphorylation of two sets of proteins of about 65-75 and 120-125 kDa. In addition, integrin ligation leads to nuclear translocation of the p50 and p65 subunits of the NF-kappa B transcription factor, to activation of a reporter gene driven by a promoter containing NF-kappa B sites, and to increased levels of mRNAs for immediate-early genes, including the cytokine interleukin (IL)-1 beta. The tyrosine kinase inhibitors genistein and herbimycin A block both integrin-mediated tyrosine phosphorylation and increases in IL-1 beta message levels, indicating a causal relationship between the two events. The components tyrosine phosphorylated subsequent to cell adhesion include paxillin, pp125FAK, and the SH2 domain containing tyrosine kinase Syk. In contrast, integrin ligation with antibodies induces tyrosine phosphorylation of Syk but not of FAK or paxillin. In adhering cells, pre-treatment with cytochalasin D suppresses tyrosine phosphorylation of FAK and paxillin but not of Syk, while IL-1 beta message induction is unaffected. These observations indicate that the Syk tyrosine kinase may be an important component of an integrin signaling pathway in monocytic cells, leading to activation of NF-kappa B and to increased levels of cytokine messages. 


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Sterol dependent LDL-receptor gene transcription in lymphocytes from normal and CML patients. 
Sterol regulatory element (SRE) has been recognized to regulate various key genes coding for especially low density lipoprotein (LDL)-receptor, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and HMG-CoA synthase known to play a crucial role in the cholesterol feedback mechanism. The deranged cholesterol feedback mechanism has been widely recognised in initiation as well as progression of various types of cancers including chronic myeloid leukaemia (CML). Consequently, the present study was addressed to understand this phenomenon and revealed the existence of a unique 47 kDa protein factor having affinity for this SRE sequence in lymphocytes from normal subjects as well as its absence in lymphocytes from untreated CML patients. However, this factor appeared when the CML patients achieved complete haematological remission (CHR) through alpha-interferon therapy. Further, an inverse relationship was also observed between sterol modulated LDL-receptor gene transcription and the binding affinity of this 47 kDa factor to the SRE sequence. Based upon these results we propose that alpha-interferon through its receptor initiates phosphatidic acid dependent signalling which in turn regulates the affinity of 47 kDa sterol regulatory element binding factor as well as LDL-receptor gene transcription in lymphocytes from CML patients. 


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Towards a molecular understanding of T-cell differentiation. 
Lymphoid differentiation is one of the best studied examples of mammalian development. Here Hans Clevers and Michael Owen describe how the cloning of the genes that encode T-cell-specific membrane proteins allows the identification of transcription factors that control the expression of these T-cell genes. Such transcription factors play a key role in the development of the mature T-cell phenotype by functioning as 'master regulators of T-cell differentiation'. 


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Identification of an ionomycin/cyclosporin A-responsive element within the human T cell receptor gamma enhancer. 
Activation through the Ca2+/calcineurin pathway is essential to the transcription of many cytokine genes. The conserved cis-acting sequence, GGAAAA, and transcription factors binding to this sequence are involved in the response to increased intracellular Ca2+ concentrations. Here we report the identification and importance of the same sequence in a non-cytokine gene, the human T cell receptor gamma (TCRG) enhancer. Results from site-directed mutations and electrophoretic mobility shift assays strongly suggest that this sequence mediates the ionomycin-induced activation of the TCRG enhancer. Our studies provide an explanation for a previous observation that TCRG mRNA levels, but not mRNA levels for T cell receptor alpha and -beta, are increased by ionomycin treatment. 


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NF-kappa B activation by tumor necrosis factor alpha in the Jurkat T cell line is independent of protein kinase A, protein kinase C, and Ca(2+)-regulated kinases. 
NF-kappa B is a DNA-binding regulatory factor able to control transcription of a number of genes, including human immunodeficiency virus (HIV) genes. In T cells, NF-kappa B is activated upon cellular treatment by phorbol esters and the cytokine tumor necrosis factor alpha (TNF alpha). In the present work, we investigated the molecular events leading to NF-kappa B activation by TNF alpha in a human T cell line (Jurkat) and its subclone JCT6, which presents a deficiency in the PKA transduction pathway. We found that in both cell lines, both phorbol ester and TNF alpha were able to activate NF-kappa B. Phorbol activation was positively modulated by Ca2+ influx while TNF alpha activation was not. Furthermore, while PMA activation was inhibited by the PKC inhibitor staurosporin, the TNF alpha effect was unchanged. TNF alpha did not activate cAMP production and its signal was not modulated by cAMP activators. Moreover, cAMP activators did not activate NF-kappa B in Jurkat cells. Thus, TNF alpha-induced NF-kappa B activation was found to be mediated by none of the major signal-mediating kinases such as protein kinase C (PKC), protein kinase A, or Ca(2+)-regulated kinases. Furthermore, we found that cytoplasmic acidification facilitated NF-kappa B activation by both TNF alpha and PKC, by a mechanism that increases NF-kappa B/I kappa B dissociation without affecting the NF-kappa B translocation step. 


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Abnormal apoptosis and cell cycle progression in humans exposed to methyl tertiary-butyl ether and benzene contaminating water. 
1. In this study we hypothesized that in individuals with certain genetic makeup, MTBE, benzene or their metabolites act as adducts and may induce programmed cell death. 2. Our study involved a group of 60 male and female subjects who were exposed to MTBE and benzene-contaminated water concentrations up to 76 PPB for MTBE and 14 PPB for benzene, for a period of 5 to 8 years. For comparison, we recruited a control group consisting of 32 healthy males and females with similar age distribution and without a history of exposure to MTBE or benzene. 3. Peripheral blood lymphocytes (PBL) of both groups were tested for the percentage of apoptotic cells and cell cycle progression using flow cytometry. 4. When apoptotic lymphocytes from exposed individuals were compared to apoptotic lymphocytes from the control group, statistically-significant differences between each mean group were detected (26.4 +/- 1.8 and 12.1 +/- 1.3, respectively), indicating an increased rate of apoptosis in 80.5% of exposed individuals (P < 0.0001, Mann-Whitney U-Test). MTBE and benzene-induced apoptosis is attributed to a discrete block within the cell cycle progression. Because cell cycle analysis showed that in PBL from chemically-exposed individuals, between 20-50% of cells were accumulated at the S-G2/M boundaries. 5. One of the signaling molecules which mediates programmed cell death is nuclear factor Kappa-B (NF-kappa B). NF-kappa B was examined as one of the many molecular mechanisms for mediating cell death by MTBE and benzene. Indeed, addition of inhibitors of NF-kappa B activation pyrrolidine dithiocarbamate (PDTC), to the lymphocytes of the chemically-exposed group was capable of inhibiting programmed cell death by 40%. This reversal of apoptosis almost to the control level by inhibitor of NF-kappa B activation may indicate involvement of this signaling molecule in MTBE and benzene induction of programmed cell death. 


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Constitutive expression of p50 homodimer in freshly isolated human monocytes decreases with in vitro and in vivo differentiation: a possible mechanism influencing human immunodeficiency virus replication in monocytes and mature macrophages. 
Human immunodeficiency virus type 1 (HIV-1) replicates more efficiently in vitro in differentiated macrophages than in freshly isolated monocytes. We investigated whether this may be partly explained by changes in expression of NF-kappaB with monocyte differentiation. We demonstrated that constitutive expression of NF-kappaB in primary human monocytes changed significantly with differentiation in vitro to monocyte-derived macrophages (MDMs) and differentiation in vivo to alveolar macrophages (AMs). Freshly isolated monocytes constitutively expressed high levels of transcriptionally inactive p50 homodimer which decreased with time in culture in favor of the transcriptionally active p50/p65 and p50/RelB heterodimers. As in MDMs, AMs constitutively expressed p50/p65 and p50/RelB although at lower levels. HIV infection of fresh monocytes failed to induce p50/p65 as seen in MDMs. The replacement of p50 homodimers with transcriptionally active heterodimers following time in culture may partially explain the progressive increase in susceptibility of monocytes to HIV infection during in vitro culture. The change in NF-kappaB components with monocyte differentiation in vivo may also explain the different transcriptional activities of these cell populations in HIV-infected individuals. 


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Involvement of Alu sequences in the cell-specific regulation of transcription of the gamma chain of Fc and T cell receptors. 
The Fc epsilon RI-gamma chains are expressed in a variety of hematopoietic cells where they play a critical role in signal transduction. They are part of the high affinity IgE receptor in mast cells, basophils, Langerhans cells, and possibly other cells; a component of the low affinity receptor for IgG (Fc gamma RIIIA or CD16) in natural killer cells and macrophages; and part of the T cell antigen receptor in subsets of T cells. Here we have investigated the transcriptional regulation of the gamma chain gene by analyzing the 2.5-kilobase sequence upstream of the transcription start site. This sequence contains a promoter specific to cells of hematopoietic lineage. However, the tissue specificity of this promoter is only partial because it is active in all of the hematopoietic cells tested here, regardless of whether they constitutively express Fc epsilon RI- gamma chain transcripts. We have identified two adjacent cis-acting regulatory elements, both of which are part of an Alu repeat. The first (-445/-366) is a positive element active in both basophils and T cells. The second (-365/-264) binds to nuclear factors, which appear to be different in basophils and T cells, and acts as a negative element in basophils and as a positive one in T cells. Thus, this Alu repeat (90% identical to Alu consensus sequences) has evolved to become both a positive and negative regulator. 


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Fcgamma receptor-mediated mitogen-activated protein kinase activation in monocytes is independent of Ras. 
Receptors for the Fc portion of immunoglobulin molecules (FcR) present on leukocyte cell membranes mediate a large number of cellular responses that are very important in host defense, including phagocytosis, cell cytotoxicity, production and secretion of inflammatory mediators, and modulation of the immune response. Cross-linking of FcR with immune complexes leads, first to activation of protein-tyrosine kinases. The molecular events that follow and that transduce signals from these receptors to the nucleus are still poorly defined. We have investigated the signal transduction pathway from Fc receptors that leads to gene activation and production of cytokines in monocytes. Cross-linking of FcR, on the THP-1 monocytic cell line, by immune complexes resulted in both activation of the transcription factor NF-kappaB and interleukin 1 production. These responses were completely blocked by tyrosine kinase inhibitors. In contrast, expression of dominant negative mutants of Ras and Raf-1, in these cells, did not have any effect on FcR-mediated nuclear factor activation, suggesting that the mitogen-activated protein kinase (MAPK) signaling pathway was not used by these receptors. However, MAPK activation was easily detected by in vitro kinase assays, after FcR cross-linking with immune complexes. Using the specific MAPK/extracellular signal-regulated kinase kinase (MAPK kinase) inhibitor PD98059, we found that MAPK activation is necessary for FcR-dependent activation of the nuclear factor NF-kappaB. These results strongly suggest that the signaling pathway from Fc receptors leading to expression of different genes important to leukocyte biology, initiates with tyrosine kinases and requires MAPK activation; but in contrast to other tyrosine kinase receptors, FcR-mediated MAPK activation does not involve Ras and Raf. 


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In vivo inhibition of NF-kappa B in T-lineage cells leads to a dramatic decrease in cell proliferation and cytokine production and to increased cell apoptosis in response to mitogenic stimuli, but not to abnormal thymopoiesis. 
To understand the role of NF-kappa B complexes in T cell development and activation, we have generated transgenic mice in which RelA and c-Rel complexes were selectively inhibited in the T-lineage cells by specific expression of a trans-dominant form of I kappa B alpha. Transgene expression did not affect the thymic development, but led to lowered numbers of splenic T cells and to a dramatic decrease in the ex vivo proliferative response of splenic T lymphocytes. Analysis of IL-2 and IL-2R alpha expression demonstrated that the perturbation of the proliferation response was not attributable to an abnormal expression of these genes. In contrast, expression of IL-4, IL-10, and IFN-gamma was strongly inhibited in the transgenic T cells. The proliferative deficiency of the transgenic T cells was associated with an increased apoptosis. These results point out the involvement of NF-kappa B/Rel family proteins in growth signaling pathways by either regulating proteins involved in the IL-2 signaling or by functionally interfering with the cell cycle progression. 


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DNA-binding studies of the Epstein-Barr virus nuclear antigen 2 (EBNA-2): evidence for complex formation by latent membrane protein gene promoter-binding proteins in EBNA-2-positive cell lines. 
The Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA-2) protein is essential for the immortalization of human primary B cells by EBV. EBNA-2 trans-activates cellular and viral genes like CD23, c-fgr, latent membrane protein 1 (LMP1) and terminal protein 1 (TP1). Trans-activation of the TP1 promoter and of the BamHI C promoter has already been investigated in detail and appears to be mediated via protein-protein interactions and not by direct binding of EBNA-2 type A (of EBV type 1) to the DNA. EBNA-2 is able to trans-activate the expression of the LMP gene in several cell lines. Various reports have delineated the cis-acting elements of the LMP promoter through which EBNA-2 mediates trans-activation. To determine whether EBNA-2 also trans-activates the LMP promoter by protein-protein interactions, we performed a series of gel retardation assays and competition experiments with LMP promoter fragments of different sizes. We determined that the protein-binding region on the LMP promoter was within a 42 bp fragment encompassing nucleotides -135 to -176 relative to the LMP transcriptional start site. None of the DNA fragments investigated indicated interaction of EBNA-2 with the DNA via protein-protein interactions. No significant differences between EBNA-2-positive and EBNA-2-negative nuclear extracts could be seen in the gel retardation assay under conditions that clearly showed binding of EBNA-2A to the TP1 promoter. However, analysis of sucrose gradient fractions in the gel retardation assay provided evidence that the LMP promoter-binding proteins form a complex of higher M(r) in EBNA-2-positive cell extracts. These complexes were destroyed by detergent. We deduce from these results that EBNA-2-positive cells might indeed contain specific complexes bound to the LMP promoter which are, however, too labile to be detected in a standard gel retardation assay. 


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Immune hyperactivation of HIV-1-infected T cells mediated by Tat and the CD28 pathway. 
Human immunodeficiency virus-type 1 (HIV-1) infection is characterized by a chronic state of immune hyperactivation in patients. Infection of human peripheral blood lymphocytes with HIV-1 in vitro resulted in increased interleukin-2 (IL-2) secretion in response to T cell activation via the CD3 and CD28 receptors. Expression of the HIV-1 transactivator Tat recapitulated this phenotype and was associated with increased IL-2 secretion in response to costimulation with CD3 plus CD28. IL-2 superinduction by Tat occurred at the transcriptional level, was mediated by the CD28-responsive element in the IL-2 promoter, and was exclusively dependent on the 29 amino acids encoded by the second exon of Tat. 


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Regulation of CD14 expression during monocytic differentiation induced with 1 alpha,25-dihydroxyvitamin D3. 
CD14, a monocyte/macrophage receptor for the complex of LPS and LPS binding protein, is a differentiation marker for the monocyte/macrophage lineage. We have analyzed the regulation of CD14 expression during 1 alpha,25-dihydroxyvitamin D3 (VitD3)-induced monocytic differentiation. Using FACS, Northern blotting, and nuclear run-on analyses, we demonstrate that the up-regulation of CD14 expression during monocytic cell maturation is regulated mainly at the level of gene transcription, and that new protein synthesis is required for CD14 induction. We have recently cloned the CD14 5' upstream sequence and demonstrated its tissue-specific promoter activity. Using stable transfection of the monocytoid U937 cell line with a series of deletion mutants of the CD14 5' upstream sequence coupled to a reporter gene construct, we show that bp -128 to -70 is the critical region for the induction of CD14 expression. This region contains two binding sites for the Sp1 transcription factor. A 3-bp mutation at the distal Sp1-binding site not only eliminates Sp1 interaction, but also abolishes most of the VitD3 induction of CD14 expression. Electrophoretic mobility shift analysis does not detect a direct interaction of the CD14 distal Sp1-binding site with the vitamin D3 receptor and its partner, the retinoid X receptor. These data demonstrate that VitD3 induces CD14 indirectly through some intermediary factor, and suggest a critical role for Sp1 in this process. 


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Overexpression of HSF2-beta inhibits hemin-induced heat shock gene expression and erythroid differentiation in K562 cells. 
Acquisition of heat shock factor 2 (HSF2) DNA binding activity is accompanied by induced transcription of heat shock genes in hemin-treated K562 cells undergoing erythroid differentiation. Previous studies revealed that HSF2 consists of two alternatively spliced isoforms, HSF2-alpha and HSF2-beta, whose relative abundance is developmentally regulated and varies between different tissues. To investigate whether the molar ratio of HSF2-alpha and HSF2-beta isoforms is crucial for the activation of HSF2 and whether the HSF2 isoforms play functionally distinct roles during the hemin-mediated erythroid differentiation, we generated cell clones expressing different levels of HSF2-alpha and HSF2-beta. We show that in parental K562 cells, the HSF2-alpha isoform is predominantly expressed and HSF2 can be activated upon hemin treatment. In contrast, when HSF2-beta is expressed at levels exceeding those of endogenous HSF2-alpha, the hemin-induced DNA binding activity and transcription of heat shock genes are repressed, whereas overexpression of HSF2-alpha results in an enhanced hemin response. Furthermore, the hemin-induced accumulation of globin, known as a marker of erythroid differentiation, is decreased in cells overexpressing HSF2-beta. We suggest that HSF2-beta acts as a negative regulator of HSF2 activity during hemin-mediated erythroid differentiation of K562 cells. 


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Activation of the human immunodeficiency virus type 1 enhancer is not dependent on NFAT-1. 
The function of a putative NFAT-1 site in the human immunodeficiency virus type 1 enhancer has been analyzed. Activation by the T-cell antigen receptor is minimal in Jurkat cells and is mediated by the kappa B sites. The putative NFAT-1 region is not required for the response to anti-CD3 or to mitogens in T-cell, B-cell, or monocyte/macrophage leukemia lines, nor is it a cis-acting negative regulatory element. 


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Involvement of intracellular Ca2+ in oxidant-induced NF-kappa B activation. 
In human Jurkat T cells and its subclone Wurzburg cells oxidant challenge elevated [Ca2+]i by mobilizing Ca2+ from intracellular stores. In Jurkat cells this effect was rapid and transient, but in Wurzburg cells the response was slow and sustained. H2O2-induced NF-kappaB activation in Wurzburg cells was not influenced by the presence of extracellular EGTA but was totally inhibited in cells that were loaded with esterified EGTA. In Jurkat cells that are not sensitive to H2O2-induced NF-kappaB activation, H2O2 potentiated NF-kappaB activation in the presence of sustained high [Ca2+]i following thapsigargin treatment. NF-kappaB regulatory effect of alpha-lipoate and N-acetylcysteine appeared to be, at least in part, due to their ability to stabilize elevation of [Ca2+]i following oxidant challenge. Results of this study indicate that a sustained elevated [Ca2+]i is a significant factor in oxidant-induced NF-kappaB activation. 


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Steel factor affects SCL expression during normal erythroid differentiation. 
Steel factor is one of the growth factors that controls the proliferation and differentiation of hematopoietic cells and SCL, also known as Tcl-5 or Tal-1, is a transcription factor involved in erythropoiesis. In this report, we studied the role of SCL in the proliferation of human peripheral blood burst-forming unit-erythroid (BFU-E) and the effects of Steel factor on SCL expression in proliferating erythroid cells. BFU-E-derived colonies increase progressively in size, as determined by cell number, from day 7 to day 14 of culture, with the greatest increase in colony size (10-fold expansion) occurring between day 7 and day 10. SCL protein levels in BFU-E-derived cells were highest in day 7 cells and decreased progressively from day 7 to day 14 of culture, suggesting an association of SCL with erythroid proliferation. In contrast, SCL mRNA levels did not decrease significantly between day 7 and day 14 cells, suggesting that posttranscriptional mechanisms are largely responsible for the decrease in SCL protein observed. The role of SCL in Steel factor-induced erythroid proliferation was then examined. In BFU-E-derived colonies cultured with Steel factor, colony size was significantly increased compared to control. In day 7 and day 10 erythroid precursors cultured with Steel factor, SCL protein was increased significantly compared to control. The increase in SCL protein levels in early erythroid precursors stimulated with Steel factor suggests one mechanism through which Steel factor may enhance normal erythroid proliferation. SCL mRNA levels assessed by Northern blot in day 7 cells did not increase significantly in response to Steel factor stimulation, suggesting that posttranscriptional mechanisms may also be important in the increase in SCL protein observed in response to Steel. 


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Regulation of gene expression with double-stranded phosphorothioate oligonucleotides. 
Alteration of gene transcription by inhibition of specific transcriptional regulatory proteins is necessary for determining how these factors participate in cellular differentiation. The functions of these proteins can be antagonized by several methods, each with specific limitations. Inhibition of sequence-specific DNA-binding proteins was achieved with double-stranded (ds) phosphorothioate oligonucleotides that contained octamer or kappa B consensus sequences. The phosphorothioate oligonucleotides specifically bound either octamer transcription factor or nuclear factor (NF)-kappa B. The modified oligonucleotides accumulated in cells more effectively than standard ds oligonucleotides and modulated gene expression in a specific manner. Octamer-dependent activation of a reporter plasmid or NF-kappa B-dependent activation of the human immunodeficiency virus (HIV) enhancer was inhibited when the appropriate phosphorothioate oligonucleotide was added to a transiently transfected B cell line. Addition of phosphorothioate oligonucleotides that contained the octamer consensus to Jurkat T leukemia cells inhibited interleukin-2 (IL-2) secretion to a degree similar to that observed with a mutated octamer site in the IL-2 enhancer. The ds phosphorothioate oligonucleotides probably compete for binding of specific transcription factors and may provide anti-viral, immunosuppressive, or other therapeutic effects. 


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Signals and nuclear factors that regulate the expression of interleukin-4 and interleukin-5 genes in helper T cells. 
Mouse thymoma line EL-4 cells produce cytokines such as interleukin (IL)-2, IL-3, IL-4, IL-10, and granulocyte-macrophage colony-stimulating factor in response to phorbol 12-myristate 13-acetate (PMA). EL-4 cells also produce low levels of IL-5 when stimulated by PMA alone; however, cAMP greatly augments PMA-dependent IL-5 production. A transient transfection assay revealed that two signals, PMA and cAMP, are required for optimal activation of the IL-5 promoter. In contrast, cAMP almost completely inhibited the PMA-dependent activation of the endogenous IL-2 gene, as well as the transfected IL-2 promoter. These results indicate that the IL-5 gene is positively regulated by cAMP in a manner opposite to that for the IL-2 gene. One of the nuclear factors (NFs) that regulates the response of the IL-5 promoter to cAMP and PMA has properties similar to NF for activated t cell. The P sequence of the IL-4 gene, defined as a responsive element for PMA and calcium ionophore (A23187), shares sequence similarity with the NF kappa B and the NF-activated T cell binding sites. We attempted to determine whether NF(P), a nuclear factor specific for the P sequence, is related to NF-kappa B and nuclear factor for activated T cell (NF-AT). In electromobility shift assays both NF-kappa B (P65 or P65/P50 heterodimer) and NF-AT bound to the P sequence. However, sequence specificity of NF-AT was more similar to that of NF(P), and only a small amount of P65 was detected in NF(P). These results indicate that a component or components of NF-AT have the potential to reconstitute NF(P), whereas NF-kappa B alone does not account for NF(P) in Jurkat crude extract. Taken together, these results suggest that NF-AT-like factors are involved in the regulation of IL-4 and IL-5 genes. 


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Octamer transcription factors and the cell type-specificity of immunoglobulin gene expression. 
Antibodies are produced exclusively in B lymphocytes. The expression of the antibody-encoding genes, the immunoglobulin (Ig) genes, is also restricted to B cells. The octamer sequence ATGCAAAT is present in the promoter and the enhancer of Ig genes, and plays an important role in its tissue-specific expression. This sequence motif is a binding site for nuclear proteins, the so-called octamer transcription factors (Oct or OTF factors). The Oct-1 protein is present in all cell types analyzed so far, whereas Oct-2A and Oct-2B are found mainly in B lymphocytes. All three proteins show the same sequence specificity and binding affinity. It appears that the B cell-specific expression of Ig genes is mediated at least in part by cell type-specific Oct factors, and that there are both quantitative and qualitative differences between Oct-1 and Oct-2 factors. Recently, a number of other octamer factor variants were identified. Many of these may be created by alternative splicing of a primary transcript of one Oct factor gene and may serve a specific function in the fine tuning of gene expression. 


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Epstein-Barr virus binding to CD21 activates the initial viral promoter via NF-kappaB induction. 
Epstein-Barr virus (EBV), an oncogenic human herpesvirus, binds to and infects normal human B lymphocytes via CD21, the CR2 complement receptor. Studies of the mechanisms that enable EBV to infect nonactivated, noncycling B cells provide compelling evidence for a sequence of events in which EBV binding to CD21 on purified resting human B cells rapidly activates the NF-kappaB transcription factor, which, in turn, binds to and mediates transcriptional activation of Wp, the initial viral latent gene promoter. Thus, EBV binding to its cellular receptor on resting B cells triggers an NF-kappaB-dependent intracellular signaling pathway which is required for infection. 


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An enhancer-blocking element between alpha and delta gene segments within the human T cell receptor alpha/delta locus. 
T cell receptor (TCR) alpha and delta gene segments are organized within a single genetic locus but are differentially regulated during T cell development. An enhancer-blocking element (BEAD-1, for blocking element alpha/delta 1) was localized to a 2.0-kb region 3' of TCR delta gene segments and 5' of TCR alpha joining gene segments within this locus. BEAD-1 blocked the ability of the TCR delta enhancer (Edelta) to activate a promoter when located between the two in a chromatin-integrated construct. We propose that BEAD-1 functions as a boundary that separates the TCR alpha/delta locus into distinct regulatory domains controlled by Edelta and the TCR alpha enhancer, and that it prevents Edelta from opening the chromatin of the TCR alpha joining gene segments for VDJ recombination at an early stage of T cell development. 


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Coupling of a signal response domain in I kappa B alpha to multiple pathways for NF-kappa B activation. 
The eukaryotic transcription factor NF-kappa B plays a central role in the induced expression of human immunodeficiency virus type 1 and in many aspects of the genetic program mediating normal T-cell activation and growth. The nuclear activity of NF-kappa B is tightly regulated from the cytoplasmic compartment by an inhibitory subunit called I kappa B alpha. This cytoplasmic inhibitor is rapidly phosphorylated and degraded in response to a diverse set of NF-kappa B-inducing agents, including T-cell mitogens, proinflammatory cytokines, and viral transactivators such as the Tax protein of human T-cell leukemia virus type 1. To explore these I kappa B alpha-dependent mechanisms for NF-kappa B induction, we identified novel mutants of I kappa B alpha that uncouple its inhibitory and signal-transducing functions in human T lymphocytes. Specifically, removal of the N-terminal 36 amino acids of I kappa B alpha failed to disrupt its ability to form latent complexes with NF-kappa B in the cytoplasm. However, this deletion mutation prevented the induced phosphorylation, degradative loss, and functional release of I kappa B alpha from NF-kappa B in Tax-expressing cells. Alanine substitutions introduced at two serine residues positioned within this N-terminal regulatory region of I kappa B alpha also yielded constitutive repressors that escaped from Tax-induced turnover and that potently inhibited immune activation pathways for NF-kappa B induction, including those initiated from antigen and cytokine receptors. In contrast, introduction of a phosphoserine mimetic at these sites rectified this functional defect, a finding consistent with a causal linkage between the phosphorylation status and proteolytic stability of this cytoplasmic inhibitor. Together, these in vivo studies define a critical signal response domain in I kappa B alpha that coordinately controls the biologic activities of I kappa B alpha and NF-kappa B in response to viral and immune stimuli. 


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Two distinct signal transmission pathways in T lymphocytes are inhibited by complexes formed between an immunophilin and either FK506 or rapamycin. 
Proliferation and immunologic function of T lymphocytes are initiated by signals from the antigen receptor that are inhibited by the immunosuppressant FK506 but not by its structural analog, rapamycin. On the other hand, interleukin 2 (IL-2)-induced signals are blocked by rapamycin but not by FK506. Remarkably, these two drugs inhibit each other's actions, raising the possibility that both act by means of a common immunophilin (immunosuppressant binding protein). We find that the dissociation constant of rapamycin to the FK506 binding protein FKBP (Kd = 0.2 nM) is close to the dissociation constant of FK506 to FKBP (Kd = 0.4 nM) and to their effective biologic inhibitory concentrations. However, an excess of rapamycin is needed to revert FK506-mediated inhibition of IL-2 production, apoptosis, and transcriptional activation of NF-AT, a T-cell-specific transcription factor necessary for IL-2 gene activation. Similarly, an excess of FK506 is needed to revert rapamycin-mediated inhibition of IL-2-induced proliferation. The drug concentrations required for antagonism may be explained by the relative affinity of the drugs to, and by the abundance of, the immunophilin FKBP. FKBP has been shown to catalyze the interconversion of the cis- and trans-rotamers of the peptidyl-prolyl amide bond of peptide substrates; here we show that rapamycin, like FK506, is a potent inhibitor of the rotamase activity of FKBP (Ki = 0.2 nM). Neither FKBP binding nor inhibition of rotamase activity of FKBP alone is sufficient to explain the biologic actions of these drugs. Rather, these findings suggest that immunophilin bound to FK506 interferes with antigen receptor-induced signals, while rapamycin bound to the immunophilin interferes with IL-2-induced signals. 


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Constitutive activation of different Jak tyrosine kinases in human T cell leukemia virus type 1 (HTLV-1) tax protein or virus-transformed cells. 
HTLV-1 infection causes an adult T cell leukemia in humans. The viral encoded protein tax, is thought to play an important role in oncogenesis. Our previous data obtained from a tax transgenic mouse model revealed that tax transforms mouse fibroblasts but not thymocytes, despite comparable levels of tax expression in both tissues. Constitutive tyrosine phosphorylation of a 130-kD protein(s) was observed in the tax transformed fibroblast B line and in HTLV-1 transformed human lymphoid lines, but not in thymocytes from Thy-tax transgenic mice. Phosphotyrosine immunoprecipitation followed by Western blot analysis with a set of Jak kinase specific antibodies, identified p130 as Jak2 in the tax transformed mouse fibroblastic cell line and Jak3 in HTLV-1 transformed human T cell lines. Phosphorylation of Jak2 in tax transformed cells resulted from high expression of IL-6. Tyrosine phosphorylation of this protein could also be induced in Balb/c3T3 cells using a supernatant from the B line, which was associated with induction of cell proliferation. Both phosphorylation and proliferation were inhibited by IL-6 neutralizing antibodies. Constitutive phosphorylation of Jak kinases may facilitate tumor growth in both HTLV-1 infected human T cells and the transgenic mouse model. 


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Regulation of interleukin-1 beta production by glucocorticoids in human monocytes: the mechanism of action depends on the activation signal. 
Glucocorticoids are known to downregulate interleukin-1 beta production in monocytic cells by two different mechanims: direct inhibition of the gene transcription and destabilization of the preformed interleukin-1 beta mRNA. Now we have examined the effect of the nature of the monocyte activating signal on these two inhibitory mechanims. When human monocytes were preincubated with dexamethasone for 1 hour and then stimulated either with bacterial lipopolysaccharide or phorbol myristate, it was found that dexamethasone inhibited the lipopolysaccharide-induced interleukin-1 beta protein production, but the phorbol myristate-induced production was increased 3-10 fold. This difference was also seen at the mRNA level. When dexamethasone was added to the cultures 3 hours after the stimulators, it clearly decreased the interleukin-1 beta mRNA levels regardless of the stimulator used (although the effect was clearly weaker on the PMA-induced mRNA). Thus these data suggest that the phorbol myristate-induced signal (prolonged protein kinase C activation?) cannot be inhibited by prior incubation with dexamethasone and it also protects the induced mRNA for the degradative action of dexamethasone. 


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Heat shock induces HIV-1 replication in chronically infected promyelocyte cell line OM10.1. 
A long period of clinical latency before development of symptoms is characteristic of human immunodeficiency virus type 1 (HIV-1) infection. OM10.1, a promyelocyte cell line latently infected with HIV-1, has been developed as a model for studying the mechanism of viral latency and the activation of virus expression. We found that this latently infected cell line with heat shock at 42 degrees C for 2 h resulted in a high level of HIV-1 production without addition of any cytokines. The mechanism of activation was analyzed by using anti-TNF-alpha antibody and various inhibitors. Although the TNF-alpha level in culture supernatants was below the sensitivity of an ELISA assay system, addition of anti-TNF-alpha antibody in culture medium could partially suppress the heat shock induced HIV-1 production. Staurosporine (PKC inhibitor), pentoxifylline (NF-kappa B inhibitor), and Ro5-3335 (HIV-1 Tat inhibitor) also inhibited significantly the heat shock induced virus activation. In particular, staurosporine achieved approximately 90% inhibition of the HIV-1 antigen expression in heat shock-treated OM10.1 at a non-toxic concentration. Although the mechanism of HIV-1 activation with heat shock has not been fully elucidated yet, it is presumed PKC plays an important role in HIV-1 activation. Thus, the present observations will provide a further insight into the pathogenesis of HIV-1 infections. 


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Cellular and molecular mechanisms of IL-5 synthesis in atopic diseases: a study with allergen-specific human helper T cells. 
BACKGROUND: Cytokines produced by helper T cells are intimately involved in chronic allergic diseases associated with eosinophilic inflammation. OBJECTIVE: We investigated the production of IL-5, a potent growth factor and chemotactic factor for eosinophils, by CD4+ T lymphocytes in patients with asthma. METHODS: Allergen-specific T cell clones and T cell hybridomas were established from the peripheral blood lymphocytes of patients with asthma, and the responses to various stimuli were determined. RESULTS: After nonspecific stimulation, IL-5 production by CD4+ T cells from both atopic and nonatopic subjects with asthma was significantly enhanced compared with that by cells from healthy controls. Peripheral blood mononuclear cells from atopic asthma patients both proliferated and produced IL-5 after incubation with mite allergen, suggesting that mite-specific helper T cells were involved in the eosinophilic inflammation of atopic asthma. A human IL-5 promoter/enhancer luciferase gene construct transfected into IL-5-producing T cell clones was clearly transcribed after stimulation, indicating that the 515 base pair IL-5 gene segment upstream of the coding region was sufficient to respond to activating signals in human helper T cells. The same gene segment was not transcribed in IL-5-nonproducing T cell clones, suggesting that human T cell IL-5 synthesis is regulated at the transcriptional level. Experiments with T cell hybridomas confirmed these findings and suggested that a unique transcription factor may be essential for human IL-5 gene transcription. CONCLUSION: Enhanced IL-5 production by helper T cells seems to cause the eosinophilic inflammation of both atopic and nonatopic asthma. Elucidation of IL-5-specific regulatory mechanisms may facilitate the development of novel treatments for allergic diseases associated with eosinophilic inflammation. 


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3-deazaadenosine, a S-adenosylhomocysteine hydrolase inhibitor, has dual effects on NF-kappaB regulation. Inhibition of NF-kappaB transcriptional activity and promotion of IkappaBalpha degradation. 
Previously we reported that 3-deazaadenosine (DZA), a potent inhibitor and substrate for S-adenosylhomocysteine hydrolase inhibits bacterial lipopolysaccharide-induced transcription of tumor necrosis factor-alpha and interleukin-1beta in mouse macrophage RAW 264.7 cells. In this study, we demonstrate the effects of DZA on nuclear factor-kappaB (NF-kappaB) regulation. DZA inhibits the transcriptional activity of NF-kappaB through the hindrance of p65 (Rel-A) phosphorylation without reduction of its nuclear translocation and DNA binding activity. The inhibitory effect of DZA on NF-kappaB transcriptional activity is potentiated by the addition of homocysteine. Taken together, DZA promotes the proteolytic degradation of IkappaBalpha, but not IkappaBbeta, resulting in an increase of DNA binding activity of NF-kappaB in the nucleus in the absence of its transcriptional activity in RAW 264.7 cells. The reduction of IkappaBalpha by DZA is neither involved in IkappaB kinase complex activation nor modulated by the addition of homocysteine. This study strongly suggests that DZA may be a potent drug for the treatment of diseases in which NF-kappaB plays a central pathogenic role, as well as a useful tool for studying the regulation and physiological functions of NF-kappaB. 


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Rel/NF-kappaB can trigger the Notch signaling pathway by inducing the expression of Jagged1, a ligand for Notch receptors. 
Jagged1 belongs to the DSL family of ligands for Notch receptors that control the proliferation and differentiation of various cell lineages. However, little is known about the transcription factors that regulate its expression. Here, we show that Jagged1 is a Rel/NF-kappaB-responsive gene. Both c-Rel and RelA induced jagged1 gene expression, whereas a mutant defective for transactivation did not. Importantly, jagged1 transcripts were also upregulated by endogenous NF-kappaB activation and this effect was inhibited by a dominant mutant of IkappaBalpha, a physiological inhibitor of NF-kappaB. Cell surface expression of Jagged1 in c-Rel-expressing cell monolayers led to a functional interaction with lymphocytes expressing the Notch1/TAN-1 receptor. This correlated with the initiation of signaling downstream of Notch, as evidenced by increased levels of HES-1 transcripts in co-cultivated T cells and of CD23 transcripts in co-cultivated B cells. Consistent with its Rel/NF-kappaB-dependent induction, Jagged1 was found to be highly expressed in splenic B cells where c-Rel is expressed constitutively. These results demonstrate that c-Rel can trigger the Notch signaling pathway in neighboring cells by inducing jagged1 gene expression, and suggest a role for Jagged1 in B-cell activation, differentiation or function. These findings also highlight the potential for an interplay between the Notch and NF-kappaB signaling pathways in the immune system. 


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IFN-gamma priming of monocytes enhances LPS-induced TNF production by augmenting both transcription and MRNA stability. 
The induction of cytokine expression in monocytes/macrophages by bacterial endotoxin or lipopolysaccharide is a critical, highly regulated host defence response. The augmentation of LPS responses by interferon gamma (IFN-gamma), referred to as priming, is well established. However, the mechanism(s) by which priming occurs is poorly defined. Using tumour necrosis factor (TNF) induction as a model, experiments were designed to analyse in detail the priming effect on the LPS response in human monocytes. Priming by IFN-gamma was primarily manifested at the level of TNF mRNA accumulation. IFN-gamma pre-treatment affected the magnitude rather than the sensitivity of the LPS response. Priming occurred after several hours of treatment, and the primed state was induced by either IFN-gamma or GM-CSF, but not M-CSF. Primed monocytes transcribed TNF mRNA at a higher rate than freshly isolated monocytes upon activation with LPS. The increased transcriptional rate correlated with a marked increase in nuclear factor-kappa B activity in these cells as determined by electrophoretic mobility shift assay using a consensus NF-kappa B oligonucleotide. An additional significant finding was than TNF mRNA induced in primed cells was much more stable than in unprimed cells (T1/2 increased 6-8-fold). Consistent with the increased mRNA stability, the duration of mRNA accumulation was longer following LPS stimulation in primed monocytes, in addition to being of greater magnitude. Finally, primed and unprimed cells possessed a differential sensitivity to the kinase inhibitor H-89. H-89 substantially suppressed LPS-induced TNF mRNA accumulation in unprimed cells, but had no effect on primed monocytes following LPS stimulation. (ABSTRACT TRUNCATED AT 250 WORDS) 


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Immunosuppression by glucocorticoids: inhibition of NF-kappa B activity through induction of I kappa B synthesis [see comments] 
Glucocorticoids are among the most potent anti-inflammatory and immunosuppressive agents. They inhibit synthesis of almost all known cytokines and of several cell surface molecules required for immune function, but the mechanism underlying this activity has been unclear. Here it is shown that glucocorticoids are potent inhibitors of nuclear factor kappa B (NF-kappa B) activation in mice and cultured cells. This inhibition is mediated by induction of the I kappa B alpha inhibitory protein, which traps activated NF-kappa B in inactive cytoplasmic complexes. Because NF-kappa B activates many immunoregulatory genes in response to pro-inflammatory stimuli, the inhibition of its activity can be a major component of the anti-inflammatory activity of glucocorticoids. 


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Calcium signalling in T cells stimulated by a cyclophilin B-binding protein. 
The immunosuppressant drug cyclosporin A blocks a calcium-dependent signal from the T-cell receptor (TCR) that normally leads to T-cell activation. When bound to cyclophilin, cyclosporin A binds and inactivates the key signalling intermediate calcineurin. To identify potential cellular homologues of cyclosporin A that might regulate calcium signalling, we have cloned human genes encoding cyclophilin B-binding-proteins using the yeast two-hybrid system. One gene product, when overexpressed in Jurkat T cells, specifically induced transcription from the interleukin-2 enhancer, by activating the T-cell-specific transcription factors NF-AT and NF-IL2A. This protein, termed calcium-signal modulating cyclophilin ligand (CAML), acts downstream of the TCR and upstream of calcineurin by causing an influx of calcium. CAML appears to be a new participant in the calcium-signal transduction pathway, implicating cyclophilin B in calcium signalling, even in the absence of cyclosporin. 


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Characterization of defensin resistance phenotypes associated with mutations in the phoP virulence regulon of Salmonella typhimurium. 
The defensin sensitivities of Salmonella typhimurium strains with mutations in the phoP/phoQ two-component virulence regulon were tested by using purified defensins NP-1 and NP-2. Strains with mutations in either gene of the regulatory pair (phoP [transcriptional activator] or phoQ [membrane sensor kinase]) had increased sensitivities to defensin. The predicted periplasmic domain of the PhoQ protein contained a markedly anionic domain that could interact with cationic proteins and that could be responsible for resistance to defensin. Because insertion mutations in phoP are polar on phoQ, we constructed strains that expressed the PhoQ protein in the absence of PhoP to test whether resistance to defensin requires only the phoQ gene product. We found that resistance to defensin requires the function of both components of this regulatory system, because strains expressing PhoQ without PhoP were still markedly sensitive to defensins. This implied that a pag (phoP-activated gene) product is responsible for defensin resistance. We also tested for the ability of defensins NP-1, NP-5, and HNP-1 to activate pag expression and found that these peptides have no effect. Defensin resistance is not the only virulence characteristic controlled by the PhoP-PhoQ regulon because mutations in pagC, as well as ones in the phoP locus that resulted in constitutive pag activation (phenotype PhoPc), had no effect on defensin resistance, even though they rendered the organism avirulent and deficient in survival within macrophages. The virulence defect conferred by mutations in the phoP-phoQ two-component regulatory system is not completely explained by alterations in resistance to cationic proteins and involves the control of other proteins necessary for S. typhimurium survival within macrophages. 


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Histamine modulates the expression of c-fos through cyclic AMP production via the H2 receptor in the human promonocytic cell line U937. 
We examined the effects of histamine and its agonists on the expression of the c-fos and c-myc proto-oncogenes at the transcriptional and translational levels in the human promonocytic U937 cell line. Histamine transiently increased cAMP and c-fos expression through H2 receptors. Dibutyryl cAMP also increased c-fos mRNA and protein, and levels remained elevated even after 12 hr of treatment. Dose-dependence studies using histamine and dimaprit showed that the EC50 values for cAMP production and c-fos increase were similar, suggesting that cAMP might be involved in c-fos induction via H2 receptors. Furthermore, studies carried out using H7, a protein kinase A/protein kinase C inhibitor, blocked c-fos induction, whereas no effect was observed with bisindolylmaleimide, a specific protein kinase C inhibitor. No modification of c-myc expression could be detected on treatment with histamine or its analogues. Nevertheless, dibutyryl cAMP induced a down-regulation of the levels of this proto-oncogene. In addition, dibutyryl cAMP inhibited cell growth in a dose-dependent manner, whereas histamine failed to affect proliferation and differentiation of U937 cells. Cells pretreated with dimaprit showed a decrease in the cAMP response to subsequent addition of H2 agonists, whereas the cAMP response to prostaglandin E2 remained unaltered. This homologous mechanism of H2 receptor desensitization was time dependent. These results indicate that histamine activates several mechanisms involved in the induction of differentiation, such as cAMP and c-fos production, but fails to promote differentiation of U937 cells, apparently due to the rapid desensitization of H2 receptors. 


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Transcription mediated by NFAT is highly inducible in effector CD4+ T helper 2 (Th2) cells but not in Th1 cells. 
Transcriptional factors of the NFAT family play an important role in regulating the expression of several cytokine genes during the immune response, such as the genes for interleukin 2 (IL-2) and IL-4, among others. Upon antigen stimulation, precursor CD4+ T helper (pTh) cells proliferate and differentiate into two populations of effector cells (eTh1 and eTh2), each one expressing a specific pattern of cytokines that distinguishes them from their precursors. eTh2 cells are the major source of IL-4, while gamma interferon is produced by eTh1 cells. Here we have used reporter transgenic mice to show that DNA binding and transcriptional activities of NFAT are transiently induced during the differentiation of pTh cells into either eTh1 or eTh2 cells to mediate the expression of IL-2 as a common growth factor in both pathways. However, although NFAT DNA binding is similarly induced in both eTh1 and eTh2 cells upon antigen stimulation, only the NFAT complexes present in eTh2 cells are able to mediate high-level transcription, and relatively little NFAT transcriptional activity was induced in eTh1 cells. In contrast to activated pTh cells, neither eTh1 nor eTh2 cells produced significant IL-2 upon stimulation, but the high levels of NFAT transcriptional activities directly correlate with the IL-4 production induced in response to antigen stimulation in eTh2 cells. These data suggest that activated NFAT is involved in the effector function of eTh2 cells and that the failure of eTh1 cells to produce IL-4 in response to an antigen is due, at least partially, to a failure to induce high-level transcription of the IL-4 gene by NFAT. Regulation of NFAT could be therefore a critical element in the polarization to eTh1 or eTh2. 


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Protein kinase C and calcineurin synergize to activate IkappaB kinase and NF-kappaB in T lymphocytes. 
The nuclear factor of kappaB (NF-kappaB) is a ubiquitous transcription factor that is key in the regulation of the immune response and inflammation. T cell receptor (TCR) cross-linking is in part required for activation of NF-kappaB, which is dependent on the phosphorylation and degradation of IkappaBalpha. By using Jurkat and primary human T lymphocytes, we demonstrate that the simultaneous activation of two second messengers of the TCR-initiated signal transduction, protein kinase C (PKC) and calcineurin, results in the synergistic activation of the IkappaBalpha kinase (IKK) complex but not of another putative IkappaBalpha kinase, p90(rsk). We also demonstrate that the IKK complex, but not p90(rsk), is responsible for the in vivo phosphorylation of IkappaBalpha mediated by the co-activation of PKC and calcineurin. Each second messenger is necessary, as inhibition of either one reverses the activation of the IKK complex and IkappaBalpha phosphorylation in vivo. Overexpression of dominant negative forms of IKKalpha and -beta demonstrates that only IKKbeta is the target for PKC and calcineurin. These results indicate that within the TCR/CD3 signal transduction pathway both PKC and calcineurin are required for the effective activation of the IKK complex and NF-kappaB in T lymphocytes. 


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Attenuated function of a variant form of the helix-loop-helix protein, Id-3, generated by an alternative splicing mechanism. 
The Id family of helix-loop-helix proteins function as negative regulators of DNA binding, basic helix-loop-helix proteins in the regulation of cell growth and differentiation. We report here on the identification of a 17 kDa variant of the 14 kDa Id-3 protein termed Id-3L (long version) which possesses a unique 60 amino acid carboxy-terminus generated by read through of a 'coding intron' and alternative splicing. Northern analysis revealed expression of a minor 1.1 kb Id-3L transcript together with the predominant 0.95 kb Id-3 transcript in the majority of adult human tissues analysed. The variant Id-3L protein is functionally distinguishable from conventional Id-3 since in in vitro DNA mobility shift assays, it was greatly impaired in its ability to abrogate binding of the basic helix-loop-helix protein, E47, to an E box recognition sequence. 


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Targeted disruption of the MyD88 gene results in loss of IL-1- and IL-18-mediated function. 
MyD88, originally isolated as a myeloid differentiation primary response gene, is shown to act as an adaptor in interleukin-1 (IL-1) signaling by interacting with both the IL-1 receptor complex and IL-1 receptor-associated kinase (IRAK). Mice generated by gene targeting to lack MyD88 have defects in T cell proliferation as well as induction of acute phase proteins and cytokines in response to IL-1. Increases in interferon-gamma production and natural killer cell activity in response to IL-18 are abrogated. In vivo Th1 response is also impaired. Furthermore, IL-18-induced activation of NF-kappaB and c-Jun N-terminal kinase (JNK) is blocked in MyD88-/- Th1-developing cells. Taken together, these results demonstrate that MyD88 is a critical component in the signaling cascade that is mediated by IL-1 receptor as well as IL-18 receptor. 


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Transcriptional regulation of the beta-casein gene by cytokines: cross-talk between STAT5 and other signaling molecules. 
The beta-casein promoter has been widely used to monitor the activation of STAT (signal transducer and activator of transcription)5 since STAT5 was originally found as a mediator of PRL-inducible beta-casein expression. However, not only is expression of the beta-casein gene regulated by STAT5 but it is also affected by other molecules such as glucocorticoid and Ras. In this report, we describe the transcriptional regulation of the beta-casein gene by cytokines in T cells. We have found that the beta-casein gene is expressed in a cytotoxic T cell line, CTLL-2, in response to interleukin-2 (IL-2), which activates STAT5. While IL-4 does not activate STAT5, it induces expression of STAT5-regulated genes in CTLL-2, i.e. beta-casein, a cytokine-inducible SH2-containing protein (CIS), and oncostatin M (OSM), suggesting that STAT6 activated by IL-4 substitutes for the function of STAT5 in T cells. IL-2-induced beta-casein expression was enhanced by dexamethasone, and this synergistic effect of Dexamethasone requires the sequence between -155 and -193 in the beta-casein promoter. Coincidentally, a deletion of this region enhanced the IL-2-induced expression of beta-casein. Expression of an active form of Ras, Ras(G12V), suppressed the IL-2-induced beta-casein and OSM gene expression, and the negative effect of Ras is mediated by the region between -105 and -193 in the beta-casein promoter. In apparent contradiction, expression of a dominant negative form of Ras, RasN17, also inhibited IL-2-induced activation of the promoter containing the minimal beta-casein STAT5 element as well as the promoters of CIS and OSM. In addition, Ras(G12V) complemented signaling by an erythropoietin receptor mutant defective in Ras activation and augmented the activation of the beta-casein promoter by the mutant erythropoietin receptor signaling, suggesting a possible role of Ras in Stat5-mediated gene expression. These results collectively reveal a complex interaction of STAT5 with other signaling pathways and illustrate that regulation of gene expression requires integration of opposing signals. 


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Induction of transcription factors in human T lymphocytes by aspirin-like drugs. 
Aspirin-like drugs (ALD) induce calcium mobilization, an essential component of T cell activation, but do not induce the biosynthesis of IL-2. To understand the extent to which ALD may mimic mitogenic stimulation, we studied cytoplasmic and nuclear signaling steps in ALD-treated T cells. We found that ALD induce a transient activation of protein kinase (PKC) but have no effect (in comparison to anti-CD3 antibodies) on protein tyrosine phosphorylation nor on PCL gamma 1 tyrosine phosphorylation. ALD-induced calcium mobilization and PKC activation are independent of tyrosine protein kinase activity as shown by the lack of effect of herbimycin, a tyrosine-protein kinase-specific inhibitor. Although we detected no IL-2 mRNA in ALD-treated cells, the nuclei of these cells contain proteins capable of binding to three regulatory sequences in the IL-2 promoter region: NFAT, NF kappa B, and AP-1. These binding activities are expressed only in activated T cells. The expression of AP-1 depended on calcium mobilization and PKC activation. These data suggest that ALD cause transient but significant changes in T cell transmembrane signaling, although some events induced by stimulation with anti-CD3 antibodies are not induced by ALD. The signal is transmitted to the nucleus and induces DNA-binding activity by several transcription factors. However, the ALD stimulus is not capable of causing complete T cell activation. 


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Induction of activator protein (AP)-1 and nuclear factor-kappaB by CD28 stimulation involves both phosphatidylinositol 3-kinase and acidic sphingomyelinase signals. 
A major obstacle in understanding the signaling events that follow CD28 receptor ligation arises from the fact that CD28 acts as a costimulus to TCR engagement, making it difficult to assess the relative contribution of CD28 signals as distinct from those of the TCR. To overcome this problem, we have exploited the observation that activated human T cell blasts can be stimulated via the CD28 surface molecule in the absence of antigenic challenge; thus, we have been able to observe the response of normal T cells to CD28 activation in isolation. Using this system, we observed that CD28 stimulation by B7-transfected CHO cells induced a proliferative response in T cells that was not accompanied by measurable IL-2 production. However, subsequent analysis of transcription factor generation revealed that B7 stimulation induced both activator protein-1 (AP-1) and nuclear factor-kappaB (NF-kappaB) complexes, but not NF-AT. In contrast, engagement of the TCR by class II MHC/superantigen, either with or without CD28 ligation, resulted in the induction of NF-AT, AP-1, and NF-kappaB as well as IL-2 production. Using selective inhibitors, we investigated the signaling pathways involved in the CD28-mediated induction of AP-1 and NF-kappaB. This revealed that NF-kappaB generation was sensitive to chloroquine, an inhibitor of acidic sphingomyelinase, but not to the phosphatidylinositol 3-kinase inhibitor, wortmannin. In contrast, AP-1 generation was inhibited by wortmannin and was also variably sensitive to chloroquine. These data suggest that in activated normal T cells, CD28-derived signals can stimulate proliferation at least in part via NF-kappaB and AP-1 generation, and that this response uses both acidic sphingomyelinase and phosphatidylinositol 3-kinase-linked pathways. 


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Different sequence requirements for expression in erythroid and megakaryocytic cells within a regulatory element upstream of the GATA-1 gene. 
The lineage-restricted transcription factor GATA-1 is required for differentiation of erythroid and megakaryocytic cells. We have localized a 317 base pair cis-acting regulatory element, HS I, associated with a hematopoietic-specific DNase I hypersensitive site, which lies approx. 3.7 kilobases upstream of the murine hematopoietic-specific GATA-1 IE promoter. HS I directs high-level expression of reporter GATA-1/lacZ genes to primitive and definitive erythroid cells and megakaryocytes in transgenic mice. Comparative sequence analysis of HS I between human and mouse shows approx. 63% nucleotide identity with a more conserved core of 169 base pairs (86% identity). This core contains a GATA site separated by 10 base pairs from an E-box motif. The composite motif binds a multi-protein hematopoietic-specific transcription factor complex which includes GATA-1, SCL/tal-1, E2A, Lmo2 and Ldb-1. Point mutations of the GATA site abolishes HS I function, whereas mutation of the E-box motif still allows reporter gene expression in both lineages. Strict dependence of HS I activity on a GATA site implies that assembly of a protein complex containing a GATA-factor, presumably GATA-1 or GATA-2, is critical to activating or maintaining its function. Further dissection of the 317 base pair region demonstrates that, whereas all 317 base pairs are required for expression in megakaryocytes, only the 5' 62 base pairs are needed for erythroid-specific reporter expression. These findings demonstrate differential lineage requirements for expression within the HS I element. 


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Anti-rheumatic compound aurothioglucose inhibits tumor necrosis factor-alpha-induced HIV-1 replication in latently infected OM10.1 and Ach2 cells. 
NF-kappaB is a potent cellular activator of HIV-1 gene expression. Down-regulation of NF-kappaB activation is known to inhibit HIV replication from the latently infected cells. Gold compounds have been effectively used for many decades in the treatment of rheumatoid arthritis. We previously reported that gold compounds, especially aurothioglucose (AuTG) containing monovalent gold ion, inhibited the DNA-binding of NF-kappaB in vitro. In this report we have examined the efficacy of the gold compound AuTG as an inhibitor of HIV replication in latently infected OM10.1 and Ach2 cells. Tumor necrosis factor (TNF)-alpha-induced HIV-1 replication in OM10.1 or Ach2 cells was significantly inhibited by non-cytotoxic doses of AuTG (>10 microM in OM10.1 cells and >25 F.M in Ach2 cells), while 25 microM of the counter-anion thioglucose (TG) or gold compound containing divalent gold ion, HAuCl3, had no effect. The effect of AuTG on NF-kappaB-dependent gene expression was confirmed by a transient CAT assay. Specific staining as well as electron microscopic examinations revealed the accumulation of metal gold in the cells, supporting our previous hypothesis that gold ions could block NF-kappaB-DNA binding by a redox mechanism. These observations indicate that the monovalent gold compound AuTG is a potentially useful drug for the treatment of patients infected with HIV. 


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T-lymphocytes from individuals with filarial inflammatory disease have increased transendothelial migration in vitro. 
The in vitro transendothelial migration of circulating filarial antigen-specific T-cells was examined in Wuchereria banerofti infection. Circulating T-cells from individuals with filaria-induced lymphatic pathology (LP) had significantly greater migration through unstimulated HUVEC monolayers than did T-cells from asymptomatic infected (MF) individuals (P = 0.04). In contrast to the MF individuals where no effect was seen, transendothelial migration of 48-hr filarial antigen stimulated T-cells from LP individuals was significantly (P = 0.01) greater than migration of 48-hr media-stimulated T-cells. In six of seven patients examined, inhibition of the VLA-4/VCAM-1 pathway resulted in greater than 50% inhibition of transendothelial migration of T-cells. 


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Tissue factor expression of human monocytes is suppressed by lysophosphatidylcholine. 
The expression of tissue factor (TF), the principal initiator of coagulation, is increased during inflammation and atherosclerosis. Both conditions are promoted by lysophosphatidylcholine (lysoPC). We observed in the present study that lysoPC (1 to 10 micromol/L) dose-dependently reduced TF activity in human monocytes, as elicited by lipopolysaccharide (LPS). Lysophosphatidylethanolamine (lysoPE) and other lysophospholipids did not affect LPS-induced TF activity of human monocytes. TF antigen expression as elicited by LPS was also lowered by lysoPC. Phospholipid analyses indicated a selective increase in the lysoPC content of the monocytes after preincubation with the lysophospholipid. LysoPC inhibited the TF activity of Mono Mac-6 cells to a similar extent as in the monocytes. LPS binding to plasma membrane receptors and internalization of LPS into monocytes were not affected by lysoPC. In contrast, LPS-mediated nuclear binding of nuclear factor-kappaB/Rel to a TF-specific kappaB site was inhibited by lysoPC. Induction of TF mRNA expression by LPS tended to be partially reduced by the lysophospholipid. Preincubation with lysoPC increased monocytic cAMP levels. Inhibition of adenylyl cyclase by pretreatment with 2'-deoxy-3'-adenosine monophosphate partially reversed the inhibition of TF activity promoted by lysoPC. In conclusion, lysoPC markedly decreases LPS-mediated TF expression of human monocytes, the effect probably being mediated by both transcriptional and posttranscriptional mechanisms. LysoPC may thus attenuate activation of coagulation during inflammation and atherosclerosis. 


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IL-2-independent activation and proliferation in human T cells induced by CD28. 
Although the role of CD28 in T cell costimulation is firmly established, the mechanisms by which it exerts its costimulatory actions are less clear. In many circumstances it is difficult to distinguish the effects of CD28 from subsequent actions of cytokines, such as IL-2, on T cell proliferation. Here, we report a model of CD28 costimulation using PMA plus the natural ligand CD80 that resulted in very limited stimulation of IL-2, as evidenced by both cytokine production and IL-2 promoter stimulation. Promoter assays revealed CD28-dependent effects on both NF-kappaB and AP-1, but not on NF-AT or the intact IL-2 promoter. In addition, T cell proliferation was completely resistant to the actions of the immunosuppressant cyclosporin A (CsA). Moreover T cell proliferation was unaffected by the addition of blocking Abs to both IL-2 and the IL-2 receptor, demonstrating that this form of costimulation by CD28 was independent of IL-2. We also investigated the effects of stimulating T cell blasts with CD80 alone and found that there was a limited requirement for IL-2 in this system. We conclude that CD28 costimulation can cause substantial T cell proliferation in the absence of IL-2, which is driven by a soluble factor independent of NF-AT transactivation. 


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Human neutrophils express GH-N gene transcripts and the pituitary transcription factor Pit-1b. 
Since GH stimulates the development and function of granulocytes, we investigated the expression of GH in granulocyte subsets. By immunocytochemistry, 25 +/- 7% of the human neutrophils were shown to express immunoreactive GH, whereas eosinophils were negative. Reversed transcription (RT)-PCR analysis demonstrated GH mRNA in neutrophils. Restriction analysis revealed that neutrophils express the GH-N gene but not the GH-V gene. Furthermore, we demonstrated by western blot analysis that neutrophils express an alternatively spliced variant of the pituitary transcription factor Pit-1, designated Pit-1b. 


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Cell growth-regulated expression of mammalian MCM5 and MCM6 genes mediated by the transcription factor E2F. 
Initiation of DNA replication requires the function of MCM gene products, which participate in ensuring that DNA replication occurs only once in the cell cycle. Expression of all mammalian genes of the MCM family is induced by growth stimulation, unlike yeast, and the mRNA levels peak at G1/S boundary. In this study, we examined the transcriptional activities of isolated human MCM gene promoters. Human MCM5 and MCM6 promoters with mutation in the E2F sites failed in promoter regulation following serum stimulation and exogenous E2F expression. In addition, we identified a novel E2F-like sequence in human MCM6 promoter which cooperates with the authentic E2F sites in E2F-dependent regulation. Forced expression of E2F1 could induce expression of all members of the endogenous MCM genes in rat embryonal fibroblast REF52 cells. Our results demonstrated that the growth-regulated expression of mammalian MCM5 and MCM6 genes, and presumably other MCM members, is primarily regulated by E2F through binding to multiple E2F sites in the promoters. 


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Cyclosporin A inhibits monocyte tissue factor activation in cardiac transplant recipients. 
BACKGROUND: Fibrin deposition and thrombosis have been implicated in both allograft rejection and vasculopathy after cardiac transplantation. Because monocytes play a pivotal role in the pathophysiology of intravascular coagulation activation through their ability to synthesize tissue factor (TF), we asked (1) whether monocyte TF activation occurs in cardiac transplant recipients and (2) whether monocyte TF expression is affected by treatment with cyclosporin A (CsA). METHODS AND RESULTS: We measured levels of TF activity in peripheral blood mononuclear cells and highly purified monocytes/macrophages from 10 consecutive cardiac transplant recipients and 10 healthy control subjects. TF activity generated by both unstimulated and endotoxin-stimulated cells was significantly higher in transplant recipients than in control subjects (P<.05). Increased monocyte TF expression in transplant recipients was shown to be adversely affected by treatment with CsA: TF induction was markedly reduced by CsA serum concentrations reaching peak CsA drug levels. Inhibition of TF induction in the presence of high CsA blood concentrations was also observed when stimulation of cells was performed with interferon-gamma or interleukin-1beta. As shown by reverse transcription-polymerase chain reaction and electrophoretic mobility shift assay, respectively, treatment with CsA leads to decreased TF mRNA expression and reduced activation of the NF-kappaB transcription factor, which is known to contribute to the induction of the TF promotor in human monocytes. CONCLUSIONS: This study demonstrates that TF activation, occurring in mononuclear cells of cardiac transplant recipients, is inhibited by treatment with CsA. Inhibition of monocyte TF induction by CsA may contribute to its successful use in cardiac transplant medicine and might be useful in managing further settings of vascular pathology also known to involve TF expression and NF-kappaB activation. 


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Activation of transcription factor NF-kappa B by phagocytic stimuli in human neutrophils. 
Phagocytosis represents an important physiological trigger for the inducible expression of several genes in human neutrophils. Here, we report that a DNA-binding activity primarily consisting of the classical NF-kappa B heterodimer, p50/RelA, is induced in phagocytosing neutrophils. Under these conditions, NF-kappa B activation was found to be a rapid and transient response, reaching a maximum by 10-15 min, and returning to near-basal levels by 30 min. In neutrophils undergoing the phagocytosis of opsonized yeasts, the onset of NF-kappa B activation was paralleled by a decline in immunoreactive I kappa B-alpha protein levels, and the cellular I kappa B-alpha pool was replenished by 30 min, in agreement with our gel shift data. We conclude that NF-kappa B activation could constitute one of the mechanisms whereby the expression of kappa B-responsive genes is enhanced in phagocytosing neutrophils. To our knowledge, this represents the first demonstration that phagocytic stimuli can induce NF-kappa B activation in human neutrophils. 


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Binding of erythroid and non-erythroid nuclear proteins to the silencer of the human epsilon-globin-encoding gene. 
To clarify the molecular mechanisms involved in the developmental control of hemoglobin-encoding genes we have been studying the expression of these genes in human cells in continuous culture. We have previously reported the presence of a transcriptional control element with the properties of a silencer extending from -392 to -177 bp relative to the cap site of the human epsilon-globin-encoding gene [Cao et al., Proc.Natl.Acad.Sci.USA 86 (1989) 5306-5309]. We also showed that this silencer has stronger inhibitory activity in HeLa cells, as compared to K562 human erythroleukemia cells. Using deletion mutants and cis-cloned synthetic oligodeoxyribonucleotides in transient expression assays, nucleotide sequences responsible for this effect have now been further delimited to 44 bp located from -294 to -251 bp. Gel electrophoresis mobility shift assays and DNaseI footprinting assays demonstrate that these negative regulatory sequences are recognized differently by proteins present in nuclear extracts obtained from HeLa and K562 cells. Two binding proteins are detected in K562 nuclear extracts, while only one is found in extracts from HeLa cells. Possible mechanisms by which these proteins may regulate transcription of the epsilon-globin-encoding gene in erythroid and non-erythroid cells are discussed. 


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A novel SP-1 site in the human interleukin-1 beta promoter confers preferential transcriptional activity in keratinocytes. 
To investigate the mechanisms of transcriptional activation of interleukin-1beta (IL-1beta) in non-monocytic cells, we constructed a series of reporter plasmids with the bacterial chloramphenicol acetyltransferase gene linked to various parts of the human IL-1beta promoter and performed transient transfection experiments. We identified a promoter segment that activates transcription most efficiently in keratinocytes. Electrophoretic mobility shift assays (EMSA) with a 43-mer oligonucleotide derived from the functionally identified cis-acting element revealed specific complexes. By competition analysis with transcription factor consensus sequence oligonucleotides and by immunosupershift, transcription factor SP-1 or a closely related protein was shown to bind to this regulatory element. The closest match to the known SP-1 consensus sequence within the respective region is a TCCCCTCCCCT motif. Mutation of this motif almost completely, and specifically, abolished the binding of two low-mobility complexes and led to a 95% decrease of constitutive transcriptional activation of a reporter construct IL-1beta (-170/+108). Likewise, activation of this reporter construct by tumor necrosis factor-alpha depended on the SP-1 site. These observations suggest that a so-far-unrecognized SP-1 site in the human IL-1beta promoter may participate in the transcriptional regulation of this gene in keratinocytes. 


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Multifactor cis-dominant negative regulation of IL-2 gene expression in anergized T cells. 
The molecular mechanism underlying IL-2 transcriptional blockade in anergic T cell clones is not fully understood. To examine whether an active negative regulatory process occurs, we created a reporter construct containing as an enhancer four copies of the NF-AT site and one copy of the octamer site (4X NF-AT-Oct). This construct was only slightly reduced (1.3-fold) in its expression when stimulated under anergic conditions, while a whole mouse IL-2 enhancer construct showed a reduction of 4.3-fold. Addition of the -176 to -96 sequence to the 4X NF-AT-Oct construct did not impart the ability to be affected by anergy, but addition of the -236 to -96 sequence did, demonstrating that anergy is an active inhibitory process and that more than the presence of the -150 AP-1 binding site (-152 to -147) is required to mediate the effect. Mutational studies of the -236 to -96 sequence indicated that the presence of both the -130 AP-1-like site (-187 to -181) and the -150 proximal AP-1 site were necessary to observe anergy. Because the -180 site is not required for trans-activation, it was possible to confirm by mutation in the normal mouse IL-2 enhancer that this site is absolutely essential for anergy induction. The simplest model to explain these results is that anergy is mediated by a complex of multiple transcription factors that exert a cis-acting dominant negative regulatory effect on the trans-activation of the IL-2 gene. 


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Disruption of alpha beta but not of gamma delta T cell development by overexpression of the helix-loop-helix protein Id3 in committed T cell progenitors. 
Enforced expression of Id3, which has the capacity to inhibit many basic helix-loop-helix (bHLH) transcription factors, in human CD34(+) hematopoietic progenitor cells that have not undergone T cell receptor (TCR) gene rearrangements inhibits development of the transduced cells into TCRalpha beta and gamma delta cells in a fetal thymic organ culture (FTOC). Here we document that overexpression of Id3, in progenitors that have initiated TCR gene rearrangements (pre-T cells), inhibits development into TCRalpha beta but not into TCRgamma delta T cells. Furthermore, Id3 impedes expression of recombination activating genes and downregulates pre-Talpha mRNA. These observations suggest possible mechanisms by which Id3 overexpression can differentially affect development of pre-T cells into TCRalpha beta and gamma delta cells. We also observed that cell surface CD4(-)CD8(-)CD3(-) cells with rearranged TCR genes developed from Id3-transduced but not from control-transduced pre-T cells in an FTOC. These cells had properties of both natural killer (NK) and pre-T cells. These findings suggest that bHLH factors are required to control T cell development after the T/NK developmental checkpoint. 


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Human CD3-CD16+ natural killer cells express the hGATA-3 T cell transcription factor and an unrearranged 2.3-kb TcR delta transcript. 
In this study we analyzed the T cell receptor(TcR) delta transcripts expressed by CD3-CD16+ cells and we investigated whether these cells expressed the hGATA-3 T cell transcription factor and the recombination-activating gene (RAG)-1. Multiple TcR delta transcripts deriving from an unrearranged TcR delta gene were detected in both polyclonal and clonal CD3-CD16+ natural killer(NK) cell lines. Two unrearranged TcR delta transcripts had a size similar to that of the functional TcR delta mRNA (2.3 and 1.3 kb) found in TcR gamma/delta+ T lymphocytes. Sequence analysis of nine different 2.3-kb cDNA clones obtained from NK-derived polyA+ RNA confirmed that they corresponded to an unrearranged TcR delta gene. These cDNA were 2343 bp long and their transcription initiation site was located 814 bp upstream from the J delta 1 segment. The sequence located upstream of the J delta 1 segment corresponded to the previously reported germ-line sequence. The J delta 1 segment was correctly spliced to C delta; in addition the four C delta exons were found to be already assembled. Two polyadenylation sites were present in the fourth C delta exon. However, only that located at the 3' end appeared to be utilized in the 2.3-kb cDNA. The expression of hGATA-3, a T cell-specific factor known to be involved in the regulation of the transcription of TcR delta locus, was analyzed by Northern blot, in cultured NK cell population and clones (but not in freshly derived cell populations). All NK clones and cell lines studied were found to express hGATA-3-specific mRNA, suggesting that hGATA-3 may be involved in the regulation of the unrearranged TcR delta gene expression in NK cells. Finally, no transcription of the RAG-1 gene could be detected in all NK cell lines or clones analyzed. 


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NF-AT activation induced by a CAML-interacting member of the tumor necrosis factor receptor superfamily. 
Activation of the nuclear factor of activated T cells transcription factor (NF-AT) is a key event underlying lymphocyte action. The CAML (calcium-modulator and cyclophilin ligand) protein is a coinducer of NF-AT activation when overexpressed in Jurkat T cells. A member of the tumor necrosis factor receptor superfamily was isolated by virtue of its affinity for CAML. Cross-linking of this lymphocyte-specific protein, designated TACI (transmembrane activator and CAML-interactor), on the surface of transfected Jurkat cells with TACI-specific antibodies led to activation of the transcription factors NF-AT, AP-1, and NFkappaB. TACI-induced activation of NF-AT was specifically blocked by a dominant-negative CAML mutant, thus implicating CAML as a signaling intermediate. 


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Distinct mechanisms for N-acetylcysteine inhibition of cytokine-induced E-selectin and VCAM-1 expression. 
We have examined the effects of N-acetyl-L-cysteine (NAC), a well-characterized, thiol-containing antioxidant, on agonist-induced monocytic cell adhesion to endothelial cells (EC). NAC inhibited interleukin-1 (IL-1 beta)-induced, but not basal, adhesion with 50% inhibition at approximately 20 mM. Monocytic cell adhesion to EC in response to tumor necrosis factor-alpha (TNF-alpha), lipopolysaccharide (LPS), alpha-thrombin, or phorbol 12-myristate 13-acetate (PMA) was similarly inhibited by NAC. Unlike published studies with pyrrolidinedithiocarbamate, which specifically inhibited vascular cell adhesion molecule 1 (VCAM-1), NAC inhibited IL-1 beta-induced mRNA and cell surface expression of both E-selectin and VCAM-1. NAC had no effect on the half-life of E-selectin or VCAM-1 mRNA. Although NAC reduced nuclear factor-kappa B (NF-kappa B) activation in EC as measured by gel-shift assays using an oligonucleotide probe corresponding to the consensus NF-kappa B binding sites of the VCAM-1 gene (VCAM-NF-kappa B), the antioxidant had no appreciable effect when an oligomer corresponding to the consensus NF-kappa B binding site of the E-selectin gene (E-selectin-NF-kappa B) was used. Because NF-kappa B has been reported to be redox sensitive, we studied the effects of NAC on the EC redox environment. NAC caused an expected dramatic increase in the reduced glutathione (GSH) levels in EC. In vitro studies demonstrated that whereas the binding affinity of NF-kappa B to the VCAM-NF-kappa B oligomer peaked at a GSH-to-oxidized glutathione (GSSG) ratio of approximately 200 and decreased at higher ratios, the binding to the E-selectin-NF-kappa B oligomer appeared relatively unaffected even at ratios > 400, i.e., those achieved in EC treated with 40 mM NAC. These results suggest that NF-kappa B binding to its consensus sequences in the VCAM-1 and E-selectin gene exhibits marked differences in redox sensitivity, allowing for differential gene expression regulated by the same transcription factor. Our data also demonstrate that NAC increases the GSH-to-GSSG ratio within the EC suggesting one possible mechanism through which this antioxidant inhibits agonist-induced monocyte adhesion to EC. 


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Dual effects of LPS antibodies on cellular uptake of LPS and LPS-induced proinflammatory functions. 
Human phagocytes recognize bacterial LPS (endotoxin) through membrane CD14 (mCD14), a proinflammatory LPS receptor. This study tested the hypothesis that anti-LPS Abs neutralize endotoxin by blocking cellular uptake through mCD14. Ab-associated changes in the uptake and cellular distribution of FITC-LPS were assessed by flow cytometry and laser scanning confocal microscopy in human CD14-transfected Chinese hamster ovary fibroblasts (CHO-CD14 cells) and human peripheral blood monocytes. LPS core- and O-side chain-specific mAbs inhibited mCD14-mediated LPS uptake by both cell types in the presence of serum. O-side chain-specific mAb concurrently enhanced complement-dependent LPS uptake by monocytes through complement receptor-1 (CR1) and uptake by CHO-CD14 cells involving another heat-labile serum factor(s) and cell-associated recognition molecule(s). Core-specific mAb inhibited mCD14-mediated uptake of homologous and heterologous LPS, while producing less concurrent enhancement of non-mCD14-mediated LPS uptake. The modulation by anti-LPS mAbs of mCD14-mediated LPS uptake was associated with inhibition of LPS-induced nuclear factor-kappaB (NF-kappaB) translocation and TNF-alpha secretion in CHO-CD14 cells and monocytes, respectively, while mAb enhancement of non-mCD14-mediated LPS uptake stimulated these activities. LPS-specific Abs thus mediate anti-inflammatory and proinflammatory functions, respectively, by preventing target cell uptake of LPS through mCD14 and augmenting uptake through CR1 or other cell receptors. 


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Increased IkappaB expression and diminished nuclear NF-kappaB in human mononuclear cells following hydrocortisone injection. 
We have recently demonstrated that hydrocortisone and other glucocorticoids inhibit reactive oxygen species (ROS) generation by mononuclear (MNC) and polymorphonuclear leucocytes (PMNL). Since NF-kappaB/IkappaB system regulates the transcription of proinflammatory genes, including those responsible for ROS generation, we tested the hypothesis that hydrocortisone may stimulate IkappaB production thus inhibiting NF-kappaB translocation from the cytosol into the nucleus in MNC, in vivo. One hundred milligram of hydrocortisone was injected intravenously into 4 normal subjects. Blood samples were obtained prior to the injection and at 1, 2, 4, 8 and 24 hr after the injection. Nuclear extracts and total cell lysates were prepared from MNC by standard techniques. IkappaB levels in MNC homogenates increased at 1 hr, peaked at 2-4 hr, started to decrease at 8 hr, and returned to baseline levels at 24 hr. NF-kappaB in MNC nuclear extracts decreased at 1 hr, reached a nadir at 4 hr, gradually increased at 8 hr and returned back to baseline levels at 24 hr. The total protein content of NF-kappaB subunit (P65) in MNC lysates also showed a decrease following hydrocortisone injection. This decrease was observed at 2 hr, reached a nadir at 4 hr, and returned to baseline levels at 24 hr. ROS generation inhibition paralleled NF-kappaB levels in the nucleus. It was inhibited at 1 hr, reached a nadir at 2-4 hr, started to increase at 8 hr, and returned to basal levels at 24 hr. Our data demonstrate that hydrocortisone induces IkappaB and suppresses NF-kappaB expression in MNC in parallel. IkappaB further reduces the translocation of NF-kappaB into the nucleus thus preventing the expression of proinflammatory genes. 


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Anaphylatoxins C5a and C3a induce nuclear factor kappaB activation in human peripheral blood monocytes. 
The anaphylatoxins C5a and C3a are involved in the regulation of cytokine production. In this study the capability of C5a and C3a to induce transcription factor activation was examined. C5a and C3a stimulation of human peripheral blood monocytes resulted in nuclear expression of a DNA binding activity with specificity to the kappaB sequence. The p50 and p65 proteins, constituents of the prototypic nuclear factor kappaB, were identified as components of the DNA-protein complexes by anti-peptide antibodies in gel supershift assays. C5a induced kappaB binding activity was detected 15 min after agonist stimulation, peaked at 30-40 min, and remained detectable at 2 h. Binding to kappaB sequence was accompanied by an initial decrease and subsequent increase in the cytoplasmic IkappaBalpha levels, as detected by Western blotting using an anti-IkappaBalpha antibody. Pertussis toxin treatment markedly decreased kappaB binding activities induced by both C5a and C3a, whereas cholera toxin displayed no inhibitory effect. Neither of the two toxins affected kappaB binding activity induced by TNFalpha in the same cells. These results imply a potential role of the anaphylatoxins C5a and C3a in regulating leukocytes gene expression through G protein-coupled transcription factor activation. 


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Platelet-activating factor stimulates transcription of the heparin-binding epidermal growth factor-like growth factor in monocytes. Correlation with an increased kappa B binding activity. 
Human peripheral blood monocytes responded to stimulation of platelet-activating factor (PAF) with up-regulation of the transcript for heparin-binding epidermal growth factor-like growth factor (HB-EGF), a potent mitogen for vascular smooth muscle cells. This function of PAF was observed at nanomolar concentrations of the ligand, starting at 30 min after stimulation. The PAF-induced up-regulation of HB-EGF mRNA was accompanied by an increase in kappa B binding activity. These functions of PAF appeared to be mediated through the cell surface PAF receptors, as two PAF receptor antagonists, WEB 2086 and L-659,989, blocked both the up-regulation of HB-EGF mRNA and kappa B binding activity induced by PAF. The antagonists, however, had no effect on phorbol ester-induced up-regulation of HB-EGF mRNA and kappa B binding activity. Pretreatment of monocytes with pertussis toxin inhibited these functions of PAF, whereas cholera toxin had no inhibitory effect. Pyrrolidine dithiocarbamate, an inhibitor for NF-kappa B activation, markedly reduced PAF-stimulated kappa B binding activity as well as up-regulation of HB-EGF mRNA. These results suggest a potential role of PAF in HB-EGF expression and provide evidence that this stimulation may occur through increased kappa B binding activity. 


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Expression of the Runt domain-encoding PEBP2 alpha genes in T cells during thymic development. 
The PEBP2 alpha A and PEBP2 alpha B genes encode the DNA-binding subunit of a murine transcription factor, PEBP2, which is implicated as a T-cell-specific transcriptional regulator. These two related genes share the evolutionarily conserved region encoding the Runt domain. PEBP2 alpha B is the murine counterpart of human AML1, which is located at the breakpoints of the 8;21 and 3;21 chromosome translocations associated with acute myeloid leukemia. Northern (RNA) blots of various adult mouse tissues revealed that the levels of expression of both genes were most prominent in the thymus. Furthermore, transcripts of PEBP2 alpha A and mouse AML1/PEBP2 alpha B were detected in T lymphocytes in the thymuses from day 16 embryos and newborns, as well as 4-week-old adult mice, by in situ hybridization. The expression of the genes persisted in peripheral lymph nodes of adult mice. The transcripts were detected in all the CD4- CD8-, CD4+ CD8+, CD4+ CD8-, and CD4- CD8+ cell populations. The results indicated that both genes are expressed in T cells throughout their development, supporting the notion that PEBP2 is a T-cell-specific transcription factor. Transcripts of mouse AML1/PEBP2 alpha B were also detected in day 12 fetal hematopoietic liver and in the bone marrow cells of newborn mice. The implication of mouse AML1/PEBP2 alpha B expression in hematopoietic cells other than those of T-cell lineage is discussed in relation to myeloid leukemogenesis. 


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Regulation of granulocyte-macrophage colony-stimulating factor and E-selectin expression in endothelial cells by cyclosporin A and the T-cell transcription factor NFAT. 
Nuclear factor of activated T cells (NFAT) was originally described as a T-cell-specific transcription factor athat supported the activation of cytokine gene expression and mediated the immunoregulatory effects of cyclosporin A (CsA). As we observed that activated endothelial cells also expressed NFAT, we tested the antiinflammatory properties of CsA in endothelial cells. Significantly, CsA completely suppressed the induction of NFAT in endothelial cells and inhibited the activity of granulocyte-macrophage colony-stimulating factor (GM-CSF) gene regulatory elements that use NFAT by 60%. CsA similarly mediated a reduction of up to 65% in GM-CSF mRNA and protein expression in activated endothelial cells. CsA also suppressed E-selectin, but not vascular cell adhesion molecule-1 (VCAM-1) expression in endothelial cells, even though the E-selectin promoter is activated by NF-kappa B rather than NFAT. Hence, induction of cell surface expression of this leukocyte adhesion molecule by tumor necrosis factor (TNF)-alpha was reduced by 40% in the presence of CsA, and this was reflected by a 29% decrease in neutrophil adhesion. The effects of CsA on endothelial cells were also detected at the chromatin structure level, as DNasel hypersensitive sites within both the GM-CSF enhancer and the E-selectin promoter were suppressed by CsA. This represents the first report of NFAT in endothelial cells and suggests mechanisms by which CsA could function as an antiinflammatory agent. 


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Suppression of a cellular differentiation program by phorbol esters coincides with inhibition of binding of a cell-specific transcription factor (NF-E2) to an enhancer element required for expression of an erythroid-specific gene. 
Induction by hemin increases, while induction with 12-O-tetradecanoylphorbol-13-acetate (TPA) represses, erythroid-specific gene expression in the human cell line K562. We analyzed the effects of hemin or TPA induction on the binding and activity of transcription factors at a regulatory element found within the transcriptional regulatory sequences of many erythroid-specific genes. TPA induction increases the binding of ubiquitous AP-1 factors to this element. TPA induction inhibits the binding of the lineage limited transcription factor NF-E2 to this transcriptional control element. Hemin induction of K562 cells does not facilitate the binding of NF-E2 to its recognition site. Hemin induction appears to nonspecifically increase the expression of transiently transfected genes in K562 cells. Beyond this nonspecific increase in gene expression, hemin induction acts to increase the activity of the lineage limited transcription factor NF-E2. The divergent effects of hemin and TPA on gene expression in K562 cells are mediated, in part, by their contrasting effects on the transcription factor NF-E2. 


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Evidence for lowered induction of nuclear factor kappa B in activated human T lymphocytes during aging. 
Transcription factor NF kappa B (nuclear factor kappa B) is induced in T lymphocytes from young individuals following activation with a variety of stimuli including anti-CD3, phorbol myristate acetate (PMA), and tumor necrosis factor-alpha (TNF-alpha). In contrast, activated T lymphocytes from older individuals show a significant reduction in the induction of NF kappa B in response to the same stimuli. The age-related decline in induction of NF kappa B could not be attributed to alteration in the composition of subunits, p50 and p65 were found to be the predominant subunits of induced NF kappa B in T cells from young as well as elderly donors. Furthermore, similar levels of NF kappa B were found in the cytosols of unactivated T cells from both young and elderly donors suggesting that precursor levels of NF kappa B remain unaltered during aging. These results suggest that an age-associated decline in the induction of NF kappa B in activated T cells from elderly individuals may be attributable to altered regulation of the inhibitor, I kappa B, and may play an important role in immune dysregulation accompanying aging. 


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Expression of Ah receptor (TCDD receptor) during human monocytic differentiation. 
We have previously found a high expression of human Ah receptor (TCDD receptor) mRNA in peripheral blood cells of individuals. In this paper, the expression of this gene in blood cells was first investigated in fractions of nucleated cells, revealing predominant expression of the Ah receptor gene in the monocyte fraction. Then the expression levels of AhR mRNA in various hematopoietic cell lines were examined together with those of Arnt and P450IA1. AhR was expressed at high levels in monocytoid U937, THP1, and HEL/S cells, and at moderate levels in promyelocytic HL60 cells and erythroblastic HEL cells. However, it was not detected in lymphoid cells MOLT4 (T cell) and BALL1 (B cell), nor in K562 erythroblasts. Furthermore, a specific induction of AhR during monocytic differentiation was investigated in HL60 and HEL cells. HL60 cells were induced to differentiate toward monocytes-macrophages by incubation with phorbol ester, showing a 5- to 2-fold increase of AhR mRNA. The incubation with transforming growth factor beta 1 and 1 alpha,25-dihydroxyvitamin D3 resulted in a 5- to 7-fold increase of AhR mRNA. The HEL cells also exhibited a similar elevation of AhR mRNA level, when they had differentiated toward monocyte-macrophage cells by these combined inducers, but little change in the mRNA level was observed when the cells were induced to differentiate into other cell types. Treatment of the differentiated HL60 cells with 3-methylcholanthrene, a ligand of AhR, induced the expression of the P450IA1 gene. These results indicated that expression of AhR mRNA was significantly induced during monocytic differentiation and that the differentiated cells were responsive to xenobiotics. Our results suggest that AhR may play an important role in the function of monocytes and also in the eventual activation of environmental carcinogens. 


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Genes encoding general initiation factors for RNA polymerase II transcription are dispersed in the human genome. 
General transcription factors are required for accurate initiation of transcription by RNA polymerase II. Human cDNAs encoding subunits of these factors have been cloned and sequenced. Using fluorescence in situ hybridization (FISH), we show here that the genes encoding the TATA-box binding protein (TBP), TFIIB, TFIIE alpha, TFIIE beta, RAP30, RAP74 and the 62 kDa subunit, of TFIIH are located at the human chromosomal bands 6q26-27, 1p21-22, 3q21-24, 8p12, 13q14, 19p13.3 and 11p14-15.1, respectively. This dispersed localization of a group of functionally related gene provides insights into the molecular mechanism of human genome evolution and their possible involvement in human diseases. 


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Interferon-gamma and the sexual dimorphism of autoimmunity. 
The sexual difference in the incidence of autoimmune diseases has remained an enigma for many years. In the examination of the induction of autoimmunity in transgenic mice, evidence has been obtained further implicating the lymphokine interferon-gamma in the etiology of autoimmunity. Sex steroid regulation of the production of this molecule, as well as other cytokines, may help explain the gender-specific differences in the immune system, including autoimmunity. 


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Alternative splicing of RNA transcripts encoded by the murine p105 NF-kappa B gene generates I kappa B gamma isoforms with different inhibitory activities. 
The gene encoding the 105-kDa protein (p105) precursor of the p50 subunit of transcription factor NF-kappa B also encodes a p70 I kappa B protein, I kappa B gamma, which is identical to the C-terminal 607 amino acids of p105. Here we show that alternative RNA splicing generates I kappa B gamma isoforms with properties different from those of p70. One 63-kDa isoform, termed I kappa B gamma-1, which lacks 59 amino acids C-terminal to ankyrin repeat 7, has a novel 35-amino acid C terminus encoded by an alternative reading frame of the p105 gene. A 55-kDa isoform, I kappa B gamma-2, lacks the 190 C-terminal amino acids of p70I kappa B gamma. In contrast to p70I kappa B gamma, which is a cytoplasmic protein, I kappa B gamma-1 is found in both the cytoplasm and nucleus, whereas I kappa B gamma-2 is predominantly nuclear. The I kappa B gamma isoforms also display differences in specificity and affinity for Rel/NF-kappa B proteins. While p70I kappa B gamma inhibits p50-, p65-, and c-Rel-mediated transactivation and/or DNA binding, both I kappa B gamma-1 and I kappa B gamma-2 are specific for p50 and have different affinities for this subunit. The absence in I kappa B gamma-1 and I kappa B gamma-2 of a protein kinase A site whose phosphorylation modulates p70I kappa B gamma inhibitory activity suggests that alternative RNA splicing may be used to generate I kappa B gamma isoforms that respond differently to intracellular signals. 


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Structure and function analysis of the human myeloid cell nuclear differentiation antigen promoter: evidence for the role of Sp1 and not of c-Myb or PU.1 in myelomonocytic lineage-specific expression. 
The human myeloid nuclear differentiation antigen (MNDA) is expressed specifically in maturing cells of the myelomonocytic lineage and in monocytes and granulocytes. Epitope enhancement was used to confirm the strict lineage- and stage-specific expression of MNDA in bone marrow as well as in other paraffin-embedded fixed tissues. A 1-kb region of the gene that includes 5' flanking sequence was reported earlier to contain functional promoter activity and was specifically demethylated in expressing cells in contrast to null cells. Further analysis has revealed that this 1-kb fragment promotes higher reporter gene activity in MNDA-expressing cells than non-expressing cells, indicating cell-specific differences in transactivation. This sequence contains consensus elements consistent with myeloid-specific gene expression, including a PU.1 consensus site near the major transcription start site and a cluster of c-Myb sites located several hundred bases upstream of this region. However, analysis of deletion mutants localized nearly all of the promoter activity to a short region (-73 to -16) that did not include the cluster of c-Myb sites. A 4-bp mutation of the core Sp1 consensus element (GC box) (-20) reduced overall promoter activity of the 1-kb fragment. Mutation of the PU.1 site did not significantly affect promoter activity. Only a small region (-35 to +22) including the Sp1 element and transcription start site, but not the PU.1 site was footprinted. The 4-bp mutation of the core Sp1 consensus element abolished footprinting at the site and an antibody super-shift reaction showed that Sp1 is one of the factors binding the consensus site. The Sp1 site also co-localizes with a DNase I hypersensitive site. The results indicate that DNA methylation, chromatin structure, and transactivation at an Sp1 site contribute to the highly restricted expression of this myelomonocytic lineage specific gene. 


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The promoter of the CD19 gene is a target for the B-cell-specific transcription factor BSAP. 
The CD19 protein is expressed on the surface of all B-lymphoid cells with the exception of terminally differentiated plasma cells and has been implicated as a signal-transducing receptor in the control of proliferation and differentiation. Here we demonstrate complete correlation between the expression pattern of the CD19 gene and the B-cell-specific transcription factor BSAP in a large panel of B-lymphoid cell lines. The human CD19 gene has been cloned, and several BSAP-binding sites have been mapped by in vitro protein-DNA binding studies. In particular, a high-affinity BSAP-binding site instead of a TATA sequence is located in the -30 promoter region upstream of a cluster of heterogeneous transcription start sites. Moreover, this site is occupied by BSAP in vivo in a CD19-expressing B-cell line but not in plasma or HeLa cells. This high-affinity site has been conserved in the promoters of both human and mouse CD19 genes and was furthermore shown to confer B-cell specificity to a beta-globin reporter gene in transient transfection experiments. In addition, BSAP was found to be the only abundant DNA-binding activity of B-cell nuclear extracts that interacts with the CD19 promoter. Together, this evidence strongly implicates BSAP in the regulation of the CD19 gene. 


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Simple derivation of TFIID-dependent RNA polymerase II transcription systems from Schizosaccharomyces pombe and other organisms, and factors required for transcriptional activation. 
Resolution of whole cell extract through two chromatographic steps yields a single protein fraction requiring only the addition of TFIID for the initiation of transcription at RNA polymerase II promoters. This approach allows the convenient generation of RNA polymerase II transcription systems from Saccharomyces cerevisiae, human lymphocytes, and Schizosaccharomyces pombe. TFIIDs from all three organisms are interchangeable among all three systems. The S. cerevisiae and Sch. pombe systems support effects of acidic activator proteins, provided a further protein fraction from S. cerevisiae is supplied. This further fraction is distinct from the mediator of transcriptional activation described previously and represents a second component in addition to general initiation factors that may facilitate a response to acidic activators. 


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I kappa B/MAD-3 masks the nuclear localization signal of NF-kappa B p65 and requires the transactivation domain to inhibit NF-kappa B p65 DNA binding. 
The active nuclear form of the NF-kappa B transcription factor complex is composed of two DNA binding subunits, NF-kappa B p65 and NF-kappa B p50, both of which share extensive N-terminal sequence homology with the v-rel oncogene product. The NF-kappa B p65 subunit provides the transactivation activity in this complex and serves as an intracellular receptor for a cytoplasmic inhibitor of NF-kappa B, termed I kappa B. In contrast, NF-kappa B p50 alone fails to stimulate kappa B-directed transcription, and based on prior in vitro studies, is not directly regulated by I kappa B. To investigate the molecular basis for the critical regulatory interaction between NF-kappa B and I kappa B/MAD-3, a series of human NF-kappa B p65 mutants was identified that functionally segregated DNA binding, I kappa B-mediated inhibition, and I kappa B-induced nuclear exclusion of this transcription factor. Results from in vivo expression studies performed with these NF-kappa B p65 mutants revealed the following: 1) I kappa B/MAD-3 completely inhibits NF-kappa B p65-dependent transcriptional activation mediated through the human immunodeficiency virus type 1 kappa B enhancer in human T lymphocytes, 2) the binding of I kappa B/MAD-3 to NF-kappa B p65 is sufficient to retarget NF-kappa B p65 from the nucleus to the cytoplasm, 3) selective deletion of the functional nuclear localization signal present in the Rel homology domain of NF-kappa B p65 disrupts its ability to engage I kappa B/MAD-3, and 4) the unique C-terminus of NF-kappa B p65 attenuates its own nuclear localization and contains sequences that are required for I kappa B-mediated inhibition of NF-kappa B p65 DNA binding activity. Together, these findings suggest that the nuclear localization signal and transactivation domain of NF-kappa B p65 constitute a bipartite system that is critically involved in the inhibitory function of I kappa B/MAD-3. Unexpectedly, our in vivo studies also demonstrate that I kappa B/MAD-3 binds directly to NF-kappa B p50. This interaction is functional as it leads to retargeting of NF-kappa B p50 from the nucleus to the cytoplasm. However, no loss of DNA binding activity is observed, presumably reflecting the unique C-terminal domain that is distinct from that present in NF-kappa B p65. 


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Evaluation of the role of ligand and thermal activation of specific DNA binding by in vitro synthesized human glucocorticoid receptor. 
We have used a DNA-binding/immunoprecipitation assay to analyze the capacity of human glucocorticoid receptor (hGR), generated in rabbit reticulocyte lysates, to bind DNA. In vitro translated hGR was indistinguishable from native hGR, as determined by migration on sodium dodecyl sulfate-polyacrylamide gels, sedimentation on sucrose density gradients, and reactivity with antipeptide antibodies generated against hGR. In addition, cell-free synthesized hGR was capable of specific binding to glucocorticoid response element (GRE)-containing DNA fragments. Using this assay system, we have evaluated the contributions of ligand binding and heat activation to DNA binding by these glucocorticoid receptors. In vitro translated hGR was capable of selective DNA binding even in the absence of glucocorticoid. Treatment with dexamethasone or the antiglucocorticoid RU486 had no additional effect on the DNA-binding capacity when receptor preparations were maintained at 0 C (no activation). In contrast, addition of either ligand or antagonist in combination with a heat activation step promoted DNA binding by approximately 3-fold over that of heat-activated unliganded receptors. Agonist (dexamethasone) was slightly more effective in supporting specific DNA binding than antagonist (RU486). DNA binding by in vitro synthesized GR was blocked by the addition of sodium molybdate to the receptor preparations before steroid addition and thermal activation. Addition of KCl resulted in less DNA binding either due to blockage of DNA-receptor complex formation or disruption of the complexes. The specificity of DNA binding by cell-free synthesized hGR was analyzed further by examining the abilities of various DNAs to compete for binding to a naturally occurring GRE found in the mouse mammary tumor virus-long terminal repeat. Oligonucleotides containing the consensus GRE were the most efficient competitors, and fragments containing regulatory sequences from glucocorticoid-repressible genes were somewhat competitive, whereas single stranded oligonucleotides were unable to compete for mouse mammary tumor virus-long terminal repeat DNA binding, except when competitor was present at extremely high concentrations. Together these studies indicate that hGR synthesized in rabbit reticulocyte lysates displays many of the same properties, including GRE-specific DNA binding, observed for glucocorticoid receptor present in cytosolic extracts of mammalian cells and tissues. Similarities between the effects of dexamethasone and RU486 suggest that the antiglucocorticoid properties of RU486 do not occur at the level of specific DNA binding. 


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Thrombin-induced p65 homodimer binding to downstream NF-kappa B site of the promoter mediates endothelial ICAM-1 expression and neutrophil adhesion. 
We investigated the mechanisms by which proinflammatory mediator, thrombin, released during intravascular coagulation and tissue injury, induces ICAM-1 (CD54) expression in endothelial cells. Stimulation of HUVEC with thrombin resulted in dose- and time-dependent increases in ICAM-1 mRNA and cell surface expression and in ICAM-1-dependent endothelial adhesivity toward polymorphonuclear leukocytes. Transient transfection of endothelial cells with ICAM-1 promoter luciferase reporter gene (ICAM-1LUC) constructs indicated that deletion of upstream NF-kappa B site (-533 bases from translation start site) had no effect on thrombin responsiveness, whereas mutation/deletion of downstream NF-kappa B site (-223 bases from the translation start site) prevented the activation of ICAM-1 promoter, indicating that the downstream NF-kappa B site is critical for thrombin inducibility. NF-kappa B-directed luciferase activity increased approximately 3-fold when cells transfected with the plasmid pNF-kappa BLUC containing five copies of consensus NF-kappa B site linked to a minimal adenovirus E1B promoter-luciferase gene were exposed to thrombin, indicating that activation of NF-kappa B was essential for thrombin response. Gel supershift assays demonstrated that thrombin induced binding of NF-kappa Bp65 (Rel A) to downstream NF-kappa B site of the ICAM-1 promoter. Thrombin receptor activation peptide, a 14-amino-acid peptide representing the new NH2 terminus of proteolytically activated receptor-1, mimicked thrombin's action in inducing ICAM-1 expression. These data indicate that thrombin activates endothelial ICAM-1 expression and polymorphonuclear leukocyte adhesion by NF-kappa Bp65 binding to the downstream NF-kappa B site of ICAM-1 promoter after proteolytically activated receptor-1 activation. 


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Constitutive expression c-fos, c-jun, and NF kappa B mRNA is in nucleated fetal blood cells and up-regulation of c-fos and c-jun with anti-CD3 stimulation. 
Fetal and neonatal lymphocytes are relatively resistant to activation and cytokine production when stimulated either via their T-cell antigen receptors or lectins. The molecular mechanism(s) responsible for this phenomenon have not been clearly elucidated. We have hypothesized that such defects in fetal/neonatal T-cell activation may be due to lack of expression of the transcriptional regulatory elements required for T-cell activation. We used reverse transcriptase-polymerase chain reaction to examine both fetal and term neonatal cord bloods for mRNA expression of three transcription factors implicated in T-cell activation: c-jun, c-fos, and NF kappa B (p50 subunit). We demonstrate that mRNAs for all three of these regulatory factors are expressed in fetal blood cells by the 27th week of gestation and in term cord bloods. Activation of term infant cord blood mononuclear cells with anti-CD3 monoclonal antibodies resulted in up-regulation of both c-jun and c-fos mRNAs within 15 min of stimulation. However, secretion of IL-2 by anti-CD3-stimulated cord blood mononuclear cells was still blunted compared with control cells from adults. We conclude that fetal nucleated blood cells constitutively express important genes for cytokine regulation and are able to increase intracellular accumulation of the mRNAs for these factors in response to anti-CD3 stimulation. Thus, qualitative differences in the capacity to regulate these factors could not be shown in fetal blood cells. Quantitative experiments comparing binding of these transcription factors to the IL-2 promoter are currently under investigation. 


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The carboxyl-terminal cytoplasmic domain of CD36 is required for oxidized low-density lipoprotein modulation of NF-kappaB activity by tumor necrosis factor-alpha. 
The binding of oxidized low-density lipoprotein (Ox LDL) by monocyte-macrophages causes pleiotropic effects, including changes in gene expression, and is thought to represent an early event in atherogenesis. The integral membrane glycoprotein CD36 appears to play a physiological role in binding and uptake of Ox LDL by monocyte-macrophages, although the molecular events associated with CD36-Ox LDL interaction are unknown. To approach this issue, we used CD36 transfected Chinese hampster ovary (CHO) cells, exposed them to Ox LDL, and determined changes in the activity of the transcription factor NF-kappaB. We report here that Ox LDL enhanced DNA binding activity of nuclear extracts to an NF-kappaB sequence following activation of CD36-producing CHO cells with the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha). This enhanced DNA binding activity was inhibited by coincubation of CD36 transfected cells with the human CD36-specific antibody OKM5. We also determined that activation of NF-kappaB DNA binding activity required an intact carboxyl-terminal cytoplasmic segment on CD36. Our results support the idea that human CD36 mediates signal transduction events in response to Ox LDL. 


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One base pair change abolishes the T cell-restricted activity of a kB-like proto-enhancer element from the interleukin 2 promoter. 
The inducible, T cell-specific enhancers of murine and human Interleukin 2 (Il-2) genes contain the kB-like sequence GGGATTTCACC as an essential cis-acting enhancer motif. When cloned in multiple copies this so-called TCEd (distal T cell element) acts as an inducible proto-enhancer element in E14 T lymphoma cells, but not in HeLa cells. In extracts of induced, Il-2 secreting El4 cells three individual protein factors bind to TCEd DNA. The binding of the most prominent factor, named TCF-1 (T cell factor 1), is correlated with the proto-enhancer activity of TCEd. TCF-1 consists of two polypeptides of about 50 kD and 105 kD; the former seems to be related to the 50 kD polypeptide of NF-kB. Purified NF-kB is also able to bind to the TCEd, but TCF-1 binds stronger than NF-kB to TCEd DNA. The conversion of the TCEd to a 'perfect' NF-kB binding site leads to a tighter binding of NF-kB to TCEd DNA and, as a functional consequence, to the activity of the 'converted' TCEd motifs in HeLa cells. Thus, the substitution of the underlined A residue to a C within the GGGATTTCACC motif abolishes its T cell-restricted activity and leads to its functioning in both El4 cells and HeLa cells. These results indicate that lymphocyte-specific factors binding to the TCEd are involved in the control of T cell specific-transcription of the Il-2 gene. 


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CD2 signalling induces phosphorylation of CREB in primary lymphocytes. 
Promoter sequences responsive to cyclic AMP (cAMP) are found in a number of cellular genes, and bind transcription factors of the cAMP response element binding protein (CREB)/activating transcription factor-1 (ATF-1) family. We have used a human T-lymphotropic virus type 1 (HTLV-1) model of cAMP response element (CRE) transcription to investigate the influence of lymphocyte activation on transcription from homologous regions in the viral promoter. We previously demonstrated increased HTLV-1 transcription following CD2 but not CD3 receptor cross-linking. We hypothesized that this increased viral transcription was mediated, in part, through the phosphorylation of CREB. Therefore, we investigated CD2 and CD3 receptor-mediated signalling in primary human peripheral blood mononuclear cells (PBMC). CD2, but not CD3, cross-linking increased cAMP detected by competitive enzyme-linked immunosorbent assay (ELISA) approximately fourfold. CD2 cross-linking concurrently increased phosphorylation of CREB detected by immunoblot assay eightfold. Consistent with post-translational regulation, no change in total level of CREB protein was observed. Phosphorylation of CREB occurred through a herbimycin A and Rp-cAMP- sensitive pathway, suggesting phosphorylation required antecedent activation of both protein tyrosine kinases (PTK) and protein kinase A (PKA). Both CD2 and CD3 cross-linking increased binding of nuclear proteins to a radiolabelled CRE oligonucleotide probe in electrophoretic mobility shift assays suggesting that lymphocyte activation enhances binding independently of phosphorylation of CREB at serine 133. These data indicate specific modulation of the CREB/ATF-1 family of transcription factors by the CD2 signalling pathway and suggest CD2 receptor modulation of CRE-mediated transcription following ligand engagement (e.g. cell-to-cell contact). 


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Human immunodeficiency virus type-1 transcription: role of the 5'-untranslated leader region (review). 
Human immunodeficiency virus type-1 (HIV-1) transcription is dependent on the interaction of host-cell transcription factors with cis-regulatory DNA elements within the viral long terminal repeat (LTR). Much attention has focused on the series of sequence elements upstream of the transcriptional initiation site in the U3 region of the LTR including the Sp1 and NF-kappaB binding sites. Recent studies, however, demonstrate that the transcribed 5'-untranslated leader region (5'-UTR) also contains important transcriptional elements. These regulatory elements situated downstream of transcription interact with constitutive and inducible transcription factors, mediate transmission of cellular activation signals, and are important for efficient HIV-1 transcription and replication. The 5'-UTR contains binding sites for the transcription factors AP-1, NF-kappaB, NF-AT, IRF, and Sp1. Mutations in these binding sites can interfere with the viral response to cell activation signals, decrease LTR transcription, and inhibit viral replication. The 5'-UTR also interacts with a specific nucleosome that is rapidly displaced during transcriptional activation of the latent provirus. We propose that the inducible transcription factor binding sites in the 5'-UTR comprise a downstream enhancer domain that can function independent of, or in concert with, the LTR promoter to rapidly increase latent proviral transcription in response to cell activation signals. In this review, we describe the host-cell transcription factors that interact with the 5'-UTR and discuss their role in the transcriptional regulation of HIV-1 gene expression. 


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T-cell expression of the human GATA-3 gene is regulated by a non-lineage-specific silencer. 
The GATA-3 transcription factor is required for development of the T-cell lineage and Th2 cytokine gene expression in CD4 T-cells. We have mapped the DNase-I-hypersensitive (HS) regions of the human GATA-3 gene in T-cells and non-T-cells and studied their transcriptional activities. HS I-III, located 5' from the transcriptional initiation site, were found in hematopoietic and non-hematopoietic cells, whereas HS IV-VII, located 3' from the transcriptional start site, were exclusively observed in T-cells. Among these hypersensitive sites, two transcriptional control elements were found, one in the first intron of the GATA-3 gene and the other between 8.3 and 5.9 kilobases 5' from the GATA-3 transcriptional initiation site. The first intron acted as a strong transcriptional activator in a position-dependent manner and with no cell-type specificity. The upstream regulatory element could confer T-cell specificity to the GATA-3 promoter activity, and analysis of this region revealed a 707-base pair silencer that drastically inhibited GATA-3 promoter activity in non-T-cells. Two CAGGTG E-boxes, located at the 5'- and 3'-ends of the silencer, were necessary for this silencer activity. The 3'-CAGGTG E-box could bind USF proteins, the ubiquitous repressor ZEB, or the basic helix-loop-helix proteins E2A and HEB, and we showed that a competition between ZEB and E2A/HEB proteins is involved in the silencer activity. 


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Analysis of the ligand-binding domain of human retinoic acid receptor alpha by site-directed mutagenesis. 
Three subtypes of retinoic acid receptors (RAR), termed RAR alpha, RAR beta, and RAR gamma, have been described. They are composed of different structural domains, including distinct domains for DNA and ligand binding. RARs specifically bind all-trans-retinoic acid (RA), 9-cis-RA, and retinoid analogs. In this study, we examined the functional role of cysteine and arginine residues in the ligand-binding domain of hRAR alpha (hRAR alpha-LBD, amino acids 154 to 462). All conserved cysteine and arginine residues in this domain were mutated by site-directed mutagenesis, and the mutant proteins were characterized by blocking reactions, ligand-binding experiments, transactivation assays, and protease mapping. Changes of any cysteine residue of the hRAR alpha-LBD had no significant influence on the binding of all-trans RA or 9-cis RA. Interestingly, residue C-235 is specifically important in antagonist binding. With respect to arginine residues, only the two single mutations of R-276 and R-394 to alanine showed a dramatic decrease of agonist and antagonist binding whereas the R272A mutation showed only a slight effect. For all other arginine mutations, no differences in affinity were detectable. The two mutations R217A and R294A caused an increased binding efficiency for antagonists but no change in agonist binding. From these results, we can conclude that electrostatic interactions of retinoids with the RAR alpha-LBD play a significant role in ligand binding. In addition, antagonists show distinctly different requirements for efficient binding, which may contribute to their interference in the ligand-inducible transactivation function of RAR alpha. 


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Abundant expression of erythroid transcription factor P45 NF-E2 mRNA in human peripheral granurocytes. 
Transcription factor NF-E2 is crucial for regulation of erythroid-specific gene expression. p45 subunit of NF-E2 contains a basic-leucine zipper domain and dimerizes with the small Maf family protein to form functional NF-E2 complex. While p45 expression was shown to be restricted to erythroid cells, megakaryocytes and mast cells in hematopoietic lineage, we found in this study that p45 mRNA is abundantly transcribed in the granulocyte fraction of human peripheral blood cells. As neutrophils occupy approximately 92% of the cells in granulocyte fraction of human peripheral blood cells. As neutrophils occupy approximately 92% of the cells in this fraction, the cells expressing p45 is most likely to be neutrophils. p45 mRNA is also expressed in HL-60 promyelocytes, albeit the expression level is much lower than that of the granulocyte fraction. HL-60 cells were found to express mafK mRNA, indicating the presence of genuine NF-E2 complex in the cells. Although p45 mRNA is transcribed from two different promoters, aNF-E2 promoter and fNF-E2 promoter, in erythroid and megakaryocytic lineage cells, p45 mRNA is transcribed only from aNF-E2 promoter. The expression of p45 megakaryocytic lineage cells, p45 mRNA is transcribed only from aNF-E2 promoter. The expression of p45 mRNA in the neutrophils declined rapidly after transfer of the cells to in vitro culture and G-CSF could not sustain the expression from the down-regulation, suggesting the E2 may also participate in the regulation of neutrophil-specific gene expression. 


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Regulation of CD95 (Fas) ligand expression by TCR-mediated signaling events. 
Stimulation of mature peripheral T cells by TCR engagement results in activation of signals that drive induction of cytokine gene expression and clonal expansion. However, under some conditions, engagement of the TCR leads instead to apoptosis. Recent studies demonstrate that TCR-stimulated apoptosis requires expression of CD95 ligand on activated T cells followed by an interaction between CD95 ligand and the CD95 receptor also expressed on this population. The experiments reported in this study were designed to address the signaling events triggered by TCR engagement that are important for regulating CD95 ligand gene expression. To approach this, we generated a luciferase reporter construct containing elements of the CD95 ligand promoter. Using a previously described mutant of the Jurkat T cell line, we show that proximal signaling events dependent on the presence of the CD45 tyrosine phosphatase are required for TCR-stimulated CD95 ligand expression. Transient transfection studies demonstrate further that TCR-stimulated activation of the Ras signaling pathway is required for optimal activation of CD95 ligand. Next, in an effort to determine critical transcription factors that regulate CD95 ligand expression, we demonstrate a cyclosporin A-sensitive nuclear factor-AT response element in the promoter region of this gene that is critical for optimal CD95 ligand reporter activity in stimulated T cells. Together, these studies begin a dissection of the biochemical events that lead to expression of CD95 ligand, a required step for TCR-induced apoptosis. 


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Dependence for the proliferative response to erythropoietin on an established erythroid differentiation program in a human hematopoietic cell line, UT-7. 
Erythroid differentiation involves the activation of a number of erythroid-specific genes, most of which, including the globin genes and the erythropoietin receptor (Epo-R) gene, are, at least in part, regulated by the transcription factor GATA-1. In order to understand the relationship, if any, between expression of GATA-1, response to Epo and erythroid differentiation, we analyzed the expression of GATA-1, Epo-R and globin genes in an Epo-dependent human cell line, UT-7 Epo. The results were compared to those obtained with the parental granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent cell line, UT-7, which has a predominantly megakaryoblastic phenotype and is unable to proliferate continuously in the presence of Epo. UT-7 Epo and UT-7 expressed similar levels of GATA-1 mRNA and binding activity. The two lines also expressed comparable levels of Epo-R mRNA while the number of Epo-binding sites on UT-7 Epo cells was one-sixth the number of UT-7 cells (2400 +/- 3 vs. 13,800 +/- 300). This difference in the number of binding sites could be due to differences in cell surface (UT-7 cells are 20% smaller than the parental UT-7 cells) or in receptor turnover. By Northern analysis, UT-7 cells expressed detectable levels of beta- and gamma-globin but not alpha-globin. In comparison, UT-7 Epo cells expressed alpha-globin and higher levels of gamma-globin (5-fold) and beta-globin (from barely to clearly detectable). Globin chains (alpha, beta and gamma) were clearly detectable by affinity chromatography in UT-7 Epo but not in UT-7 cells. The frequency of the cells which expressed beta- and gamma- globin genes in the two cell populations was measured by immunofluorescence with beta- and gamma-specific antibodies. The number of gamma-positive cells and their fluorescence intensity were higher in UT-7 Epo than in UT-7 cells (0 to 17% barely positive cells and 23 to 40% clearly positive cells, respectively), indicating that the increase in globin mRNA observed in UT-7 Epo is due to both an increase of gene expression per cell and an increase in numbers of cells containing gamma-globin. The levels of GATA-1, Epo-R and globin mRNA expressed were not affected by a 24-hour incubation of either cell line with Epo, GM-CSF or interleukin-3 (IL-3). (ABSTRACT TRUNCATED AT 400 WORDS) 


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Epstein-Barr viral latency is disrupted by the immediate-early BRLF1 protein through a cell-specific mechanism. 
Epstein-Barr virus (EBV), the causative agent of infectious mononucleosis, is a human herpesvirus associated with epithelial cell malignancies (nasopharyngeal carcinoma) as well as B-cell malignancies. Understanding how viral latency is disrupted is a central issue in herpesvirus biology. Epithelial cells are the major site of lytic EBV replication within the human host, and viral reactivation occurs in EBV-associated nasopharyngeal carcinomas. It is known that expression of a single viral immediate-early protein, BZLF1, is sufficient to initiate the switch from latent to lytic infection in B cells. Cellular regulation of BZLF1 transcription is therefore thought to play a key role in regulating the stringency of viral latency. Here we show that, unexpectedly, expression of another viral immediate-early protein, BRLF1, can disrupt viral latency in an epithelial cell-specific fashion. Therefore, the mechanisms leading to disruption of EBV latency appear to be cell-type specific. 


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Regulation of c-jun mRNA expression by hydroxyurea in human K562 cells during erythroid differentiation [published erratum appears in Biochim Biophys Acta 1995 Dec 27;1264(3):409] 
Hydroxyurea (HU) is an antitumor agent which also induces hemoglobinization during erythroid differentiation. In addition, HU stimulates the synthesis of fetal hemoglobin in sickle cell anemia patients. To further understand its mechanism of action, we investigated the effects of HU on regulation of c-jun expression prior to the onset of erythroid differentiation of K562 cells. HU induced a dose-dependent stimulation of c-jun synthesis. The levels of c-jun mRNA was elevated 4 to 7.5-fold by HU within 2 h. This was followed by a gradual decline to the basal level by 24 h. Both nuclear run-on and actinomycin D pulse experiments strongly indicate that HU regulates c-jun mRNA expression by increasing the rate of synthesis as well as stabilizing the c-jun mRNA. In addition, the level of jun protein was elevated by 2 to 5-fold within 4 h in HU treated cells. Furthermore, concentrations of HU below 250 microM slightly increased the 5X AP-1/CAT activity. These results strongly suggest that HU induces both transcriptional and post-transcription regulation of c-jun during erythroid differentiation. 


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Cross-linking of Fc gamma receptors activates HIV-1 long terminal repeat-driven transcription in human monocytes. 
Elevation of the levels of circulating immune complexes frequently accompanies HIV-1 infection and is a prognostic indicator of clinical progression from asymptomatic infection to AIDS. Here we report that cross-linking of Fc gamma RI or Fc gamma RII by adherent human IgG or by specific anti-Fc gamma R mAb activates HIV-1 gene expression in the human monocytic cell line BF24 and increased HIV RNA expression in monocytes from HIV infected patients as assayed by reverse transcription-PCR. In THP-1 cells, Fc gamma R cross-linking induced NF-kappa B, which is known to bind to the regulatory region of the long terminal repeat (LTR) of HIV-1 and to activate HIV-1 transcription. Anti-TNF-alpha antibody but not anti-IL-1 beta antibody strongly inhibited both the induction of HIV-1-LTR-driven transcription and the induction of NF-kappa B by Fc gamma R cross-linking. These results indicate that Fc gamma R can mediate a TNF-alpha-dependent induction of HIV-1 gene transcription and suggest that immune complexes may contribute to the pathophysiology of HIV-1 infection by augmenting viral replication in monocytes. 


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Adherence-dependent increase in human monocyte PDGF(B) mRNA is associated with increases in c-fos, c-jun, and EGR2 mRNA. 
Adherence is an important initial step in the transition of a circulating monocyte to a tissue macrophage. This differentiation is accompanied by an augmented capacity to generate growth factors. We hypothesized that adherence itself might be an important trigger for a sequence of gene activation culminating in cells with increased mRNA encoding profibrotic growth factors such as platelet-derived growth factor B subunit (PDGF[B]) and transforming growth factor-beta (TGF-beta). After in vitro adherence, human monocytes had a biphasic increase in PDGF(B) mRNA with peaks at 6 h and 13 d. No increase in TGF-beta mRNA was observed. The 6-h increase in PDGF(B) mRNA was adherence dependent, and in addition, was abrogated when the cytoskeletal integrity was compromised by cytochalasin D. The 6-h increase in PDGF(B) mRNA was unaltered by adherence in the presence of the monocyte stimulus lipopolysaccharide. Adherence to either fibronectin or collagen-coated plastic had little consistent effect on PDGF(B) mRNA accumulation. The increased PDGF(B) mRNA observed in adherent monocytes was accompanied by increases in mRNAs of the early growth response genes c-fos (maximal at 20 min), c-jun, and EGR2 (maximal at 6-24 h). The increase in c-jun and EGR2, but not c-fos, mRNA was also abrogated by cytochalasin D. These observations suggest that adherence results in increases of c-fos, c-jun, EGR2, and PDGF(B) mRNA. In addition, the increases in c-jun, EGR2, and PDGF(B) may depend on cytoskeletal rearrangement. Modulation of these events at the time of adherence offers a mechanism by which differential priming of the cells may be accomplished. 


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NF-kappaB-mediated up-regulation of Bcl-x and Bfl-1/A1 is required for CD40 survival signaling in B lymphocytes. 
Activation of CD40 is essential for thymus-dependent humoral immune responses and rescuing B cells from apoptosis. Many of the effects of CD40 are believed to be achieved through altered gene expression. In addition to Bcl-x, a known CD40-regulated antiapoptotic molecule, we identified a related antiapoptotic molecule, A1/Bfl-1, as a CD40-inducible gene. Inhibition of the NF-kappaB pathway by overexpression of a dominant-active inhibitor of NF-kappaB abolished CD40-induced up-regulation of both the Bfl-1 and Bcl-x genes and also eliminated the ability of CD40 to rescue Fas-induced cell death. Within the upstream promoter region of Bcl-x, a potential NF-kappaB-binding sequence was found to support NF-kappaB-dependent transcriptional activation. Furthermore, expression of physiological levels of Bcl-x protected B cells from Fas-mediated apoptosis in the absence of NF-kappaB signaling. Thus, our results suggest that CD40-mediated cell survival proceeds through NF-kappaB-dependent up-regulation of Bcl-2 family members. 


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Regulation of low shear flow-induced HAEC VCAM-1 expression and monocyte adhesion. 
We recently reported that prolonged exposure of human aortic endothelial cells (HAEC) to low shear stress flow patterns is associated with a sustained increase in the activated form of the transcriptional regulator nuclear factor-kappaB (NF-kappaB). Here we investigate the hypothesis that low shear-induced activation of NF-kappaB is responsible for enhanced expression of vascular cell adhesion molecule (VCAM-1) resulting in augmented endothelial cell-monocyte (EC-Mn) adhesion and that this activation is dependent on intracellular oxidant activity. Before exposure to low shear (2 dyn/cm2) for 6 h, HAEC were preincubated with or without the antioxidants pyrrolidine dithiocarbamate (PDTC) or N-acetyl-L-cysteine (NAC). PDTC strongly inhibited low shear-induced activation of NF-kappaB, expression of VCAM-1, and EC-Mn adhesion. Paradoxically, NAC exerted a positive effect on low shear-induced VCAM-1 expression and EC-Mn adhesion and only slightly downregulated NF-kappaB activation. However, cytokine-induced NF-kappaB activation and VCAM-1 expression are blocked by both PDTC and NAC. These data suggest that NF-kappaB plays a key role in low shear-induced VCAM-1 expression and that pathways mediating low shear- and cytokine-induced EC-Mn adhesion may be differentially regulated. 


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The small GTP-binding protein Rho potentiates AP-1 transcription in T cells. 
The Rho family of small GTP-binding proteins is involved in the regulation of cytoskeletal structure, gene transcription, specific cell fate development, and transformation. We demonstrate in this report that overexpression of an activated form of Rho enhances AP-1 activity in Jurkat T cells in the presence of phorbol myristate acetate (PMA), but activated Rho (V14Rho) has little or no effect on NFAT, Oct-1, and NF-kappaB enhancer element activities under similar conditions. Overexpression of a V14Rho construct incapable of membrane localization (CAAX deleted) abolishes PMA-induced AP-1 transcriptional activation. The effect of Rho on AP-1 is independent of the mitogen-activated protein kinase pathway, as a dominant-negative MEK and a MEK inhibitor (PD98059) did not affect Rho-induced AP-1 activity. V14Rho binds strongly to protein kinase Calpha (PKCalpha) in vivo; however, deletion of the CAAX site on V14Rho severely diminished this association. Evidence for a role for PKCalpha as an effector of Rho was obtained by the observation that coexpression of the N-terminal domain of PKCalpha blocked the effects of activated Rho plus PMA on AP-1 transcriptional activity. These data suggest that Rho potentiates AP-1 transcription during T-cell activation. 


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Fibrinogen activates NF-kappa B transcription factors in mononuclear phagocytes. 
Adhesion to extracellular matrices is known to modulate leukocyte activation, although the mechanisms are not fully understood. Mononuclear phagocytes are exposed to fibrinous provisional matrix throughout migration into inflammatory foci, so this study was undertaken to determine whether fibrinogen triggers activation of NF-kappa B transcription factors. U937 cells differentiated with PMA in nonadherent culture were shown to express two fibrinogen-binding integrins, predominately CD11b/CD18, and to a lesser extent, CD11c/CD18. Cells stimulated with fibrinogen (10-100 microg/ml)/Mn2+ (50 microM) for 2 h were examined by electrophoretic mobility shift assay. NF-kappa B activation, minimal in unstimulated cells, was substantially up-regulated by fibrinogen. Fibrinogen also caused activation of AP-1, but not SP1 or cAMP response element-binding protein (CREB) factors. Blocking mAbs against CD18 and CD11b abrogated fibrinogen-induced NF-kappa B activation. To determine the effects on transcriptional regulation, U937 cells were transfected with a plasmid containing the HIV-1 enhancer (bearing two NF-kappa B sites) coupled to a chloramphenicol acetyltransferase (CAT) reporter. Cells were subsequently stimulated with 1) PMA for 24 h, inducing CAT activity by 2.6-fold, 2) fibrinogen/Mn2+ for 2 h, inducing CAT activity by 3.2-fold, or 3) costimulation with fibrinogen and PMA, inducing 5.7-fold the CAT activity induced by PMA alone. We conclude that contact with fibrinogen-derived proteins may contribute to mononuclear phagocyte activation by signaling through CD11b/CD18, resulting in selective activation of transcriptional regulatory factors, including NF-kappa B. 


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Alteration of a single serine in the basic domain of the Epstein-Barr virus ZEBRA protein separates its functions of transcriptional activation and disruption of latency. 
The ZEBRA protein from Epstein-Barr virus (EBV) activates a switch from the latent to the lytic expression program of the virus. ZEBRA, a member of the bZIP family of DNA-binding proteins, is a transcriptional activator capable of inducing expression from viral lytic cycle promoters. It had previously been thought that ZEBRA's capacity to disrupt EBV latency resided primarily in its ability to activate transcription of genes that encode products required for lytic replication. We generated a point mutant of ZEBRA, Z(S186A), that was not impaired in its ability to activate transcription; however, this mutation abolished its ability to initiate the viral lytic cascade. The mutant, containing a serine-to-alanine substitution in the DNA-binding domain of the protein, bound to several known ZEBRA-binding sites and activated transcription from reporters bearing known ZEBRA-responsive promoters but did not disrupt latency in EBV-infected cell lines. Therefore, initiation of the EBV lytic cycle by the ZEBRA protein requires a function in addition to transcriptional activation; a change of serine 186 to alanine in the DNA-binding domain of ZEBRA abolished this additional function and uncovered a new role for the ZEBRA protein in disruption of EBV latency. The additional function that is required for initiation of the lytic viral life cycle is likely to require phosphorylation of serine 186 of the ZEBRA protein, which may influence either DNA recognition or transcriptional activation of lytic viral promoters in a chromatinized viral episome. 


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Erythropoietin-dependent induction of hemoglobin synthesis in a cytokine-dependent cell line M-TAT. 
M-TAT is a cytokine-dependent cell line with the potential to differentiate along the erythroid and megakaryocytic lineages. We cultured M-TAT cells long term (> 1 year) in the continuous presence of erythropoietin (EPO), granulocyte-macrophage colony-stimulating factor (GM-CSF), or stem cell factor (SCF). These long term cultures are referred to as M-TAT/EPO, M-TAT/GM-CSF, and M-TAT/SCF cells, respectively. Hemoglobin concentration and gamma-globin and erythroid delta-aminolevulinate synthase mRNA levels were significantly higher in M-TAT/EPO cells than in M-TAT/GM-CSF cells. When the supplemented cytokine was switched from GM-CSF to EPO, hemoglobin synthesis in M-TAT/GM-CSF cells increased rapidly (within 5 h), and the level of GATA-1 mRNA increased. In contrast, the addition of GM-CSF to the M-TAT/EPO cell culture decreased the amount of hemoglobin, even in the presence of EPO, indicating that the EPO signal for erythroid differentiation is suppressed by GM-CSF. Thus, erythroid development of M-TAT cells is promoted by EPO and suppressed by GM-CSF. These results support the hypothesis that EPO actively influences the programming of gene expression required for erythroid progenitor cell differentiation. 


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Transcription factor NF-kappaB regulation of renal fibrosis during ureteral obstruction. 
Irrespective of the etiology, many kidney diseases result in inflammation and fibrosis of the tubulointerstitium, with the subsequent loss of renal function. To initiate any disease process or for any disease process to progress, there must be changes in the transcription of genes within the affected tissue. The nuclear factor-kappa B (NF-kappaB) family of transcription factors regulates genes involved in inflammation, cell proliferation, and cell differentiation. This review discusses the NF-kappaB transcription factor family in general and the association of NF-kappaB activation with cellular/molecular events of renal inflammation and fibrosis. 


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Regulation of transcription of the human erythropoietin receptor gene by proteins binding to GATA-1 and Sp1 motifs. 
Erythropoietin (Epo), the primary regulator of the production of erythroid cells, acts by binding to a cell surface receptor (EpoR) on erythroid progenitors. We used deletion analysis and transfection assays with reporter gene constructs to examine the transcription control elements in the 5' flanking region of the human EpoR gene. In erythroid cells most of the transcription activity was contained in a 150 bp promoter fragment with binding sites for transcription factors AP2, Sp1 and the erythroid-specific GATA-1. The 150 bp hEpoR promoter exhibited high and low activity in erythroid OCIM1 and K562 cells, respectively, reflecting the high and low levels of constitutive hEpoR expression. The GATA-1 and Sp1 binding sites in this promoter lacking a TATA sequence were necessary for a high level of transcription activation. Protein-DNA binding studies suggested that Sp1 and two other CCGCCC binding proteins from erythroid and non-erythroid cells could bind to the Sp1 binding motif. By increasing GATA-1 levels via co-transfection, we were able to transactivate the hEpoR promoter in K562 cells and non-erythroid cells, but not in the highly active OCIM1 cells, although GATA-1 mRNA levels were comparable in OCIM1 and K562. Interestingly, when we mutated the Sp1 site, resulting in a marked decrease in hEpoR promoter activity, we could restore transactivation by increasing GATA-1 levels in OCIM1 cells. These data suggest that while GATA-1 can transactivate the EpoR promoter, the level of hEpoR gene expression does not depend on GATA-1 alone. Rather, hEpoR transcription activity depends on coordination between Sp1 and GATA-1 with other cell-specific factors, including possibly other Sp1-like binding proteins, to provide high level, tissue-specific expression. 


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Alcohol-induced regulation of nuclear regulatory factor-kappa beta in human monocytes. 
Acute ethanol exposure has the capacity to modulate immune functions, particularly, to down regulate monocyte production of inflammatory cytokines. However, the intracellular mechanisms for these effects of ethanol are yet to be understood. Considering that nuclear regulatory factor-kappa beta (NF-kappa B)/Rel is a common regulatory element of the promoter region of the inflammatory cytokine genes, herein, we tested the hypothesis that acute ethanol affects NF-kappa B activation in human monocytes. Adherence-isolated monocytes showed constitutive DNA binding activity of NF-kappa B. A clinically relevant dose (25 mM) of acute ethanol treatment in vitro increased NF-kappa B binding activity in monocytes with a preferential induction of the inhibitory, p50/p50, NF-kappa B/Rel homodimer, and resulted in no induction of the p65/p50 heterodimer. In contrast, lipopolysaccharide stimulation primarily induced the p65/p50 heterodimer that has been shown to result in gene activation. Thus, such unique activation of the inhibitory p50/p50 homodimer by acute ethanol treatment may result in inhibition rather than activation of NF-kappa B-regulated inflammatory cytokine genes. Consequently, these results suggest that physiologically relevant concentrations of ethanol may affect production of inflammatory cytokines, such as tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-6 by disrupting NF-kappa B signaling in monocytes. 


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Signal transduction pathways triggered by the FcepsilonRIIb receptor (CD23) in human monocytes lead to nuclear factor-kappaB activation. 
BACKGROUND: Alveolar macrophages play a key role in the initiation of the inflammatory reaction of allergic asthma. Alveolar macrophages and peripheral blood monocytes are activated when IgE/allergen immune complexes bind to the CD23 receptor, which leads to the production of inflammatory cytokines. OBJECTIVE: We sought to investigate the molecular mechanisms regulating this early inflammatory response. We have focused on the study of the signal transduction pathways triggered by CD23 in human monocytes and the promonocytic cell line U937. METHODS: CD23 was cross-linked in human monocytes and U937 cells with IgE immune complexes. Surface expression of CD23 was determined by FACS analysis. Transcription factor activation and gene transcription were studied by gel-shift assays and Northern blot analysis, respectively. IkappaBalpha phosphorylation and degradation was analyzed by Western blot. RESULTS: Nuclear factor (NF)-kappaB is the main transcription factor involved in the gene activation that follows CD23 cross-linking in monocytes. CD23-induced NF-kappaB is a heterodimer composed of p65/p50 subunits. NF-kappaB nuclear translocation is secondary to the phosphorylation and subsequent degradation of the NF-kappaB inhibitory molecule IkappaBalpha. Tyrosine kinase-dependent, and not protein kinase C-dependent, pathways mediate CD23-triggered NF-kappaB activation but do not participate in the direct phosphorylation of IkappaBalpha. IkappaBalpha degradation and NF-kappaB nuclear translocation correlate with transcriptional activation of the inflammatory cytokines TNF-alpha and IL-1beta. CONCLUSIONS: NF-kappaB is the main transcription factor involved in the signal transduction pathway of CD23 in monocytes. 


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Role of Egr-2 in up-regulation of Fas ligand in normal T cells and aberrant double-negative lpr and gld T cells. 
We previously identified a Fas ligand regulatory element (FLRE) in the Fas ligand (fasL) promoter that binds Egr family proteins and demonstrated that Egr-3 (PILOT) but not Egr-1 (NGFI-A, Krox-24, Tis-8, and Zif-268) induces transcription of fasL. The aberrant CD4(-)CD8(-) T cells from lpr/lpr and gld/gld mice, which have mutations in the genes encoding Fas and FasL, respectively, have an activated phenotype and constitutively express high levels of fasL mRNA, prompting us to ask what role if any the FLRE and Egr family proteins have in this aberrant expression of fasL. Unstimulated MRL-lpr/lpr and C3H-gld/gld CD4(-)CD8(-) T cells constitutively contained high levels of two proteins that bound to the FLRE. Supershift analysis revealed these proteins to be Egr-1 and Egr-2 (Krox-20); Egr-3 was not detected. Activation of normal lymph node cells resulted in increased expression of Egr-1, -2, and -3. As with egr-3, expression of egr-2 was blocked by cyclosporin A. Although overexpressed Egr-1 was ineffective, overexpressed Egr-2 was as potent as Egr-3 in inducing fasL promoter-dependent reporter constructs in T cell hybridomas and HeLa cells, and both up-regulated endogenous fasL mRNA in HeLa cells. FasL-dependent reporter constructs in MRL-lpr/lpr and C3H-gld/gld CD4(-)CD8(-) T cells were constitutively active, and this activity was largely prevented by mutation of the critical Egr family binding element. Thus, Egr-2, in addition to Egr-3, regulates FasL expression in activated normal T cells, and Egr-2 is likely to play a direct role in aberrant fasL up-regulation in lpr/lpr and gld/gld CD4(-)CD8(-) T cells. 


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Cortivazol mediated induction of glucocorticoid receptor messenger ribonucleic acid in wild-type and dexamethasone-resistant human leukemic (CEM) cells. 
Cortivazol is a phenylpyrazolo glucocorticoid of high potency and unusual structure. In both wild-type and highly dexamethasone(dex)-resistant clones of the human leukemic cell line CEM, exposure to cortivazol leads to cell death. It has been shown recently that in wild-type CEM cells but not in a dex-resistant, glucocorticoid receptor(GR)-defective clone ICR-27 TK-3, dex induces GR mRNA. To test the hypothesis that cortivazol acts in dex-resistant cells by making use of the residual GR found there, wild-type and dex-resistant clones were treated with various concentrations of cortivazol and induction of GR mRNA was studied. Cortivazol significantly induced GR mRNA in the normal CEM-C7 as well as in two classes of dex-resistant clones, although the dex-resistant clones needed at least 10 times more cortivazol than the normal cells for significant GR mRNA induction. Increased levels of GR mRNA were noticed as early as 3 h after treatment. A general correlation between induction of GR mRNA and lysis of the normal and dex-resistant cells was found. Positive induction of GR mRNA might be one of the earliest crucial steps in the lysis of normal and dex-resistant CEM cells, or might serve as a marker for the process. However, the lysis pathway in the dex-resistant cells is defective in that dex-resistant clones needed significantly more cortivazol than the normal cells for lysis of the cells. 


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Expression of PILOT, a putative transcription factor, requires two signals and is cyclosporin A sensitive in T cells. 
Few known genes (IL-2, members of the IL-8 family, interferon-gamma) are induced in T cells only through the combined effect of phorbol myristic acetate (PMA) and a Ca(2+)-ionophore, and expression of only these genes can be fully suppressed by Cyclosporin A (CyA). We have identified a putative transcription factor, designated PILOT, with an identical dual signal requirement for expression. Induction of the PILOT gene is detectable in human T cells 20 min following activation in the presence of cycloheximide and is fully suppressed by CyA. The PILOT protein has a calculated M(r) of 42.6 kDa and contains three zinc fingers of the C2H2-type at the carboxyl-terminus which are highly homologous to the zinc finger regions of the transcription factors EGR1, EGR2, and pAT 133. In contrast to T cells, in fibroblasts PILOT gene expression requires only one signal (PMA) and is not affected by CyA. This observation directly demonstrates the existence of a Ca2+ signal-dependent regulatory element obligatory for expression of some genes in T cells but not in fibroblasts. This differential expression model will be valuable in the dissection of the dual signal pathway in T cells and the effects of CyA upon it. 


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ras protein activity is essential for T-cell antigen receptor signal transduction. 
In a Jurkat cell model of T-cell activation an interleukin-2 promoter/reporter gene construct was activated by antigen receptor agonism in combination with the lymphokine interleukin-1. Antigen receptor signals could be mimicked by suboptimal activation of protein kinase C (PKC) with phorbol esters in combination with calcium mobilization by an ionophore. In cotransfection experiments, oncogenic rats obviated the need for PKC stimulation but did not replace either the calcium signal or interleukin-1. Activated ras expression also replaced the requirement for PKC stimulation in activation of the T-cell transcription factor NF-AT. A dominant inhibitory ras mutant specifically blocked antigen receptor agonism, indicating that ras activity is required for antigen receptor signaling. In addition, an inhibitor of PKC blocked both activated ras and phorbol ester stimulation, suggesting a role for ras upstream of PKC. 


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Differential induction of interferon (IFN)-inducible protein 10 following differentiation of a monocyte, macrophage cell lineage is related to the changes of nuclear proteins bound to IFN stimulus response element and kappaB sites. 
We examined chemokine gene expression following the differentiation of a monocyte, macrophage cell lineage. The human monoblastic cell line, U937 was differentiated to macrophages by the treatment with either phorbol 12-myristate 13-acetate (PMA), retinoic acid (RA), or vitamin D3 (VitD3). The gene expression of interferon (IFN)-inducible protein 10 (IP-10) (a CXC chemokine) was markedly augmented by the IFNgamma treatment in PMA- or RA-differentiated U937 cells, but only marginally in undifferentiated or VitD3-treated cells. In contrast, another inducible gene expression of monocyte chemotactic protein-1 (a CC chemokine) and the activation of the transcriptional factor (FcRFgamma) bound to the gamma response region were similarly or less abundantly induced by IFNgamma treatment in PMA- or RA-differentiated U937 cells, indicating that increased IP-10 mRNA induction was not due to the augmented ability of the cells to respond to the presence of IFNgamma. Increased expression of IFNgamma-induced IP-10 mRNA following the differentiation of U937 cells was mediated largely by augmented transcriptional activity of the gene and was related to differentiation-dependent changes of the proteins bound to IFN stimulus response element (ISRE) and kB sites, suggesting that these nuclear proteins may determine the IP-10 mRNA inducibility by IFNgamma. 


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Cloning and characterization of the beta subunit of human proximal sequence element-binding transcription factor and its involvement in transcription of small nuclear RNA genes by RNA polymerases II and III. 
The proximal sequence element (PSE)-binding transcription factor (PTF), which binds the PSE of both RNA polymerase II- and RNA polymerase III-transcribed mammalian small nuclear RNA (snRNA) genes, is essential for their transcription. We previously reported the purification of human PTF, a complex of four subunits, and the molecular cloning and characterization of PTF gamma and delta subunits. Here we describe the isolation and expression of a cDNA encoding PTF beta, as well as functional studies using anti-PTF beta antibodies. Native PTF beta, in either protein fractions or a PTF-Oct-1-DNA complex, can be recognized by polyclonal antibodies raised against recombinant PTF beta. Immunodepletion studies show that PTF beta is required for transcription of both classes of snRNA genes in vitro. In addition, immunoprecipitation analyses demonstrate that substantial and similar molar amounts of TATA-binding protein (TBP) and TFIIIB90 can weakly associate with PTF at low salt conditions, but this association is dramatically reduced at high salt concentrations. Along with our previous demonstration of both physical interactions between PTF gamma/PTF delta and TBP and the involvement of TFIIIB90 in the transcription of class III snRNA genes, these results are consistent with the notion that a TBP-containing complex related to TFIIIB is required for the transcription of class III snRNA genes, and acts through weak interaction with the four-subunit PTF. 


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Tandem AP-1-binding sites within the human beta-globin dominant control region function as an inducible enhancer in erythroid cells. 
A powerful enhancer has been mapped to an 18-bp DNA segment located 11 kb 5' to the human epsilon-globin gene within the dominant control or locus-activating region. This enhancer is inducible in K562 human erythroleukemia cells, increasing linked gamma-globin promoter/luciferase gene expression to 170-fold over an enhancerless construct. The enhancer consists of tandem AP-1-binding sites, phased 10 bp apart, which are both required for full activity. DNA-protein binding assays with nuclear extracts from induced cells demonstrate a high molecular weight complex on the enhancer. The formation of this complex also requires both AP-1 sites and correlates with maximal enhancer activity. Induction of the enhancer may have a role in the increase in globin gene transcription that characterizes erythroid maturation. Enhancer activity appears to be mediated by the binding of a complex of proteins from the jun and fos families to tandem AP-1 consensus sequences. 


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Activation of NF-kappa B by phosphatase inhibitors involves the phosphorylation of I kappa B alpha at phosphatase 2A-sensitive sites. 
Activation of NF-kappa B by various cellular stimuli involves the phosphorylation and subsequent degradation of its inhibitor, I kappa B alpha, although the underlying mechanism remains unclear. In the present study, the role of serine/threonine phosphatases in the regulation of I kappa B alpha phosphorylation was investigated. Our studies demonstrate that incubation of human T cells with low concentrations (approximately 1-5 nM) of calyculin A or okadaic acid, potent inhibitors of protein phosphatase type 1 (PP-1) and type 2A (PP-2A), induces the phosphorylation of I kappa B alpha even in the absence of any cellular stimulus. This action of the phosphatase inhibitors, which is associated with the activation of the RelA.p50 NF-kappa B heterodimer, is not affected by agents that block the induction of I kappa B alpha phosphorylation by tumor necrosis factor alpha (TNF-alpha). Furthermore, the phosphorylated I kappa B alpha from calyculin A-treated cells, but not that from TNF-alpha-stimulated cells, is sensitive to PP-2A in vitro, suggesting the existence of fundamental differences in the phosphorylation of I kappa B alpha induced by the two different NF-kappa B inducers. However, induction of I kappa B alpha phosphorylation by both TNF-alpha and the phosphatase inhibitors is associated with the subsequent degradation of I kappa B alpha. We further demonstrate that TNF-alpha- and calyculin A-induced I kappa B alpha degradation exhibits similar but not identical sensitivities to a proteasome inhibitor. Together, these results suggest that phosphorylation of I kappa B alpha, mediated through both the TNF-alpha-inducible and the PP-2A-opposing kinases, may serve to target I kappa B alpha for proteasome-mediated degradation. 


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LPS-Induced NF-kappaB activation and TNF-alpha release in human monocytes are protein tyrosine kinase dependent and protein kinase C independent. 
BACKGROUND: Tumor necrosis factor alpha (TNF-alpha) is an important mediator of septic shock. Endotoxin (LPS) signal transduction in human monocytes leads to activation of nuclear factor-kappa B (NF-kappaB) and TNF-alpha release. Previous studies have implicated activation of both protein kinase C (PKC) and protein tyrosine kinases (PTK) in LPS-induced NF-kappaB activation and TNF-alpha production. We hypothesized that inhibition of either PKC or PTK would decrease LPS-induced NF-kappaB DNA binding and TNF-alpha release in human monocytes. MATERIALS AND METHODS: Human monocytes were stimulated with PMA (50 ng/ml) alone or LPS (100 ng/ml) with and without a nonspecific serine/threonine protein kinase inhibitor staurosporine (Stauro), a specific pan-PKC inhibitor bisindolylmaleimide (Bis), or an inhibitor of PTK genistein (Gen). TNF-alpha release in culture supernatants was measured by an ELISA. NF-kappaB DNA binding was evaluated by electrophoretic mobility shift assay. RESULTS: LPS increased NF-kappaB DNA binding and TNF-alpha release in human monocytes. Nonspecific protein kinase inhibition inhibited NF-kappaB activation and TNF-alpha release, while specific PKC inhibition with Bis had no effect on LPS-induced NF-kappaB DNA binding or TNF-alpha release. PTK inhibition with Gen attenuated both LPS-induced NF-kappaB DNA binding and TNF-alpha production in human monocytes. Direct activation of PKC with PMA induced both NF-kappaB activation and TNF-alpha production by human monocytes. CONCLUSIONS: These results suggest that LPS-induced NF-kappaB activation and TNF-alpha release in human monocytes are independent of PKC activity. Furthermore, our results provide evidence that PTK plays a role in LPS-induced NF-kappaB activation and TNF-alpha release in human monocytes and thus could be a potential therapeutic target in inflammatory states. Copyright 1999 Academic Press. 


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Inhibition of HIV-1 replication by combination of a novel inhibitor of TNF-alpha with AZT. 
The small molecule S9a was derived from an established tumor necrosis factor-alpha (TNF-alpha) inhibitor (Canventol) by replacement of the isopropylidine group with a phenyl ring. S9a at 10 to 100 nM inhibited HIV production as potently as 3'-azido-3'-deoxythymidine (AZT), an inhibitor of viral reverse transcriptase. Furthermore, S9a and AZT in combination, at noncytoxic concentrations strongly inhibited HIV-1 replication that was more than additive and substantially prolonged the appearance of virus both in acutely infected CD4+ lymphocytes (SupT) in culture and in peripheral blood mononuclear cells (PBMCs) infected with a primary HIV-1 isolate. S9a inhibited TNF-alpha promoter-driven reporter gene activity. It was proposed that the mechanism of antiviral action of S9a was on the host cell, by blocking TNF-alpha transcription via a Tat-induced tar-independent loop, which decreases downstream NF-kappaB activation of HIV-1 long terminal repeat (LTR). S9a was superior to the first generation compound Canventol, which was superior to the natural compound sarcophytol A, demonstrating that further structure-based enhancement of potency of these compounds is feasible. This study suggests a therapeutic approach against AIDS by application of two drugs, one against a cellular and the other a viral target, which may provide an approach to the problem of frequent emergence of resistant variants to combinations of drugs that target only HIV genes. 


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Oct2 transactivation from a remote enhancer position requires a B-cell-restricted activity. 
Previous cotransfection experiments had demonstrated that ectopic expression of the lymphocyte-specific transcription factor Oct2 could efficiently activate a promoter containing an octamer motif. Oct2 expression was unable to stimulate a multimerized octamer enhancer element in HeLa cells, however. We have tested a variety of Oct2 isoforms generated by alternative splicing for the capability to activate an octamer enhancer in nonlymphoid cells and a B-cell line. Our analyses show that several Oct2 isoforms can stimulate from a remote position but that this stimulation is restricted to B cells. This result indicates the involvement of either a B-cell-specific cofactor or a specific modification of a cofactor or the Oct2 protein in Oct2-mediated enhancer activation. Mutational analyses indicate that the carboxy-terminal domain of Oct2 is critical for enhancer activation. Moreover, this domain conferred enhancing activity when fused to the Oct1 protein, which by itself was unable to stimulate from a remote position. The glutamine-rich activation domain present in the amino-terminal portion of Oct2 and the POU domain contribute only marginally to the transactivation function from a distal position. 


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Characterization of an immediate-early gene induced in adherent monocytes that encodes I kappa B-like activity. 
We have cloned a group of cDNAs representing mRNAs that are rapidly induced following adherence of human monocytes. One of the induced transcripts (MAD-3) encodes a protein of 317 amino acids with one domain containing five tandem repeats of the cdc10/ankyrin motif, which is 60% similar (46% identical) to the ankyrin repeat region of the precursor of NF-kappa B/KBF1 p50. The C-terminus has a putative protein kinase C phosphorylation site. In vitro translated MAD-3 protein was found to specifically inhibit the DNA-binding activity of the p50/p65 NF-kappa B complex but not that of the p50/p50 KBF1 factor or of other DNA-binding proteins. The MAD-3 cDNA encodes an I kappa B-like protein that is likely to be involved in regulation of transcriptional responses to NF-kappa B, including adhesion-dependent pathways of monocyte activation. 


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Inhibition of NF-kappa B activation in human T-cell lines by anetholdithiolthione. 
Nuclear factor (NF)-kappa B is a redox sensitive cytosolic transcription factor. Redox regulation of NF-kappa B has been implicated in the activation of the human immuno-deficiency virus (HIV). Therefore, inhibition of NF-kappa B activation may be an effective strategy for acquired immunodeficiency syndrome therapy. Anetholdithiolthione (ADT, 5-[p-methoxyphenyl]-3H-1,2-dithiol-3-thione) is an antioxidant which has been used to protect against acetaminophen- and CCl4-induced hepatotoxicity, lipid peroxidation, radiation injury, and also has been used clinically as an anti-choleretic agent. The present study examined the effect of ADT pretreatment on NF-kappa B activation in response to a variety of stimuli such as H2O2, phorbol myristate acetate (PMA) or tumor necrosis factor alpha (TNF alpha). PMA and TNF alpha induced activation of (NF)-kappa B in human Jurkat T-cells was partially inhibited by ADT (0.1 mM) pretreatment. ADT (0.1 mM) also inhibited H2O2 induced activation of the transcription factor in the peroxide sensitive human Wurzburg T-cells. Furthermore, ADT treated Wurzburg cells had significantly higher glutathione levels as compared with untreated cells. H2O2 induced lipid peroxidation in Wurzburg cells was remarkably inhibited by ADT pretreatment. ADT, a pro-glutathione antioxidant, was observed to be capable of modulating NF-kappa B activation. 


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A lymphoid cell-specific nuclear factor containing c-Rel-like proteins preferentially interacts with interleukin-6 kappa B-related motifs whose activities are repressed in lymphoid cells. 
The proto-oncoprotein c-Rel is a member of the nuclear factor kappa B transcription factor family, which includes the p50 and p65 subunits of nuclear factor kappa B. We show here that c-Rel binds to kappa B sites as homodimers as well as heterodimers with p50. These homodimers and heterodimers show distinct DNA-binding specificities and affinities for various kappa B motifs. In particular, the c-Rel homodimer has a high affinity for interleukin-6 (IL-6) and beta interferon kappa B sites. In spite of its association with p50 in vitro, however, we found a lymphoid cell-specific nuclear factor in vivo that contains c-Rel but not p50 epitopes; this factor, termed IL-6 kappa B binding factor II, appears to contain the c-Rel homodimer and preferentially recognizes several IL-6 kappa B-related kappa B motifs. Although it has been previously shown that the IL-6 kappa B motif functions as a potent IL-1/tumor necrosis factor-responsive element in nonlymphoid cells, its activity was found to be repressed in lymphoid cells such as a Jurkat T-cell line. We also present evidence that IL-6 kappa B binding factor II functions as a repressor specific for IL-6 kappa B-related kappa B motifs in lymphoid cells. 


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The hematopoietic transcription factor PU.1 is downregulated in human multiple myeloma cell lines. 
PU.1 is a hematopoietic transcription factor belonging to the Ets-family. It is identical to the Spi-1 oncogene, which is implicated in spleen focus-forming virus-induced murine erythroleukemias. PU.1 seems to be required for early development of multiple hematopoietic lineages, but its expression in mature cells is preferentially observed in cells of the B-cell-and monocyte/macrophage-differentiation lineage. It binds the so-called Pu box, an important tissue-specific regulatory DNA element present in a number of genes expressed in these cell lineages. We have analyzed the expression and activity of PU.1 during human B-cell development using a panel of B-cell lines representing different stages of maturation, from early precursors to differentiated plasma cells. PU.1 mRNA expression and PU.1 DNA binding activity, as measured by Northern blot analysis and electrophoretic mobility shift assay, respectively, were evident in cell lines representing pro-B, pre-B, and mature B cells. We could also show Pu box-dependent transactivation of a reporter gene in transient transfections in these cell lines. In contrast, in a number of multiple myeloma cell lines, representing differentiated, plasma cell-like B cells, PU.1 DNA binding activity, mRNA expression, and Pu box-dependent transactivation were absent or detectable at a very low level. In lymphoblastoid cell lines, which exemplify an intermediate stage of B-cell differentiation, a reduced expression and activity were observed. The findings in the human multiple myeloma cell lines represent the first examples of B cells with downregulated PU.1 expression and apparently contradict observations in the murine system in which PU.1 is expressed and active in plasmacytoma cell lines. At present, it is unclear whether the lack of PU.1 expression and activity in human multiple myeloma cell lines represents a malignancy-associated defect in these cells or exemplifies a normal developmental regulation in terminally differentiated B cells. 


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Expression of v-src in T cells correlates with nuclear expression of NF-kappa B. 
NF-kappa B is a rapidly inducible transcriptional activator that responds to a variety of signals and influences the expression of many genes involved in the immune response. Protein tyrosine kinases transmit signals from cytokine and immune receptors. Very little information exists linking these two important classes of signaling molecules. We now demonstrate that v-src expression correlates with nuclear expression of a kappa B binding complex similar to that induced by phorbol ester and ionomycin, as detected by electrophoretic mobility shift assay using a variety of kappa B sites. This complex was blocked by the tyrosine kinase inhibitor, herbimycin A. The v-src-induced complex comprised the p50 and p65 components of NF-kappa B, as determined by supershift and immunoblot analysis. As a functional correlate of this finding, transient co-transfection of HIV-1 LTR reporter constructs in a different T cell line demonstrated that v-src activated this promoter in a kappa B-dependent manner. We found that transactivation of the HIV-1 LTR by v-src was more sensitive to mutations of the proximal, rather than the distal, kappa B element. The implications for T cell receptor signaling and HIV-1 gene expression are considered. 


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Comparison of constitutive and inducible transcriptional enhancement mediated by kappa B-related sequences: modulation of activity in B cells by human T-cell leukemia virus type I tax gene. 
The kappa B sequence (GGGACTTTCC) binds a factor, NF-kappa B, that is constitutively found in its functional, DNA binding form only in B lymphocytes. A factor with apparently indistinguishable sequence specificity can be induced in many other cell types, where it is used to regulate inducible gene expression. For example, kappa B-related sequences have been shown to be important for the transcription of a few inducible genes, such as the interleukin 2 receptor alpha-chain gene and the beta-interferon gene. However, these genes are not constitutively active in B lymphocytes, suggesting that other regulatory mechanisms must play a role in determining the patterns of expression. We have investigated the constitutive and inducible transcriptional activity mediated by five kappa B-related sequence elements in two different cell types. We show that in S194 plasma cells the activity of each element correlates well with the relative affinity of B-cell-derived NF-kappa B for that element. This leads to significantly lower transcription enhancement by sites derived from the interleukin 2 receptor or T-cell receptor genes in S194 cells. However, in either EL-4 (T) cells or S194 cells, both lower-affinity sites can be significantly induced by the tax gene product of human T-cell leukemia virus type I, showing that NF-kappa B activity can be modulated even in a B-cell line that constitutively expresses this factor. 


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Cell-specific differences in activation of NF-kappa B regulatory elements of human immunodeficiency virus and beta interferon promoters by tumor necrosis factor. 
Three aspects of the involvement of tumor necrosis factor in human immunodeficiency virus (HIV) pathogenesis were examined. Tumor necrosis factor alpha (TNF-alpha) mRNA production was analyzed by polymerase chain reaction amplification in monocytic U937 cells and in a chronically HIV infected U937 cell line (U9-IIIB). TNF-alpha RNA was undetectable in U937 cells, whereas a low constitutive level was detected in U9-IIIB cells. Paramyxovirus infection induced a 5- to 10-fold increase in the steady-state level of TNF-alpha RNA in U9-IIIB cells compared with U937 cells, suggesting that HIV-infected monocytic cells produced higher levels of TNF-alpha than did normal cells after a secondary virus infection. The effects of TNF-alpha on gene expression were examined by transient expression assays using reporter chloramphenicol acetyltransferase plasmids linked to regulatory elements from the HIV long terminal repeat (LTR) and the beta interferon promoter. In U937 and Jurkat T lymphoid cells, the inducibility of the different hybrid promoters by TNF-alpha or phorbol ester varied in a cell type- and promoter context-specific manner; the levels of gene activity of NF-kappa B-containing plasmids correlated directly with induction of NF-kappa B DNA-binding activity. Although the intact beta interferon promoter was only weakly stimulated by phorbol ester or TNF-alpha, multimers of the PRDII NF-kappa B-binding domain were inducible by both agents. TNF-alpha was able to increase expression of the HIV LTR in T cells, but in monocytic cells, TNF-alpha did not induce the HIV LTR above a constitutive level of activity. This level of NF-kappa B-independent activity appears to be sufficient for virus multiplication, since TNF-alpha treatment had no effect on the kinetics of de novo HIV type 1 (HIV-1) infection and viral RNA production in U937 cells. However, in Jurkat cells, TNF-alpha dramatically enhanced the spread of HIV-1 through the cell population and increased viral RNA synthesis, indicating that in T cells HIV-1 multiplication was stimulated by TNF-alpha treatment. 


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Polymorphic nucleotides within the human IL-4 promoter that mediate overexpression of the gene. 
Atopy, which predisposes individuals to develop asthma, severe systemic anaphylaxis, and atopic dermatitis, is usually associated with dramatically elevated total serum IgE levels and is thought to be controlled by a major susceptibility gene and multiple minor susceptibility genes. A recent sib-pair analysis revealed a tight linkage between markers on 5q31.1 and a major susceptibility gene controlling total serum IgE levels. Due to its location within this cluster and its biologic role in Ig class switching and Th2 cell differentiation, the IL-4 gene has emerged as one major candidate for the atopy gene. In one model, polymorphisms within IL-4 regulatory elements might result in overexpression of the gene, amplifying Th2 cell differentiation and class switching to IgE. In support of this model, we report that the human IL-4 promoter exists in multiple allelic forms that exhibit distinct transcriptional activities in IL-4-positive T cells. A particular allele has an unusually high transcriptional activity. A nucleotide substitution within a recently described OAP40 element located just upstream of an NF-AT site (P sequence) appears to be largely responsible for the increased promotor strength of this particular allelic form of the IL-4 promoter. In EMSAs, this substitution results in a markedly enhanced affinity for sequence-specific complexes exhibiting an AP-1 specificity. The identification of allelic nucleotides, which results in overexpression of the IL-4 gene, provides specific targets for a comprehensive screening of atopic and nonatopic individuals and may provide a clue for genetic predisposition for atopy. 


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Comparative analysis of NFAT (nuclear factor of activated T cells) complex in human T and B lymphocytes. 
Nuclear factor of activated T cells (NFAT) is a transcriptional activator that binds to sequences in the interleukin-2 (IL-2) promoter and is thought to be largely responsible for the T cell-specific inducibility of IL-2 expression. Electrophoretic mobility shift assays (EMSA) showed that specific NFAT binding activity could also be induced in human B cells. The B cell NFAT complex, however, was not functional, since it failed to activate transcription from an NFAT-driven chloramphenicol acetyltransferase (CAT) construct. Competition with an AP-1 motif or with anti-Jun and anti-Fos antibodies abolished binding to the NFAT motif in both T and B cells, indicating that Jun and Fos are critical for NFAT complex formation in both cell types. Purified recombinant Jun and Fos proteins failed to bind directly to the NFAT motif. However, when combined with unstimulated B or T cell extracts, full-length, but not truncated, Jun/Fos heterodimers were able to form an NFAT complex, indicating the presence of a constitutively expressed nuclear factor(s) in B and T cells necessary for the formation of the NFAT complex in both cell types. An NFAT oligonucleotide carrying mutations in the 5' purine-rich part of the NFAT sequence failed to form a complex and to compete with the wild type motif for NFAT complex formation in both T and B cells. We therefore propose a model whereby a core NFAT complex consisting of Jun, Fos, and a constitutive nuclear factor is formed in both T and B cells, but an additional factor and/or post-translational modification of a factor, missing in B cells, might be required for transactivation by NFAT. 


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Distinctive gene expression patterns in human mammary epithelial cells and breast cancers. 
cDNA microarrays and a clustering algorithm were used to identify patterns of gene expression in human mammary epithelial cells growing in culture and in primary human breast tumors. Clusters of coexpressed genes identified through manipulations of mammary epithelial cells in vitro also showed consistent patterns of variation in expression among breast tumor samples. By using immunohistochemistry with antibodies against proteins encoded by a particular gene in a cluster, the identity of the cell type within the tumor specimen that contributed the observed gene expression pattern could be determined. Clusters of genes with coherent expression patterns in cultured cells and in the breast tumors samples could be related to specific features of biological variation among the samples. Two such clusters were found to have patterns that correlated with variation in cell proliferation rates and with activation of the IFN-regulated signal transduction pathway, respectively. Clusters of genes expressed by stromal cells and lymphocytes in the breast tumors also were identified in this analysis. These results support the feasibility and usefulness of this systematic approach to studying variation in gene expression patterns in human cancers as a means to dissect and classify solid tumors. 


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Association between expression of intercellular adhesion molecule-1 and integration of human T-cell-leukemia virus type 1 in adult T-cell leukemia cells. 
It is known that the expression levels of intercellular adhesion molecule-1 (ICAM-1) in adult T cell leukemia(ATL) cells are high, whereas those in T-lymphoid cells are not. In order to investigate the factors that influence the induction of ICAM-1 molecules, Northern blot analysis to measure the expression level of ICAM-1 mRNAs and Southern blot hybridization to analyze the integration of human T-cell-leukemia virus type 1 (HTLV-1) provirus were done. The levels of ICAM-1 mRNA expression of ATL cells were generally higher than those of T-lymphoid cells. However, ILT-mat cells and ATL16T(-) cells, although they were ATL cells, showed rather low surface ICAM-1 expression and ICAM-1 mRNA expression. Southern blot hybridization showed that only two and four bands were found in ILT-mat and ATL16T(-) cells, respectively, whereas > 10 bands were detected in other ATL cells. These results suggest that monoclonal integration of HTLV-1 provirus to the genome of T cell, especially the number of integration sites, is one of the factors for induction of ICAM-1 molecules. 


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Induction of nuclear factor kappa B/Rel nuclear activity in human peripheral blood T lymphocytes by anti-HLA class I monoclonal antibodies. 
Monoclonal antibodies against either monomorphic or polymorphic determinants of class I antigen induced in PBMC and highly purified T lymphocytes the nuclear activity of NF-kappa B/Rel complexes. These included both p50/p50 and p50/p65 dimers, recognized by specific antibodies in EMSA. The induced complexes were detectable in extracts of cells incubated with anti-class I monoclonal antibody (mAb) for 1.5 h; the induction was maximal at 5 h, persistent at 16 h and no longer observed at 40 h. The mAb failed to induce NF-kappa B/Rel nuclear activity in cells incubated in the presence of 3,4-dichloroisocoumarin, an inhibitor of I kappa B-alpha degradation. Together, these results suggest that class I triggering can induce the activity of NF-kappa B/Rel nuclear activity in peripheral blood T lymphocytes, thereby modulating the expression of genes regulated by these transcription factors. 


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Regulation of gene expression at early stages of B-cell and T-cell differentiation. 
The expression of distinct sets of genes at different stages of B-lymphocyte and T-lymphocyte differentiation is controlled at the level of transcription. A number of recent studies have described interactions between transcription factors in lymphocytes that provide new insights into mechanisms regulating gene expression. These mechanisms include the assembly of higher order nucleoprotein complexes and other protein-protein interactions that enhance the functional specificity of transcriptional regulators in lymphocytes. 


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Oleic acid inhibits endothelial activation : A direct vascular antiatherogenic mechanism of a nutritional component in the mediterranean diet. 
Because oleic acid is implicated in the antiatherogenic effects attributed to the Mediterranean diet, we investigated whether this fatty acid can modulate endothelial activation, ie, the concerted expression of gene products involved in leukocyte recruitment and early atherogenesis. We incubated sodium oleate with human umbilical vein endothelial cells for 0 to 72 hours, followed by coincubation of oleate with human recombinant tumor necrosis factor, interleukin (IL)-1alpha, IL-1beta, IL-4, Escherichia coli lipopolysaccharide (LPS), or phorbol 12-myristate 13-acetate for a further 6 to 24 hours. The endothelial expression of vascular cell adhesion molecule-1 (VCAM-1), E-selectin, and intercellular adhesion molecule-1 was monitored by cell surface enzyme immunoassays or flow cytometry, and steady-state levels of VCAM-1 mRNA were assessed by Northern blot analysis. At 10 to 100 micromol/L for >24 hours, oleate inhibited the expression of all adhesion molecules tested. After a 72-hour incubation with oleate and a further 16-hour incubation with oleate plus 1 microg/mL LPS, VCAM-1 expression was reduced by >40% compared with control. Adhesion of monocytoid U937 cells to LPS-treated endothelial cells was reduced concomitantly. Oleate also produced a quantitatively similar reduction of VCAM-1 mRNA levels on Northern blot analysis and inhibited nuclear factor-kappaB activation on electrophoretic mobility shift assays. Incubation of endothelial cells with oleate for 72 hours decreased the relative proportions of saturated (palmitic and stearic) acids in total cell lipids and increased the proportions of oleate in total cell lipids without significantly changing the relative proportions of polyunsaturated fatty acids. Although less potent than polyunsaturated fatty acids in inhibiting endothelial activation, oleic acid may contribute to the prevention of atherogenesis through selective displacement of saturated fatty acids in cell membrane phospholipids and a consequent modulation of gene expression for molecules involved in monocyte recruitment. 


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Activation of a novel gene in 3q21 and identification of intergenic fusion transcripts with ecotropic viral insertion site I in leukemia. 
We have identified a novel gene, GR6, located within the leukemia breakpoint region of 3q21, that is normally expressed in early fetal development but not in adult peripheral blood. GR6 is activated in the UCSD-AML1 cell line and in a leukemic sample, both of which carry a t(3;3)(q21;q26). In UCSD-AML1, we have also identified fusion transcripts between the ecotropic viral insertion site I (EVI1) gene in 3q26 and GR6 and between EVI1 and Ribophorin I that maps 30 kb telomeric to GR6 in 3q21. All fusions splice the 5' ends of the 3q21 genes into exon 2 of the EVI1 gene, an event that is similar to the normal intergenic splicing of MDS1-EVI1 and to those previously documented in leukemias with t(3;21) and t(3;12), in which acute myelogenous leukemia 1-EVI1 fusions and ETV6-EVI1 fusions, respectively, occur. The Ribophorin I-EVI1 fusion in particular may be a common occurrence in t(3;3). 


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Human immunodeficiency virus type 1 long terminal repeat quasispecies differ in basal transcription and nuclear factor recruitment in human glial cells and lymphocytes. 
The generation of genomic diversity during the course of infection has the potential to affect all aspects of HIV-1 replication, including expression of the proviral genome. To gain a better understanding of the impact of long terminal repeat (LTR) sequence diversity on LTR-directed gene expression in cells of the central nervous system (CNS) and immune system, we amplified and cloned LTRs from proviral DNA in HIV-1-infected peripheral blood. Sequence analysis of nineteen LTRs cloned from 2 adult and 3 pediatric patients revealed an average of 33 nucleotide changes (with respect to the sequence of the LAI LTR) within the 455-bp U3 region. Transient expression analyses in cells of neuroglial and lymphocytic origin demonstrated that some of these LTRs had activities which varied significantly from the LAI LTR in U-373 MG cells (an astrocytoma cell line) as well as in Jurkat cells (a CD4-positive lymphocyte cell line). While LTRs which demonstrated the highest activities in U-373 MG cells also yielded high activities in Jurkat cells, the LTRs were generally more active in Jurkat cells when compared to the LAI LTR. Differences in LTR sequence also resulted in differences in transcription factor recruitment to cis-acting sites within the U3 region of the LTR, as demonstrated by electrophoretic mobility shift assays. In particular, naturally occurring sequence variation impacted transcription factor binding to an activating transcription factor/cAMP response element binding (ATF/CREB) binding site (located between the LEF-1 and distal NF-kappaB transcription factor binding sites) that we identified in previous studies of the HIV-1 LTR. These findings suggest that LTR sequence changes can significantly affect basal LTR function and transcription factor recruitment, which may, in turn, alter the course of viral replication in cells of CNS and immune system origin. 


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Inhibition of NF-kappa B by sodium salicylate and aspirin [see comments] 
The transcription factor nuclear factor-kappa B (NF-kappa B) is critical for the inducible expression of multiple cellular and viral genes involved in inflammation and infection including interleukin-1 (IL-1), IL-6, and adhesion molecules. The anti-inflammatory drugs sodium salicylate and aspirin inhibited the activation of NF-kappa B, which further explains the mechanism of action of these drugs. This inhibition prevented the degradation of the NF-kappa B inhibitor, I kappa B, and therefore NF-kappa B was retained in the cytosol. Sodium salicylate and aspirin also inhibited NF-kappa B-dependent transcription from the Ig kappa enhancer and the human immunodeficiency virus (HIV) long terminal repeat (LTR) in transfected T cells. 


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Costimulation of peripheral blood T cell activation by human endothelial cells. Enhanced IL-2 transcription correlates with increased c-fos synthesis and increased Fos content of AP-1. 
Endothelial cells (EC) act as APC for resting PBL in vitro, and may have important roles in vivo in the pathogenesis of allograft rejection and delayed hypersensitivity. We previously reported that human umbilical vein EC provide costimulatory signals to PHA-stimulated PBL via CD2:lymphocyte function-associated Ag-3 and an unidentified ligand pair, resulting in a three- to eight-fold enhancement of IL-2 production. The physiologic relevance of this increase was demonstrated by the proliferative advantage provided by EC to PBL suboptimally stimulated with mAb OKT3. We now report that EC costimulation causes increased levels of IL-2 mRNA as a result of increased IL-2 transcription in PBL. We therefore examined the effects of EC on T cell nuclear factors known to regulate IL-2 transcription, including c-jun and c-fos-two components of the transcription factor AP-1, NFAT, and others. PBL constitutively express c-jun transcripts, and the level of c-jun mRNA is not altered by PHA activation in the absence or presence of EC. In contrast, c-fos mRNA is absent from resting T cells and is induced on PHA activation. EC alone do not induce c-fos mRNA but augment the level of c-fos mRNA in PHA-activated T cells by 3- to 10-fold. This effect is largely independent of the CD2:lymphocyte function-associated Ag-3 pathway. Gel-shift analysis reveals the constitutive presence of nuclear factors in resting PBL that bind to the proximal AP-1 site of the IL-2 promoter and that contain immunoreactive c-Jun but not c-Fos protein. In contrast, AP-1 from PHA-activated cells contains c-Jun and low levels of c-Fos. Strikingly, costimulation with EC results in a dramatic increase (up to 15-fold) in the c-Fos content of AP-1. Levels of other nuclear factors involved in IL-2 regulation were not altered by EC, although NFAT-DNA complexes migrated at a slightly different mobility. In summary, our data suggest that changes in the composition of transcription factor AP-1 is a key molecular mechanism for increasing IL-2 transcription and may underlie the phenomenon of costimulation by EC. 


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Inhibition of human immunodeficiency virus type 1 replication by a Tat-activated, transduced interferon gene: targeted expression to human immunodeficiency virus type 1-infected cells. 
We have examined the feasibility of using interferon (IFN) gene transfer as a novel approach to anti-human immunodeficiency virus type 1 (HIV-1) therapy in this study. To limit expression of a transduced HIV-1 long terminal repeat (LTR)-IFNA2 (the new approved nomenclature for IFN genes is used throughout this article) hybrid gene to the HIV-1-infected cells, HIV-1 LTR was modified. Deletion of the NF-kappa B elements of the HIV-1 LTR significantly inhibited Tat-mediated transactivation in T-cell lines, as well as in a monocyte line, U937. Replacement of the NF-kappa B elements in the HIV-1 LTR by a DNA fragment derived from the 5'-flanking region of IFN-stimulated gene 15 (ISG15), containing the IFN-stimulated response element, partially restored Tat-mediated activation of LTR in T cells as well as in monocytes. Insertion of this chimeric promoter (ISG15 LTR) upstream of the human IFNA2 gene directed high levels of IFN synthesis in Tat-expressing cells, while this promoter was not responsive to tumor necrosis factor alpha-mediated activation. ISG15-LTR-IFN hybrid gene inserted into the retrovirus vector was transduced into Jurkat and U937 cells. Selected transfected clones produced low levels of IFN A (IFNA) constitutively, and their abilities to express interleukin-2 and interleukin-2 receptor upon stimulation with phytohemagglutinin and phorbol myristate acetate were retained. Enhancement of IFNA synthesis observed upon HIV-1 infection resulted in significant inhibition of HIV-1 replication for a period of at least 30 days. Virus isolated from IFNA-producing cells was able to replicate in the U937 cells but did not replicate efficiently in U937 cells transduced with the IFNA gene. These results suggest that targeting IFN synthesis to HIV-1-infected cells is an attainable goal and that autocrine IFN synthesis results in a long-lasting and permanent suppression of HIV-1 replication. 


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Protein kinase C-zeta mediates NF-kappa B activation in human immunodeficiency virus-infected monocytes. 
The molecular mechanisms regulating human immunodeficiency virus (HIV) persistence in a major cell reservoir such as the macrophage remain unknown. NF-kappa B is a transcription factor involved in the regulation of the HIV long terminal repeat and is selectively activated following HIV infection of human macrophages. Although little information as to what signal transduction pathways mediate NF-kappa B activation in monocytes-macrophages is available, our previous work indicated that classical protein kinase C (PKC) isoenzymes were not involved in the HIV-mediated NF-kappa B activation. In this study, we have focused on atypical PKC isoenzymes. PKC-zeta belongs to this family and is known to be an important step in NF-kappa B activation in other cell systems. Immunoblotting experiments with U937 cells demonstrate that PKC-zeta is present in these cells, and its expression can be downmodulated by antisense oligonucleotides (AO). The HIV-mediated NF-kappa B activation is selectively reduced by AO to PKC-zeta. In addition, cotransfection of a negative dominant molecule of PKC-zeta (PKC-zeta mut) with NF-kappa B-dependent reporter genes selectively inhibits the HIV- but not phorbol myristate acetate- or lipopolysaccharide-mediated activation of NF-kappa B. That PKC-zeta is specific in regulating NF-kappa B is concluded from the inability of PKC-zeta(mut) to interfere with the basal or phorbol myristate acetate-inducible CREB- or AP1-dependent transcriptional activity. Lastly, we demonstrate a selective inhibition of p24 production by HIV-infected human macrophages when treated with AO to PKC-zeta. Altogether, these results suggest that atypical PKC isoenzymes, including PKC-zeta, participate in the signal transduction pathways by which HIV infection results in the activation of NF-kappa B in human monocytic cells and macrophages. 


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Regulation of I kappa B alpha and p105 in monocytes and macrophages persistently infected with human immunodeficiency virus. 
The mechanisms regulating human immunodeficiency virus (HIV) persistence in human monocytes/macrophages are partially understood. Persistent HIV infection of U937 monocytic cells results in NF-kappa B activation. Whether virus-induced NF-kappa B activation is a mechanism that favors continuous viral replication in macrophages remains unknown. To further delineate the molecular mechanisms involved in the activation of NF-kappa B in HIV-infected monocytes and macrophages, we have focused on the regulation of the I kappa B molecules. First, we show that persistent HIV infection results in the activation of NF-kappa B not only in monocytic cells but also in macrophages. In HIV-infected cells, I kappa B alpha protein levels are decreased secondary to enhanced protein degradation. This parallels the increased I kappa B alpha synthesis secondary to increased I kappa B alpha gene transcription, i.e., increased RNA and transcriptional activity of its promoter-enhancer. Another protein with I kappa B function, p105, is also modified in HIV-infected cells: p105 and p50 steady-state protein levels are increased as a result of increased synthesis and proteolytic processing of p105. Transcriptional activity of p105 is also increased in infected cells and is also mediated by NF-kappa B through a specific kappa B motif. These results demonstrate the existence of a triple autoregulatory loop in monocytes and macrophages involving HIV, p105 and p50, and MAD3, with the end result of persistent NF-kappa B activation and viral persistence. Furthermore, persistent HIV infection of monocytes and macrophages provides a useful model with which to study concomitant modifications of different I kappa B molecules. 


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Characterization of 5' end of human thromboxane receptor gene. Organizational analysis and mapping of protein kinase C--responsive elements regulating expression in platelets. 
Platelet thromboxane receptors are acutely and reversibly upregulated after acute myocardial infarction. To determine if platelet thromboxane receptors are under transcriptional control, we isolated and characterized human genomic DNA clones containing the 5' flanking region of the thromboxane receptor gene. The exon-intron structure of the 5' portion of the thromboxane receptor gene was determined initially by comparing the nucleotide sequence of the 5' flanking genomic clone with that of a novel human uterine thromboxane receptor cDNA that extended the mRNA 141 bp further upstream than the previously identified human placental cDNA. A major transcription initiation site was located in three human tissues approximately 560 bp upstream from the translation initiation codon and 380 bp upstream from any previously identified transcription initiation site. The thromboxane receptor gene has neither a TATA nor a CAAT consensus site. Promoter function of the 5' flanking region of the thromboxane receptor gene was evaluated by transfection of thromboxane receptor gene promoter/chloramphenicol acetyltransferase (CAT) chimera plasmids into platelet-like K562 cells. Thromboxane receptor promoter activity, as assessed by CAT expression, was relatively weak but was significantly enhanced by phorbol ester treatment. Functional analysis of 5' deletion constructs in transfected K562 cells and gel mobility shift localized the major phorbol ester-responsive motifs in the thromboxane receptor gene promoter to a cluster of activator protein-2 (AP-2) binding consensus sites located approximately 1.8 kb 5' from the transcription initiation site. These studies are the first to determine the structure and organization of the 5' end of the thromboxane receptor gene and demonstrate that thromboxane receptor gene expression can be regulated by activation of protein kinase C via induction of an AP-2-like nuclear factor binding to upstream promoter elements. These findings strongly suggest that the mechanism for previously described upregulation of platelet thromboxane receptors after acute myocardial infarction is increased thromboxane receptor gene transcription in platelet-progenitor cells. 


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The effect of Toremifene on the expression of some genes in human mononuclear cells. 
Toremifene exerts multiple and varied effects on the gene expression of human peripheral mononuclear cells. After short-term, in vitro exposure to therapeutical levels, distinct changes in P-glycoprotein, steroid receptors, p53 and Bcl-2 expression take place. In view of the increasing use of antiestrogens in cancer therapy and prevention, there is obvious merit in long-term in vivo studies to be conducted. 


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Role of HIV-1 Nef expression in activation pathways in CD4+ T cells. 
The role of the human immunodeficiency virus (HIV-1) Nef protein in T cell activation pathways was investigated using a Jurkat CD4+ cell line stably transfected with a Nef expression vector. Secretion of IL-2 and TNF-alpha, surface expression of IL-2R, and DNA-binding activity of NF-kappa B and AP-1 (Fos/Jun) complex in response to phorbol myristate acetate, TNF-alpha, or immobilized antibodies to CD3 were monitored. These parameters were not modified by Nef expression in Jurkat cells, whereas stimulation with the same stimuli resulted in partial inhibition of LTR activation in Nef+ Jurkat cells. This inhibition was not mediated through Nef phosphorylation on Thr-15 or GTP-binding activity because mutations in critical sites did not alter this inhibition. Analysis of truncated LTRs confirmed that inhibition of LTR activation was not mediated through NF-kappa B-binding activity but through the region containing the negative responding elements (NREs). These results suggest that Nef downmodulates LTR activation without significantly inhibiting the capacity of T cells to respond to immunological activations. 


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Transcriptional activation of the vascular cell adhesion molecule-1 gene in T lymphocytes expressing human T-cell leukemia virus type 1 Tax protein. 
Recruitment and extravasation of T cells through the blood-brain barrier are favored by adhesion molecule-mediated interactions of circulating T cells with endothelial cells. Since a common pathological finding in human T-cell leukemia virus type 1 (HTLV-1)-associated diseases is the infiltration of HTLV-1-infected T lymphocytes into various organs, we have looked for the profile of adhesion molecules expressed by HTLV-1-transformed T cells. Flow cytometry analysis indicated that these cells were expressing high levels of vascular cell adhesion molecule 1 (VCAM-1 [CD106]), a 110-kDa member of the immunoglobulin gene superfamily, first identified on endothelial cells stimulated with inflammatory cytokines. This adhesion molecule was also expressed by T cells obtained from one patient with HTLV-1-associated myelopathy/tropical spastic paraparesis but not by activated T cells isolated from one normal blood donor. The role of the viral trans-activator Tax protein in the induction of VCAM-1 was first indicated by the detection of this adhesion molecule on Jurkat T-cell clones stably expressing the tax gene. The effect of Tax on VCAM-1 gene transcription was next confirmed in JPX-9 cells, a subclone of Jurkat cells, carrying the tax sequences under the control of an inducible promoter. Furthermore, deletion and mutation analyses of the VCAM-1 promoter performed with chloramphenicol acetyltransferase constructs revealed that Tax was trans activating the VCAM-1 promoter via two NF-kappaB sites present at bp -72 and -57 in the VCAM-1 gene promoter, with both of them being required for the Tax-induced expression of this adhesion molecule. Finally, gel mobility shift assays demonstrated the nuclear translocation of proteins specifically bound to these two NF-kappaB motifs, confirming that VCAM-1 was induced on Tax-expressing cells in a kappaB-dependent manner. Collectively, these results therefore suggest that the exclusive Tax-induced expression of VCAM-1 on T cells may represent a pivotal event in the progression of HTLV-1-associated diseases. 


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Rel/NF-kappa B transcription factors and the control of apoptosis. 
The process of apoptosis is used to eliminate unwanted cells from a wide variety of organisms. Various extracellular signals, often converging in common intracellular pathways, can induce apoptosis in a cell-type-specific fashion. Recent work from several laboratories has demonstrated that Rel/NF-kappa B transcription factors regulate apoptosis in many cell types. In most cells, Rel/NF-kappa B transcription factors appear to mediate survival signals that protect cells from apoptosis; however, under some circumstances, activation of these factors may also promote apoptosis. 


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Reactive oxygen species and antioxidants in inflammatory diseases. 
This paper aims to review the role of free radical-induced tissue damage and antioxidant defence mechanisms in inflammatory diseases that involve pathogenic processes similar to the periodontal diseases. There is a clearly defined and substantial role for free radicals or reactive oxygen species (ROS) in periodontitis, but little research has been performed in this area. This paper reviews the considerable data available relating ROS activity and antioxidant defence to inflammatory diseases and attempts to draw parallels with periodontitis, in an effort to stimulate more periodontal research in this important area. The recent discovery of the transcription factor nuclear factor kappa B (NF-kappa B) is reviewed and several potential pathways for cytokine-induced periodontal tissue damage, mediated by NF-kappa B1 are discussed. Emphasis is placed on cytokines that have been studied in periodontitis, principally TNF-alpha, IL-1, IL-6, IL-8 and beta-interferon. The link between cellular production of such important mediators of inflammation and the antioxidant (AO) thiols, cysteine and reduced glutathione (GSH), is discussed and it is hypothesised that NF-kappa B antagonists may offer important therapeutic benefits. 


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A novel mitogen-inducible gene product related to p50/p105-NF-kappa B participates in transactivation through a kappa B site. 
A Rel-related, mitogen-inducible, kappa B-binding protein has been cloned as an immediate-early activation gene of human peripheral blood T cells. The cDNA has an open reading frame of 900 amino acids capable of encoding a 97-kDa protein. This protein is most similar to the 105-kDa precursor polypeptide of p50-NF-kappa B. Like the 105-kDa precursor, it contains an amino-terminal Rel-related domain of about 300 amino acids and a carboxy-terminal domain containing six full cell cycle or ankyrin repeats. In vitro-translated proteins, truncated downstream of the Rel domain and excluding the repeats, bind kappa B sites. We refer to the kappa B-binding, truncated protein as p50B by analogy with p50-NF-kappa B and to the full-length protein as p97. p50B is able to form heteromeric kappa B-binding complexes with RelB, as well as with p65 and p50, the two subunits of NF-kappa B. Transient-transfection experiments in embryonal carcinoma cells demonstrate a functional cooperation between p50B and RelB or p65 in transactivation of a reporter plasmid dependent on a kappa B site. The data imply the existence of a complex family of NF-kappa B-like transcription factors. 


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MEK1 and the extracellular signal-regulated kinases are required for the stimulation of IL-2 gene transcription in T cells. 
TCR engagement stimulates the activation of the protein kinase Raf-1. Active Raf-1 phosphorylates and activates the mitogen-activated protein (MAP) kinase/extracellular signal-regulated kinase kinase 1 (MEK1), which in turn phosphorylates and activates the MAP kinases/extracellular signal regulated kinases, ERK1 and ERK2. Raf-1 activity promotes IL-2 production in activated T lymphocytes. Therefore, we sought to determine whether MEK1 and ERK activities also stimulate IL-2 gene transcription. Expression of constitutively active Raf-1 or MEK1 in Jurkat T cells enhanced the stimulation of IL-2 promoter-driven transcription stimulated by a calcium ionophore and PMA, and together with a calcium ionophore the expression of each protein was sufficient to stimulate NF-AT activity. Expression of MEK1-interfering mutants inhibited the stimulation of IL-2 promoter-driven transcription and blocked the ability of constitutively active Ras and Raf-1 to costimulate NF-AT activity with a calcium ionophore. Expression of the MAP kinase-specific phosphatase, MKP-1, which blocks ERK activation, inhibited IL-2 promoter and NF-AT-driven transcription stimulated by a calcium ionophore and PMA, and in addition, MKP-1 neutralized the transcriptional enhancement caused by active Raf-1 and MEK1 expression. We conclude that the MAP kinase signal transduction pathway consisting of Raf-1, MEK1, and ERK1 and ERK2 functions in the stimulation IL-2 gene transcription in activated T lymphocytes. 


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Tpl-2 induces IL-2 expression in T-cell lines by triggering multiple signaling pathways that activate NFAT and NF-kappaB. 
The Tpl-2 kinase activates the nuclear factor of activated T cells (NFAT) and induces IL-2 expression in T-cell lines. Here we show that the activation of the IL-2 promoter by Tpl-2 is inhibited by mutant signaling molecules that inhibit the mitogen-activated protein kinase (MAPK) or the calcineurin/NFAT pathways and is promoted by combinations of signaling molecules that activate these pathways. We, therefore, conclude that signals generated by the convergence of the MAPK and the calcineurin/NFAT pathway are necessary and sufficient for the activation of the IL-2 promoter by Tpl-2. The activation of both the IL-2 promoter and an NFAT-driven minimal promoter were shown to depend on signals transduced by Raf1. However, it was only the IL-2 promoter whose activation by Tpl-2 was fully blocked by the dominant negative mutant MEK1S218/222A and the MEK1/MEK2 inhibitor PD098059. Since the activation of NFAT is MAPK-dependent these findings suggested that the activation of MAPK by Tpl-2 is either independent or only partially dependent on MEK1 and MEK2. In addition, they suggested that the activation of the IL-2 promoter is under the control of not only NFAT but also a second factor whose activation is MEK-dependent. Experiments in COS-1 and EL-4 cells confirmed both hypotheses and revealed that the second factor activated by Tpl-2 is NF-kappaB. While the activation of the IL-2 promoter and an NFAT-driven minimal promoter by Tpl-2 was fully blocked by the dominant negative mutant NFAT delta418, it was only partially blocked by the calcineurin inhibitor cyclosporin A suggesting that the Tpl-2-mediated NFAT activation is under the control of a combination of calcineurin-dependent and independent pathways. Both pathways were fully blocked by Bcl-2 or Bcl-X(L). 


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Regulation of cytokine and cytokine receptor expression by glucocorticoids. 
Glucocorticoids (GCS) profoundly inhibit several aspects of T cell immunity largely through inhibition of cytokine expression at the transcriptional and posttranscriptional levels. GCS were also reported to act indirectly by inducing transforming growth factor-beta expression, which in turn blocks T cell immunity. In exerting their antiproliferative effects, GCS diffuse into target cells where they bind their cytoplasmic receptor, which in turn translocates to the nucleus where it inhibits transcription of cytokine genes through direct binding to the glucocorticoid response elements (GRE), which are located in the promoter region of cytokine genes or, alternatively, through antagonism of the action of transcription factors required for optimal transcriptional activation. In contrast to their inhibitory effects on cytokine expression, GCS up-regulate cytokine receptor expression that correlates with enhanced cytokine effects on target cells. In this review, we summarize the current state of knowledge of the mechanism of action of GCS, including the phenomenon of steroid-induced rebound, which ensues upon GCS withdrawal. 


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Selenium-mediated inhibition of transcription factor NF-kappa B and HIV-1 LTR promoter activity. 
The eukaryotic transcription factor NF-kappa B is involved in the inducible expression of various inflammatory genes as well as in HIV-1 replication. Activation of NF-kappa B is induced by prooxidants and several stimuli eliciting oxidative stress, such as cytokines, lipopolysaccharide, UV irradiation and other mediators. Various antioxidants inhibit NF-kappa B activation in response to these stimuli. In this study, we have investigated the effects of selenium, an integral component of glutathione peroxidase (GPX), on NF-kappa B activation. In selenium-deprived Jurkat and ESb-L T lymphocytes, supplementation of selenium led to a substantial increase of GPX activity. Analysis of DNA binding revealed that NF-kappa B activation in response to TNF was significantly inhibited under these conditions. Likewise, reporter gene assays using luciferase constructs driven by the HIV-1 long terminal repeat showed a dose-dependent inhibition of NF-kappa B controlled gene expression by selenium. The effects of selenium were specific for NF-kappa B, since the activity of the transcription factor AP-1 was not suppressed. These data suggest that selenium supplementation may be used to modulate the expression of NF-kappa B target genes and HIV-1. 


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Soluble factors secreted by activated T-lymphocytes modulate the transcription of the immunosuppressive cytokine TGF-beta 2 in glial cells. 
Coordination of the immune response to injury or disease in the brain is postulated to involve bi-directional discourse between the immune system and the central nervous system. This cross communication involves soluble mediators, including various growth factors, cytokines, and neuropeptides. In this report, we demonstrate that the supernatant from activated T-lymphocytes is able to induce the transcription of a potent cytokine, TGF-beta 2 in glial cells. The activating stimulus invokes signaling mechanisms distinct from known kinase or protease pathways. Activation of TGF-beta 2 transcription correlates with the loss of binding activity for an 80 kDA glial labile repressor protein, GLRP, to a responsive region within the TFG-beta 2 promoter. Although GLRP shares some characteristics with the inducible transcription factor AP-1, it appears to be distinct from known AP-1 family members. These data along with previous observations demonstrating the potent immunosuppressive activity of TGF-beta 2, support a model for a feedback mechanism between the activated T-lymphocytes and astrocytes via TGF-beta 2 to regulate the immune response. 


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Distinct DNase-I hypersensitive sites are associated with TAL-1 transcription in erythroid and T-cell lines. 
The tal-1 gene, frequently activated in human T-cell acute lymphoblastic leukemia (T-ALL), is expressed in the erythroid, megakaryocytic, and mast cell lineages during normal hematopoiesis. To gain further insight into the molecular mechanisms that control tal-1 expression, we investigated tal-1 chromatin structure in erythroid/megakaryocytic cell lines and in T-cell lines either with or without tal-1 rearrangements. Tal-1 transcription was shown to be monoallelic in Jurkat, a T-cell line that expresses tal-1 in the absence of apparent genomic alteration of the locus. Methylation studies indicated that the tal-15' GC-rich region behaves like a CpG island, hypomethylated in normal cells, and methylated de novo on transcriptionally inactive alleles in established cell lines. Five major DNase-I hypersensitive sites (HS) were mapped in the tal-1 locus. HS I, IV, and V were exclusively observed in the erythroid/megakaryocytic cell lines that express tal-1 from the promoters 1a and 1b. HS II was weak in hematopoietic cell lines, absent in Hela, and greatly enhanced in Jurkat, suggesting that this region might be implicated in the cis-activation of tal-1 promoter 1b in this cell line. HS III was weak in HEL and Jurkat, and greatly enhanced in DU528, a T-cell line that bears a t (1;14) and initiates tal-1 transcription within exon 4. These results suggest that distinct regulatory elements are associated with the use of the different tal-1 promoters. 


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The tax protein of human T-cell leukemia virus type 1 mediates the transactivation of the c-sis/platelet-derived growth factor-B promoter through interactions with the zinc finger transcription factors Sp1 and NGFI-A/Egr-1. 
Transcriptional up-regulation of the c-sis/platelet-derived growth factor-B (PDGF-B) proto-oncogene by the Tax protein of human T-cell leukemia virus type 1 has been implicated as one possible mechanism of cellular transformation by human T-cell leukemia virus type 1. In previous work, we identified an essential site in the c-sis/PDGF-B promoter, Tax-responsive element 1 (TRE1), necessary for transactivation by Tax. We also identified Sp1, Sp3, and NGFI-A/Egr-1 as the primary nuclear transcription factors binding to TRE1 which mediate Tax responsiveness. In the present work, we have investigated the mechanism(s) whereby Tax transactivates the c-sis/PDGF-B proto-oncogene. In vitro transcription assays showed that Tax was able to significantly increase the transcriptional activity of a template containing the -257 to +74 region of the c-sis/PDGF-B promoter. Electrophoretic mobility shift assay analysis showed that Tax increased the DNA binding activity of both Sp1 and NGFI-A/Egr-1 using a TRE1 probe. Analysis of Tax mutants showed that two mutants, IEXC29S and IEXL320G, were unable to significantly transactivate the c-sis/PDGF-B promoter. Finally, co-immunoprecipitation analysis revealed that Tax is able to stably bind to both Sp1 and NGFI-A/Egr-1. Interestingly, co-immunoprecipitation analysis also revealed that Tax mutant IEXC29S is unable to interact with NGFI-A/Egr-1, whereas Tax mutant IEXL320G is able to interact with NGFI-A/Egr-1. 


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Effects of CD45 on NF-kappa B. Implications for replication of HIV-1. 
Increased levels of replication of the HIV type 1 are observed after the activation of infected T cells through the TCR. However, anti-CD45 antibodies inhibit these effects in cells from infected individuals. In this study, we examined interrelationships between CD45 and HIV-1 further. We measured effects on the HIV-1 LTR in T cell lines that were stimulated with antibodies against CD45 and in those that lacked the expression of CD45 on their surfaces. First, anti-CD45 antibodies did not affect basal but decreased activated levels of expression from the HIV-1 LTR. Second, T cells, which lack CD45 and cannot signal via the TCR, supported higher levels of viral replication and gene expression. This was due to the presence of active NF-kappa B complexes in the nucleus of CD45- T cells. Additionally, infected T cells displayed lower levels of CD45 on their surfaces. Thus, CD45 plays an active role in the physiology of T cells and in the replication of HIV-1. 


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Astrocytes and glioblastoma cells express novel octamer-DNA binding proteins distinct from the ubiquitous Oct-1 and B cell type Oct-2 proteins. 
The 'octamer' sequence, ATGCAAAT or its complement ATTTGCAT, is a key element for the transcriptional regulation of immunoglobulin genes in B-lymphocytes as well as a number of housekeeping genes in all cell types. In lymphocytes, the octamer-binding protein Oct-2A and variants thereof are thought to contribute to the B-cell specific gene expression, while the ubiquitous protein Oct-1 seems to control general octamer site-dependent transcription. Various other genes, for example interleukin-1 and MHC class II genes, contain an octamer sequence in the promoter and are expressed in cells of both the immune and nervous systems. This prompted us to analyze the octamer-binding proteins in the latter cells. Using the electrophoretic mobility shift assay, at least six novel octamer binding proteins were detected in nuclear extracts of cultured mouse astrocytes. These proteins are differentially expressed in human glioblastoma and neuroblastoma cell lines. The nervous system-derived (N-Oct) proteins bound to the octamer DNA sequence in a manner which is indistinguishable from the Oct-1 and Oct-2A proteins. The relationship of the N-Oct proteins to Oct-1 and Oct-2A was analyzed by proteolytic clipping bandshift assays and by their reactivity towards antisera raised against recombinant Oct-1 and Oct-2A proteins. On the basis of these assays, all N-Oct-factors were found to be distinct from the ubiquitous Oct-1 and the lymphoid-specific Oct-2A proteins. In melanoma cells that contain the N-Oct-3 factor, a transfected lymphocyte-specific promoter was neither activated nor was it repressed upon contransfection with an Oct-2A expression vector. We therefore speculate that N-Oct-3 and other N-Oct factors have a specific role in gene expression in cells of the nervous system. 


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CD40 is a functional activation antigen and B7-independent T cell costimulatory molecule on normal human lung fibroblasts. 
CD40 is an important signaling and activation Ag found on certain bone marrow-derived cells. Recently, CD40 also has been shown to be expressed by mesenchymal cells, including human fibroblasts. Little is known about the role of CD40 in fibroblasts. The current study investigates the hypothesis that CD40 expressed on lung fibroblasts is an activation structure and mechanism for interaction with hemopoietic cells. Communication between resident tissue fibroblasts and T cells is necessary for normal wound healing, and can be pathologic, resulting in tissue fibrosis. Signaling through CD40 with soluble CD40 ligand stimulated fibroblast activation, as evidenced by mobilization of nuclear factor-kappaB and by induction of the proinflammatory and chemoattractant cytokines IL-6 and IL-8. IFN-gamma-primed lung fibroblasts costimulate T lymphocyte proliferation utilizing CD40, but not the well-studied costimulatory molecules B7-1 and B7-2. Data reported herein support the hypothesis that cognate interactions between tissue fibroblasts and infiltrating T lymphocytes, via the CD40/CD40L pathway, augment inflammation and may promote fibrogenesis by activating both cell types. 


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A factor that regulates the class II major histocompatibility complex gene DPA is a member of a subfamily of zinc finger proteins that includes a Drosophila developmental control protein. 
A novel DNA sequence element termed the J element involved in the regulated expression of class II major histocompatibility complex genes was recently described. To study this element and its role in class II gene regulation further, a cDNA library was screened with oligonucleotide probes containing both the S element and the nearby J element of the human DPA gene. Several DNA clones were obtained by this procedure, one of which, clone 18, is reported and characterized here. It encodes a protein predicted to contain 688 amino acid residues, including 11 zinc finger motifs of the C2H2 type in the C-terminal region, that are Kruppel-like in the conservation of the H/C link sequence connecting them. The 160 N-terminal amino acids in the nonfinger region of clone 18 are highly homologous with similar regions of several other human, mouse, and Drosophila sequences, defining a subfamily of Kruppel-like zinc finger proteins termed TAB (tramtrack [ttk]-associated box) here. One of the Drosophila sequences, ttk, is a developmental control gene, while a second does not contain a zinc finger region but encodes a structure important in oocyte development. An acidic activation domain is located between the N-terminal conserved region of clone 18 and its zinc fingers. This protein appears to require both the S and J elements, which are separated by 10 bp for optimal binding. Antisense cDNA to clone 18 inhibited the expression of a reporter construct containing the DPA promoter, indicating its functional importance in the expression of this class II gene. 


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Activation of nuclear factor-kappa B by beta-amyloid peptides and interferon-gamma in murine microglia. 
An increasing body of evidence suggests that amyloid-beta (A beta) peptides and microglia are crucially involved in the pathogenesis of Alzheimer's disease. In an effort to further elucidate the biological effects of A beta towards microglia, we investigated the ability of A beta peptides to activate nuclear factor (NF)-kappa B in the N9 murine microglial cell line. Co-stimulation of microglia with suboptimal concentrations of A beta(25-35) and 100 U/ml IFN gamma resulted in the detection of a specific NF-kappa B DNA-binding activity in nuclear extracts, as determined in gel mobility shift assays. This response required at least 120 min to be evident and supershift experiments revealed that the NF-kappa B complex contains both RelA and p50. Accordingly, immunoblot experiments showed that amongst NF-kappa B/Rel proteins, RelA and p50 are mobilized to the nucleus following microglial cell stimulation with A beta(25-35) plus IFN gamma. Higher concentrations of A beta(25-35) were effective by themselves in inducing NF-kappa B activation, both in the N9 microglial cell line and in rat primary microglia, as well as in human monocytes. For purposes of comparison, microglia were also stimulated with bacterial LPS, a known NF-kappa B inducer. As expected, LPS strongly induced the formation of two NF-kappa B DNA-binding activities, one of which was identified as RelA/p50. The LPS response was also more rapid, as it was already evident by 40 min and remained sustained for up to 3 h. Collectively, these findings indicate that NF-kappa B activation might constitute one of the mechanisms underlying the inducible expression of kappa B-dependent genes in microglia stimulated by A beta peptides and IFN gamma, or by LPS. 


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Potent inhibition of HIV type 1 replication by an antiinflammatory alkaloid, cepharanthine, in chronically infected monocytic cells. 
Cepharanthine is a biscoclaurine alkaloid isolated from Stephania cepharantha Hayata and has been shown to have antiinflammatory, antiallergic, and immunomodulatory activities in vivo. As several inflammatory cytokines and oxidative stresses are involved in the pathogenesis of HIV-1 infection, we investigated the inhibitory effects of cepharanthine on tumor necrosis factor alpha (TNF-alpha)- and phorbol 12-myristate 13-acetate (PMA)-induced HIV-1 replication in chronically infected cell lines. Two chronically HIV-1-infected cell lines, U1 (monocytic) and ACH-2 (T lymphocytic), were stimulated with TNF-alpha or PMA and cultured in the presence of various concentrations of the compound. HIV-1 replication was determined by p24 antigen level. The inhibitory effects of cepharanthine on HIV-1 long terminal repeat (LTR)-driven gene expression and nuclear factor kappaB (NF-kappaB) activation were also examined. Cepharanthine dose dependently inhibited HIV-1 replication in TNF-alpha- and PMA-stimulated U1 cells but not in ACH-2 cells. Its 50% effective and cytotoxic concentrations were 0.016 and 2.2 microg/ml in PMA-stimulated U1 cells, respectively. Cepharanthine was found to suppress HIV-1 LTR-driven gene expression through the inhibition of NF-kappaB activation. These results indicate that cepharanthine is a highly potent inhibitor of HIV-1 replication in a chronically infected monocytic cell line. Since biscoclaurine alkaloids, containing cepharanthine as a major component, are widely used for the treatment of patients with various inflammatory diseases in Japan, cepharanthine should be further pursued for its chemotherapeutic potential in HIV-1-infected patients. 


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Activation of Stat 5b in erythroid progenitors correlates with the ability of ErbB to induce sustained cell proliferation. 
Self renewal of normal erythroid progenitors is induced by the receptor tyrosine kinase c-ErbB, whereas other receptors (c-Kit/Epo-R) regulate erythroid differentiation. To address possible mechanisms that could explain this selective activity of c-ErbB, we analyzed the ability of these receptors to activate the different members of the Stat transcription factor family. Ligand activation of c-ErbB induced the tyrosine phosphorylation, DNA-binding, and reporter gene transcription of Stat 5b in erythroblasts. In contrast, ligand activation of c-Kit was unable to induce any of these effects in the same cells. Activation of the erythropoietin receptor caused specific DNA-binding of Stat 5b, but failed to induce reporter gene transcription. These biochemical findings correlate perfectly with the selective ability of c-ErbB to cause sustained self renewal in erythroid progenitors. 


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Identification of human TR2 orphan receptor response element in the transcriptional initiation site of the simian virus 40 major late promoter [published erratum appears in J Biol Chem 1995 Nov 3;270(44):26721] 
A DNA response element (TR2RE-SV40) for the TR2 orphan receptor, a member of the steroid-thyroid hormone receptor superfamily, has been identified in the simian virus 40 (SV40) +55 region (nucleotide numbers 368-389, 5'-GTTAAGGTTCGTAGGTCATGGA-3'). Electrophoretic mobility shift assay, using in vitro translated TR2 orphan receptor with a molecular mass of 67 kilodaltons, showed a specific binding with high affinity (dissociation constant = 9 nM) for this DNA sequence. DNA-swap experiments using chloramphenicol acetyl-transferase assay demonstrated that androgen can suppress the transcriptional activities of SV40 early promoter via the interaction between this TR2RE-SV40 and the chimeric receptor AR/TR2/AR with the DNA-binding domain of the TR2 orphan receptor flanked by the N-terminal and androgen-binding domains of the androgen receptor. In addition, this TR2RE-SV40 can function as a repressor to suppress the transcriptional activities of both SV40 early and late promoters. Together, these data suggest the TR2RE-SV40 may represent the first identified natural DNA response element for the TR2 orphan receptor that may function as a repressor for the SV40 gene expression. 


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T cell priming enhances IL-4 gene expression by increasing nuclear factor of activated T cells. 
The repetitive activation of T cells (priming) enhances the expression of many cytokines, such as IL-4, but not others, such as IL-2. Molecular mechanisms underlying selective expression of cytokines by T cells remain poorly understood. Here we show that priming of CD4 T cells selectively enhances IL-4 expression relative to IL-2 expression by a transcriptional mechanism involving nuclear factor of activated T cells (NFAT) proteins. As detected by in vivo footprinting, priming markedly increases the activation-dependent engagement of the P0 and P1 NFAT-binding elements of the IL-4 promoter. Moreover, each proximal P element is essential for optimal IL-4 promoter activity. Activated primed CD4 T cells contain more NFAT1 and support greater NFAT-directed transcription than unprimed CD4 T cells, while activator protein 1 binding and activator protein 1-mediated transcription by both cell types is similar. Increased expression of wild-type NFAT1 substantially increases IL-4 promoter activity in unprimed CD4 T cells, suggesting NFAT1 may be limiting for IL-4 gene expression in this cell type. Furthermore, a truncated form of NFAT1 acts as a dominant-negative, reducing IL-4 promoter activity in primed CD4 T cells and confirming the importance of endogenous NFAT to increased IL-4 gene expression by effector T cells. NFAT1 appears to be the major NFAT family member responsible for the initial increased expression of IL-4 by primed CD4 T cells. 


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Host defense mechanisms triggered by microbial lipoproteins through toll-like receptors. 
The generation of cell-mediated immunity against many infectious pathogens involves the production of interleukin-12 (IL-12), a key signal of the innate immune system. Yet, for many pathogens, the molecules that induce IL-12 production by macrophages and the mechanisms by which they do so remain undefined. Here it is shown that microbial lipoproteins are potent stimulators of IL-12 production by human macrophages, and that induction is mediated by Toll-like receptors (TLRs). Several lipoproteins stimulated TLR-dependent transcription of inducible nitric oxide synthase and the production of nitric oxide, a powerful microbicidal pathway. Activation of TLRs by microbial lipoproteins may initiate innate defense mechanisms against infectious pathogens. 


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Interleukin (IL)-10 inhibits nuclear factor kappa B (NF kappa B) activation in human monocytes. IL-10 and IL-4 suppress cytokine synthesis by different mechanisms. 
Our previous studies in human monocytes have demonstrated that interleukin (IL)-10 inhibits lipopolysaccharide (LPS)-stimulated production of inflammatory cytokines, IL-1 beta, IL-6, IL-8, and tumor necrosis factor (TNF)-alpha by blocking gene transcription. Using electrophoretic mobility shift assays (EMSA), we now show that, in monocytes stimulated with LPS or TNF alpha, IL-10 inhibits nuclear stimulation of nuclear factor kappa B (NF kappa B), a transcription factor involved in the expression of inflammatory cytokine genes. Several other transcription factors including NF-IL-6, AP-1, AP-2, GR, CREB, Oct-1, and Sp-1 are not affected by IL-10. This selective inhibition by IL-10 of NF kappa B activation occurs rapidly and in a dose-dependent manner and correlates well with IL-10's cytokine synthesis inhibitory activity in terms of both kinetics and dose responsiveness. Furthermore, compounds such as tosylphenylalanyl chloromethyl ketone and pyrrolidinedithiocarbamate that are known to selectively inhibit NF kappa B activation block cytokine gene transcription in LPS-stimulated monocytes. Taken together, these results suggest that inhibition of NF kappa B activation may be an important mechanism for IL-10 suppression of cytokine gene transcription in human monocytes. IL-4, another cytokine that inhibits cytokine mRNA accumulation in monocytes, shows little inhibitory effect on LPS-induced NF kappa B activation. Further examination reveals that, unlike IL-10, IL-4 enhances mRNA degradation and does not suppress cytokine gene transcription. These data indicate that IL-10 and IL-4 inhibit cytokine production by different mechanisms. 


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Role of ascorbate in the activation of NF-kappaB by tumour necrosis factor-alpha in T-cells. 
The first product of ascorbate oxidation, the ascorbate free radical (AFR), acts in biological systems mainly as an oxidant, and through its role in the plasma membrane redox system exerts different effects on the cell. We have investigated the role of ascorbate, AFR and dehydroascorbate (DHA) in the activation of the NF-kappaB transcription factor in Jurkat T-cells stimulated by tumour necrosis factor-alpha (TNF-alpha). Here we show, by electrophoretic mobility shift assays, that ascorbate increases the binding of NF-kappaB to DNA in TNF-alpha-stimulated Jurkat cells. The ability of ascorbate to enhance cytoplasmic inhibitory IkBalpha protein degradation correlates completely with its capacity to induce NF-kappaB binding to DNA and to potentiate NF-kappaB-mediated transactivation of the HIV-1 long terminal repeat promoter in TNF-alpha-stimulated Jurkat cells but not in cells stimulated with PMA plus ionomycin. AFR behaves like ascorbate, while DHA and ascorbate phosphate do not affect TNF-alpha-mediated NF-kappaB activation. These results provide new evidence for a possible relationship between the activation of the electron-transport system at the plasma membrane by ascorbate or its free radical and redox-dependent gene transcription in T-cells. 


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Cross-linking CD40 on B cells rapidly activates nuclear factor-kappa B. 
The B cell-associated surface molecule CD40 functions to regulate B cell responses. Cross-linking CD40 on B cells can lead to homotypic cell adhesion, IL-6 production, and, in combination with cytokines, to Ig isotype switching. Tyrosine kinase activity is increased shortly after engagement of this receptor. Little is known about how the very early events induced by CD40 cross-linking link to cellular responses. In this study, we demonstrate that nuclear factor (NF)-kappa B and NF-kappa B-like transcription factors are activated after cross-linking CD40 on resting human tonsillar B cells and on B cell lines. The activation is rapid and is mediated through a tyrosine kinase-dependent pathway. The complexes detected in electrophoretic mobility shift assays contain p50, p65 (RelA), c-Rel, and most likely other components. By using transient transfection assays, we found that cross-linking CD40 supports NF-kappa B-dependent gene expression. Our results define the NF-kappa B system as an intermediate event in CD40 signaling and suggest that the CD40 pathway can influence the expression of B cell-associated genes with NF-kappa B consensus sites. 


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A negative regulatory region containing a glucocorticosteroid response element (nGRE) in the human interleukin-1beta gene. 
Interleukin-1 beta (IL-1beta) is one of the most important inflammatory mediators in human inflammatory and immunological diseases. The regulation of human IL-1beta gene expression has been studied for several years, and a few regulatory elements have been discovered in the promoter region. However, little is known about negative regulation of IL-1beta expression at the transcriptional level, which may play an important role in anti-inflammatory and immunosuppressive effects. We have identified a negative regulatory element located in the region between -685 and -395. Within this region, a 19-bp nuclear factor binding site (-570 to -552) was characterized by DNase I footprinting and electromobility shift assay. A consensus sequence for a negative glucocorticoid response element (nGRE) and a transcription activator protein-2 binding site were noted within this footprint. Functional studies showed a 2.5-fold increase in promoter activity when this 19-bp binding site was deleted in the reporter constructs IL-1beta/CAT and IL-1beta/SV40 promoter/CAT. Dexamethasone (10(-8) M) repressed chloramphenicol acetyltransferase (CAT) production by 75% in the wild-type fragment but not in a deletion mutant lacking the 19-bp site. A protein of about 150 kD that bound to this negative regulatory sequence was identified by UV cross-linking. This is the first description of a negative regulatory region responsive to glucocorticoids in a cytokine gene. 


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Differences in transcriptional enhancers of HIV-1 and HIV-2. Response to T cell activation signals. 
T cell activation results in high levels of HIV replication and is thought to be one mechanism leading to the conversion from latent to active viral infection. In HIV-1, the sequences that respond to these signaling events are found in the long terminal repeat (LTR) and comprise the transcriptional enhancer, which contains two conserved binding sites for the nuclear factor kappa B (NF kappa B). The corresponding region in the second AIDS retrovirus, HIV-2, contains a conserved and a divergent NF kappa B binding site. We demonstrate that the HIV-1 LTR responds better than the HIV-2 LTR to T cell activation signals. These qualitative differences in the response to T cell activation are reproduced not only when HIV-1 or HIV-2 enhancers are placed upstream of a heterologous promoter but also when these enhancers are switched between their respective LTR. In electrophoretic mobility shift assays, NF kappa B binds to both conserved sites in the HIV-1 transcriptional enhancer and only to the single conserved site in the HIV-2 transcriptional enhancer. Instead of NF kappa B, the activator protein 3 binds to the divergent site in HIV-2. In conclusion, HIV-1 and HIV-2 are differentially regulated by T cell activation signals, and this difference may account for the longer period of viral latency observed with HIV-2 than with HIV-1 infection. 


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Defining therapeutic targets by using adenovirus: blocking NF-kappaB inhibits both inflammatory and destructive mechanisms in rheumatoid synovium but spares anti-inflammatory mediators. 
The role of the transcription factor NF-kappaB in the pathogenesis of rheumatoid arthritis has long been a subject of controversy. We used an adenoviral technique of blocking NF-kappaB through overexpression of the inhibitory subunit IkappaBalpha, which has the advantage that it can be used in the diseased tissue itself, with >90% of the synovial macrophages, fibroblasts, and T cells infected. We found that the spontaneous production of tumor necrosis factor alpha and other pro-inflammatory cytokines is NF-kappaB-dependent in rheumatoid synovial tissue, in contrast to the main anti-inflammatory mediators, like IL-10 and -11, and the IL-1 receptor antagonist. Of even more interest, IkappaBalpha overexpression inhibited the production of matrix metalloproteinases 1 and 3 while not affecting their tissue inhibitor. Blocking NF-kappaB in the rheumatoid joint thus has a very beneficial profile, reducing both the inflammatory response and the tissue destruction. The adenoviral technique described here has widespread applicability, allowing rapid testing of the effects of blocking a potential therapeutic target in either cultures of normal cells or in the diseased tissue itself. 


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The control of lytic replication of Epstein-Barr virus in B lymphocytes (Review). 
Uncontrolled replication of a virus, which is harmful to the host is also disadvantageous to the virus. Most viruses cannot compete with the various immune mechanisms and become eliminated in the course of infection. Therefore, only the time between infection and eradication remains for these viruses to proliferate. A few viruses, like the Herpesviruses or the papillomaviruses, however, have developed a sophisticated strategy for persisting lifelong, usually asymptomatically in the host, hiding from the immune system and producing infectious progeny at the same time. This strategy depends on a separation of latency and the lytic replication, either by time due to differentiation-dependent mechanisms or by spatial separation as the result of different host cell types. Both are true for the Epstein-Barr virus (EBV). B cells and epithelial cells have a pivotal role in the life cycle of the virus. The former can become latently infected and are thought to be the virus reservoir in vivo, whereas the latter were shown to be permissive for lytic replication. However, replication of EBV in vivo is controlled primarily by host immune mechanisms selecting for cells that are not permissive for viral replication as the result of a particular set of transcription factors. These factors control the activity of the regulatory immediate-early genes and, in addition, lytic and latent cycle regulatory genes negatively interfere with each other and thus link cellular and viral gene regulatory mechanisms. Disturbance of both the immune surveillance as well as viral gene regulation may result in EBV-associated disease. 


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Identification of a region which directs the monocytic activity of the colony-stimulating factor 1 (macrophage colony-stimulating factor) receptor promoter and binds PEBP2/CBF (AML1). 
The receptor for the macrophage colony-stimulating factor (or colony-stimulating factor 1 [CSF-1]) is expressed from different promoters in monocytic cells and placental trophoblasts. We have demonstrated that the monocyte-specific expression of the CSF-1 receptor is regulated at the level of transcription by a tissue-specific promoter whose activity is stimulated by the monocyte/B-cell-specific transcription factor PU.1 (D.-E.Zhang, C.J.Hetherington, H.-M.Chen, and D.G.Tenen, Mol.Cell. Biol.14:373-381, 1994). Here we report that the tissue specificity of this promoter is also mediated by sequences in a region II (bp -88 to - 59), which lies 10 bp upstream from the PU.1-binding site. When analyzed by DNase footprinting, region II was protected preferentially in monocytic cells. Electrophoretic mobility shift assays confirmed that region II interacts specifically with nuclear proteins from monocytic cells. Two gel shift complexes (Mono A and Mono B) were formed with separate sequence elements within this region. Competition and supershift experiments indicate that Mono B contains a member of the polyomavirus enhancer-binding protein 2/core-binding factor (PEBP2/CBF) family, which includes the AML1 gene product, while Mono A is a distinct complex preferentially expressed in monocytic cells. Promoter constructs with mutations in these sequence elements were no longer expressed specifically in monocytes. Furthermore, multimerized region II sequence elements enhanced the activity of a heterologous thymidine kinase promoter in monocytic cells but not other cell types tested. These results indicate that the monocyte/B-cell-specific transcription factor PU.1 and the Mono A and Mono B protein complexes act in concert to regulate monocyte-specific transcription of the CSF-1 receptor. 


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Transcriptional repression of the interleukin-2 gene by vitamin D3: direct inhibition of NFATp/AP-1 complex formation by a nuclear hormone receptor. 
T-lymphocyte proliferation is suppressed by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the active metabolite of vitamin D3, and is associated with a decrease in interleukin 2 (IL-2), gamma interferon, and granulocyte-macrophage colony-stimulating factor mRNA levels. We report here that 1,25(OH)2D3-mediated repression in Jurkat cells is cycloheximide resistant, suggesting that it is a direct transcriptional repressive effect on IL-2 expression by the vitamin D3 receptor (VDR). We therefore examined vitamin D3-mediated repression of activated IL-2 expression by cotransfecting Jurkat cells with IL-2 promoter/reporter constructs and a VDR overexpression vector and by DNA binding. We delineated an element conferring both DNA binding by the receptor in vitro and 1,25(OH)2D3-mediated repression in vivo to a short 40-bp region encompassing an important positive regulatory element, NF-AT-1, which is bound by a T-cell-specific transcription factor, NFATp, as well as by AP-1. VDR DNA-binding mutants were unable to either bind to this element in vitro or repress in vivo; the VDR DNA-binding domain alone, however, bound the element but also could not repress IL-2 expression. These results indicate that DNA binding by VDR is necessary but not sufficient to mediate IL-2 repression. By combining partially purified proteins in vitro, we observed the loss of the bound NFATp/AP-1-DNA complex upon inclusion of VDR or VDR-retinoid X receptor. Order of addition and off-rate experiments indicate that the VDR-retinoid X receptor heterodimer blocks NFATp/AP-1 complex formation and then stably associates with the NF-AT-1 element. This direct inhibition by a nuclear hormone receptor of transcriptional activators of the IL-2 gene may provide a mechanistic explanation of how vitamin derivatives can act as potent immunosuppressive agents. 


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Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-kappa B transcription factor and HIV-1. 
Hydrogen peroxide and oxygen radicals are agents commonly produced during inflammatory processes. In this study, we show that micromolar concentrations of H2O2 can induce the expression and replication of HIV-1 in a human T cell line. The effect is mediated by the NF-kappa B transcription factor which is potently and rapidly activated by an H2O2 treatment of cells from its inactive cytoplasmic form. N-acetyl-L-cysteine (NAC), a well characterized antioxidant which counteracts the effects of reactive oxygen intermediates (ROI) in living cells, prevented the activation of NF-kappa B by H2O2. NAC and other thiol compounds also blocked the activation of NF-kappa B by cycloheximide, double-stranded RNA, calcium ionophore, TNF-alpha, active phorbol ester, interleukin-1, lipopolysaccharide and lectin. This suggests that diverse agents thought to activate NF-kappa B by distinct intracellular pathways might all act through a common mechanism involving the synthesis of ROI. ROI appear to serve as messengers mediating directly or indirectly the release of the inhibitory subunit I kappa B from NF-kappa B. 


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Processing of the precursor of NF-kappa B by the HIV-1 protease during acute infection. 
Transcription of the human immunodeficiency virus type-1 (HIV-1) genome is regulated in part by cellular factors and is stimulated by activation of latently infected T cells. T-cell activation also correlates with the induction of the factor NF-kappa B which binds to two adjacent sites in the HIV-1 long terminal repeat. This factor consists of two DNA-binding subunits of relative molecular mass 50,000 (50K) associated with two 65K subunits. It is located in the nucleus in mature B cells, but is present in other cell types as an inactive cytoplasmic complex. External stimuli, including those that activate T cells, result in nuclear translocation of active NF-kappa B. The cloning of the complementary DNA for the 50K subunit helped to identify an exclusively cytoplasmic 105K precursor (p105) (V.B., P.K. and A.I., manuscript submitted). The expression of active NF-kappa B might therefore also be regulated by the extent of processing of p105. Because HIV-1 requires active NF-kappa B for efficient transcription, we tested the effect of HIV-1 infection on the processing of the human 105K precursor. We show here that the HIV-1 protease can process p105 and increases levels of active nuclear NF-kappa B complex. 


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Differential RNA display identifies novel genes associated with decreased vitamin D receptor expression. 
To characterize further the function of the intracellular vitamin D receptor (VDR), we have developed stable transfectant variants of a vitamin D-responsive cell line (U937) which express either decreased or increased numbers of VDR. In this study we have analyzed changes in gene expression associated with this variable VDR expression. Initial experiments indicated that a 50% decrease in VDR levels was associated with a 2-fold increase in cell proliferation and a similar rise in c-myc mRNA expression. Further studies were carried out using differential RNA display (DD). Sequence analysis of DD products revealed two cDNAs with identity to known gene products: the catalytic sub-unit of DNA-protein kinase (DNA-PK(CS)), and the peroxisomal enzyme 17beta-hydroxysteroid dehydrogenase type IV (17beta-HSD IV). Northern analysis confirmed that expression of both mRNAs was reduced in cells with decreased numbers of VDR. Down-regulation of 17beta-HSD IV mRNA expression was associated with enhanced estradiol inactivation by U937 cells, suggesting a link between estrogenic pathways and cell proliferation. Further Northern analyses indicated that there was no significant change in 17beta-HSD IV or DNA-PK(CS) mRNA levels following treatment with 1,25(OH)2D3, although expression of both genes varied with changes in cell proliferation. These data suggest that, in addition to its established role as a hormone-dependent trans-activator, VDR may influence gene expression by ligand-independent mechanisms. 


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Mapping of the transcriptional repression domain of the lymphoid-specific transcription factor oct-2A. 
The lymphoid-specific transcription factor Oct-2a is implicated in B cell-specific transcriptional activity via the octamer motif. Structure/function analysis of various Oct-2a effector regions in the context of the GAL4 DNA-binding domain revealed that Oct-2a contains two functionally different activation domains at the N and the C termini. The transcriptional activity of both domains is strongly potentiated by interactions with distinct B cell-specific coactivators. Recently, we have identified a repression domain located within the N terminus of Oct-2a (amino acids 2-99). When this domain was transferred to a potent activator, transcription was strongly inhibited. In this study we present a deletion analysis of the N-terminal region of Oct-2a to determine the minimal repression domain. We identified a stretch of 23 amino acids, rich in serine and threonine residues, which was responsible for most of the repression activity. We show that repression is strongly dependent on the type of enhancer present in the reporter plasmid as well as on the cell line tested. The possibility that Oct-2a can act as an activator and/or a repressor may have important consequences for the function of Oct-2a in B cell differentiation and other developmental processes. 


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Regulation of cell-type-specific interleukin-2 receptor alpha-chain gene expression: potential role of physical interactions between Elf-1, HMG-I(Y), and NF-kappa B family proteins. 
The interleukin 2 receptor alpha-chain (IL-2R alpha) gene is rapidly and potently induced in T cells in response to mitogenic stimuli. Previously, an inducible enhancer between nucleotides -299 and -228 that contains NF-kappa B and CArG motifs was identified. We now report the characterization of a second essential positive regulatory element located between nucleotides -137 and -64 that binds Elf-1 and HMG-I(Y). This element had maximal activity in lymphoid cells, paralleling the cell type specificity of Elf-1 expression. Transcription from the IL-2R alpha promoter was inhibited when either the Elf-1 or the HMG-I(Y) binding site was mutated. Coexpression of both proteins activated transcription of the -137 to -64 element in COS-7 cells. Elf-1 physically associated with HMG-I and with NF-kappa B p50 and c-Rel in vitro, suggesting that protein-protein interactions might functionally coordinate the actions of the upstream and downstream positive regulatory elements. This is the first report of a physical interaction between an Ets family member and NF-kappa B family proteins. These findings provide significant new insights into the protein-protein and protein-DNA interactions that regulate cell-type-specific and inducible IL-2R alpha gene expression and also have implications for other genes regulated by Elf-1 and NF-kappa B family proteins. 


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LMP-1 activates NF-kappa B by targeting the inhibitory molecule I kappa B alpha. 
LMP-1, an Epstein-Barr virus membrane protein expressed during latent infection, has oncogenic properties, as judged from its ability to transform B lymphocytes and rodent fibroblasts. LMP-1 induces the expression of bcl2, an oncogene which protects cells from apoptosis, as well as of genes encoding other proteins involved in cell regulation and growth control. The mechanisms by which LMP-1 upregulates these proteins is unknown, but it is plausible that LMP-1 modifies signal transduction pathways that result in the activation of one or more transcription factors that ultimately regulate transcription of oncogenic genes. NF-kappa B, a transcription factor controlling the expression of genes involved in cell activation and growth control, has been shown to be activated by LMP-1. The mechanism(s) regulating this activation remains unknown. Our data indicate that increased NF-kappa B DNA binding and functional activity are present in B-lymphoid cells stably or transiently expressing LMP-1. I kappa B alpha is selectively modified in LMP-1-expressing B cells. A phosphorylated form of I kappa B alpha and increased protein turnover-degradation correlate with increased NF-kappa B nuclear translocation. This results in increased transcription of NF-kappa B-dependent-genes, including those encoding p105 and I kappa B alpha (MAD3). These results indicate that LMP-1 activates NF-kappa B in B-cell lines by targeting I kappa B alpha. Identification of the pathways activated by LMP-1 to result in posttranslational modifications of I kappa B alpha will aid in determining the role of this virus-host cell protein interaction in Epstein-Barr virus-mediated oncogenesis. 


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Granulocyte colony-stimulating factor activates a 72-kDa isoform of STAT3 in human neutrophils. 
Granulocyte colony-stimulating factor (G-CSF) signaling involves activation of STATs, proteins that serve the dual function of signal transduction and activation of transcription. We previously demonstrated that G-CSF activated a distinct Stat3-like protein in immature and mature normal myeloid cells, StatG. StatG in normal immature human myeloid cells, i.e. adult CD34+ bone marrow cells, was composed of Stat3beta. This investigation was undertaken to determine the composition of StatG in mature normal human myeloid cells, i.e. polymorphonuclear neutrophilic granulocytes (PMN). These studies revealed that the major protein in extracts of PMN activated by G-CSF to bind the high-affinity serum-inducible element (hSIE) is a 72-kDa protein that cross-reacts with Stat3 monoclonal antibody, which we have designated Stat3gamma. Stat3gamma is derived from Stat3alpha by limited proteolysis and lacks the carboxyl-terminal portion of Stat3alpha. Because this region of Stat3alpha is involved in transcriptional activation, our findings suggest the possibility that Stat3gamma may be transcriptionally inactive and may compete with Stat3alpha for Stat3 binding sites in these terminally differentiated myeloid cells. 


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NF-kappa B-independent suppression of HIV expression by ascorbic acid. 
Ascorbic acid (ascorbate or vitamin C) has been shown to suppress the induction of HIV in latently infected T lymphocytic cells following stimulation with a tumor promoter (PMA) and inflammatory cytokine (TNF-alpha). To assess whether this inhibition was mediated via modulation of the cellular transcription factor, NF-kappa B, we carried out gel shift analysis on nuclear extracts prepared under different conditions of cell stimulation in the presence or absence of ascorbate, N-acetylcysteine (NAC), or zidovudine (AZT). Pretreatment of ACH-2 T cells by NAC followed by stimulation with PMA, TNF-alpha, or hydrogen peroxide (H2O2) resulted in strong suppression of NF-kappa B activation. In contrast, neither ascorbate nor AZT affected NF-kappa B activity under all three induction conditions in the ACH-2 cell line. Ascorbate and AZT also had no effect on NF-kappa B activation following TNF-alpha- or PMA-induced stimulation of U1 promonocytic cells. These results suggest that the molecular mechanism of HIV inhibition by ascorbate is not mediated via NF-kappa B inhibition, unlike that seen with other antioxidants. 


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Paradoxical priming effects of IL-10 on cytokine production. 
IL-10 is a well-known immunosuppressive and/or anti-inflammatory cytokine. However, we report in vitro experimental studies in which IL-10 primed leukocytes and led to an enhanced production of tumor necrosis factor (TNF) upon further stimulation by lipopolysaccharide (LPS). Monocytes and peripheral blood mononuclear cells (PBMC) prepared from whole blood maintained for 20 h at 37 degrees C in the presence of recombinant human IL-10 had an enhanced capacity to produce TNF in response to LPS. In addition to TNF, LPS-induced IL-6 and spontaneous IL-1ra production were also enhanced. When isolated PBMC were first cultured for 20 h in the presence of IL-10 on Teflon to prevent adherence, washed to remove IL-10 and then further cultured in plastic dishes for an additional 20 h in the presence of LPS or IL-1beta, an enhanced release of TNF was observed. This was not the case when PBMC were pre-cultured in plastic multidishes in the presence of IL-10. TNF mRNA expression induced by LPS was decreased when the pre-treatment of PBMC with IL-10 was performed on plastic, whereas this was not the case when cells were pre-cultured with IL-10 on Teflon. Furthermore, NFkappaB translocation following LPS activation was higher after IL-10 pre-treatment on Teflon than on plastic. Interestingly, an enhanced frequency of CD16 and CD68(+) cells among the CD14(+) cells was observed in the presence of IL-10, independently of the pre-culture conditions of the PBMC. Altogether, these results indicate that the IL-10-induced up-regulation of cytokine production depends on the prevention of monocyte adherence by red cells in the whole blood assays or by cultures of PBMC on Teflon. In contrast, the adherence parameter has no effect on the IL-10-induced modulation of some monocyte surface markers. 


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Sequence analysis and expression in cultured lymphocytes of the human FOSB gene (G0S3). 
G0S3 is a member of a set of putative G0/G1 switch regulatory genes (G0S genes) selected by screening cDNA libraries prepared from human blood mononuclear cells cultured for 2 hr with lectin and cycloheximide. The sequence shows high homology with the murine FOSB gene, which encodes a component of the AP1 transcriptional regulator. Comparison of cDNA and genomic sequences reveals a 4-exon structure characteristic of the FOS family of genes. Freshly isolated cells show high levels of FOSB/G0S3 and FOS/G0S7 mRNAs, which decline rapidly during incubation in culture medium. The kinetics of expression suggest that the high initial levels are caused by the isolation procedure, and do not reflect constitutive expression. In cells preincubated for a day, levels of FOS mRNA reach a maximum 20 min after the addition of lectin and decline to control levels over the next 3 hr. Levels of FOSB mRNA reach a maximum 40 min after the addition of lectin and decline to control levels over the next 6 hr. In freshly isolated cells, both FOS and FOSB mRNAs increase dramatically in response to the protein synthesis inhibitor cycloheximide. In preincubated cells, the cycloheximide response is decreased, especially in the case of FOSB. These differences in expression of FOS and FOSB suggest different roles and regulation. Regions of low base order-dependent stem-loop potential in the region of the gene are defined. These indicate where base order has been adapted for purposes other than stem-loop stability (e.g., encoding proteins or gene regulation). Regions of low potential in a 68.5-kb genomic segment containing the FOSB gene suggest that the potential may help locate genes in uncharted DNA sequences. 


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Biphasic control of NF-kappa B activation induced by the triggering of HLA-DR antigens expressed on B cells. 
The regulation of NF-kappa B activation following the triggering of HLA-DR antigens by mAb L243 has been studied at various times in Raji cells. Electrophoretic mobility shift assays demonstrated a strong increase of NF-kappa B DNA binding after triggering of HLA-DR antigens. Using TNF-alpha-activity neutralizing antibodies, the authors demonstrated that the upregulation of NF-kappa B was found to depend, at later time point, on an autocrine effect of TNF-alpha secreted following triggering of HLA-DR antigens. In contrast, it was found to be TNF-alpha independent in the early time point. Moreover, the upregulation of NF-kappa B binding activity is regulated by the triggering of selected epitopes of HLA-DR antigens. In fact, mAb L243 but not the staphylococcal superantigens, staphylococcal exotoxin toxic shock syndrome toxin-I or staphylococcal enterotoxin B, regulate the NF-kappa B binding activity. 


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Characterization of a new isoform of the NFAT (nuclear factor of activated T cells) gene family member NFATc [published erratum appears in J Biol Chem 1996 Dec 27;271(52):33705] 
The cyclosporin A (CsA)/FK506-sensitive nuclear factor of activated T cells (NFAT) plays a key role in the inducible expression of cytokine genes in T cells. Although NFAT has been recently shown to be inducible in several non-T immune cells, the NFAT gene family members characterized to date have been isolated only from T cells. To further characterize NFAT function in human B cells and to demonstrate cytokine gene specificity of NFAT proteins, we report here the isolation and characterization of a cDNA clone from the Raji B cell line. The cDNA clone encodes a new isoform, NFATc.beta, of the NFAT gene family member NFATc (designated here NFATc.alpha). The amino acid sequence of NFATc.beta differs from that of NFATc.alpha in the first NH2-terminal 29 residues and contains an additional region of 142 residues at the COOH terminus. Northern analysis using a probe encompassing a common region of both isoforms showed two mRNA species of 2.7 and 4.5 kilobase pairs, while an NFATc.beta-specific probe detected only the 4.5-kilobase pair mRNA which was preferentially expressed in the spleen. Transient expression of NFATc.beta was capable of activating an interleukin-2 NFAT-driven reporter gene in stimulated Jurkat cells in a CsA-sensitive manner. However, NFATc.beta neither bound to the kappa3 element ( an NFAT-binding site ) in the tumor necrosis factor-alpha promoter nor activated the tumor necrosis factor-alpha promoter in cotransfection assays. These data suggest that different members or isoforms of NFAT gene family may regulate inducible expression of different cytokine genes. 


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Activation of nuclear factor kappa B in human neuroblastoma cell lines. 
The nuclear factor kappa B (NF-kappa B) is a eukaryotic transcription factor. In B cells and macrophages it is constitutively present in cell nuclei, whereas in many other cell types, NF-kappa B translocates from cytosol to nucleus as a result of transduction by tumor necrosis factor alpha (TNF alpha), phorbol ester, and other polyclonal signals. Using neuroblastoma cell lines as models, we have shown that in neural cells NF-kappa B was present in the cytosol and translocated into nuclei as a result of TNF alpha treatment. The TNF alpha-activated NF-kappa B was transcriptionally functional. NF-kappa B activation by TNF alpha was not correlated with cell differentiation or proliferation. However, reagents such as nerve growth factor (NGF) and the phorbol ester phorbol 12-myristate 13-acetate (PMA), which induce phenotypical differentiation of the SH-SY5Y neuroblastoma cell line, activated NF-kappa B, but only in that particular cell line. In a NGF-responsive rat pheochromocytoma cell line, PC12, PMA activated NF-kappa B, whereas NGF did not. In other neuroblastoma cell lines, such as SK-N-Be(2), the lack of PMA induction of differentiation was correlated with the lack of NF-kappa B activation. We found, moreover, that in SK-N-Be(2) cells protein kinase C (PKC) enzymatic activity was much lower compared with that in a control cell line and that the low PKC enzymatic activity was due to low PKC protein expression. NF-kappa B was not activated by retinoic acid, which induced morphological differentiation of all the neuroblastoma cell lines used in the present study. Thus, NF-kappa B activation was not required for neuroblastoma cell differentiation. Furthermore, the results obtained with TNF alpha proved that NF-kappa B activation was not sufficient for induction of neuroblastoma differentiation. 


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Tap: a novel cellular protein that interacts with tip of herpesvirus saimiri and induces lymphocyte aggregation. 
Tip of herpesvirus saimiri associates with Lck and down-regulates Lck-mediated activation. We identified a novel cellular Tip-associated protein (Tap) by a yeast two-hybrid screen. Tap associated with Tip following transient expression in COS-1 cells and stable expression in human Jurkat-T cells. Expression of Tip and Tap in Jurkat-T cells induced dramatic cell aggregation. Aggregation was likely caused by the up-regulated surface expression of adhesion molecules including integrin alpha, L-selectin, ICAM-3, and H-CAM. Furthermore, NF-kappaB transcriptional factor of aggregated cells had approximately 40-fold higher activity than that of parental cells. Thus, Tap is likely to be an important cellular mediator of Tip function in T cell transformation by herpesvirus saimiri. 


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Cell-type-specific regulation of the human tumor necrosis factor alpha gene in B cells and T cells by NFATp and ATF-2/JUN. 
The human tumor necrosis factor alpha (TNF-alpha) gene is one of the earliest genes transcribed after the stimulation of a B cell through its antigen receptor or via the CD-40 pathway. In both cases, induction of TNF-alpha gene transcription can be blocked by the immunosuppressants cyclosporin A and FK506, which suggested a role for the NFAT family of proteins in the regulation of the gene in B cells. Furthermore, in T cells, two molecules of NFATp bind to the TNF-alpha promoter element kappa 3 in association with ATF-2 and Jun proteins bound to an immediately adjacent cyclic AMP response element (CRE) site. Here, using the murine B-cell lymphoma cell line A20, we show that the TNF-alpha gene is regulated in a cell-type-specific manner. In A20 B cells, the TNF-alpha gene is not regulated by NFATp bound to the kappa 3 element. Instead, ATF-2 and Jun proteins bind to the composite kappa 3/CRE site and NFATp binds to a newly identified second NFAT site centered at -76 nucleotides relative to the TNF-alpha transcription start site. This new site plays a critical role in the calcium-mediated, cyclosporin A-sensitive induction of TNF-alpha in both A20 B cells and Ar-5 cells. Consistent with these results, quantitative DNase footprinting of the TNF-alpha promoter using increasing amounts of recombinant NFATp demonstrated that the -76 site binds to NFATp with a higher affinity than the kappa 3 site. Two other previously unrecognized NFATp-binding sites in the proximal TNF-alpha promoter were also identified by this analysis. Thus, through the differential use of the same promoter element, the composite kappa 3/CRE site, the TNF-alpha gene is regulated in a cell-type-specific manner in response to the same extracellular signal. 


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Cell type specificity and activation requirements for NFAT-1 (nuclear factor of activated T-cells) transcriptional activity determined by a new method using transgenic mice to assay transcriptional activity of an individual nuclear factor. 
Nuclear factor of activated T-cells (NFAT-1) is a transcription factor which is considered to be an important regulator in early T-cell activation. We have developed a system to monitor the transcriptional activity of NFAT-1 at the single cell level in whole animals. The system is based on the use of an oligomerized NFAT-1 binding motif that directs transcription of SV40 T-antigen in transgenic mice. This report represents the first demonstration that a multimerized short binding motif can function appropriately in transgenic mice. NFAT-1 activity had previously been thought to be confined to activated T-lymphocytes upon release of intracellular calcium. By targeting NFAT-1-dependent gene expression in transgenic mice we discovered new sites of NFAT-1 activity. Besides in T-lymphocytes NFAT-1 activity could also be induced in T-lymphocyte-depleted spleen cells and purified B-lymphocytes and requires agents that both release intracellular calcium and activate protein kinase C. A difference in the time course of appearance of NFAT-1 activity between T-lymphocytes and non-T-lymphocytes was revealed. Constitutive expression was observed in a small population of cells in the dermis and some mice have developed skin lesions. Interestingly, the tissue pattern of expression of the NFAT-1 activity resembles the expression pattern described for HIV-LTR/tat transgenic mice (Vogel, J., Hinrichs, S. H., Reynolds, R. K., Luciw, P. A., and Jay, G. (1988) Nature 335, 606-611). This similarity in expression and the fact that NFAT-1 has been shown to bind functional sequences in HIV-LTR suggest a role for NFAT-1 in dermal activation of the HIV-LTR. 


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The ubiquitous octamer-binding protein(s) is sufficient for transcription of immunoglobulin genes. 
All immunoglobulin genes contain a conserved octanucleotide promoter element, ATGCAAAT, which has been shown to be required for their normal B-cell-specific transcription. Proteins that bind this octamer have been purified, and cDNAs encoding octamer-binding proteins have been cloned. Some of these proteins (referred to as OTF-2) are lymphoid specific, whereas at least one other, and possibly more (referred to as OTF-1), is found ubiquitously in all cell types. The exact role of these different proteins in directing the tissue-specific expression of immunoglobulin genes is unclear. We have identified two human pre-B-cell lines that contain extremely low levels of OTF-2 yet still express high levels of steady-state immunoglobulin heavy-chain mRNA in vivo and efficiently transcribe an immunoglobulin gene in vitro. Addition of a highly enriched preparation of OTF-1 made from one of these pre-B cells or from HeLa cells specifically stimulated in vitro transcription of an immunoglobulin gene. Furthermore, OFT-1 appeared to have approximately the same transactivation ability as OTF-2 when normalized for binding activity. These results suggest that OTF-1, without OTF-2, is sufficient for transcription of immunoglobulin genes and that OTF-2 alone is not responsible for the B-cell-specific regulation of immunoglobulin gene expression. 


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alpha-Tocopheryl succinate inhibits monocytic cell adhesion to endothelial cells by suppressing NF-kappa B mobilization. 
The adherence of monocytes to activated endothelium is an early event in atherogenesis. Because antioxidants have been considered to be of antiatherosclerotic potential, we investigated the effects of alpha-tocopherol (TCP) and its acetate and succinate esters on monocyte adhesion to cytokine-stimulated human umbilical vein endothelial cells (HUVEC). Endothelial cells were treated with TCP, alpha-tocopherol acetate (TCP acetate), or alpha-tocopheryl succinate (TCP succinate) before stimulation with tumor necrosis factor-alpha (TNF-alpha; 10 U/ml, 6 h) or interleukin-1 beta (IL-1 beta; 10 U/ml, 6 h). Cytokine-stimulated cell surface expression of vascular cell adhesion molecule-1 (VCAM-1, CD106) and E-selectin (ELAM-1, CD62E), but not of intercellular adhesion molecule-1 (ICAM-1, CD54), was time- and dose-dependently inhibited by TCP succinate but not by TCP or TCP acetate. TCP succinate (200 microM, 24 h) reduced TNF-induced VCAM-1 and E-selectin expression from a specific mean fluorescence intensity of 151 +/- 28 to 12 +/- 4 channels and from 225 +/- 38 to 79 +/- 21 channels, respectively. Succinate alone had no effect. Decreased adhesion molecule expression was associated with a reduction of monocytic cell adhesion. TCP succinate (20 microM, 72 h), but not TCP (200 microM, 72 h), reduced U-937 cell adhesion to TNF-alpha-stimulated (10 U/ml, 6 h) HUVEC by 30% (P < 0.025) and to IL-1 beta-stimulated HUVEC by 56% (P < 0.010). Electrophoretic mobility-shift assays of HUVEC nuclear proteins revealed a decrease in TNF-alpha-stimulated nuclear factor-kappa B (NF-kappa B) activation after pretreatment of HUVEC with TCP succinate but not with TCP, TCP acetate, or succinate alone. In conclusion, we demonstrate that the vitamin E derivative TCP succinate prevents monocytic cell adhesion to cytokine-stimulated endothelial cells by inhibiting the activation of NF-kappa B, further emphasizing the antiatherosclerotic potential of lipid soluble antioxidants. 


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Activation of nuclear factor-kappaB by lipopolysaccharide in mononuclear leukocytes is prevented by inhibitors of cytosolic phospholipase A2. 
In monocytes, lipopolysaccharide induces synthesis and activity of the 85-kDa cytosolic phospholipase A2. This enzyme releases arachidonic acid and lyso-phospholipids from membranes which are metabolized to eicosanoids and platelet-activating-factor. These lipid mediators increase activity of transcription factors and expression of cytokine genes indicating a function for cytosolic phospholipase A2 in signal transduction and inflammation. We have shown previously that trifluoromethylketone inhibitors of cytosolic phospholipase A2 suppressed interleukin-1beta protein and steady-state mRNA levels in human lipopolysaccharide-stimulated peripheral blood mononuclear leukocytes. In this study, the subcellular mechanisms were analyzed by which trifluoromethylketones interfere with gene expression. We found that they reduced the initial interleukin-1beta mRNA transcription rate through prevention of degradation of inhibitor-kappaB alpha. Consequently, cytosolic activation, nuclear translocation and DNA-binding of nuclear factor-kappaB were decreased. Trifluoromethylketones ameliorate chronic inflammation in vivo. Thus, this therapeutic potency may reside in retention of inactive nuclear factor-kappaB in the cytosol thereby abrogating interleukin-1beta gene transcription. 


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The ability of BHRF1 to inhibit apoptosis is dependent on stimulus and cell type. 
The development of resistance to host defense mechanisms such as tumor necrosis factor (TNF)- and Fas-mediated apoptosis of transformed or virus-infected cells may be a critical component in the development of disease. To find genes that protect cells from apoptosis, we used an expression cloning strategy and identified BHRF1, an Epstein-Barr virus (EBV) early-lytic-cycle protein with distant homology to Bcl-2, as an anti-apoptosis protein. Expression of BHRF1 in MCF-Fas cells conferred nearly complete resistance against both anti-Fas antibody and TNF-mediated apoptosis. In addition, BHRF1 protected these cells from monocyte-mediated killing but failed to protect them from killing mediated by lymphokine-activated killer cells. The ability of BHRF1 to protect MCF-Fas cells from apoptosis induced by various stimuli was identical to that of Bcl-2 and Bcl-xL. Moreover, the mechanism of action of BHRF1 resembled that of Bcl-2 and Bcl-xL as it inhibited TNF- and anti-Fas-induced activation of two enzymes participating in the apoptosis pathway, cytosolic phospholipase A2 and caspase-3/CPP32, but did not interfere with the activation of NF-kappaB-like transcription factors. A putative function of BHRF1 in EBV-infected epithelial cells may be to protect virus-infected cells from TNF- and/or anti-Fas- induced cell death in order to maximize virus production. Surprisingly, expression of neither BHRF1 nor Bcl-2 in a B-cell line, BJAB, protected the cells from anti-Fas-mediated apoptosis even though they increased the survival of serum-starved cells. Thus, the protective role of BHRF1 against apoptosis resembles that of Bcl-2 in being cell type specific and dependent on the apoptotic stimulus. 


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In vitro suppression of programmed cell death of B cells by tissue inhibitor of metalloproteinases-1. 
Cellular pathways for induction of programmed cell death (PCD) have been identified, but little is known about specific extracellular matrix processes that may affect apoptosis along those pathways. In this study, a series of Burkitt's lymphoma (BL) cell lines were assayed for their expression of tissue inhibitor of metalloproteinases (TIMP)-1. Results indicate that TIMP-1-positive BL lines show resistance to cold-shock-induced apoptosis. Furthermore, recombinant TIMP-1, but not TIMP-2 or a synthetic metalloproteinase inhibitor (BB-94), confers resistance to apoptosis induced by both CD95-dependent and -independent (cold shock, serum deprivation, and gamma-radiation) pathways in TIMP-1-negative BL lines. TIMP-1 suppression of PCD is not due to metalloproteinase inhibition, as reduction and alkylation of the TIMP-1 did not abolish this activity. Retroviral induction of TIMP-1 not only resulted in cell survival but also in continued DNA synthesis for up to 5 d in the absence of serum, while controls underwent apoptosis. This resistance to apoptosis is reversed by anti-TIMP-1 antibodies, demonstrating that secreted TIMP-1 is active in blocking apoptosis. Furthermore, TIMP-1 upregulation induced expression of Bcl-XL but not Bcl-2 as well as decreased NF-kappaB activity as compared with controls. These results demonstrate that TIMP-1 suppresses apoptosis in B cells and suggests a novel activity for TIMP-1 in tissue homeostasis. 


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Cooperation between core binding factor and adjacent promoter elements contributes to the tissue-specific expression of interleukin-3. 
Tissue-specific expression of interleukin-3 (IL-3) is mediated via cis-acting elements located within 315 base pairs of the transcription start. This is achieved in part through the positive activities of the AP-1 and Elf-1 sites in the IL-3 promoter. The contribution to T cell-specific expression by other promoter sites was assessed in a transient expression assay with IL-3 promoter constructs linked to a luciferase gene, focusing initially on the core binding factor (CBF) site, which is footprinted in vivo upon T cell activation. Activity of the CBF site is shown to be critically dependent on the adjacent activator site Act-1. Together the Act-1 and CBF sites form a functional unit (AC unit) with dual activity. The AC unit is demonstrated to enhance basal activity of promoters both in fibroblasts and T cells. This activity is further inducible in activated T cells, but not in fibroblasts. In addition to the already identified NIP repressor site, evidence is presented for a second repressor region that restricts promoter activity in fibroblasts. Finally, a novel positive regulatory element has been mapped in the IL-3 promoter between nucleotide -180 and -210 that leads to increased expression in T cells. Together these results demonstrate that T cell expression of IL-3 is not specified by the activity of a single tissue-specific element, but instead involves multiple interacting elements that provide both specific positive regulation in T cells and specific negative regulation in fibroblasts. 


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Relief of cyclin A gene transcriptional inhibition during activation of human primary T lymphocytes via CD2 and CD28 adhesion molecules. 
Cyclin A transcription is cell cycle regulated and induced by cell proliferative signals. To understand the mechanisms underlined in this regulation in normal human cells, we have analysed in vivo protein-DNA interactions at the Cyclin A locus in primary T lymphocytes. Stimulation of purified T lymphocytes by a combination of monoclonal antibodies directed at CD2 and CD28 adhesion molecules gives rise to a long lasting proliferation in the absence of accessory cells. Cyclin A was observed after 4 days of costimulation with anti CD2 + CD28 whereas stimulation by anti CD2 or anti CD28 alone was not effective. In vivo genomic DMS footprinting revealed upstream of the major transcription initiation sites, the presence of at least three protein binding sites, two of which were constitutively occupied. They bind in vitro respectively ATF-1 and NF-Y proteins. The third site was occupied in quiescent cells or in cells stimulated by anti CD2 or anti CD28 alone. The mitogenic combination of anti CD2 + anti CD28 released the footprint as cells were committed to proliferation. Consistent with theses results, nuclear extracts prepared from quiescent cells formed a specific complex with this element, whereas extracts prepared from cells treated with anti CD2 + anti CD28 failed to do so after cells entered a proliferative state. 


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A factor known to bind to endogenous Ig heavy chain enhancer only in lymphocytes is a ubiquitously active transcription factor. 
The transcriptional enhancer located in the first intron of the immunoglobulin heavy chain constant region is a major determinant of B-cell-specific expression of immunoglobulin genes. Like other enhancers, the Ig heavy chain enhancer contains several short sequence motifs that bind specific transcription factors. Each binding site contributes to the overall activity of the enhancer, however no single element seems absolutely required for activity. For a better understanding of the Ig heavy chain enhancer components, we have cloned and analyzed individual sequence elements. We find that the factor that binds to the E3 enhancer motif, CATGTGGC, is a ubiquitous transcription factor. It is present in an active form in both B cells and non-B cells, where it can mediate transcriptional activation in vitro and in vivo. However, despite its ability to activate transcription of a transfected reporter gene, the factor is apparently unable to bind to the endogenous Ig heavy chain enhancer in non-lymphoid cells: In previous experiments by others, the characteristic in vivo footprint of this factor, designated NF-muE3, was detected in B cells but not in non-B cells. From this and other findings the picture emerges that there are at least three categories of factors which mediate cell-type-specific transcription in B lymphocytes: (a) cell-specific factors such as Oct-2A and Oct-2B that are not expressed in most other cell types: (b) ubiquitous factors such as NF-kappa B that are constitutively active in B cells but are sequestered in an inactive form in other cells; (c) ubiquitously active factors, exemplified by the one binding to the E3 sequence motif. This factor is present in an active form in a variety of cell types but is apparently unable to bind to the endogenous Ig heavy chain enhancer in non-B cells, perhaps due to a non-permissive chromatin structure of the Ig heavy chain locus. 


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Direct interaction of hematopoietic transcription factors PU.1 and GATA-1: functional antagonism in erythroid cells. 
Malignant transformation usually inhibits terminal cell differentiation but the precise mechanisms involved are not understood. PU.1 is a hematopoietic-specific Ets family transcription factor that is required for development of some lymphoid and myeloid lineages. PU.1 can also act as an oncoprotein as activation of its expression in erythroid precursors by proviral insertion or transgenesis causes erythroleukemias in mice. Restoration of terminal differentiation in the mouse erythroleukemia (MEL) cells requires a decline in the level of PU.1, indicating that PU.1 can block erythroid differentiation. Here we investigate the mechanism by which PU.1 interferes with erythroid differentiation. We find that PU.1 interacts directly with GATA-1, a zinc finger transcription factor required for erythroid differentiation. Interaction between PU.1 and GATA-1 requires intact DNA-binding domains in both proteins. PU.1 represses GATA-1-mediated transcriptional activation. Both the DNA binding and transactivation domains of PU.1 are required for repression and both domains are also needed to block terminal differentiation in MEL cells. We also show that ectopic expression of PU.1 in Xenopus embryos is sufficient to block erythropoiesis during normal development. Furthermore, introduction of exogenous GATA-1 in both MEL cells and Xenopus embryos and explants relieves the block to erythroid differentiation imposed by PU.1. Our results indicate that the stoichiometry of directly interacting but opposing transcription factors may be a crucial determinant governing processes of normal differentiation and malignant transformation. 


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Signal transduction pathways activated in endothelial cells following infection with Chlamydia pneumoniae. 
Chlamydia pneumoniae is an important respiratory pathogen. Recently, its presence has been demonstrated in atherosclerotic lesions. In this study, we characterized C. pneumoniae-mediated activation of endothelial cells and demonstrated an enhanced expression of endothelial adhesion molecules followed by subsequent rolling, adhesion, and transmigration of leukocytes (monocytes, granulocytes). These effects were blocked by mAbs against endothelial and/or leukocyte adhesion molecules (beta1 and beta2 integrins). Additionally, activation of different signal transduction pathways in C. pneumoniae-infected endothelial cells was shown: protein tyrosine phosphorylation, up-regulation of phosphorylated p42/p44 mitogen-activated protein kinase, and NF-kappaB activation/translocation occurred within 10-15 min. Increased mRNA and surface expression of E-selectin, ICAM-1, and VCAM-1 were noted within hours. Thus, C. pneumoniae triggers a cascade of events that could lead to endothelial activation, inflammation, and thrombosis, which in turn may result in or may promote atherosclerosis. 


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A functional T-cell receptor signaling pathway is required for p95vav activity. 
Stimulation of the T-cell antigen receptor (TCR) induces activation of multiple tyrosine kinases, resulting in phosphorylation of numerous intracellular substrates. One substrate is p95vav, which is expressed exclusively in hematopoietic and trophoblast cells. It contains a number of structural motifs, including Src homology 2, Src homology 3, and pleckstrin homology domains and a putative guanine nucleotide exchange domain. The role of p95vav in TCR-mediated signaling processes is unclear. Here, we show that overexpression of p95vav alone in Jurkat T cells leads to activation of the nuclear factors, including NFAT, involved in interleukin-2 expression. Furthermore, p95vav synergizes with TCR stimulation in inducing NFAT- and interleukin-2-dependent transcription. In contrast, NFAT activation by a G-protein-coupled receptor is not modulated by p95vav overexpression, suggesting that the effect is specific to the TCR signaling pathways. Although removal of the first 67 amino acids of p95vav activates its transforming potential in NIH 3T3 cells, this region appears to be required for its function in T cells. We further demonstrate that the p95vav-induced NFAT activation is not mimicked by Ras activation, though its function is dependent upon Ras and Raf. Furthermore, the activating function of p95vav is blocked by FK506, suggesting that its activity also depends on calcineurin. To further dissect p95vav involvement in TCR signaling, we analyzed various Jurkat mutants deficient in TCR signaling function or TCR expression and showed that an intact TCR signaling pathway is required for p95vav to function. However, overexpression of p95vav does not appear to influence TCR-induced protein tyrosine phosphorylation or increases in cytoplasmic free calcium. Taken together, our data suggest that p95vav plays an important role at an yet unidentified proximal position in the TCR signaling cascade. 


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Separation of oxidant-initiated and redox-regulated steps in the NF-kappa B signal transduction pathway. 
Studies presented here show that overall NF-kappa B signal transduction begins with a parallel series of stimuli-specific pathways through which cytokines (tumor necrosis factor alpha), oxidants (hydrogen peroxide and mitomycin C), and phorbol ester (phorbol 12-myristate 13-acetate) individually initiate signaling. These initial pathways culminate in a common pathway through which all of the stimulating agents ultimately signal NF-kappa B activation. We distinguish the stimuli-specific pathways by showing that the oxidative stimuli trigger NF-kappa B activation in only one of two human T-cell lines (Wurzburg but not Jurkat), whereas tumor necrosis factor alpha and phorbol 12-myristate 13-acetate readily stimulate in both lines. We propose the common pathway as the simplest way of accounting for the common requirements and properties of the signaling pathway. We include a redox-regulatory mechanism(s) in this common pathway to account for the previously demonstrated redox regulation of NF-kappa B activation in Jurkat cells (in which oxidants don't activate NF-kappa B); we put tyrosine phosphorylation in the common pathway by showing that kinase activity (inhibitable by herbimycin A and tyrphostin 47) is required for NF-kappa B activation by all stimuli tested in both cell lines. Since internal sites of oxidant production have been shown to play a key role in the cytokine-stimulated activation of NF-kappa B, and since tyrosine kinase and phosphatase activities are known to be altered by oxidants, these findings suggest that intracellular redox status controls NF-kappa B activation by regulating tyrosine phosphorylation event(s) within the common step of the NF-kappa B signal transduction pathway. 


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CD30 ligation induces nuclear factor-kappa B activation in human T cell lines. 
CD30 is a recently described member of the tumor necrosis factor/nerve growth factor receptor superfamily. In this report, we show that following incubation of L540 cells (Hodgkin's disease-derived, T cell-like, CD30+ cells) with the agonistic anti-CD30 monoclonal antibodies (mAb) M44 and M67, two nuclear factor (NF)-kappa B DNA binding activities were induced in nuclear extracts, as determined in gel retardation assays. The effect of the mAb towards NF-kappa B activation was rapid, as it occurred within 20 min, and was sustained for up to 6 h. By comparison, an isotype-matched antibody had no effect on NF-kappa B activation. Moreover, in human T helper (Th) clones functionally characterized as being of the type 0, type 1 and type 2 (28%, < 1% und 93% CD30+, respectively), the extent of CD30-mediated NF-kappa B activation correlated with the proportion of CD30+ cells. In all cell lines investigated, the NF-kappa B complexes induced following CD30 engagement were shown to contain p50 NF-kappa B1, p65 RelA, and possibly other transcription factors. Collectively, our results demonstrate that nuclear translocation and activation of NF-kappa B rank among the short-term cellular responses elicited following CD30 ligation. 


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Downregulation of Wilms' tumor gene (WT1) is not a prerequisite for erythroid or megakaryocytic differentiation of the leukemic cell line K562. 
The Wilms' tumor gene (WT1) encodes a transcription factor of the zinc finger type. A high expression of WT1 has been detected in a range of acute leukemias, and WT1 is downregulated during induced differentiation of some leukemic cell lines. Overexpression of WT1 in some myeloid cell lines confers resistance to differentiation induction. These observations suggest that a high WT1 expression in hematopoietic cells is incompatible with differentiation. In this study, each of the four different isoforms of WT1 was constitutively overexpressed in the leukemic cell line K562. K562 cells express endogenous WT1, which is downregulated as a response to induced differentiation along the erythroid and megakaryocytic pathways. We now demonstrate that a forced exogenous expression of the four different isoforms of WT1 in K562 does not affect the differentiation response, as judged by accumulation of hemoglobin in response to hemin or the expression of megakaryocytic cell surface markers in response to 12-O-tetradecanoylphorbol-13-acetate (TPA). We conclude that downregulation of WT1 during induced differentiation of K562 cells is not a prerequisite for erythroid or megakaryocytic differentiation of these cells. 


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A T cell-specific enhancer in the interleukin-3 locus is activated cooperatively by Oct and NFAT elements within a DNase I-hypersensitive site. 
Interleukin-3 (IL-3) is a cytokine that is expressed primarily in activated T cells. Here we identified an inducible T cell-specific enhancer 14 kb upstream of the IL-3 gene that responded to activation of T cell receptor signaling pathways. The IL-3 enhancer spanned an inducible cyclosporin A-sensitive DNase I-hypersensitive site found only in T cells. Four NFAT-like elements exist within the enhancer. The two most active NFAT-like elements were located at the center of the DNase I-hypersensitive site. One of these NFAT-like elements encompassed overlapping Oct- and NFATp/c-binding sites, which functioned in a highly synergistic manner. We suggest that the T cell-specific expression of the IL-3 gene is partly controlled through the enhancer by cooperation between Oct and NFAT family proteins. 


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Interleukin-10 stabilizes inhibitory kappaB-alpha in human monocytes. 
Interleukin-10 (IL-10) protects animals from lethal endotoxemia. This beneficial effect is mediated, in part, by inhibition of inflammatory cytokine production, including tumor necrosis factor-alpha (TNF-alpha). Evidence suggests that IL-10 may inhibit activation of the transcription factor nuclear factor-kappaB (NF-kappaB) through an unknown mechanism. NF-kappaB activation in response to inflammatory signals is dependent upon degradation of its associated inhibitory peptide, inhibitory kappaB-alpha (IkappaB-alpha). We hypothesized that IL-10 prevents human monocyte NF-kappaB activation and resultant TNF-alpha production by stabilization of IkappaB-alpha. The purpose of this study was to determine the effect of IL-10 on lipopolysaccharide (LPS)-induced human monocyte TNF-alpha production, NF-kappaB activation, and IkappaB-alpha degradation. Monocytes were isolated from human donors. Cells were stimulated with endotoxin (LPS, 100 ng/mL) with and without human IL-10 (10 ng/mL). Following stimulation, TNF-alpha was measured in cell supernatants by ELISA, NF-kappaB activity by electrophoretic mobility shift assay, and IkappaB-alpha levels by Western blot. We observed that after LPS stimulation of human monocytes, TNF-alpha increased to 798+/-67 pg/mL (p < .001 versus control). IL-10 attenuated LPS-stimulated TNF-alpha production (297+/-54; p < .001 versus LPS alone). After LPS stimulation in human monocytes, IkappaB-alpha protein levels decreased, and NF-kappaB DNA binding increased. IL-10 pretreatment prevented LPS-induced decreases in IkappaB-alpha protein levels and attenuated NF-kappaB DNA binding. IL-10 appears to prevent activation of NF-kappaB by preserving IkappaB-alpha protein levels, leading to a reduction in TNF-alpha release. 


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Human alveolar macrophages are markedly deficient in REF-1 and AP-1 DNA binding activity. 
Although many functions of human alveolar macrophages are altered compared with their precursor cell, the blood monocyte (monocyte), the reason(s) for these functional changes have not been determined. We recently reported that human alveolar macrophages do not express AP-1 DNA binding activity (Monick, M. M., Carter, A. B., Gudmundsson, G., Geist, L. J., and Hunninghake, G. W. (1998) Am. J. Physiol. 275, L389-L397). To determine why alveolar macrophages do not express AP-1 DNA binding activity, we first showed that there was not a decrease in expression of the FOS and JUN proteins that make up the AP-1 complex. There was, however, a significant difference in the amounts of the nuclear protein, REF-1 (which regulates AP-1 DNA binding by altering the redox status of FOS and JUN proteins), in alveolar macrophages compared with monocytes. In addition, in vitro differentiation of monocytes to a macrophage-like cell resulted in decreased amounts of REF-1. Finally, addition of REF-1 from activated monocytes to alveolar macrophage nuclear proteins resulted in a marked increase in AP-1 DNA binding. These studies strongly suggest that the process of differentiation of monocytes into alveolar macrophages is associated with a loss of REF-1 and AP-1 activity. This observation may explain, in part, some of the functional differences observed for alveolar macrophages compared with monocytes. 


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Induction of immediate early response genes by macrophage colony-stimulating factor in normal human monocytes. 
A group of coordinately induced protooncogenes, cytoskeletal, and extracellular matrix genes have been termed immediate early response genes, and their induction has been associated with growth factor-stimulated cell proliferation. We have investigated the induction of these genes by macrophage-CSF (M-CSF) in human monocytes that do not proliferate in response to M-CSF but require the factor for optimal cell differentiation. Normal human monocytes were isolated, carefully washed, and incubated for 36 to 48 h in fetal bovine serum-containing medium. At the end of this incubation the resting cells were stimulated with M-CSF, and RNA was isolated for analysis by Northern blotting. RNA from control resting cells contained low to undetectable levels of c-jun, fibronectin receptor, and actin mRNA. Within 15 to 30 min of addition of M-CSF, however, there was a dramatic coordinate induction of these genes. The c-jun gene expression was very transient and was not detectable by 60 min after M-CSF addition. In contrast, the expression of actin and fibronectin receptor mRNA was more sustained, and the expression of these genes remained elevated at 24 to 48 h after M-CSF addition. We also observed the induction of the myelomonocytic specific tyrosine kinase hck gene simultaneously with the other immediate early response genes. The protein synthesis inhibitor cycloheximide did not block the induction of any of these genes, and in fact, super-induced the expression of c-jun and hck. Nuclear run on transcription of the c-jun, hck, and actin genes. Therefore, in normal human monocytes M-CSF induces immediate early response genes without inducing cell proliferation. These genes may then play a role in altering the physiologic status of the cells in response to CSF. 


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Cloning of the novel human myeloid-cell-specific C/EBP-epsilon transcription factor. 
Chicken NF-M transcription factor, in cooperation with either c-Myb or v-Myb, is active in the combinatorial activation of myeloid-cell-specific genes in heterologous cell types, such as embryonic fibroblasts. In humans, similar effects were observed with homologous members of the CCAAT/enhancer-binding protein (C/EBP) family of transcriptional regulators, especially the human homolog of chicken NF-M, C/EBP-beta (NF-IL6). However, the NF-IL6 gene is expressed in a variety of nonmyeloid cell types and is strongly inducible in response to inflammatory stimuli, making it an unlikely candidate to have an exclusive role as a combinatorial differentiation switch during myelopoiesis in human cells. By using a reverse transcription-PCR-based approach and a set of primers specific for the DNA-binding domains of highly homologous members of the C/EBP family of transcriptional regulators, we have cloned a novel human gene encoding a member of the C/EBP gene family, identified as the human homolog of CRP1, C/EBP-epsilon. A 1.2-kb cDNA encoding full-length human C/EBP-epsilon was cloned from a promyelocyte-late myeloblast-derived lambda gt11 library. Molecular analysis of the cDNA and genomic clones indicated the presence of two exons encoding a protein with an apparent molecular mass of 32 kDa and a pI of 9.5. Primer extension analysis of C/EBP-epsilon mRNA detected a single major transcription start site approximately 200 bp upstream of the start codon. The putative promoter area is similar to those of several other myeloid-cell-specific genes in that it contains no TATAAA box but has a number of purine-rich stretches with multiple sites for the factors of the Ets family of transcriptional regulators. Northern blot analyses indicated a highly restricted mRNA expression pattern, with the strongest expression occurring in promyelocyte and late-myeloblast-like cell lines. Western blot and immunoprecipitation studies using rabbit anti-C/EBP-epsilon antibodies raised against the N-terminal portion of C/EBP-epsilon (amino acids 1 to 115) showed that C/EBP-epsilon is a 32-kDa nuclear phosphoprotein. The human C/EBP-epsilon protein exhibited strong and specific binding to double-stranded DNA containing consensus C/EBP sites. Cotransfection of the C/EBP-epsilon sense and antisense expression constructs together with chloramphenicol acetyltransferase reporter vectors containing myeloid-cell-specific c-mim and human myeloperoxidase promoters suggested a role for C/EBP-epsilon transcription factor in the regulation of a subset of myeloid-cell-specific genes. Transient tranfection of a promyelocyte cell line (NB4) with a C/EBP-epsilon expression plasmid increased cell growth by sevenfold, while antisense C/EBP-epsilon caused a fivefold decrease in clonal growth of these cells. 


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Regulation of the Ets-related transcription factor Elf-1 by binding to the retinoblastoma protein. 
The retinoblastoma gene product (Rb) is a nuclear phosphoprotein that regulates cell cycle progression. Elf-1 is a lymphoid-specific Ets transcription factor that regulates inducible gene expression during T cell activation. In this report, it is demonstrated that Elf-1 contains a sequence motif that is highly related to the Rb binding sites of several viral oncoproteins and binds to the pocket region of Rb both in vitro and in vivo. Elf-1 binds exclusively to the underphosphorylated form of Rb and fails to bind to Rb mutants derived from patients with retinoblastoma. Co-immunoprecipitation experiments demonstrated an association between Elf-1 and Rb in resting normal human T cells. After T cell activation, the phosphorylation of Rb results in the release of Elf-1, which is correlated temporally with the activation of Elf-1-mediated transcription. Overexpression of a phosphorylation-defective form of Rb inhibited Elf-1-dependent transcription during T cell activation. These results demonstrate that Rb interacts specifically with a lineage-restricted Ets transcription factor. This regulated interaction may be important for the coordination of lineage-specific effector functions such as lymphokine production with cell cycle progression in activated T cells. 


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Human cytomegalovirus induces interleukin-8 production by a human monocytic cell line, THP-1, through acting concurrently on AP-1- and NF-kappaB-binding sites of the interleukin-8 gene. 
Cytomegalovirus (CMV) infection induced interleukin-8 (IL-8) gene transcription in a human monocytic cell line, THP-1 cells, leading to IL-8 secretion. The functional analysis of the IL-8 gene revealed that both AP-1- and NF-kappaB factor-binding elements were involved in conferring the responsiveness to CMV. Moreover, electrophoretic mobility shift assays demonstrated that CMV induced the formation of NF-kappaB and AP-1 complexes. These results suggest that CMV activates these transcriptional factors, resulting in IL-8 gene expression. 


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The NF kappa B independent cis-acting sequences in HIV-1 LTR responsive to T-cell activation. 
The rate of transcription initiation directed by the long terminal repeat (LTR) of HIV-1 increases in response to mitogenic stimuli of T cells. Here we show that the response of the HIV-1 LTR may be governed by two independent sequences located 5' to the site of transcription initiation sequences that bind either NFAT-1 or NF kappa B. The rate of LTR-directed gene expression increased in response to treatment with either a phorbol ester or tumor necrosis factor alpha if either the NFAT-1 or NF kappa B binding sites were deleted, but failed to respond to these mitogenic stimuli if both sequences were absent. The HIV-1 mutant virus containing both NF kappa B and NFAT-1 deletion was able to replicate although at a much decreased growth rate, while the deletion of NFAT-1 alone increased the viral growth rate in Jurkat cells. Neither deletion of NF kappa B nor deletion of NFAT-1 decreased activation of viral replication by phorbol ester. 


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Control of NF-kappa B activity by the I kappa B beta inhibitor. 
The transcription factor NF-kappa B is maintained in an inactive cytoplasmic state by I kappa B inhibitors. In mammalian cells, I kappa B alpha and I kappa B beta proteins have been purified and shown to be the inhibitors of NF-kappa B through their association with the p65 or c-Rel subunits. In addition, we have isolated a third NF-kappa B inhibitor, I kappa B epsilon (1). Upon treatment with a large variety of inducers, I kappa B alpha, I kappa B beta are proteolytically degraded, resulting in NF-kappa B translocation into the nucleus. Here we show that in E29.1 T cell hybridoma I kappa B alpha and I kappa B beta are equally associated with p65 and that I kappa B beta is degraded in response to TNF alpha in contrast to what has been originally published. Our data also suggest that, unlike I kappa B alpha, I kappa B beta is constitutively phosphorylated and resynthesized as a hypophosphorylated form. The absence of slow migrating forms of I kappa B beta following stimulation suggests that the phosphorylation does not necessarily constitute the signal-induced event which targets the molecule for proteolysis. 


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Transcription of a minimal promoter from the NF-IL6 gene is regulated by CREB/ATF and SP1 proteins in U937 promonocytic cells. 
NF-IL6 is an important transcriptional regulator of genes induced in activated monocytes/macrophages, and NF-IL6 is the only CCAAT/enhancer-binding protein (C/EBP) family member whose steady-state mRNA levels increase upon activation of monocytes (1). We show that increased transcription of the NF-IL6 gene is responsible, at least in part, for induction of NF-IL6 mRNA following activation of U937 promonocytic cells. We have identified a 104-bp minimal promoter region of the NF-IL6 gene that is sufficient for basal and activation-dependent induction of transcription in U937 cells. This region contains binding sites for the cAMP response element-binding protein/activation transcription factor (CREB/ATF) and Sp1 families of transcription factors. Each site is functionally important and contributes independently to transcription of the NF-IL6 gene in U937 cells. 


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Identification of nucleotide sequences that regulate transcription of the MCF13 murine leukemia virus long terminal repeat in activated T cells. 
The region downstream of the enhancer (DEN) of the long terminal repeat of the mink cell focus-forming murine leukemia virus is important for viral pathogenicity. Another important activity of DEN is its control of transcription in activated T cells, and we have determined that an NF-kappaB site is critical for this activity. 


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Lymphoid specific gene expression of the adenovirus early region 3 promoter is mediated by NF-kappa B binding motifs. 
A primary site of infection by human adenoviruses is lymphoid cells. However, analysis of the viral control elements and the cellular factors that regulate adenoviral gene expression in lymphocytes has not been reported. The adenovirus early region 3 (ES) gene products are involved in the maintenance of viral persistence by complexing with the class I MHC antigens, thus preventing their cell surface expression with a resultant decrease in host immunologic destruction. To determine whether different cellular factors were involved in E3 regulation in lymphocytes as compared with HeLa cells, both DNA binding and transfection analysis with the E3 promoter in both cell types were performed. These studies detected two novel domains referred to as L1 and L2 with a variety of lymphoid but not HeLa extracts. Each of these domains possessed strong homology to motifs previously found to bind the cellular factor NF-kappa B. Transfections of E3 constructs linked to the chloramphenicol acetyltransferase gene revealed that mutagenesis of the distal NF-kappa B motif (L2) had minimal effects on promoter expression in HeLa cells, but resulted in dramatic decreases in expression by lymphoid cells. In contrast, mutagenesis of proximal NF-kappa B motif (L1) had minimal effects on gene expression in both HeLa cells and lymphoid cells but resulted in a small, but reproducible, increase in gene expression in lymphoid cells when coupled to the L2 mutation. Reversing the position and subsequent mutagenesis of the L1 and L2 domains indicated that the primary sequence of these motifs rather than their position in the E3 promoter was critical for regulating gene expression. (ABSTRACT TRUNCATED AT 250 WORDS) 


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The Megakaryocyte/Platelet-specific enhancer of the alpha2beta1 integrin gene: two tandem AP1 sites and the mitogen-activated protein kinase signaling cascade. 
The alpha2beta1 integrin, a collagen receptor on platelets and megakaryocytes, is required for normal platelet function. Transcriptional regulation of the alpha2 integrin gene in cells undergoing megakaryocytic differentiation requires a core promoter between bp -30 and -92, a silencer between bp -92 and -351, and megakaryocytic enhancers in the distal 5' flank. We have now identified a 229-bp region of the distal 5' flank of the alpha2 integrin gene required for high-level enhancer activity in cells with megakaryocytic features. Two tandem AP1 binding sites with dyad symmetry are required for enhancer activity and for DNA-protein complex formation with members of the c-fos/c-jun family. The requirement for AP1 activation suggested a role for the mitogen-activated protein kinase (MAPK) signaling pathway in regulating alpha2 integrin gene expression. Inhibition of the MAP kinase cascade with PD98059, a specific inhibitor of MAPK kinase 1, prevented the expression of the alpha2 integrin subunit in cells induced to become megakaryocytic. We provide a model of megakaryocytic differentiation in which expression of the alpha2 integrin gene requires signaling via the MAP kinase pathway to activate two tandem AP1 binding sites in the alpha2 integrin enhancer. 


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The evolutionarily conserved sequence upstream of the human Ig heavy chain S gamma 3 region is an inducible promoter: synergistic activation by CD40 ligand and IL-4 via cooperative NF-kappa B and STAT-6 binding sites. 
Germline C gamma gene transcription is a crucial event in the process that leads to switch DNA recombination to IgG, but its regulation in the human is poorly understood. We took advantage of our monoclonal model of germinal center B cell differentiation, IgM+ IgD+ CL-01 cells, to define the role of the I gamma 3 evolutionarily conserved sequence (ECS) in the germline transcriptional activation of the human C gamma 3 gene. The I gamma 3 ECS lies upstream of the major I gamma 3 transcription initiation site and displays more than 90% identity with the corresponding human I gamma 1, I gamma 2, and I gamma 4 regions. Reporter luciferase gene vectors containing the human gamma 3 ECS were used to transfect CL-01 cells, which have been shown to undergo Smu-->S gamma 3 DNA recombination, upon engagement of CD40 by CD40 ligand (CD40L) and exposure to IL-4. In these transfected CL-01 cells, CD40:CD40L engagement and exposure to IL-4 synergistically induced gamma 3 ECS-dependent luciferase reporter gene activation. Targeted mutational analysis demonstrated that a tandem NF-kappa B/Rel binding motif is critical for the gamma 3 ECS responsiveness to both CD40L and IL-4, while a STAT-6-binding site is additionally required for IL-4 inducibility. Electrophoretic mobility shift assays showed that p50/p65/c-Rel and STAT-6 are effectively induced by CD40L and IL-4, respectively, and bind to specific DNA motifs within the ECS. These partially overlapping CD40L and IL-4 responsive elements are functionally cooperative as the disruption of one of them prevents synergistic promoter activation. Thus, the gamma 3 ECS is an inducible promoter containing cis elements that critically mediate CD40L and IL-4-triggered transcriptional activation of the human C gamma 3 gene. 


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Transcriptional control of the IL-5 gene by human helper T cells: IL-5 synthesis is regulated independently from IL-2 or IL-4 synthesis. 
BACKGROUND: IL-5 is fundamentally involved in eosinophilic inflammation. Control of IL-5 production may be effective for the management of allergic diseases. OBJECTIVE: We aimed to find the transcriptional mechanisms that regulate the IL-5 gene to selectively control IL-5 synthesis. METHODS: Allergen-specific T-cell clones and T-cell hybridomas were established from the peripheral blood lymphocytes of patients with asthma, and the transcriptional regulation of the IL-5 gene was investigated with transient transfection and electrophoretic mobility shift analysis. RESULTS: A human IL-5 promoter/enhancer-luciferase gene construct, pIL-5(-511)Luc, was transcribed on activation of IL-5-producing T-cell clones, but not IL-5-nonproducing clones. pIL-5(-511)Luc was transcribed by T-cell hybridomas derived from fusion between IL-5-producing T-cell clones and an IL-5 gene-nonexpressing T-cell line, but not by hybridomas derived from IL-5-nonproducing T-cell clones. IL-5 synthesis was not only induced by T-cell receptor stimulation but also by IL-2 receptor stimulation. Binding of NF-AT, NF-kappaB, and AP-1 was induced by T-cell receptor (TcR) stimulation, although there was no significant upregulation of binding by IL-2 stimulation. CONCLUSION: IL-5 synthesis by human helper T cells is regulated at the transcriptional level. A unique transcriptional mechanism distinct from those regulating the IL-2 or IL-4 genes seems to control the IL-5 gene. Selective regulation of IL-5 gene transcription may be useful for treating eosinophlic inflammation. 


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Molecular mechanisms of neutrophil-endothelial cell adhesion induced by redox imbalance. 
Previous studies have implicated a role for intracellular thiols in the activation of nuclear factor-kappaB and transcriptional regulation of endothelial cell adhesion molecules. This study was designed to determine whether changes in endothelial cell glutathione (GSH) or oxidized glutathione (GSSG) can alter neutrophil adhesivity and to define the molecular mechanism that underlies this GSSG/GSH-induced adhesion response. Treatment of human umbilical vein endothelial cell (HUVEC) monolayers for 6 hours with 0.2 mmol/L diamide and 1 mmol/L buthionine sulfoximine (BSO) decreased GSH levels and increased the ratio of GSSG to GSH without cell toxicity. These redox changes are similar to those observed with anoxia/reoxygenation. Diamide plus BSO-induced thiol/disulfide imbalance was associated with a biphasic increase in neutrophil adhesion to HUVECs with peak responses observed at 15 minutes (phase 1) and 240 minutes (phase 2). N-Acetylcysteine treatment attenuated neutrophil adhesion in both phases, which indicated a role for GSH in the adhesion responses. Interestingly, phase 1 adhesion was inversely correlated with GSH levels but not with the GSSG/GSH ratio, whereas phase 2 neutrophil adhesion was positively correlated with GSSG/GSH ratio but not with GSH levels. Intercellular adhesion molecule-1 and P-selectin-specific monoclonal antibodies attenuated the increased neutrophil adhesion during both phases, whereas an anti-E-selectin monoclonal antibody also attenuated the phase 2 response. Pretreatment with actinomycin D and cycloheximide or with competing ds-oligonucleotides that contained nuclear factor-kappaB or activator protein-1 cognate DNA sequences significantly attenuated the phase 2 response, which implicated a role for de novo protein synthesis. Surface expression of intercellular adhesion molecule-1, P-selectin, and E-selectin on HUVECs correlated with the phase 1 and 2 neutrophil adhesion responses. This study demonstrates that changes in endothelial cell GSSG/GSH cause transcription-independent and transcription-dependent surface expression of different endothelial cell adhesion molecules, which leads to a 2-phase neutrophil-endothelial adhesion response. 


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Characterization of NF(P), the nuclear factor that interacts with the regulatory P sequence (5'-CGAAAATTTCC-3') of the human interleukin-4 gene: relationship to NF-kappa B and NF-AT. 
The P sequence of the human interleukin-4 (IL-4) gene, which was defined as a responsive element for phorbol 12-myristate 13-acetate and calcium ionophore (A23187) in Jurkat T cells, shares sequence similarity with the NF-kappa B and the NF-AT binding sites. We examined whether NF(P), a nuclear factor specific for the P sequence, is related to NF-kappa B and NF-AT. NF-kappa B (P65 or P65/P50 heterodimer) bound to the P sequence in electrophoretic mobility shift assays (EMSA) and activated transcription through the P sequence when expression plasmids were cotransfected with P sequence-driven reporter plasmids in Jurkat T cells. In EMSAs, NF(P) binding was inhibited by the unlabeled NF-AT binding site but not by the unlabeled AP1 binding site and purified NF-AT contained an activity that bound to the P sequence. Both mobility shift and sequence specificity of NF-AT were similar to those of NF(P) and only a small amount of P65 was detected in NF(P) in crude nuclear extracts. These results indicate that the component(s) of NF-AT has the potential to reconstitute NF(P) whereas NF-kappa B alone cannot account for NF(P) in crude extracts. Unlike NF-AT, NF(P) does not contain AP1 as its DNA binding component. 


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Activation of early growth response 1 gene transcription and pp90rsk during induction of monocytic differentiation. 
The present work has studied mechanisms responsible for induction of early growth response 1 (EGR-1) gene expression during monocytic differentiation of U-937 myeloid leukemia cells. Differentiation of U-937 cells with 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of the serine/threonine protein kinase C, was associated with transcriptional activation of EGR-1 promoter-reporter constructs. The EGR-1 promoter contains six CC(A/T)6GG (CArG) motifs. The two 5'-most distal CArG sequences conferred TPA inducibility. In contrast, there was little effect of TPA on EGR-1 transcription in a TPA-resistant U-937 cell variant, designated TUR. Treatment of both U-937 and TUR cells with okadaic acid, an inhibitor of serine/threonine protein phosphatases 1 and 2A, was associated with induction of monocytic differentiation and EGR-1 transcription through the 5'-most CArG element. Since these findings supported the involvement of serine/threonine protein phosphorylation in the regulation of EGR-1 expression, we studied activation of the 40S ribosomal protein S6 serine/threonine kinases, pp70S6K and pp90rsk. Although both kinases participate in regulating cell growth, there was no detectable activation of pp70S6K during TPA- or okadaic acid-induced monocytic differentiation. Moreover, rapamycin, an inhibitor of pp70S6K activation, had no effect on induction of EGR-1 expression. In contrast, analysis of pp90rsk activity by phosphorylation of a peptide derived from S6 protein demonstrated stimulation of this kinase in TPA-treated U-937, and not TUR, cells. Okadaic acid treatment of both cell types was associated with activation of pp90rsk. 


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Pyrrolidine dithiocarbamate, a potent inhibitor of nuclear factor kappa B (NF-kappa B) activation, prevents apoptosis in human promyelocytic leukemia HL-60 cells and thymocytes. 
We examined the effect of pyrrolidine dithiocarbamate (PDTC), which potently blocks the activation of nuclear factor kappa B (NF-kappa B), on the induction of apoptosis by a variety of agents. Treatment of a human promyelocytic leukemia cell line, HL-60, with 10 micrograms/mL etoposide or 2 microM 1-beta-D-arabinofuranosylcytosine induced NF-kappa B activation within 1 hr and subsequently caused apoptosis within 3-4 hr. The simultaneous addition of 50-500 microM PDTC with these agents blocked NF-kappa B activation and completely abrogated both morphologically apoptotic changes and internucleosomal DNA fragmentation for up to 6 hr. However, PDTC failed to inhibit the endonuclease activity contained in the whole cell lysates. The inhibitory effect of PDTC was also observed in etoposide- and dexamethasone-induced apoptosis in human thymocytes at a concentration of 1-10 microM. Since PDTC has both antioxidant and metal-ion chelating activities, we tested the effects of N-acetyl-L-cysteine (NAC) (antioxidant) or o-phenanthroline (OP) (metal-ion chelator) on the induction of apoptosis. Pretreatment of HL-60 cells or thymocytes with 100-500 microM OP for 2 hr, but not 10-60 mM NAC, suppressed subsequent occurrence of apoptosis induced by etoposide. These results suggest that the activation of NF-kappa B plays an important role in the apoptotic process of human hematopoietic cells. 


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LPS tolerance in monocytes/macrophages: three 3' cytosins are required in the DNA binding motif for detection of upregulated NF-kappa B p50 homodimers. 
When monocytes are stimulated with LPS (lipopolysaccharide) repeatedly then the initially high expression of the TNF (tumor necrosis factor) gene is only very low, i.e. the cells are tolerant to LPS. Tolerant cells still express the CD14 receptor and they can still be activated to mobilize NF-kappa B into nucleus. Analysis of the binding proteins employing the -605 motif of the human TNF promoter (GGGGCTGTCCC) revealed that in tolerant cells of the human monocytic cell line Mono Mac 6 there is a predominance of p50p50 of NF-kappa B. We now show that a mutant motif that exchanges the terminal 3' C for a G fails to bind the p50 homodimer that is upregulated in LPS toler ant human Mono Mac 6 cells. The same is true for nuclear extracts taken from the murine P388D1 macrophage cell line when tested with the -516 motif of the murine TNF promoter (GGGGGCTTTCCC). Here the wild type motif gives efficient binding of p50p50 that again is upregulated in tolerant cells whereas a mutant with a 3' G shows hardly any binding of p50p50. Conversely, the murine kappa light chain enhancer motif (GGGGACTTTCCG) does not efficiently bind the nuclear p50p50 from tolerant murine P388 macrophages. Binding is, however, readily detected when the 3' G is replaced by a C. These data show that the detection of upregulated p50 homodimers in LPS tolerant cells is dependent on subtle differences in the sequence of the DNA binding motif. 


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Induction of Mn SOD in human monocytes without inflammatory cytokine production by a mutant endotoxin. 
Endotoxin selectively induces monocyte Mn superoxide dismutase (SOD) without affecting levels of Cu,Zn SOD, catalase, or glutathione peroxidase. However, little is known about the structure-activity relationship and the mechanism by which endotoxin induces Mn SOD. In this study we demonstrated that a mutant Escherichia coli endotoxin lacking myristoyl fatty acid at the 3' R-3-hydroxymyristate position of the lipid A moiety retained its full capacity to coagulate Limulus amoebocyte lysate compared with the wild-type E. coli endotoxin and markedly stimulated the activation of human monocyte nuclear factor-kappaB and the induction of Mn SOD mRNA and enzyme activity. However, in contrast to the wild-type endotoxin, it failed to induce significant production of tumor necrosis factor-alpha and macrophage inflammatory protein-1alpha by monocytes and did not induce the phosphorylation and nuclear translocation of mitogen-activated protein kinase. These results suggest that 1) lipid A myristoyl fatty acid, although it is important for the induction of inflammatory cytokine production by human monocytes, is not necessary for the induction of Mn SOD, 2) endotoxin-mediated induction of Mn SOD and inflammatory cytokines are regulated, at least in part, through different signal transduction pathways, and 3) failure of the mutant endotoxin to induce tumor necrosis factor-alpha production is, at least in part, due to its inability to activate mitogen-activated protein kinase. 


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Of the GATA-binding proteins, only GATA-4 selectively regulates the human IL-5 gene promoter in IL-5 producing cells which express multiple GATA-binding proteins. 
Interleukin-5 (IL-5) is produced by T lymphocytes and known to support B cell growth and eosinophilic differentiation of the progenitor cells. Using ATL-16T cells which express IL-5 mRNA, we have identified a region, within the human IL-5 gene promoter, that regulates IL-5 gene transcription. This cis-acting sequence contains the core binding motif, (A/T)GATA(A/G), for GATA-binding family proteins and thus suggests the involvement of these family members. In this report, we describe the cloning of human GATA-4 (hGATA-4) and show that hGATA-4 selectively interacts with the -70 GATA site within the IL-5 proximal promoter region. By promoter deletion and mutation analyses, we established this region as a positive regulatory element. Cotransfection experiments revealed that both hGATA-4 and PMA/A23187 stimulation are necessary for the IL-5 promoter activation. The requirement of another regulatory element called CLE0, which lies downstream of the -70 GATA site, was also demonstrated. ATL-16T cells express mRNA of three GATA-binding proteins, hGATA-2, hGATA-3 and hGATA-4, and each of them has a potential to bind to the consensus (A/T)GATA(G/ A) motif. However, using ATL-16T nuclear extract, we demonstrated that GATA-4 is the only GATA-binding protein that forms specific DNA-protein complex with the -70 GATA site. The electrophoretic mobility shift assay with extracts of COS cells expressing GATA-binding proteins showed that GATA-4 has the highest binding affinity to the -70 GATA site among the three GATA-binding proteins. When the transactivation ability was compared among the three, GATA-4 showed the highest activity. These results demonstrate the selective role of GATA-4 in the transcriptional regulation of the IL-5 gene in a circumstance where multiple members of the GATA-binding proteins are expressed. 


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Expression of erythroid-specific genes in megakaryoblastic disorders. 
Currently available data indicate that erythroid and megakaryocytic differentiation pathways are closely related to each other, and there may exist progenitor cells common to those two lineages may exist. Acute megakaryoblastic leukemia (AML-M7) and transient myeloproliferative disorder in Down's syndrome (TMD) are characterized by rapid growth of abnormal blast cells which express megakaryocytic markers. These blast cells express lineage-specific transcription factors such as GATA-1 common to these lineages and frequently express erythroid-specific mRNAs such as gamma-globin and erythroid delta-aminolevulinate synthase (ALAS-E), indicating that most of the blasts in M7 and TMD cases have erythroid and megakaryocytic phenotypes. These results suggest that blasts in M7 and TMD may correspond to progenitors of both erythroid and megakaryocytic lineages. 


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Replication of human immunodeficiency virus-1 in primary human T cells is dependent on the autocrine secretion of tumor necrosis factor through the control of nuclear factor-kappa B activation. 
Tumor necrosis factor (TNF)-alpha controls T-cell activation and is a major inducer of human immunodeficiency virus (HIV)-1 replication in chronically infected cells. Therefore, we have investigated its role in primary cultures of HIV-infected human T lymphocytes by using neutralizing anti-TNF-alpha antibodies or TNF-alpha. Primary resting T lymphocytes produced TNF-alpha and supported HIV replication after T-cell receptor activation. Addition of neutralizing anti-TNF-alpha antibodies drastically reduced p24 antigen release and prevented CD4+ cell depletion associated with infection. Anti-TNF-alpha also prevented nuclear factor-kappa B (NF-kappa B) activation, and a good correlation between this inhibition and inhibition of HIV replication was observed. Moreover, supplementing the cultures with high doses of IL-2 reverted anti-TNF-alpha inhibition of cell proliferation but did not affect the inhibition of HIV p24 antigen release or NF-kappa B activation in the same cultures. Moreover, anti-TNF-alpha inhibited HIV-1 long terminal repeat (LTR)-driven transcription of a reporter gene in primary T cells in response to activation, either in the presence or the absence of HIV-1 Tat. Our results support an important role for autocrine TNF-alpha secretion in controlling HIV replication in primary T cells because of its ability to maintain NF-kappa B elevated in the nucleus of T cells. 


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A novel lipopolysaccharide-induced transcription factor regulating tumor necrosis factor alpha gene expression: molecular cloning, sequencing, characterization, and chromosomal assignment. 
Lipopolysaccharide (LPS) is a potent stimulator of monocytes and macrophages, causing secretion of tumor necrosis factor alpha (TNF-alpha) and other inflammatory mediators. Given the deleterious effects to the host of TNF-alpha, it has been postulated that TNF-alpha gene expression must be tightly regulated. The nature of the nuclear factor(s) that control TNF-alpha gene transcription in humans remains obscure, although NF-kappaB has been suggested. Our previous studies pertaining to macrophage response to LPS identified a novel DNA-binding domain located from -550 to -487 in the human TNF-alpha promoter that contains transcriptional activity, but lacks any known NF-kappaB-binding sites. We have used this DNA fragment to isolate and purify a 60-kDa protein binding to this fragment and obtained its amino-terminal sequence, which was used to design degenerate probes to screen a cDNA library from THP-1 cells. A novel cDNA clone (1.8 kb) was isolated and fully sequenced. Characterization of this cDNA clone revealed that its induction was dependent on LPS activation of THP-1 cells; hence, the name LPS-induced TNF-alpha factor (LITAF). Inhibition of LITAF mRNA expression in THP-1 cells resulted in a reduction of TNF-alpha transcripts. In addition, high level of expression of LITAF mRNA was observed predominantly in the placenta, peripheral blood leukocytes, lymph nodes, and the spleen. Finally, chromosomal localization using fluorescence in situ hybridization revealed that LITAF mapped to chromosome 16p12-16p13.3. Together, these findings suggest that LITAF plays an important role in the activation of the human TNF-alpha gene and proposes a new mechanism to control TNF-alpha gene expression. 


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Human T cell activation through the activation-inducer molecule/CD69 enhances the activity of transcription factor AP-1. 
The induction of the AP-1 transcription factor has been ascribed to the early events leading to T cell differentiation and activation. We have studied the regulation of AP-1 activity in human peripheral blood T lymphocytes stimulated through the activation inducer molecule (AIM)/CD69 activation pathway. Phorbol esters are required to induce AIM/CD69 cell-surface expression as well as for triggering the proliferation of T cells in conjunction with anti-AIM mAb. Mobility shift assays showed that addition of anti-AIM mAb to PMA-treated T lymphocytes markedly enhanced the binding activity of AP-1 to its cognate sequence, the phorbol ester response element. In contrast, anti-AIM mAb did not induce any change in the binding activity of NF-kappa B, a transcription factor whose activity is also regulated by protein kinase C. The increase in AP-1-binding activity was accompanied by the marked stimulation of the transcription of c-fos but not that of c-jun. Blockade of the DNA-binding complexes with an anti-Fos mAb demonstrated a direct participation of c-Fos in the AP-1 complexes induced by anti-AIM mAb. Most of the AP-1 activity could be eliminated when the anti-AIM mAb was added to the culture medium in the presence of cycloheximide, suggesting that de novo protein synthesis is crucial for the induction of AP-1-binding activity. These data provide the evidence that activation of human peripheral blood T cells through the AIM activation pathway regulate the activity of AP-1. Therefore, this pathway appears as a crucial step in the initiation of early T cell activation events. 


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A critical role of Sp1- and Ets-related transcription factors in maintaining CTL-specific expression of the mouse perforin gene. 
This study was designed to determine the potential cis-elements involved in transcriptional regulation of the mouse perforin gene. DNase I hypersensitive site (DHS) mapping revealed that the perforin locus contained six DHS within 7.0 kb of the 5' upstream sequence (-7.0 kb) and two DHS in intron 2. The six 5' upstream and one intronic DHS were detected in only perforin-expressing lymphocytes. Chloramphenicol acetyltransferase (CAT) activities directed by 5' upstream promoter were detected preferentially in perforin-expressing cell lines. A construct termed PFP5a containing -795 bp exhibited the highest CAT activity, and PFP9a20 containing only -73 bp also produced significantly high CAT activity in CTLL-R8 cells. The proximal region in PFP9a20 contained two potential Sp1 binding sites (GC box and GT box) and one Ets binding site (EBS). Electrophoretic mobility shift assay showed that each of the cis-elements bound specific protein factors. When single-point mutation was introduced to each GC box, EBS, and GT box in PFP9a20, at least 3-fold less CAT activity was observed in CTLL-R8 cells. To confirm the importance of the three cis-acting elements in the perforin gene expression, point mutation was introduced again to each proximal GC box, EBS, and GT box of PFP5a. The point mutations resulted in a 2.5- to 3-fold reduction of CAT activity. The results suggest that a combination of the three proximal cis-acting elements may constitute a minimal region responsible for CTL-specific expression of perforin. 


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Retinoic acid downmodulates erythroid differentiation and GATA1 expression in purified adult-progenitor culture. 
All-trans retinoic acid (RA) is an important morphogen in vertebrate development, a normal constituent in human adult blood and is also involved in the control of cell growth and differentiation in acute promyelocytic leukemia. We have examined the effects of RA on normal hematopoiesis by using early hematopoietic progenitor cells (HPC) stringently purified from adult peripheral blood. In clonogenetic fetal calf serum-supplemented (FCS+) or -nonsupplemented (FCS-) culture treated with saturating levels of interleukin-3 (IL-3) granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin (Ep) (combined with c-kit ligand in FCS(-)-culture conditions), RA induces a dramatic dose-dependent shift from erythroid to granulomonocytic colony formation, the latter colonies being essentially represented by granulocytic clones. This shift is apparently not caused by a recruitment phenomenon, because in FCS+ culture, the total number of colonies is not significantly modified by RA addition. In FCS- liquid-suspension culture supplemented with saturating Ep level and low-dose IL-3/GM-CSF, adult HPC undergo unilineage erythropoietic differentiation: Here again, treatment with high-dose RA induces a shift from the erythroid to granulocytic differentiation pathway. Studies on RA time-response or pulse treatment in semisolid or liquid culture show that early RA addition is most effective, thus indicating that early but not late HPC are sensitive to its action. We then analyzed the expression of the master GATA1 gene, which encodes a finger transcription factor required for normal erythroid development; addition of RA to HPC stimulated into unilineage erythropoietic differentiation in liquid culture caused a virtually complete inhibition of GATA1 mRNA induction. These results indicate that RA directly inhibits the erythroid differentiation program at the level of early adult HPC, and may lead to a shift from the erythroid to granulocytic differentiation pathway. This phenomenon is correlated with inhibition of GATA1 induction in the early stages of erythropoietic differentiation. 


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STAT-related transcription factors are constitutively activated in peripheral blood cells from acute leukemia patients. 
A signal transduction pathway activated by many cytokines has recently been elaborated. The JAK kinases and the signal transducers and activators of transcription (STAT) factors have been found to be essential components. In this report, we describe the presence of constitutively activated STAT factors in peripheral blood cells from patients with acute leukemia. We used oligonucleotide probes from the beta-casein and IRF-1 gene promoters and the ISRE probe to detect STAT proteins in nuclear extracts from acute leukemia cells in bandshift assays. Specific DNA protein complex formation was observed with the probes from the beta-casein and IRF-1 gene promoters, but not with the ISRE oligonucleotide probe, when cell extracts from acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) were investigated. We used nonradioactive oligonucleotides as competitors to show the specificity of the complex formation. Specific antibodies directed against the individual STAT proteins were used in supershift experiments. STAT5- and STAT1-related factors were detected in ALL and STAT1-, STAT3-, and STAT5-related proteins were present in nuclear cell extracts from AML. Since the cells were not treated with cytokines before the nuclear proteins were extracted, we conclude that these factors are constitutively activated in vivo. It is likely that the constitutive activation of STAT proteins is a part of the events of leukemogenesis. 


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Functional antagonism between vitamin D3 and retinoic acid in the regulation of CD14 and CD23 expression during monocytic differentiation of U-937 cells. 
1,25 alpha-Dihydroxicholecalciferol (VitD3) and retinoic acid (RA) are important regulators of the proliferation and differentiation of several cell types. This paper describes how the expression of the monocyte-macrophage Ag, CD14, and the low affinity Fc receptor for IgE, CD23, were inversely regulated during VitD3- and RA-induced monocytic differentiation of human U-937 monoblasts. PMA induced the expression of both CD14 and CD23 mRNA and protein. Exposure to VitD3 rapidly induced the de novo expression of CD14 mRNA and protein. The addition of cycloheximide completely blocked the VitD3 induction of CD14 mRNA expression, indicating that the induction was dependent on ongoing protein synthesis. While inducing CD14 expression, VitD3 concomitantly suppressed the basal, PMA-, and RA-inducible CD23 expression in a dose-dependent manner. In contrast, U-937 cells induced by RA strongly increased their expression of CD23 mRNA and protein, whereas they completely lacked detectable CD14 cell surface or mRNA expression. Furthermore, the VitD3- and the PMA-induced CD14 expression was inhibited as a temporal consequence of the RA-induced differentiation. The results suggest that there exists a functional antagonism between VitD3 and RA that may have important implications for the regulation of certain immune and inflammatory responses through their inverse effects on CD14 and CD23 gene expression. 


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Peripheral blood mononuclear cells isolated from patients with diabetic nephropathy show increased activation of the oxidative-stress sensitive transcription factor NF-kappaB. 
Increased oxidative stress and subsequent activation of the transcription factor NF-kappaB has been linked to the development of late diabetic complications. To determine whether oxidative stress dependent NF-kappaB activation is evident in patients with diabetic nephropathy we used an Electrophoretic Mobility Shift Assay based semiquantitative detection system which enabled us to determine NF-kappaB activation in ex vivo isolated peripheral blood mononuclear cells. We examined 33 patients with diabetes mellitus (Type I and Type II). Patients with diabetic nephropathy showed higher NF-kappaB binding activity in Electrophoretic Mobility Shift Assays and stronger immunohistological staining for activated NF-kappaBp65 than patients without renal complications. NF-kappaB binding activity correlated with the degree of albuminuria (r = 0.316) and with thrombomodulin plasma concentrations (r = 0.33), indicative for albuminuria associated endothelial dysfunction. In a 3 day intervention study in which 600 mg of the antioxidant thioctic acid (alpha-lipoic acid) per day were given to nine patients with diabetic nephropathy oxidative stress in plasma samples was decreased by 48% and NF-kappaB binding activity in ex vivo isolated peripheral blood mononuclear cells by 38%. In conclusion, activation of the transcription factor NF-kappaB in ex vivo isolated peripheral blood mononuclear cells of patients with diabetes mellitus correlates with the degree of diabetic nephropathy. NF-kappaB activation is at least in part dependent on oxidative stress since thioctic acid (alpha-lipoic acid) reduced NF-kappaB binding activity. 


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Induction of NF-kappa B during monocyte differentiation is associated with activation of HIV-gene expression. 
Cells of the monocyte-macrophage lineage are important targets of HIV infection. We report here that the phenotypic differentiation of monocyte cell lines induced by phorbol esters or tumour necrosis factor alpha (TNF alpha) is associated with expression of nuclear factor kappa B (NF-kappa B). In parallel with such differentiation, HIV transcription, monitored using an HIV long terminal repeat reporter gene construct, is activated in such cells under the influence of enhanced NF-kappa B expression. Also, in a promonocyte cell line chronically infected with HIV, NF-kappa B expression and HIV transcription were enhanced on stimulation with phorbol ester or TNF alpha. Thus, stimulation of monocyte cell lines by phorbol esters or TNF alpha induces cell differentiation and activates HIV transcription. Such a process may have fundamental implications in AIDS pathogenesis in vivo and may be important in disease progression induced by opportunistic infections directly or indirectly involving macrophages. 


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Hypoxia causes the activation of nuclear factor kappa B through the phosphorylation of I kappa B alpha on tyrosine residues. 
The response of mammalian cells to stress is controlled by transcriptional regulatory proteins such as nuclear factor kappa B (NF-kappa B) to induce a wide variety of early response genes. In this report, we show that exposure of cells to hypoxia (0.02% O2) results in I kappa B alpha degradation, increased NF-kappa B DNA binding activity, and transactivation of a reporter gene construct containing two NF-kappa B DNA binding sites. Pretreatment of cells with protein tyrosine kinase inhibitors and the dominant negative allele of c-Raf-1 (Raf 301) inhibited I kappa B alpha degradation, NF-kappa B binding, and transactivation of kappa B reporter constructs by hypoxia. To demonstrate a direct link between changes in the phosphorylation pattern of I kappa B alpha with NF-kappa B activation, we immunoprecipitated I kappa B alpha after varying times of hypoxic exposure and found that its tyrosine phosphorylation status increased during hypoxic exposure. Inhibition of the transfer of tyrosine phosphoryl groups onto I kappa B alpha prevented I kappa B alpha degradation and NF-kappa B binding. In comparison to other activators of NF-kappa B such as phorbol myristate acetate or tumor necrosis factor, we did not detect changes in the tyrosine phosphorylation status of I kappa B alpha following treatment with either of these agents. These results suggest that tyrosine phosphorylation of I kappa B alpha during hypoxia is an important proximal step which precedes its dissociation and degradation from NF-kappa B. 


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NF-kappaB activation is required for C5a-induced interleukin-8 gene expression in mononuclear cells. 
C5a, a potent peptide chemoattractant, stimulates interleukin-8 (IL-8) secretion from peripheral blood mononuclear cells (PBMC). Experiments were conducted to understand the mechanisms for C5a-induced IL-8 production, which was 14-fold greater than that in unstimulated cells by 2 hours. IL-8 secretion was accompanied by accumulation of IL-8 mRNA in the cytosol and by nuclear expression of a kappaB DNA binding activity within 30 minutes. AP-1 but not NF-IL-6 DNA binding activity was also detected in C5a-stimulated PBMC; however, its delayed expression (maximal at 4 hours) suggested a less important role in the rapid production of IL-8. The correlation between C5a-induced kappaB binding activity and IL-8 gene expression was examined in the RAW264.7 macrophage cells using reporter genes directed by the kappaB sequence from IkappaBalpha and IL-8 promoter regions. C5a-induced reporter gene expression was abolished by introducing mutations into the kappaB sites and by coexpression of a dominant negative IkappaBalpha construct resistant to agonist-induced phosphorylation. Pertussis toxin, which ADP-ribosylates the Gi proteins known to couple to the C5a receptor, produced minimal inhibition of C5a-induced IL-8 expression and had little effect on C5a-induced calcium mobilization in RAW264.7 cells. These results suggest that NF-kappaB activation is required for C5a-induced IL-8 gene expression and that this response is mediated primarily through a pertussis toxin-insensitive pathway. 


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CIITA-induced occupation of MHC class II promoters is independent of the cooperative stabilization of the promoter-bound multi-protein complexes. 
Precise regulation of MHC class II expression plays a crucial role in the control of the immune response. The transactivator CIITA behaves as a master controller of constitutive and inducible MHC class II gene activation, but its exact mechanism of action is not known. Activation of MHC class II promoters requires binding of at least three distinct multi-protein complexes (RFX, X2BP and NF-Y). It is known that the stability of this binding results from cooperative interactions between these proteins. We show here that expression of CIITA in MHC class II- cells triggers occupation of the promoters by these complexes. This observation raised the possibility that the effect of CIITA on promoter occupation is mediated by an effect on the cooperative stabilization of the DNA-bound multi-protein complexes. We show, however, that the presence of CIITA does not affect the stability of the higher-order protein complex formed on DNA by RFX, X2BP and NF-Y. This suggests other mechanisms for CIITA-induced promoter occupancy, such as an effect on chromatin structure leading to increased accessibility of MHC class II promoters. This ability of CIITA to facilitate promoter occupation is undissociable from its transactivation potential. Finally, we conclude that this effect of CIITA is cell-type specific, since expression of CIITA is not required for normal occupation of MHC class II promoters in B lymphocytes. 


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Activation of distinct transcription factors in neutrophils by bacterial LPS, interferon-gamma, and GM-CSF and the necessity to overcome the action of endogenous proteases. 
Human neutrophils can be induced to actively transcribe a number of early-response genes, in particular those encoding cytokines, chemokines, and the high-affinity surface receptor for IgG, FcgammaRI. Although little is known to date about the regulation of gene transcription in neutrophils, several indications point to a role for distinct transcription factors, such as members of the NF-kappaB and STAT families. In this study, we investigated whether these transcription factors become activated under stimulatory conditions which are known to induce gene transcription in neutrophils. Unexpectedly, we found that conventional procedures employed to prepare cellular extracts cause the release of proteolytic activities that are normally stored in intracellular granules, resulting in the degradation of various NF-kappaB/Rel and STAT proteins. To circumvent this problem, we developed an alternative procedure which allowed us to show that in neutrophils, LPS and TNFalpha induce a NF-kappaB DNA-binding activity which essentially consists of p50/RelA dimers, and that IFNgamma promotes the binding of STAT1 homodimers to the IFNgamma response region of the FcgammaRI promoter. Moreover, we report that neutrophil stimulation with GM-CSF results in the formation of a STAT5-containing DNA-binding activity. Collectively, the current findings open new perspectives about mechanisms that are likely to regulate gene transcription in neutrophils. In addition, the procedure described herein could prove useful in other cell types that express high levels of endogenous proteases. 


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Low CD3+CD28-induced interleukin-2 production correlates with decreased reactive oxygen intermediate formation in neonatal T cells. 
The capacity of neonatal T cells to secrete interleukin-2 (IL-2) has been reported to be variable. We analysed IL-2 production in purified neonatal and adult T cells using polyclonal activator phorbol ester + calcium ionophore (PDBu + iono) or receptor-mediated anti-CD3/anti-CD3+ anti-CD28 stimulation. PDBu + iono induced equally high IL-2 levels in both groups and, when stimulated with plate-bound anti-CD3 monoclonal antibody (mAb), the IL-2 secretion by neonatal cells was undetectable and adult cells produced low amounts of IL-2 (mean 331 +/- 86 pg/ml). The addition of anti-CD28 mAb to anti-CD3-stimulated cells markedly increased IL-2 production in both cell types, but levels of IL-2 in neonatal T cells remained clearly lower than those of adult T cells (respective mean values: 385 +/- 109 pg/ml and 4494 +/- 1199 pg/ml). As NF-kappa B is a critical transcription factor in the control of IL-2 expression, we next analysed its nuclear translocation in neonatal and adult T cells using the electrophoretic mobility shift assay and, because induction of reactive oxygen intermediates (ROI) is required for the activation of NF-kappa B, we also analysed levels of intracellular ROI in these cells using the ROI-reactive fluorochrome DCFH-DA and flow cytometry. In neonatal T cells NF-kappa B activation and ROI formation after anti-CD3 stimulation were low compared with adult T cells and, although addition of anti-CD28 mAb increased induction of NF-kappa B and ROI formation, levels similar to those of adults were not achieved. After PDBu + iono stimulation, the cells showed similar ROI formation and IL-2 secretion. Our results suggest that reduced IL-2 production by neonatal T cells is specific for anti-CD3 and anti-CD3+ anti-CD28-mediated stimulation and that these activators cannot effectively activate the ROI-NF-kappa B signalling pathway in neonatal T cells. 


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Aspirin inhibits nuclear factor-kappa B mobilization and monocyte adhesion in stimulated human endothelial cells. 
BACKGROUND: The induction of vascular cell adhesion molecule-1 (VCAM-1) and E-selectin by tumor necrosis factor-alpha (TNF) is mediated by mobilization of the transcription factor nuclear factor-kappa B (NF-kappa B). Since salicylates have been reported to inhibit NF-kappa B activation by preventing the degradation of its inhibitor I kappa B, we studied a potential inhibition of this pathway by acetylsalicylate (aspirin) in human umbilical vein endothelial cells (HUVECs). METHODS AND RESULTS: Gel-shift analyses demonstrated dose-dependent inhibition of TNF-induced NF-kappa B mobilization by aspirin at concentrations ranging from 1 to 10 mmol/L. Induction of VCAM-1 and E-selectin surface expression by TNF was dose-dependently reduced by aspirin over the same range, while induction of intercellular adhesion molecule-1 (ICAM-1) was hardly affected. Aspirin appeared to prevent VCAM-1 transcription, since it dose-dependently inhibited induction of VCAM-1 mRNA by TNF. As a functional consequence, adhesion of U937 monocytes to TNF-stimulated HUVECs was markedly reduced by aspirin due to suppression of VCAM-1 and E-selectin upregulation. These effects of aspirin were not related to the inhibition of cyclooxygenase activity, since indomethacin was ineffective. CONCLUSIONS: Our data suggest that aspirin inhibits NF-kappa B mobilization, induction of VCAM-1 and E-selectin, and subsequent monocyte adhesion in endothelial cells stimulated by TNF, thereby providing an additional mechanism for therapeutic effects of aspirin. 


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T-cell functional regions of the human IL-3 proximal promoter. 
The human interleukin-3 (IL-3) gene is expressed almost exclusively in activated T cells. Its expression is regulated at both the transcriptional and post-transcriptional level. We have previously shown that treatment of Jurkat T cells with phytohemaglutinin (PHA) and the phorbol ester, PMA, activated transcription initiation from the IL-3 gene. To define the regions of the gene required for transcription activation, we generated a series of reporter constructs containing different regions of the IL-3 gene 5' and 3' flanking sequences. Both positive and negative regulatory elements were identified in the proximal 5' flanking region of the IL-3 gene. The promoter region between -173 and -60 contained the strongest activating elements. The transcription factor AP-1 could bind to this positive activator region of the promoter. We also examined the function of the IL-3 CK-1/CK-2 elements that are present in many cytokine genes and found that they acted as a repressor of basal level expression when cloned upstream of a heterologous promoter but were also inducible by PMA/PHA. 


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Negative regulation of human immunodeficiency virus type 1 expression in monocytes: role of the 65-kDa plus 50-kDa NF-kappa B dimer. 
Although monocytic cells can provide a reservoir for viral production in vivo, their regulation of human immunodeficiency virus type 1 (HIV-1) transcription can be either latent, restricted, or productive. These differences in gene expression have not been molecularly defined. In THP-1 cells with restricted HIV expression, there is an absence of DNA-protein binding complex formation with the HIV-1 promoter-enhancer associated with markedly less viral RNA production. This absence of binding was localized to the NF-kappa B region of the HIV-1 enhancer; the 65-kDa plus 50-kDa NF-kappa B heterodimer was preferentially lost. Adding purified NF-kappa B protein to nuclear extracts from cells with restricted expression overcomes this lack of binding. In addition, treatment of these nuclear extracts with sodium deoxycholate restored their ability to form the heterodimer, suggesting the presence of an inhibitor of NF-kappa B activity. Furthermore, treatment of nuclear extracts from these cells that had restricted expression with lipopolysaccharide increased viral production and NF-kappa B activity. Antiserum specific for NF-kappa B binding proteins, but not c-rel-specific antiserum, disrupted heterodimer complex formation. Thus, both NF-kappa B-binding complexes are needed for optimal viral transcription. Binding of the 65-kDa plus 50-kDa heterodimer to the HIV-1 enhancer can be negatively regulated in monocytes, providing one mechanism restricting HIV-1 gene expression. 


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Cooperation of binding sites for STAT6 and NF kappa B/rel in the IL-4-induced up-regulation of the human IgE germline promoter. 
Ig heavy chain class switching is directed by cytokines inducing transcription from unrearranged CH genes. Subsequently, such primed cells can undergo switch recombination to express the selected new isotype. In the case of IgE class switching, IL-4 activates the IgE germline promoter by inducing the interaction of the transcription factor STAT6 (IL-4STAT) with a responsive DNA element in the proximal region of the promoter. This study describes the characterization of two additional cis-acting elements that interact with members of the NF kappa B/rel transcription factor family in an IL-4-independent fashion. Electrophoretic mobility shift assays show that the nucleoprotein complex formed on the upstream site (NF kappa B1) contains the classical p50/p65 heterodimer. The complex on the proximal site (NF kappa B2) appears to be composed of p50 and relB. IgE germline promoter reporter gene constructs carrying point mutations in the NF kappa B2 site were largely unresponsive to IL-4 stimulation in transient transfection experiments, while plasmids with similar mutations in the NF kappa B1 site responded to cytokine stimulation better than the wild-type promoter. The NF kappa B2 effect was dependent on the presence of the STAT6 binding site, demonstrating that the NF kappa B2 motif is necessary but not sufficient for mediating cytokine up-regulation. In addition, the combination of a NF kappa B/rel binding site and the STAT6 response element conferred IL-4 inducibility to a heterologous minimal promoter, while the individual sites had no effect. The available data suggest that the NF kappa B2 nucleoprotein complex may cooperate with DNA-bound STAT6 to achieve IL-4-dependent activation of the human IgE germline gene. 


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Interferons inhibit activation of STAT6 by interleukin 4 in human monocytes by inducing SOCS-1 gene expression. 
Interferons (IFNs) inhibit induction by IL-4 of multiple genes in human monocytes. However, the mechanism by which IFNs mediate this inhibition has not been defined. IL-4 activates gene expression by inducing tyrosine phosphorylation, homodimerization, and nuclear translocation of the latent transcription factor, STAT6 (signal transducer and activator of transcription-6). STAT6-responsive elements are characteristically present in the promoters of IL-4-inducible genes. Because STAT6 activation is essential for IL-4-induced gene expression, we examined the ability of type I and type II IFNs to regulate activation of STAT6 by IL-4 in primary human monocytes. Pretreatment of monocytes with IFN-beta or IFN-gamma, but not IL-1, IL-2, macrophage colony-stimulating factor, granulocyte/macrophage colony-stimulating factor, IL-6, or transforming growth factor beta suppressed activation of STAT6 by IL-4. This inhibition was associated with decreased tyrosine phosphorylation and nuclear translocation of STAT6 and was not evident unless the cells were preincubated with IFN for at least 1 hr before IL-4 stimulation. Furthermore, inhibition by IFN could be blocked by cotreatment with actinomycin D and correlated temporally with induction of the JAK/STAT inhibitory gene, SOCS-1. Forced expression of SOCS-1 in a macrophage cell line, RAW264, markedly suppressed trans-activation of an IL-4-inducible reporter as well as IL-6- and IFN-gamma-induced reporter gene activity. These findings demonstrate that IFNs inhibit IL-4-induced activation of STAT6 and STAT6-dependent gene expression, at least in part, by inducing expression of SOCS-1. 


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Protein kinase C is not a downstream effector of p21ras in activated T cells. 
The aim of this present study was to investigate the role of protein kinase C (PKC), downstream of p21ras, in activating interleukin-2 (IL-2) gene expression. It has been reported that PKC is an effector of p21ras in T cells. Data is presented, using the potent and selective PKC inhibitor Ro 31-8425 and transient expression of a constitutively active ras mutant, which clearly shows that PKC is not downstream of p21ras in the induction of NF-AT and AP-1 transcriptional activity and in the expression of IL-2 in human Jurkat T cells. Reporter gene experiments demonstrated that NF-kappa B transcriptional activity is not affected by expression of activated p21ras. The signaling pathways involving PKC activation, calcium mobilization and ras activation combine to provide the necessary components for production of IL-2 during T cell activation. 


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Non-steroidal anti-inflammatory drugs inhibit the expression of cytokines and induce HSP70 in human monocytes. 
Recent studies have shown that the non-steroidal anti-inflammatory drugs (NSAIDs) activate heat shock transcription factor (HSF1) from a latent cytoplasmic form to a nuclear, DNA binding state. As HSF1 can function as both an activator of heat shock genes and a repressor of non-heat shock genes such as IL1B and c- fos, we have examined the potential role of HSF1 in the effects of NSAIDs on gene expression in a human monocytic cell line THP-1. We found that two members of the NSAIDs, sodium salicylate and sulindac repress the IL1B promoter to similar degree to heat shock or HSF1 overexpression. In addition, sodium salicylate and additional NSAIDs used at concentrations that activate HSF1 also inhibited the expression of other monocytic genes (TNF-alpha, IL-1beta, IL-6, IL-8, IL-10, ICAM-1) activated by exposure to a pro-inflammatory stimulus (lipopolysaccharide, LPS). At least in the case of the IL1B promoter, repression did not seem to involve another factor whose activity is affected by the NSAIDs, NFkappaB as the IL1B promoter fragment used in our studies is not NFkappaB responsive and binds specifically to HSF1. Exposure to NSAIDs had a complex effect on HSP gene expression and while sulindac activated the stress responsive HSP70B promoter, sodium salicylate did not. In addition, only a subset of the NSAIDs induced HSP70 mRNA species. These findings reflect the properties of HSF1 which can be activated to at least two DNA binding forms only one of which activates heat shock promoters and suggest that individual NSAID family members may differentially induce one or other of these forms. Overall therefore, exposure to NSAIDs leads to a profound switch in gene expression in monocytic cells, with suppression of genes involved in macrophage activation and induction of stress genes and HSF1 appears to play a regulatory role in these effects. Copyright 1999 Academic Press. 


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Modulation of mRNA expression of a novel human myeloid-selective CCAAT/enhancer binding protein gene (C/EBP epsilon). 
Human C/EBP epsilon is a newly cloned gene coding for a CCAAT/enhancer binding protein that may be involved in the regulation of myeloid differentiation. Our studies showed that levels of C/EBP epsilon mRNA were markedly increased in NB4 cells (promyelocytic leukemia line), because they were induced by 9-cis retinoic acid (9-cis RA) to differentiate towards granulocytes. Accumulation of C/EBP epsilon mRNA occurred as early as 1 hour after exposure of NB4 cells to 9-cis RA (5 x 10(-7) mol/L); and at 48 hours, levels were increased by 5.1-fold. Dose-response studies showed that 10(-7) to 10(-6) mol/L 9-cis RA (12 hours) resulted in peak levels of C/EBP epsilon mRNA; but even 10(-10) mol/L 9-cis RA increased levels of these transcripts. NB4 cells pulse-exposed (30 minutes) to all-trans retinoic acid (ATRA), washed, and cultured (3 days) with either dimethylsulfoxide (DMSO) or hexamethylene bisacetamide (HMBA) had a prominent increase in levels of C/EBP epsilon mRNA and an increase in granulocytic differentiation, but exposure to either DMSO or HMBA alone had no effect on base levels of C/EBP epsilon and did not induce differentiation. Macrophage-differentiation of NB4 reduced levels of C/EBP epsilon mRNA. Nuclear run-off assays and half-life studies showed that accumulation of C/EBP epsilon mRNA by 9-cis RA was due to enhanced transcription. Furthermore, this C/EBP epsilon mRNA accumulation did not require synthesis of new protein factors because 9-cis RA induced C/EBP epsilon mRNA accumulation in the absence of new protein synthesis. ATRA also induced expression of C/EBP epsilon protein in NB4 cells, as shown by Western blotting. In contrast to the increase of C/EBP epsilon in 9-cis RA-mediated granulocytic differentiation, the DMSO-induced differentiation of HL-60 cells down the granulocytic pathway was associated with an initial reduction of C/EBP epsilon mRNA levels. In summary, we have discovered that expression of C/EBP epsilon mRNA is markedly enhanced as the NB4 promyelocytes are induced by retinoids to differentiate towards granulocytes. This induction of C/EBP epsilon mRNA expression is transcriptionally mediated and occurs in the absence of synthesis of additional protein factors. We suspect that the C/EBP epsilon promoter/enhancer contains a retinoic acid-response element that is directly stimulated by retinoids. 


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Ubiquitin-mediated processing of NF-kappa B transcriptional activator precursor p105. Reconstitution of a cell-free system and identification of the ubiquitin-carrier protein, E2, and a novel ubiquitin-protein ligase, E3, involved in conjugation. 
In most cases, the transcriptional factor NF-kappa B is a heterodimer consisting of two subunits, p50 and p65, which are encoded by two distinct genes of the Rel family. p50 is translated as a precursor of 105 kDa. The C-terminal domain of the precursor is rapidly degraded, forming the mature p50 subunit consisted of the N-terminal region of the molecule. The mechanism of generation of p50 is not known. It has been suggested that the ubiquitin-proteasome system is involved in the process; however, the specific enzymes involved and the mechanism of limited proteolysis, in which half of the molecule is spared, have been obscure. Palombella and colleagues (Palombella, V.J., Rando, O.J., Goldberg, A.L., and Maniatis, T.(1994) Cell 78, 773-785) have shown that ubiquitin is required for the processing in a cell-free system of a truncated, artificially constructed, 60-kDa precursor. They have also shown that proteasome inhibitors block the processing both in vitro and in vivo. In this study, we demonstrate reconstitution of a cell-free processing system and demonstrate directly that: (a) the ubiquitin-proteasome system is involved in processing of the intact p105 precursor, (b) conjugation of ubiquitin to the precursor is an essential intermediate step in the processing, (c) the recently discovered novel species of the ubiquitin-carrier protein, E2-F1, that is involved in the conjugation and degradation of p53, is also required for the limited processing of the p105 precursor, and (d) a novel, approximately 320-kDa species of ubiquitin-protein ligase, is involved in the process. This novel enzyme is distinct from E6-AP, the p53-conjugating ligase, and from E3 alpha, the "N-end rule" ligase. 


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The severe phenotype of females with tiny ring X chromosomes is associated with inability of these chromosomes to undergo X inactivation. 
Mental retardation and a constellation of congenital malformations not usually associated with Turner syndrome are seen in some females with a mosaic 45,X/46,X,r(X) karyotype. Studies of these females show that the XIST locus on their tiny ring X chromosomes is either not present or not expressed. As XIST transcription is well correlated with inactivation of the X chromosome in female somatic cells and spermatogonia, nonexpression of the locus even when it is present suggests that these chromosomes are transcriptionally active. We examined the transcriptional activity of ring X chromosomes lacking XIST expression (XISTE-), from three females with severe phenotypes. The two tiny ring X chromosomes studied with an antibody specific for the acetylated isoforms of histone H4 marking transcribed chromatin domains were labeled at a level consistent with their being active. We also examined tow of the XISTE- ring chromosomes to determine whether genes that are normally silent on an inactive X are expressed from these chromosomes. Analyses of hybrid cells show that TIMP, ZXDA, and ZXDB loci on the proximal short arm, and AR and PHKA1 loci on the long arm, are well expressed from the tiny ring X chromosome lacking XIST DNA. Studies of the ring chromosome that has XIST DNA but does not transcribe it show that its AR allele is transcribed along with the one on the normal X allele. (ABSTRACT TRUNCATED AT 250 WORDS) 


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Inhibition of IL-4-inducible gene expression in human monocytes by type I and type II interferons. 
The Th2-type cytokines, interleukin-4 (IL-4) and interleukin-13 (IL-13), induce expression of a distinct subset of genes in human monocytes, including FcepsilonRIIb (CD23), 15-lipoxygenase, IL-1 receptor antagonist (IL-1ra), and type I and type II IL-1 receptors (IL-1R). Type I interferons (IFN-alpha and IFN-beta) and type II interferon (IFN-gamma) inhibit induction of these genes by IL-4 and IL-13. However, the mechanism by which IFNs mediate this inhibition has not been defined. In this overview, we discuss the role of the transcription factor, STAT6 (signal transducer and activator of transcription-6) in mediating IL-4- and IL-13-induced gene expression in monocytes. We also discuss our recent findings that type I and type II IFNs suppress IL-4/IL-13-inducible gene expression by inhibiting tyrosine phosphorylation and nuclear translocation of STAT6. The ability of type I and type II IFNs to inhibit IL-4/IL-13-induced STAT6 activity is dose- and time-dependent, and is not unique to monocytes because IFNs induce the same effects in fibroblasts. Inhibition of STAT6 activity is not evident unless cells are preincubated with IFN for at least 1 h before IL-4 stimulation. Furthermore, inhibition can be blocked by actinomycin D, indicating a requirement for de novo transcription. We propose a model in which stimulation of monocytes by IFN activates de novo synthesis of an inhibitory factor, possibly one or more members of the SOCS/ SSI/CIS gene family, capable of suppressing activation of STAT6 by IL-4 and IL-13. Because STAT6 activation plays an essential role in IL-4/IL-13-induced gene expression, the ability of IFN-beta and IFN-gamma to inhibit STAT6 activity provides an explanation for how IFNs can suppress IL-4/IL-13-inducible gene expression. 


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Positive and negative regulation of IL-2 gene expression: role of multiple regulatory sites. 
Interleukin 2 (IL-2) is an important lymphokine required in the process of T cell activation, proliferation, clonal expansion and differentiation. The IL-2 gene displays both T cell specific and inducible expression: it is only expressed in CD4+ T cells after antigenic or mitogenic stimulation. Several cis-acting regulatory sites are required for induction of the IL-2 gene after stimulation. In this study, we have analysed the function of these cis-acting regulatory sites in the context of the native IL-2 enhancer and promoter sequence. The results of this study suggest that the NFAT (-276 to -261), the distal octamer (-256 to -248) and the proximal octamer (-75 to -66) sites not only act as enhancers of IL-2 gene transcription in the presence of cellular stimulation, but also have a silencing effect on IL-2 gene expression in resting cells. Two other sites display disparate effects on IL-2 gene expression in different T leukemia cell lines: the distal purine box (-291 to -277) and the proximal purine box sites (-145 to -128). Finally, the AP-1 (-186 to -176) and the kappa B sites (-206 to -195) respond to different cellular activation in EL4 cells. The AP-1 site mediated the response to PMA stimulation while the kappa B site responded to IL-1 stimulation. These data suggest that the regulation of IL-2 gene expression is a complex process and multiple cis-acting regulatory sites interact to exert different effects in T cells representative of alternative stages of differentiation. 


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Nuclear transcription factors that bind to elements of the IL-2 promoter. Induction requirements in primary human T cells. 
Prior studies have identified several elements that contribute to the activity of the IL-2 promoter in the stimulated T cell line, Jurkat. The sites and their corresponding nuclear binding factors include: NF-kappa B, AP-1, AP-3, OCT-1, and NF-AT. The latter "nuclear factor for activated T cells" likely contributes to the tissue specificity of IL-2 gene expression. Using electrophoretic mobility shift assays, we have studied these transcription factors in primary T cells from human blood to verify their presence in a physiologic setting and to identify the signals that stimulate factor activity. All factors are induced in the nuclei of T cells upon activation with mitogens but not with exogenous IL-2 growth factor. However, the signaling requirements and sensitivity to protein synthesis inhibitors differ considerably. Only the activities for NF-AT and AP-1 sites require two signals for optimal induction, i.e., PMA plus either lectin or antibody to the CD3 or CD28 surface molecules. Other factors are induced by lectin, antibody, and/or PMA alone. After appropriate stimulation, both NF-AT and AP-1 are peculiarly sensitive to the protein synthesis inhibitor anisomycin. Our data correlate the activity of NF-AT and AP-1 in gel shift assays with the two signals requirements for IL-2 gene expression. 


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Expression of transcription factor genes after influenza A virus infection. 
Infection of human monocytes with influenza A virus induces a broad range of proinflammatory cytokines and mononuclear cell attracting chemokines before the infected cells undergo apoptosis. The underlying mechanisms by which the corresponding genes are transcriptionally initiated after virus infection are still poorly understood. Activation of NF-kappa B seems to play an important role in the regulation of many proinflammatory cytokine genes, but cannot be the only mechanism, since several cytokine genes lack respective binding sites in their promoter regions. Therefore, we additionally investigated other transcription factors of possible importance such as CREB, CTF, OTF-1, and OTF-2. To explore long-term regulatory mechanisms, we investigated the induction of transcription factors on the gene expression level which may be important to substitute for metabolized transcription factor proteins after their activation. We identified a cell-type-specific differential response: CREB, CTF, OTF-1, OFT-2, and NF-kappa B genes were strongly induced 1 to 4 hours after influenza A virus infection in the monocytic cell line Mono Mac 6, while in freshly prepared human monocytes no significant changes were detected. In infected monocytes, which die by apoptosis, the expression of CREB, CTF, and OTF-2 was rather suppressed 8 hours after infection. In conclusion, the long-term regulation of transcription factor gene expression in non-proliferating cells seems to be of minor importance after influenza infection since in apoptosisprone cells an immediate availability of transcription factor proteins is required. 


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Cell-specific expression of helix-loop-helix transcription factors encoded by the E2A gene. 
The E2A gene encodes transcription factors of the helix-loop-helix family that are implicated in cell-specific gene expression as part of dimeric complexes that interact with E box enhancer elements. It has previously been shown that transcripts of the E2A gene can be detected in a wide range of cell types. We have now examined expression of the mouse E2A gene at the protein level using polyclonal antisera directed against distinct portions of the E2A protein to probe blots of cellular extracts. A 73 kDa protein was identified by this analysis: this protein is highly enriched in cell lines of B lymphoid origin as compared to pancreatic beta-cells and fibroblast cells. The detection of this protein selectively in extracts of lymphoid cells correlates with the presence of the E box-binding activity LEF1/BCF1 in these cells; this binding activity was previously shown to be efficiently recognized by antiserum directed against E2A gene products. Transfection of cells with full length E2A cDNA leads to appearance of protein co-migrating with the 73 kDa protein on SDS gel electrophoresis and co-migrating with LEF1/BCF1 on mobility shift analysis. Our results are consistent with the view that the DNA-binding activity LEF1/BCF1 is a homodimer of E2A proteins; the selective appearance of this putative cell-specific transcription factor in B lymphoid cells seems to be attributable, at least in part, to the elevated E2A protein concentrations in these cells. 


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Inducible nitric oxide: an autoregulatory feedback inhibitor of vascular inflammation. 
Inducible nitric oxide (iNO) is produced at sites of vascular inflammation by resident and nonresident vascular wall cells, but its role in the inflammatory process is not known. In this study, we show that a novel function of iNO is to terminate inflammatory processes. We find that iNO produced by murine macrophage-like cells, RAW264.7, can inhibit cytokine-induced endothelial cell activation in a separated and mixed endothelial-RAW264.7 coculture system. Both iNO production and endothelial VCAM-1 expression were induced simultaneously with bacterial LPS and murine-specific IFN-gamma. Inhibition of iNO synthase (iNOS) activity with N omega-monomethyl-L-arginine in endothelial-RAW264.7 cocultures, stimulated with murine-specific IFN-gamma and LPS, decreased iNO production by 86%, augmented VCAM-1 and iNOS expression in endothelial and RAW264.7 cells, respectively, and increased monocyte adhesion to the endothelial cell surface. Transient transfection studies using various VCAM-1 promoter constructs demonstrated that inhibitory effects of iNO on VCAM-1 gene transcription were mediated, in part, by inhibitory effects of iNO on kappa B cis-acting elements. Immunofluorescence studies using an Ab to the RelA (p65) subunit of nuclear factor-kappa B revealed that iNO inhibited the activation of nuclear factor-kappa B. These studies indicate that iNO attenuates iNOS expression in macrophages and inhibits monocyte adhesion to endothelial cells, and suggest that endogenously derived iNO may be an important autoregulatory inhibitor of vascular inflammation. 


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Evidence that calcineurin is rate-limiting for primary human lymphocyte activation. 
Cyclosporine (CsA) is both a clinical immunosuppressive drug and a probe to dissect intracellular signaling pathways. In vitro, CsA inhibits lymphocyte gene activation by inhibiting the phosphatase activity of calcineurin (CN). In clinical use, CsA treatment inhibits 50-75% of CN activity in circulating leukocytes. We modeled this degree of CN inhibition in primary human leukocytes in vitro in order to study the effect of partial CN inhibition on the downstream signaling events that lead to gene activation. In CsA-treated leukocytes stimulated by calcium ionophore, the degree of reduction in CN activity was accompanied by a similar degree of inhibition of each event tested: dephosphorylation of nuclear factor of activated T cell proteins, nuclear DNA binding, activation of a transfected reporter gene construct, IFN-gamma and IL-2 mRNA accumulation, and IFN-gamma production. Furthermore, the degree of CN inhibition was reflected by a similar degree of reduction in lymphocyte proliferation and IFN-gamma production in the allogeneic mixed lymphocyte cultures. These data support the conclusion that CN activity is rate-limiting for the activation of primary human T lymphocytes. Thus, the reduction of CN activity observed in CsA-treated patients is accompanied by a similar degree of reduction in lymphocyte gene activation, and accounts for the immunosuppression observed. 


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Identification of upstream regulatory elements that repress expression of adult beta-like globin genes in a primitive erythroid environment. 
Our investigations have focused on localizing cis-elements responsible for the down regulation of the adult beta-like globin genes (delta and beta) in immature, or primitive erythroid tissues. We studied their activity after transfection into K562 cells, an erythroleukemia cell line with an embryonic-fetal phenotype. Analyzed DNA sequences included delta and beta 5' flanking regions extending from approximately -500 to +50bp (promoter regions), truncated delta and beta 5' flanking regions extending from approximately -250 to +50 bp, and chimeric promoter constructions, which consisted of a distal delta or beta fragment fused to a proximal beta or delta sequence. In CAT reporter constructions no appreciable level of CAT activity was supported by the beta globin promoter, and only low level activity by the delta promoter. Truncation of the beta globin promoter led to a 2-3 fold increase in promoter activity. In contrast, deletion of the upstream portion of the delta promoter led to a 10 fold decrease in expression. Coupling of the upstream beta globin sequence from approximately -500 to -250 bp to the truncated delta promoter fragment led to complete extinction of transcription activity, consistent with a negative regulatory effect of the beta globin gene upstream element(s). Fusion of the upstream portion of the delta promoter to the truncated beta globin promoter yielded a modest increase in promoter strength relative to the truncated beta gene promoter, indicating the presence of a positive transcriptional element(s) in the upstream delta globin regulatory region. Site-directed mutagenesis of binding sites for the repressor proteins BP1 and BP2 in the upstream portion of the beta globin gene flanking region led to a 4-6 fold increase in promoter activity. DNase I footprinting of the upstream delta-globin region revealed protected sequences corresponding to consensus binding sites for GATA-1 and BP2. These results confirm that sequences in the upstream promoter region of the adult beta globin gene contribute to its factor-mediated suppression early in development and then may modulate its expression at a later stage. 


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Characterization of the human immunodeficiency virus type 1 enhancer-binding proteins from the human T-cell line Jurkat. 
The transcription of the human immunodeficiency virus type 1 (HIV-1) is under the control of cellular proteins that bind to the viral long terminal repeat (LTR). Among the protein-binding regions of the HIV-1 LTR is the transcription-enhancer region. We show that at least one inducible, C1, and one constitutive, C2, protein can bind to the HIV enhancer in Jurkat cells. The two proteins differ in their surface charge, since they are separable by anion-exchange chromatography. Bivalent cations such as Mg2+ and Zn2+ differentially affect their binding to oligonucleotides which contain the HIV-enhancer domain. Both C1 and C2 proteins also bind to a similar sequence found in the interleukin-2-receptor alpha-subunit enhancer. The inducible C1 protein was partially purified by three chromatographic steps and characterized by u.v. cross-linking as a 47 kDa protein. 


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Mechanisms of transactivation by nuclear factor of activated T cells-1. 
Nuclear factor of activated T cells-family proteins (NFAT1/NFATp, NFATc, NFAT3, and NFAT4/NFATx/NFATc3) play a key role in the transcription of cytokine genes and other genes during the immune response. We have defined the mechanisms of transactivation by NFAT1. NFAT1 possesses two transactivation domains whose sequences are not conserved in the other NFAT-family proteins, and a conserved DNA-binding domain that mediates the recruitment of cooperating nuclear transcription factors even when it is expressed in the absence of other regions of the protein. The activity of the NH2-terminal transactivation domain is modulated by an adjacent regulatory region that contains several conserved sequence motifs represented only in the NFAT family. Our results emphasize the multiple levels at which NFAT-dependent transactivation is regulated, and predict significant differences in the architecture of cooperative transcription complexes containing different NFAT-family proteins. 


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Activation protein 1-dependent transcriptional activation of interleukin 2 gene by Ca2+/calmodulin kinase type IV/Gr. 
The Ca2+/calmodulin-dependent protein kinase (CaMK) type IV/Gr is selectively expressed in T lymphocytes and is activated after signaling via the T cell antigen receptor (TCR), indicating that it mediates some of the Ca(2+)-dependent transcriptional events that follow TCR engagement. Here we show that CaMKIV/Gr induces the transcription factor activation protein 1 (AP-1) alone or in synergy with T cell mitogens and with the p21ras oncoprotein. CaMKIV/ Gr signaling is associated with transcriptional activation of c-fos but is independent of p21ras or calcineurin. AP-1 is an integral component of the nuclear factor of activated T cells (NFAT) transcriptional complex, which is required for interleukin 2 gene expression in T cells. We demonstrate that CaMKIV/Gr reconstitutes the capacity of the cytosolic component of NFAT to direct transcription from NFAT sites in non-T cells. These results reveal a central role for CaMKIV/Gr as a Ca(2+)-regulated activator of gene transcription in T lymphocytes. 


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Transactivation of the interleukin-1alpha promoter by human T-cell leukemia virus type I and type II Tax proteins. 
Human T-cell leukemia virus type I (HTLV-I)-infected T-cell lines constitutively produce high levels of interleukin-1alpha (IL-1alpha). To analyze the mechanisms that lead to the expression of IL-1alpha in HTLV-I-infected cell lines, we studied regulatory regions of the human IL-1alpha promoter involved in activation of the IL-1alpha gene. IL-1alpha promoter constructs drive transcription of the chloramphenicol acetyltransferase (CAT) reporter gene in HTLV-I-positive MT-2 cells, which constitutively produce IL-1alpha. In a cotransfection assay, the Tax protein of both HTLV-I and HTLV-II specifically activated transcription from the IL-1alpha promoter in an uninfected Jurkat cell line. A mutant Tax protein deficient in transactivation of genes by the nuclear factor (NF)-kappaB pathway was unable to induce transcriptional activity of IL-1alpha promoter-CAT constructs, but was rescued by exogenous provision of p65/p50 NF-kappaB. We found that two IL-1alpha kappaB-like sites (positions -1,065 to -1,056 and +646 to +655) specifically formed a complex with NF-kappaB-containing nuclear extract from MT-2 cells and that NF-kappaB bound with higher affinity to the 3' NF-kappaB binding site than to the 5' NF-kappaB site. Moreover, deletion of either 5' or 3' NF-kappaB sites reduced IL-1alpha promoter activity in MT-2 cells and transactivation of the IL-1alpha promoter by exogenous NF-kappaB and Tax in Jurkat cells. These data suggest a general role for Tax induction of IL-1alpha gene transcription by the NF-kappaB pathway. Expression of IL-1alpha by HTLV-I productively infected cells may be important in the hypercalcemia, osteolytic bone lesions, neutrophilia, elevation of C-reactive protein, and fever frequently seen in patients with HTLV-I-induced adult T-cell leukemia/lymphoma. 


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USF-related transcription factor, HIV-TF1, stimulates transcription of human immunodeficiency virus-1. 
The transcription factor HIV-TF1, which binds to a region about 60 bp upstream from the enhancer of the human immunodeficiency virus-1 (HIV-1), was purified from human B cells. HIV-TF1 had a molecular weight of 39,000. Binding of HIV-TF1 to the HIV long terminal repeat (LTR) activated transcription from the HIV promoter in vitro. The HIV-TF1-binding site in HIV LTR was similar to the site recognized by upstream stimulatory factor (USF) in the adenovirus major late promoter. DNA-binding properties of HIV-TF1 suggested that HIV-TF1 might be identical or related to USF. Interestingly, treatment of purified HIV-TF1 by phosphatase greatly reduced its DNA-binding activity, suggesting that phosphorylation of HIV-TF1 was essential for DNA binding. The disruption of HIV-TF1-binding site induced a 60% decrease in the level of transcription from the HIV promoter in vivo. These results suggest that HIV-TF1 is involved in transcriptional regulation of HIV-1. 


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An essential role for NF-kappaB in human CD34(+) bone marrow cell survival. 
The transcription factor, NF-kappaB, is important for T-cell activation, B-cell maturation, and human immunodeficiency virus transcription and plays a role in alternatively mediating and protecting against apoptosis in a variety of cell types. However, a role for NF-kappaB in human CD34(+) bone marrow cells has not been described. We provide evidence here that virtually all human CD34(+) bone marrow cells express NF-kappaB that can be activated by exposure to phorbol 12-myristate 13-acetate and a variety of cytokines, eg, tumor necrosis factor alpha, interleukin-3, and granulocyte-macrophage colony-stimulating factor. In addition, we demonstrate that NF-kappaB may be required for human CD34(+) bone marrow cell clonogenic function and survival. These results offer insight into a new role for NF-kappaB in maintaining survival and function in hematopoietic stem and progenitor cells and suggest that proposed strategies involving inhibition of NF-kappaB activation as an adjunct to cancer chemotherapy should be approached with caution. 


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Interleukin-2 promoter activity in Epstein-Barr virus-transformed B lymphocytes is controlled by nuclear factor-chi B. 
The regulation of interleukin (IL)-2 gene expression has been investigated mainly in T lymphocytes, the predominant producers of IL-2. However, B cells can also synthesize IL-2. In the present study we analyzed the control of IL-2 promoter activity in Epstein-Barr virus (EBV)-transformed B cell clones which are capable of secreting IL-2 at a low level after stimulation with phorbol 12-myristate 13-acetate and the Ca2+ ionophore ionomycin. Transient transfections using reporter constructs with multiples of transcription factor binding sites from the IL-2 promoter [distal nuclear factor (NF)-AT, proximal NF-AT, AP-1/Octamer (UPS) or NF-chi B (TCEd) sites] were performed. In EBV-transformed B clones, the chi B site exerted the strongest inducible activity; the NF-AT binding sites showed either no or only weak activity compared to Jurkat T cells. An IL-2 promoter bearing a defective NF-chi B site was completely inactive in EBV-transformed B cells, while it still had activity in Jurkat T cells. In seven EBV-B cell clones or lines differing in their capacity to secrete IL-2, the activity of the IL-2 promoter correlated well with the status of IL-2 secretion. Similarly, a human immunodeficiency virus promoter, whose activity is controlled through chi B factors, was found to be active in the IL-2 producing EBV-B cells, but inactive in the non-IL-2-producing cells. Electrophoretic mobility shift assays using protein extracts from EBV-B cells and the IL-2 NF-chi B probe revealed the constitutive generation of chi B complexes in IL-2-secreting cells consisting mainly of heterodimeric p50/p65 complexes. A weaker chi B complex formation and faster-migrating complexes were detected in non-IL-2-secreting cells. These results demonstrate that the IL-2 NF-chi B site is indispensable for the activity of the IL-2 promoter in EBV-transformed B cells, whereas other transcription factors appear to be less important for IL-2 expression in these cells. 


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Prostaglandin E2 Up-regulates HIV-1 long terminal repeat-driven gene activity in T cells via NF-kappaB-dependent and -independent signaling pathways. 
Replication of human immunodeficiency virus type-1 (HIV-1) is highly dependent on the state of activation of the infected cells and is modulated by interactions between viral and host cellular factors. Prostaglandin E2 (PGE2), a pleiotropic immunomodulatory molecule, is observed at elevated levels during HIV-1 infection as well as during the course of other pathogenic infections. In 1G5, a Jurkat-derived T cell line stably transfected with a luciferase gene driven by HIV-1 long terminal repeat (LTR), we found that PGE2 markedly enhanced HIV-1 LTR-mediated reporter gene activity. Experiments have been conducted to identify second messengers involved in this PGE2-dependent up-regulating effect on the regulatory element of HIV-1. In this study, we present evidence indicating that signal transduction pathways induced by PGE2 necessitate the participation of cyclic AMP, protein kinase A, and Ca2+. Experiments conducted with different HIV-1 LTR-based vectors suggested that PGE2-mediated activation effect on HIV-1 transcription was transduced via both NF-kappaB-dependent and -independent signaling pathways. The involvement of NF-kappaB in the PGE2-dependent activating effect on HIV-1 transcription was further confirmed using a kappaB-regulated luciferase encoding vector and by electrophoretic mobility shift assays. Results from Northern blot and flow cytometric analyses, as well as the use of a selective antagonist indicated that PGE2 modulation of HIV-1 LTR-driven reporter gene activity in studied T lymphoid cells is transduced via the EP4 receptor subtype. These results suggest that secretion of PGE2 by macrophages in response to infection or inflammatory activators could induce signaling events resulting in activation of proviral DNA present into T cells latently infected with HIV-1. 


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Inhibition of HIV-1 replication and NF-kappa B activity by cysteine and cysteine derivatives. 
HIV-1 proviral DNA contains two binding sites for the transcription factor NF-kappa B. HIV-1-infected individuals have, on average, abnormally high levels of tumour necrosis factor alpha (TNF alpha) and abnormally low plasma cysteine levels. We therefore investigated the effects of cysteine and related thiols on HIV-1 replication and NF-kappa B expression. The experiments in this report show that cysteine or N-acetylcysteine (NAC) raise the intracellular glutathione (GSH) level and inhibit HIV-1 replication in persistently infected Molt-4 and U937 cells. However, inhibition of HIV-1 replication appears not to be directly correlated with GSH levels. Cysteine and NAC also inhibit NF-kappa B activity as determined by electrophoretic mobility shift assays and chloramphenicol acetyl-transferase (CAT) gene expression under control of NF-kappa B binding sites in uninfected cells. This suggests that the cysteine deficiency in HIV-1-infected individuals may cause an over-expression of NF-kappa B-dependent genes and enhance HIV-1 replication. NAC may be considered for the treatment of HIV-1-infected individuals. 


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Stimulation of HIV replication in mononuclear phagocytes by leukemia inhibitory factor. 
This study examined the effects of leukemia inhibitory factor (LIF) on human immunodeficiency virus (HIV) replication in mononuclear phagocytes (MNP). LIF induced a dose-dependent increase in p24 antigen production in the chronically infected promonocytic cell line U1. The magnitude and time kinetics of the LIF effects were similar to interleukin 1 (IL-1), IL-6, and tumor necrosis factor (TNF), other cytokines known to induce HIV replication in this cell line. To characterize mechanisms responsible for these LIF effects, levels of HIV mRNA, activation of the DNA binding protein nuclear factor (NF)-kB, signal transduction pathways, and potential interactions with other cytokines were analyzed. LIF increased steady-state levels of HIV mRNA at 2.0, 4.3, and 9.2 kB. This was detectable by 24 h and persisted until 72 h. The DNA binding protein NF-kB is a central mediator in cytokine activation of HIV transcription. NF-kB levels were higher in unstimulated U1 cells as compared to the parent cell line U937. In both cell lines LIF increased NF-kB activity. Induction of NF-kB and HIV replication by cytokines are at least in part dependent on reactive oxygen intermediates. The oxygen radical scavenger N-acetyl-L-cysteine, but not an inhibitor of nitric oxide synthase, inhibited LIF-induced HIV replication. LIF induces the production of other cytokines in monocytes but its effects on HIV replication were not inhibited by antibodies to IL-1, TNF, or IL-6. These results identify LIF as a stimulus of HIV replication. (ABSTRACT TRUNCATED AT 250 WORDS) 


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Characterization of the murine cyclin-dependent kinase inhibitor gene p27Kip1. 
The cyclin-dependent kinase inhibitor p27Kip1 plays an important role in regulating cell-cycle progression. p27Kip1 directly inhibits the catalytic activity of cyclin/cdks (cyclin-dependent kinase) complexes and/or interferes physically with cyclin/cdks activation by CAK. Interestingly, the expression level of p27Kip1 mRNA was maximal in resting Go T-cells and rapidly declined following anti-CD3 activation. We report here the cloning of p27Kip1 gene from murine genomic DNA and the functional analysis of the promoter of the p27Kip1 gene. The gene consists of at least three exons and spans more than 5.6 kb of DNA. Primer extension and nuclease S1 protection analysis revealed two major transcription initiation sites. The promoter region lacked a TATA box but contained potential binding sites for the transcriptional factors including two Sp1, CRE, Myb and NFkB located at positions -153, -178, -286, -875, and -1011, respectively. To analyze the regulatory mechanisms controlling p27Kip1 gene expression, we characterized the 5'-flanking region from nt -1609 to +178. The -326 to -615 region contained positive regulatory elements. 


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Nuclear factor of activated T cells and AP-1 are insufficient for IL-2 promoter activation: requirement for CD28 up-regulation of RE/AP. 
IL-2 gene transcription in T cells requires both TCR and costimulatory signals. IL-2 promoter activation in Jurkat T cells stimulated with superantigen presented by Raji B cells requires CD28 activation. The addition of rCTLA4Ig, which blocks CD28 binding to its ligand, to the cultures decreased IL-2 promoter activation by >80%. Interestingly, CTLA4Ig did not significantly inhibit the activation of either NF of activated T cells (NFAT) or AP-1 reporters. Therefore, activation of NFAT and AP-1 is insufficient for IL-2 promoter activation. In contrast, an RE/AP reporter was blocked by CTLA4Ig by >90%. Thus, the requirement for CD28 in IL-2 promoter activation appears to be due to RE/AP and not the NFAT or AP-1 sites. In addition, these data suggest that transcriptional activation of RE/AP is not mediated by NFAT, because activation of a NFAT reporter is not affected by the addition of CTLA4Ig. 


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Anoxia/reoxygenation-induced tolerance with respect to polymorphonuclear leukocyte adhesion to cultured endothelial cells. A nuclear factor-kappaB-mediated phenomenon. 
Exposing human umbilical vein endothelial cells (HUVECs) to anoxia/reoxygenation (A/R) results in an increase in polymorphonuclear leukocyte (PMN) adhesion to HUVECs. This A/R-induced hyperadhesion is completely prevented by a previous (24 hours earlier) exposure of HUVECs to A/R. This phenomenon has been termed "A/R tolerance." Exposing HUVECs to A/R induces an increase in nuclear factor kappaB (NF-kappaB) in HUVEC nuclei within 4 hours. Interfering with either NF-kappaB activation (proteasome inhibitor) or translocation (double-stranded oligonucleotides containing NF-kappaB binding sequence) prevents the development of A/R tolerance (ie, the increase in A/R-induced PMN adhesion to HUVECs is the same after the first and second A/R challenges). NO production by HUVECs is increased after the second A/R challenge, but not after the first A/R challenge. Inhibition of NO synthase (NOS) during the second A/R challenge prevents the development of A/R tolerance with respect to PMN adhesion. However, while HUVECs contained endothelial NOS protein, no inducible NOS was detected in either tolerant or nontolerant cells. Further studies indicated that inhibition of GTP-cyclohydrolase I (an enzyme involved in de novo synthesis of an important cofactor for NOS activity, tetrahydrobiopterin) prevented the generation of NO in A/R-tolerant cells. Extracellular generation of NO (NO donor) did not effect the hyperadhesion response induced by the initial A/R challenge. A/R also induced an oxidant stress in naive HUVECs, but not in A/R-tolerant HUVECs. Inhibition of NOS during the second A/R insult results in the generation of an oxidant stress similar to that observed after the first A/R challenge. Taken together, the findings of the present study are consistent with a role for NF-kappaB in the development of A/R tolerance (with respect to PMN adhesion), perhaps by transcriptional regulation of GTP-cyclohydrolase. The increased NO production during the second A/R insult reduces PMN adhesion most likely by reducing the intracellular oxidant stress induced by A/R. 


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Regulation of interleukin-2 receptor alpha chain expression and nuclear factor.kappa B activation by protein kinase C in T lymphocytes. Autocrine role of tumor necrosis factor alpha. 
The regulation of interleukin-2 receptor alpha chain (IL-2R alpha) expression and nuclear factor (NF) activation by protein kinase C (PKC) in resting T cells, has been studied. Treatment of human resting T cells with phorbol esters strongly induced the expression of IL-2R alpha and the activation of NF.kappa B. This activation was due to the translocation of p65 and c-Rel NF.kappa B proteins from cytoplasmic stores to the nucleus, where they bound the kappa B sequence of the IL-2R alpha promoter either as p50.p65 or as p50.c-Rel heterodimers. Interestingly, all of those events were largely indirect and mediated by endogenously secreted tumor necrosis factor alpha (TNF alpha), as they were strongly inhibited by a neutralizing anti-TNF alpha monoclonal antibody. Furthermore, cyclosporin A, which blocked TNF alpha production induced by PKC, strongly inhibited IL-2R alpha and NF.kappa B activation. The addition of either TNF alpha or IL-2 partially recovered cyclosporin A-induced IL-2R alpha inhibition, but only TNF alpha completely recovered NF.kappa B activation. Those results indicate that, in resting T cells, PKC activation has only a triggering role, whereas the endogenously secreted TNF alpha plays an essential role in the quantitative control of the expression of IL-2R alpha chain or NF.kappa B activation. 


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Octamer binding factors and their coactivator can activate the murine PU.1 (spi-1) promoter. 
PU.1 (spi-1), a member of the Ets transcription factor family, is predominantly expressed in myeloid and B cells, activates many B cell and myeloid genes, and is critical for development of both of these lineages. Our previous studies (Chen, H.M., Ray-Gallet, D., Zhang, P., Hetherington, C.J., Gonzalez, D.A., Zhang, D.-E., Moreau-Gachelin, F., and Tenen, D.G.(1995) Oncogene 11, 1549-1560) demonstrate that the PU.1 promoter directs cell type-specific reporter gene expression in myeloid cell lines, and that PU.1 activates its own promoter in an autoregulatory loop. Here we show that the murine PU.1 promoter is also specifically and highly functional in B cell lines as well. Oct-1 and Oct-2 can bind specifically to a site at base pair -55 in vitro, and this site is specifically protected in B cells in vivo. We also demonstrate that two other sites contribute to promoter activity in B cells; an Sp1 binding site adjacent to the octamer site, and the PU.1 autoregulatory site. Finally, we show that the B cell coactivator OBF-1/Bob1/OCA-B is only expressed in B cells and not in myeloid cells, and that OBF-1/Bob1/OCA-B can transactivate the PU.1 promoter in HeLa and myeloid cells. This B cell restricted coactivator may be responsible for the B cell specific expression of PU.1 mediated by the octamer site. 


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Central nervous system-derived cells express a kappa B-binding activity that enhances human immunodeficiency virus type 1 transcription in vitro and facilitates TAR-independent transactivation by Tat. 
The Tat protein of human immunodeficiency virus type 1 (HIV-1) is a potent activator of long terminal repeat-directed transcription. While in most cell types, activation requires interaction of Tat with the unusual transcription element TAR, astrocytic glial cells support TAR-independent transactivation of HIV-1 transcription by Tat. This alternative pathway of Tat activation is mediated by the viral enhancer, a kappa B domain capable of binding the prototypical form of the transcription factor nuclear factor kappa B (NF-kappa B) present in many cell types, including T lymphocytes. Tat transactivation mediated by the kappa B domain is sufficient to allow replication of TAR-deleted mutant HIV-1 in astrocytes. The present study demonstrates the existence of kappa B-specific binding factors present in human glial astrocytes that differ from prototypical NF-kappa B. The novel astrocyte-derived kappa B-binding activity is retained on an HIV-1 Tat affinity column, while prototypical NF-kappa B from Jurkat T cells is not. In vitro transcription studies demonstrate that astrocyte-derived kappa B-binding factors activate transcription of the HIV-1 long terminal repeat and that this activation is dependent on the kappa B domain. Moreover, TAR-independent transactivation of HIV-1 transcription is reproduced in vitro in an astrocyte factor-dependent manner which correlates with kappa B-binding activity. The importance of the central nervous system-enriched kappa B transcription factor in the regulation of HIV-1 expression is discussed. 


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Binding of YY1 and Oct1 to a novel element that downregulates expression of IL-5 in human T cells. 
BACKGROUND: IL-5 controls development of eosinophilia and has been shown to be involved in the pathogenesis of allergic diseases. In both atopic and nonatopic asthma, elevated IL-5 has been detected in peripheral blood and the airways. IL-5 is produced mainly by activated T cells, and its expression is regulated at the transcriptional level. OBJECTIVE: This study focuses on the functional analysis of the human IL-5 (hIL-5) promoter and characterization of cis -regulatory elements and transcription factors involved in the suppression of IL-5 transcription in T cells. METHODS: Methods used in this study include DNase I footprint assays, electrophoretic mobility shift assays, and functional analysis by mammalian cell transfection involving deletion analysis and site-directed mutagenesis. RESULTS: We identified 5 protein binding regions (BRs) located within the proximal hIL-5 promoter. Functional analysis indicates that the BRs are involved in control of hIL-5 promoter activity. Two of these regions, BR3 and BR4 located at positions -102 to -73, have not previously been described as regulators of IL-5 expression in T cells. We show that the BR3 sequence contains a novel negative regulatory element located at positions -90 to -79 of the hIL-5 promoter, which binds Oct1, octamer-like, and YY1 nuclear factors. Substitution mutations, which abolished binding of these proteins to the BR3 sequence, significantly increased hIL-5 promoter activity in activated T cells. CONCLUSION: We suggest that Oct1, YY1, and octamer-like factors binding to the -90/-79 sequence within the proximal IL-5 promoter are involved in suppression of IL-5 transcription in T cells. 


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The role of BSAP (Pax-5) in B-cell development. 
The hierarchy of transcriptional control in B-cell development has recently been analyzed by targeted gene inactivation in the mouse. In this manner, the paired box containing gene Pax-5, encoding the B cell specific transcription factor BSAP, has been shown to play a key role in early B lymphopoiesis. Other experimental strategies have implicated BSAP in the control of cell proliferation, isotype switching and transcription of the immunoglobulin heavy-chain gene at late stages of B-cell differentiation. 


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Elf-1 and Stat5 bind to a critical element in a new enhancer of the human interleukin-2 receptor alpha gene [published erratum appears in Mol Cell Biol 1997 Apr;17(4):2351] 
The interleukin 2 receptor alpha-chain (IL-2R alpha) gene is a key regulator of lymphocyte proliferation. IL-2R alpha is rapidly and potently induced in T cells in response to mitogenic stimuli. Interleukin 2 (IL-2) stimulates IL-2R alpha transcription, thereby amplifying expression of its own high-affinity receptor. IL-2R alpha transcription is at least in part controlled by two positive regulatory regions, PRRI and PRRII. PRRI is an inducible proximal enhancer, located between nucleotides -276 and -244, which contains NF-kappaB and SRE/CArG motifs. PRRII is a T-cell-specific enhancer, located between nucleotides -137 and -64, which binds the T-cell-specific Ets protein Elf-1 and HMG-I(Y) proteins. However, none of these proximal regions account for the induction of IL-2R alpha transcription by IL-2. To find new regulatory regions of the IL-2R alpha gene, 8.5 kb of the 5' end noncoding sequence of the IL-2R alpha gene have been sequenced. We identified an 86-nucleotide fragment that is 90% identical to the recently characterized murine IL-2-responsive element (mIL-2rE). This putative human IL-2rE, designated PRRIII, confers IL-2 responsiveness on a heterologous promoter. PRRIII contains a Stat protein binding site that overlaps with an EBS motif (GASd/EBSd). These are essential for IL-2 inducibility of PRRIII/CAT reporter constructs. IL-2 induced the binding of Stat5a and b proteins to the human GASd element. To confirm the physiological relevance of these findings, we carried out in vivo footprinting experiments which showed that stimulation of IL-2R alpha expression correlated with occupancy of the GASd element. Our data demonstrate a major role of the GASd/EBSd element in IL-2R alpha regulation and suggest that the T-cell-specific Elf-1 factor can serve as a transcriptional repressor. 


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Identification of an inducible regulator of c-myb expression during T-cell activation. 
Resting T cells express very low levels of c-Myb protein. During T-cell activation, c-myb expression is induced and much of the increase in expression occurs at the transcriptional level. We identified a region of the c-myb 5' flanking sequence that increased c-myb expression during T-cell activation. In vivo footprinting by ligation-mediated PCR was performed to correlate in vivo protein binding with functional activity. A protein footprint was visible over this region of the c-myb 5' flanking sequence in activated T cells but not in unactivated T cells. An electrophoretic mobility shift assay (EMSA) with nuclear extract from activated T cells and an oligonucleotide of this binding site demonstrated a new protein-DNA complex, referred to as CMAT for c-myb in activated T cells; this complex was not present in unactivated T cells. Because the binding site showed some sequence similarity with the nuclear factor of activated T cells (NFAT) binding site, we compared the kinetics of induction of the two binding complexes and the molecular masses of the two proteins. Studies of the kinetics of induction showed that the NFAT EMSA binding complex appeared earlier than the CMAT complex. The NFAT protein migrated more slowly in a sodium dodecyl sulfate-polyacrylamide gel than the CMAT protein did. In addition, an antibody against NFAT did not cross-react with the CMAT protein. The appearance of the CMAT binding complex was inhibited by both cyclosporin A and rapamycin. The CMAT protein appears to be a novel inducible protein involved in the regulation of c-myb expression during T-cell activation. 


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Unicellular-unilineage erythropoietic cultures: molecular analysis of regulatory gene expression at sibling cell level. 
In vitro studies on hematopoietic control mechanisms have been hampered by the heterogeneity of the analyzed cell populations, ie, lack of lineage specificity and developmental stage homogeneity of progenitor/precursor cells growing in culture. We developed unicellular culture systems for unilineage differentiation of purified hematopoietic progenitor cells followed by daughter cell analysis at cellular and molecular level. In the culture system reported here, (1) the growth factor (GF) stimulus induces cord blood (CB) progenitor cells to proliferate and differentiate/mature exclusively along the erythroid lineage; (2) this erythropoietic wave is characterized by less than 4% apoptotic cells; (3) asymmetric divisions are virtually absent, ie, nonresponsive hematopoietic progenitors with no erythropoietic potential are forced into apoptosis; (4) the system is cell division controlled (cdc), ie, the number of divisions performed by each cell is monitored. Single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was applied to this culture system to investigate gene expression of diverse receptors, markers of differentiation, and transcription factors (EKLF, GATA-1, GATA-2, p45 NF-E2, PU.1, and SCL/Tal1) at discrete stages of erythropoietic development. Freshly isolated CD34(+) cells expressed CD34, c-kit, PU.1, and GATA-2 but did not express CD36, erythropoietin receptor (EpoR), SCL/Tal1, EKLF, NF-E2, GATA-1, or glyocophorin A (GPA). In early to intermediate stages of erythroid differentiation we monitored the induction of CD36, Tal1, EKLF, NF-E2, and GATA-1 that preceeded expression of EpoR. In late stages of erythroid maturation, GPA was upregulated, whereas CD34, c-kit, PU.1, and GATA-2 were barely or not detected. In addition, competitive single-cell RT-PCR was used to assay CD34 mRNA transcripts in sibling CD34(+) CD38(-) cells differentiating in unilineage erythroid cultures: this analysis allowed us to semiquantitate the gradual downmodulation of CD34 mRNA from progenitor cells through their differentiating erythroid progeny. It is concluded that this novel culture system, coupled with single-cell RT-PCR analysis, may eliminate the ambiguities intrinsic to molecular studies on heterogeneous populations of hematopoietic progenitors/precursors growing in culture, particularly in the initial stages of development. 


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Nuclear factor-kappaB-dependent induction of interleukin-8 gene expression by tumor necrosis factor alpha: evidence for an antioxidant sensitive activating pathway distinct from nuclear translocation. 
Tumor necrosis factor alpha (TNFalpha) is a pluripotent activator of inflammation by inducing a proinflammatory cytokine cascade. This phenomenon is mediated, in part, through inducible expression of the CXC chemokine, interleukin-8 (IL-8). In this study, we investigate the role of TNFalpha-inducible reactive oxygen species (ROS) in IL-8 expression by "monocyte-like" U937 histiocytic lymphoma cells. TNFalpha is a rapid activator of IL-8 gene expression by U937, producing a 50-fold induction of mRNA within 1 hour of treatment. In gene transfection assays, the effect of TNFalpha requires the presence of an inducible nuclear factor-kappaB (NF-kappaB) (Rel A) binding site in the IL-8 promoter. TNFalpha treatment induces a rapid translocation of the 65 kD transcriptional activator NF-kappaB subunit, Rel A, whose binding in the nucleus occurs before changes in intracellular ROS. Pretreatment (or up to 15 minutes posttreatment) relative to TNFalpha with the antioxidant dimethyl sulfoxide (DMSO) (2% [vol/vol]) blocks 80% of NF-kappaB-dependent transcription. Surprisingly, however, DMSO has no effect on inducible Rel A binding. Similar selective effects on NF-kappaB transcription are seen with the unrelated antioxidants, N-acetylcysteine (NAC) and vitamin C. These data indicate that TNFalpha induces a delayed ROS-dependent signalling pathway that is required for NF-kappaB transcriptional activation and is separable from that required for its nuclear translocation. Further definition of this pathway will yield new insights into inflammation initiated by TNFalpha signalling. 


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Epithelial cell-initiated inflammation plays a crucial role in early tissue damage in amebic infection of human intestine. 
BACKGROUND & AIMS: Entamoeba histolytica infection of the intestine can induce severe gut inflammation. The aims of this study were to assess the role of the host inflammatory response in the tissue damage observed with amebiasis and the role of the intestinal epithelial cell in initiating that response. METHODS: E. histolytica infection was established in human intestinal xenografts in severe combined immunodeficient (SCID-HU-INT) mice. Human intestinal epithelial cell inflammatory responses to amebic infection were inhibited by the intraluminal administration of an antisense oligonucleotide to the human p65 subunit of nuclear factor kappaB, and the role of neutrophils in tissue damage observed with amebiasis was studied by depleting neutrophils from SCID-HU-INT mice. RESULTS: Administration of the antisense oligonucleotide blocked the production of human interleukin 1beta and interleukin 8 by intestinal epithelial cells and inhibited neutrophil influx into the E. histolytica-infected intestinal xenografts. Inhibition of the gut inflammatory response by the antisense oligonucleotide or the depletion of neutrophils from SCID-HU- INT mice blocked the increase in intestinal permeability observed with amebic infection. CONCLUSIONS: Intestinal epithelial cells initiate an inflammatory response with resulting neutrophil-mediated tissue damage in response to E. histolytica infection; this inflammatory cascade can be blocked by inhibiting the transcription of genes regulated by nuclear factor kappaB. 


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A novel heterodimerization partner for thyroid hormone receptor. Peroxisome proliferator-activated receptor. 
Retinoid-like receptors play a central role in hormonal responses by forming heterodimers with other nuclear hormone receptors. In this study we have identified the peroxisome proliferator-activated receptor (PPAR) as a new thyroid hormone receptor (THR) auxiliary nuclear protein, heterodimerizing with THR in solution. Although these heterodimers do not recognize a classical thyroid hormone response element (TRE) characterized by direct repeat separated by four nucleotides (DR+4), PPAR behaves as a dominant negative regulator of thyroid hormone (TH) action. However, a TH-dependent positive effect is elicited by selective interaction of the THR beta-PPAR but not the THR alpha-PPAR heterodimer with a novel TRE (DR+2). The critical region of THR beta was mapped to 3 amino acids in the distal box of the DNA binding domain. Hence, PPAR can positively or negatively influence TH action depending on TRE structure and THR isotype. 


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Induction of CIITA and modification of in vivo HLA-DR promoter occupancy in normal thymic epithelial cells treated with IFN-gamma: similarities and distinctions with respect to HLA-DR-constitutive B cells. 
In this study, the IFN-gamma induction of MHC class II gene expression in primary cultures of thymic epithelial cells (TEC) was analyzed. This cellular system offers the advantage that MHC class II induction is studied in a "physiologic" cell lineage that, as a result of this expression within the thymus, is thought to participate to the selection and maturation of the T cells. It was found that the MHC class II gene expression was associated with the de novo transcription of the gene encoding the CIITA trans-activator, a crucial MHC class II gene regulatory factor. Furthermore, the anatomy of interaction between the MHC class II DRA promoter and corresponding binding factors was analyzed by in vivo DNAse I footprint. It was found that treatment with IFN-gamma induces changes in the occupancy of the DRA gene regulatory sequences by nuclear factors. The resulting occupancy displays strong similarities with the one observed in the MHC class II-constitutive B cells, represented by both the Burkitt lymphoma line Raji and normal tonsil- derived B cells. However, some peculiar differences were observed between the TEC, either IFN-gamma-induced or not, and the constitutive B cells. These results suggest that both common mechanisms, such as the one mediated by the CIITA trans-activator, and distinct tissue-specific constraints contribute to the transcriptional control of constitutive and IFN-gamma-induced MHC class II gene expression. 


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Glucocorticoid-mediated repression of cytokine gene transcription in human arteritis-SCID chimeras. 
Giant cell arteritis (GCA) is a vasculitic syndrome that preferentially affects medium and large-sized arteries. Glucocorticoid therapy resolves clinical symptoms within hours to days, but therapy has to be continued over several years to prevent disease relapses. It is not known whether and how glucocorticoids affect the function of the inflammatory infiltrate or why the disease persists subclinically despite chronic treatment. GCA is self-sustained in temporal arteries engrafted into SCID mice, providing a model in which the mechanisms of action and limitations of glucocorticoid therapy can be examined in vivo. Administration of dexamethasone to temporal artery-SCID chimeras for 1 wk induced a partial suppression of T cell and macrophage function as indicated by the reduced tissue concentrations of IL-2, IL-1beta, and IL-6 mRNA, and by the diminished expression of inducible NO synthase. In contrast, synthesis of IFN-gamma mRNA was only slightly decreased, and expression of TGF-beta1 was unaffected. These findings correlated with activation of the IkappaBalpha gene and blockade of the nuclear translocation of NFkappaB in the xenotransplanted tissue. Dose-response experiments suggested that steroid doses currently used in clinical medicine are suboptimal in repressing NFkappaB-mediated cytokine production in the inflammatory lesions. Chronic steroid therapy was able to deplete the T cell products IL-2 and IFN-gamma, whereas the activation of tissue-infiltrating macrophages was only partially affected. IL-1beta transcription was abrogated; in contrast, TGF-beta1 mRNA synthesis was steroid resistant. The persistence of TGF-beta1-transcribing macrophages, despite paralysis of T cell function, may provide an explanation for the chronicity of the disease, and may identify a novel therapeutic target in this inflammatory vasculopathy. 


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Human tumor necrosis factor alpha gene regulation in phorbol ester stimulated T and B cell lines. 
The minimal region of the human tumor necrosis factor alpha (TNF-alpha) gene promoter necessary for its transcriptional induction by phorbol esters (PMA) in human T and B lymphocyte cell lines has been localized between -52 and +89 nucleotides (nt) relative to the gene's transcriptional start site. Comparison of these sequences to those required to mediate virus or lipopolysaccharide (LPS) induction of the gene reveal significant differences, and thus, the sequence requirements for PMA induction are distinct from those that mediate induction by virus or LPS. Although three sites in the TNF-alpha promoter (kappa 1, kappa 2, and kappa 3) specifically bind the transcription factor NF-kappa B in lymphoid nuclear extracts, TNF-alpha mRNA induction by PMA does not correlate with NF-kappa B binding activities displayed by different T and B cell lines. Moreover, kappa 1-kappa 3 can each be deleted from the TNF-alpha promoter with little effect on the gene's inducibility by PMA. Therefore, TNF-alpha mRNA induction by PMA, like its induction by virus and LPS, is not primarily mediated by NF-kappa B, but rather is mediated through other sequences and protein factors. Surprisingly, multimers of kappa 1-kappa 3 can confer PMA inducibility on a heterologous promoter in a B (Raji), but not a T (HUT78) cell line. However they are not functional on a truncated TNF-alpha promoter, indicating that promoter context and cell type specificity influence the PMA inducible function of these NF-kappa B binding sites. 


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Differential expression and phosphorylation of CTCF, a c-myc transcriptional regulator, during differentiation of human myeloid cells. 
CTCF is a transcriptional repressor of the c-myc gene. Although CTCF has been characterized in some detail, there is very little information about the regulation of CTCF activity. Therefore we investigated CTCF expression and phosphorylation during induced differentiation of human myeloid leukemia cells. We found that: (i) both CTCF mRNA and protein are down-regulated during terminal differentiation in most cell lines tested; (ii) CTCF down-regulation is retarded and less pronounced than that of c-myc; (iii) CTCF protein is differentially phosphorylated and the phosphorylation profiles depend on the differentiation pathway. We concluded that CTCF expression and activity is controlled at transcriptional and post-transcriptional levels. 


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Mapping of the interaction site of the defective transcription factor in the class II major histocompatibility complex mutant cell line clone-13 to the divergent X2-box. 
We have previously described a mutant B lymphoblastoid cell line, Clone-13, that expresses HLA-DQ in the absence of HLA-DR and -DP. Several criteria indicated that the defect in this cell line influences the activity of an isotype-specific transcription factor. Indeed, transient transfection of HLA-DRA and DQB reporter constructs indicated that the affected factor operates via cis-elements located between -141 base pairs and the transcription initiation site. A series of hybrid DRA/DQB reporter constructs was generated to further map the relevant cis-elements in this system. Insertion of oligonucleotides spanning the DQB X-box (but not the DQB-W region or the DQB Y-box) upstream of -141 in a DRA reporter plasmid rescued expression to nearly wild-type levels. Substitution promoters were then generated where the entire X-box, or only the X1- or X2-boxes of HLA-DRA were replaced with the analogous regions of HLA-DQB. The DQB X2-box was able to restore expression to the silent DRA reporter construct. Moreover, replacement of the DQB X2-box with the DRA X2-box markedly diminished the activity of the DQB promoter in the mutant cell. None of the hybrid reporter constructs were defective when transfected into the wild-type, HLA-DR/-DQ positive parental cell line, Jijoye. These studies suggest that the divergent X2-box of the class II major histocompatibility complex promoters plays an important role in influencing differential expression of the human class II isotypes. 


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Multiple transcription factors are required for activation of human interleukin 9 gene in T cells. 
The genetic elements and regulatory mechanisms responsible for human interleukin 9 (IL-9) gene expression in a human T cell leukemia virus type I-transformed human T cell line, C5MJ2, were investigated. We demonstrated that IL-9 gene expression is controlled, at least in part, by transcriptional activation. Transient expression of the luciferase reporter gene linked to serially deleted sequences of the 5'-flanking region of the IL-9 gene has revealed several positive and negative regulatory elements involved in the basal and inducible expression of the IL-9 gene in C5MJ2 cells. An AP-1 site at -146 to -140 was shown to be involved in the expression of the IL-9 gene. A proximal region between -46 and -80 was identified as the minimum sequence for the basal and inducible expression of the IL-9 gene in C5MJ2 cells. Within this region, an NF-kappaB site at -59 to -50 and its adjacent 20-base pair upstream sequence were demonstrated to play a critical role for the IL-9 promoter activity. DNA-protein binding studies indicated that NF-kappaB, c-Jun, and potentially novel proteins (around 35 kDa) can bind to this important sequence. Mutations at different sites within this proximal promoter region abolished the promoter activity as well as the DNA binding. Taken together, these results suggest that the cooperation of different transcription factors is essential for IL-9 gene expression in T cells. 


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AML1 (CBFalpha2) cooperates with B cell-specific activating protein (BSAP/PAX5) in activation of the B cell-specific BLK gene promoter. 
AML1 plays a critical role during hematopoiesis and chromosomal translocations involving AML1 are commonly associated with different forms of leukemia, including pre-B acute lymphoblastic leukemia. To understand the function of AML1 during B cell differentiation, we analyzed regulatory regions of B cell-specific genes for potential AML1-binding sites and have identified a putative AML1-binding site in the promoter of the B cell-specific tyrosine kinase gene, blk. Gel mobility shift assays and transient transfection assays demonstrate that AML1 binds specifically to this site in the blk promoter and this binding site is important for blk promoter activity. Furthermore, in vitro binding analysis revealed that the AML1 runt DNA-binding domain physically interacts with the paired DNA-binding domain of BSAP, a B cell-specific transcription factor. BSAP has been shown previously to be important for B cell-specific regulation of the blk gene. Physical interaction of AML1 with BSAP correlates with functional cooperativity in transfection studies where AML1 and BSAP synergistically activate blk promoter transcription by more than 50-fold. These results demonstrate physical and functional interactions between AML1 and BSAP and suggest that AML1 is an important factor for regulating a critical B cell-specific gene, blk. 


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A shortened life span of EKLF-/- adult erythrocytes, due to a deficiency of beta-globin chains, is ameliorated by human gamma-globin chains. 
Using homologous recombination, both EKLF alleles in murine embryonic stem (ES) cells were inactivated. These EKLF-/- ES cells were capable of undergoing in vitro differentiation to form definitive erythroid colonies that were similar in size and number to those formed by wild-type ES cells. However, the EKLF-/- colonies were poorly hemoglobinized and enucleated erythrocytes in these colonies contained numerous Heinz bodies. Reverse transcriptase-polymerase chain reaction (RT-PCR) analyses revealed that adult and embryonic globin genes were appropriately regulated, with the exception of beta h1-globin, which continued to be expressed at a very low level. The ratio of adult beta-globin/alpha-globin mRNA in the mutant ES cells was 1/15 of that in wild-type ES cells. When the EKLF-/- cells were injected into blastocysts, they did not contribute at a detectable level to the mature erythrocyte compartment of the chimeric animals, based on analysis of glucose phosphate isomerase-1 (GPI-1) isozymes and hemoglobins that distinguish ES cell-derived erythrocytes from host blastocyst-derived erythrocytes. In contrast, semiquantitative RT-PCR analysis of RNA from reticulocytes of the same chimeric animals suggested that the ES cell-derived reticulocytes were present at a level of 6% to 8%. This indicated that the EKLF-/- erythrocytes in adult animals must be short-lived, apparently due to the imbalance of beta- versus alpha-globin chains, leading to the precipitation of excess alpha-globin chains to form Heinz bodies. Consistent with this hypothesis, the short life span was ameliorated by introduction into the EKLF-/- ES cells of a human LCR/gamma-globin gene, as evidenced by the presence of ES cell-derived reticulocytes as well as mature erythrocytes in the blood of the chimeric animals. 


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Identification of transcriptional suppressor proteins that bind to the negative regulatory element of the human immunodeficiency virus type 1. 
Two different proteins which independently bound to neighboring sequences within the negative regulatory element (NRE) of human immunodeficiency virus type 1 (HIV-1) were detected in the nuclear extract of a virus-infected human T cell line. One of the factors bound to a novel dyad symmetrical sequence. This sequence is well conserved in various HIV-1 isolates and partial homology was found with the promoter region of the human retinoblastoma gene. Similar DNA binding activity was detected in a variety of virus-uninfected human T cell lines and HeLa cells by means of a gel mobility shift assay. The other factor bound to a putative AP-1 recognition sequence predicted for the HIV-1 NRE. However, this factor did not bind to a typical AP-1 site. The insertion of multiple copies of the binding site for the former or latter factor into a heterologous promoter reduced the promoter activity to one-tenth or one-third, respectively. Thus, each factor may function as a novel negative regulator of transcription. 


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E3, a hematopoietic-specific transcript directly regulated by the retinoic acid receptor alpha. 
Retinoic acid (RA)-induced maturation mediated by the retinoic acid receptor alpha (RAR alpha) has been implicated in myeloid development. We have used differential hybridization analysis of a cDNA library constructed from the murine RA-inducible MPRO promyelocyte cell line to identify immediate-early genes induced by RA during granulocytic differentiation. E3, one of nine sequences identified, was upregulated in an immediate-early manner, with transcript levels peaking after 60 minutes exposure to RA. E3 transcripts were RA-inducible in HL60 cells, but not in an RA-resistant subclone, HL60R, that harbors a mutated RAR alpha gene. However, when HL60R cells were transduced with a functional copy of the RAR alpha gene, RA induced a 10-fold increase in E3 mRNA levels. E3 transcripts are present in the myeloid, B-lymphoid, and erythroid lineages, absent in nonhematopoietic cells, and encode a highly hydrophobic, potentially phosphorylated polypeptide of unknown function with significant homology to a putative protein expressed in myeloid cells. The murine E3 promoter harbors a single bipartite retinoic acid response element which in transient transfection assays conferred RA sensitivity. These results indicate that E3 is a hematopoietic-specific gene that is an immediate target for the activated RAR alpha during myelopoiesis. 


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Signaling through the lymphotoxin-beta receptor stimulates HIV-1 replication alone and in cooperation with soluble or membrane-bound TNF-alpha. 
The level of ongoing HIV-1 replication within an individual is critical to HIV-1 pathogenesis. Among host immune factors, the cytokine TNF-alpha has previously been shown to increase HIV-1 replication in various monocyte and T cell model systems. Here, we demonstrate that signaling through the TNF receptor family member, the lymphotoxin-beta (LT-beta) receptor (LT-betaR), also regulates HIV-1 replication. Furthermore, HIV-1 replication is cooperatively stimulated when the distinct LT-betaR and TNF receptor systems are simultaneously engaged by their specific ligands. Moreover, in a physiological coculture cellular assay system, we show that membrane-bound TNF-alpha and LT-alpha1beta2 act virtually identically to their soluble forms in the regulation of HIV-1 replication. Thus, cosignaling via the LT-beta and TNF-alpha receptors is probably involved in the modulation of HIV-1 replication and the subsequent determination of HIV-1 viral burden in monocytes. Intriguingly, surface expression of LT-alpha1beta2 is up-regulated on a T cell line acutely infected with HIV-1, suggesting a positive feedback loop between HIV-1 infection, LT-alpha1beta2 expression, and HIV-1 replication. Given the critical role that LT-alpha1beta2 plays in lymphoid architecture, we speculate that LT-alpha1beta2 may be involved in HIV-associated abnormalities of the lymphoid organs. 


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Calcium/calmodulin-dependent protein kinase II downregulates both calcineurin and protein kinase C-mediated pathways for cytokine gene transcription in human T cells. 
Engagement of the T cell receptor for antigen activates phospholipase C resulting in an increase in intracellular free calcium concentration ([Ca2+]i) and activation of protein kinase C (PKC). Increased [Ca2+]i activates Ca2+/calmodulin-dependent kinases including the multifunctional Ca2+/calmodulin-dependent protein kinase II (CaM-K II), as well as calcineurin, a type 2B protein phosphatase. Recent studies have identified calcineurin as a key enzyme for interleukin (IL)-2 and IL-4 promoter activation. However, the role of CaM-K II remains unknown. We have used mutants of these kinases and phosphatases (gamma B*CaM-K and delta CaM-AI, respectively) to explore their relative role in cytokine gene transcription and their interactions with PKC-dependent signaling systems. gamma B*CaM-K and delta CaM-AI, known to exhibit constitutive Ca(2+)-independent activity, were cotransfected (alone or in combination) in Jurkat T cells with a plasmid containing the intact IL-2 promoter driving the expression of the chloramphenicol acetyltransferase reporter gene. Cotransfection of gamma B*CaM-K with the IL-2 promoter construct downregulated its transcription in response to stimulation with ionomycin and phorbol myristate acetate (PMA). The inhibitory effect of CaM-K II on IL-2 promoter was associated with decreased transcription of its AP-1 and NF-AT transactivating pathways. Under the same conditions, delta CaM-AI superinduced IL-2 promoter activity (approximately twofold increase). When both mutants were used in combination, gamma B*CaM-K inhibited the induction of the IL-2 promoter by delta CaM-AI. Similar results were obtained when a construct containing the IL-4 promoter also was used. gamma B*CaM-K also downregulated the activation of AP-1 in response to transfection with a constitutively active mutant of PKC or stimulation with PMA. These results suggest that CaM-K II may exert negative influences on cytokine gene transcription in human T cells, and provide preliminary evidence for negative cross-talk with the calcineurin- and PKC- dependent signaling systems. 


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HIV does not replicate in naive CD4 T cells stimulated with CD3/CD28. 
In this report, we demonstrate that the T cell tropic strain of HIV, LAI, does not replicate in naive CD4 T cells stimulated by cross-linking CD3 and CD28. In contrast, LAI replicates well in memory CD4 T cells stimulated in the same way. Unlike this physiologically relevant stimulation, PHA stimulates productive LAI replication in both naive and memory T cells. These studies were conducted with highly purified (FACS-isolated) subsets of CD4 T cells identified by expression of both CD45RA and CD62L. Remixing of purified T cells showed that naive T cells do not suppress LAI replication in memory T cells and that memory T cells do not restore LAI expression in naive T cells. The suppression of productive LAI replication in naive T cells is not due to differential expression of viral coreceptors, nor is it due to inhibition of activation of the important HIV transcription factors, nuclear factor-kappaB and activator protein-1. The inherent resistance of naive T cells to productive HIV infection, coupled with their proliferative advantage as demonstrated here, provides a sound basis for proposed clinical therapies using ex vivo expansion and reinfusion of CD4 T cells from HIV-infected adults. 


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Activation of the signal transducer and transcription (STAT) signaling pathway in a primary T cell response. Critical role for IL-6. 
The T cell activation is initiated by interaction of specific Ags with TCR, followed by activation of intracellular biochemical events leading to activation of several genes. The activation of signal transducer and activator of transcription (STAT) proteins in a primary TCR-mediated activation of T cells have been explored. In purified human peripheral blood T cells, nuclear STAT proteins were activated approximately 3 h after activation by cross-linked anti-CD3 Abs. These STAT proteins were detected by using the IFN-gamma-activated sequence (GAS) and related oligonucleotides as probes in electrophoretic mobility shift assay. Analysis of the nuclear extracts with anti-STAT Abs indicated that they contained STAT-3 and additional proteins crossreactive with the STAT family. The induction of STAT activity was inhibited completely by pretreatment with either cycloheximide or cyclosporin A, thus indicating that the induction was due to a secondary factor produced by the activated T cells. As neutralizing anti-IL-6 Abs effectively down-regulated the early induction of STAT proteins and as exogenously added IL-6 rapidly activated DNA binding similar to TCR-mediated bindings, it can be concluded that IL-6 is the factor responsible for the activation of STAT proteins in a primary T cell response. 


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Characterization of the human platelet/endothelial cell adhesion molecule-1 promoter: identification of a GATA-2 binding element required for optimal transcriptional activity. 
Platelet/endothelial cell adhesion molecule-1 (PECAM-1) is a 130-kD member of the Ig gene superfamily that is expressed on platelets, endothelial cells, and certain leukocyte subsets. To examine the factors controlling vascular-specific expression of PECAM-1, we cloned the 5'-flanking region of the PECAM-1 gene and analyzed its transcriptional activity. 5'-Rapid amplification of cDNA ends (5'-RACE) analysis showed that transcription initiation occurred at several closely spaced nearby sites originating approximately 204 bp upstream from the translation start site. Analysis of the sequence immediately upstream from the transcription initiation site (TIS) showed no canonical TATA or CAAT elements, however an initiator element commonly found in TATA-less promoters encompassed the TIS. 5'-serially truncated PECAM-1 promoter segments cloned in front of a luciferase reporter drove transcription in both a lineage- and orientation-specific manner. Putative cis-acting control elements present within a 300-bp core promoter included two ets sites, an Sp1 site, tandem E-box domains, two GATA-associated sites (CACCC), an AP-2 binding site, and a GATA element at -24. Mutational analysis showed that optimal transcriptional activity required the GATA sequence at position -24, and gel-shift assays further showed that the GATA-2 transcription factor, but not GATA-1, bound to this region of the PECAM-1 promoter. Understanding the cis- and transacting factors that regulate the tissue-specific expression of PECAM-1 should increase our understanding of the mechanisms by which vascular-specific gene expression is achieved. 


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Ectopic expression of a conditional GATA-2/estrogen receptor chimera arrests erythroid differentiation in a hormone-dependent manner. 
The GATA factors are a family of transcriptional regulatory proteins in eukaryotes that share extensive homology in their DNA-binding domains. One enigmatic aspect of GATA factor expression is that several GATA proteins, which ostensibly share the same DNA-binding site specificity, are coexpressed in erythroid cells. To elucidate the roles of individual GATA factors in erythropoiesis, conditional alleles of GATA-1, GATA-2, and GATA-3 were prepared by fusing each of the factors to the hormone-binding domain of the human estrogen receptor (ER). These GATA/ER chimeric factors were shown to be hormone-inducible trans-activating proteins in transient transfection assays. When stably introduced into primary erythroblasts or conditionally transformed erythroid progenitors cells, exogenous GATA-2/ER promoted proliferation and inhibited terminal differentiation in an estrogen-dependent manner. These phenotypic effects are specifically attributable to the action of ectopically expressed GATA-2/ER because erythroblasts expressing exogenous GATA-2 are constitutively arrested in differentiation and because erythroid progenitors expressing either Gal/ER or GATA-3/ER do not display a hormone-responsive block in differentiation. Thus, the GATA-2 transcription factor appears to play a role in regulating the self-renewal capacity of early erythroid progenitor cells. 


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Bcl-2-mediated drug resistance: inhibition of apoptosis by blocking nuclear factor of activated T lymphocytes (NFAT)-induced Fas ligand transcription. 
Bcl-2 inhibits apoptosis induced by a variety of stimuli, including chemotherapy drugs and glucocorticoids. It is generally accepted that Bcl-2 exerts its antiapoptotic effects mainly by dimerizing with proapoptotic members of the Bcl-2 family such as Bax and Bad. However, the mechanism of the antiapoptotic effects is unclear. Paclitaxel and other drugs that disturb microtubule dynamics kill cells in a Fas/Fas ligand (FasL)-dependent manner; antibody to FasL inhibits paclitaxel-induced apoptosis. We have found that Bcl-2 overexpression leads to the prevention of chemotherapy (paclitaxel)-induced expression of FasL and blocks paclitaxel-induced apoptosis. The mechanism of this effect is that Bcl-2 prevents the nuclear translocation of NFAT (nuclear factor of activated T lymphocytes, a transcription factor activated by microtubule damage) by binding and sequestering calcineurin, a calcium-dependent phosphatase that must dephosphorylate NFAT to move to the nucleus. Without NFAT nuclear translocation, the FasL gene is not transcribed. Thus, it appears that paclitaxel and other drugs that disturb microtubule function kill cells at least in part through the induction of FasL. Furthermore, Bcl-2 antagonizes drug-induced apoptosis by inhibiting calcineurin activation, blocking NFAT nuclear translocation, and preventing FasL expression. The effects of Bcl-2 can be overcome, at least partially, through phosphorylation of Bcl-2. Phosphorylated Bcl-2 cannot bind calcineurin, and NFAT activation, FasL expression, and apoptosis can occur after Bcl-2 phosphorylation. 


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NF-kappaB activation is a critical regulator of human granulocyte apoptosis in vitro. 
During beneficial inflammation, potentially tissue-damaging granulocytes undergo apoptosis before being cleared by phagocytes in a non-phlogistic manner. Here we show that the rate of constitutive apoptosis in human neutrophils and eosinophils is greatly accelerated in both a rapid and concentration-dependent manner by the fungal metabolite gliotoxin, but not by its inactive analog methylthiogliotoxin. This induction of apoptosis was abolished by the caspase inhibitor zVAD-fmk, correlated with the inhibition of nuclear factor-kappa B (NF-kappaB), and was mimicked by a cell permeable inhibitory peptide of NF-kappaB, SN-50; other NF-kappaB inhibitors, curcumin and pyrrolidine dithiocarbamate; and the proteasome inhibitor, MG-132. Gliotoxin also augmented dramatically the early (2-6 h) pro-apoptotic effects of tumor necrosis factor-alpha (TNF-alpha) in neutrophils and unmasked the ability of TNF-alpha to induce eosinophil apoptosis. In neutrophils, TNF-alpha caused a gliotoxin-inhibitable activation of an inducible form of NF-kappaB, a response that may underlie the ability of TNF-alpha to delay apoptosis at later times (12-24 h) and limit its early killing effect. Furthermore, cycloheximide displayed a similar capacity to enhance TNF-alpha induced neutrophil apoptosis even at time points when cycloheximide alone had no pro-apoptotic effect, suggesting that NF-kappaB may regulate the production of protein(s) which protect neutrophils from the cytotoxic effects of TNF-alpha. These data shed light on the biochemical and molecular mechanisms regulating human granulocyte apoptosis and, in particular, indicate that the transcription factor NF-kappaB plays a crucial role in regulating the physiological cell death pathway in granulocytes. 


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Downstream activation of a TATA-less promoter by Oct-2, Bob1, and NF-kappaB directs expression of the homing receptor BLR1 to mature B cells. 
The chemokine receptor, BLR1, is a major regulator of the microenvironmental homing of B cells in lymphoid organs. In vitro studies identify three essential elements of the TATA-less blr1 core promoter that confer cell type- and differentiation-specific expression in the B cells of both humans and mice, a functional promoter region (-36 with respect to the transcription start site), a NF-kappaB motif (+44), and a noncanonical octamer motif (+157). The importance of these sites was confirmed by in vivo studies in gene-targeted mice deficient of either Oct-2, Bob1, or both NF-kappaB subunits p50 and p52. In all of these animals, the expression of BLR1 was reduced or absent. In mice deficient only of p52/NF-kappaB, BLR1 expression was unaffected. Thus our data demonstrate that BLR1 is a target gene for Oct-2, Bob1, and members of the NF-kappaB/Rel family and provides a link to the impaired B cell functions in mice deficient for these factors. 


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Defective transcription of the IL-2 gene is associated with impaired expression of c-Fos, FosB, and JunB in anergic T helper 1 cells. 
Anergic CD4+ Th cells do not produce IL-2 when challenged with Ag-pulsed accessory cells because of a transcriptional defect. In this work, we report that these anergic T cells are defective in their ability to up-regulate protein binding and transactivation at two critical IL-2 DNA enhancer elements: NF-AT (nuclear factor of activated T cells; a sequence that binds a heterotrimeric NFATp, Fos, and Jun protein complex) and Activator Protein-1 (AP-1) (that binds Fos and Jun heterodimers). Western blot analysis of nuclear extracts showed that the impaired DNA-protein interactions in anergic T cells were associated with poor expression of the inducible AP-1 family members c-Fos, FosB, and JunB. However, the reduced expression of these proteins was not the result of a global TCR/CD3-signaling defect because CD3 cross-linking induced an equivalent increase in intracellular-free calcium ions, as well as NFATp dephosphorylation, translocation to the nucleus, and DNA binding in both normal and anergic T cells. Thus, defective IL-2 gene transcription appears to be due, at least in part, to a selective block in the expression of the AP-1 Fos and Jun family members in anergic T cells. 


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Human T-cell leukemia virus type 1 tax protein abrogates interleukin-2 dependence in a mouse T-cell line. 
Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia. Tax, the viral protein, is thought to be crucial in the development of the disease, since it transforms healthy T cells in vitro and induces tumors in transgenic animals. We examined the effect of Tax activity on the growth of the interleukin-2 (IL-2)-dependent T-cell line CTLL-2. Stable expression of Tax in CTLL-2 transformed cell growth from being IL-2 dependent to IL-2 independent. Tax stimulated transcription through NF-kappaB and the cyclic AMP-responsive element-like sequence in the HTLV-1 promoter. The finding of Tax mutants segregating these two pathways suggested that the NF-kappaB pathway was essential for IL-2-independent growth of CTLL-2 cells while the CRE pathway was unnecessary. However, both pathways were necessary for another transformation-related activity (colony formation in soft agar) of CTLL-2/Tax. Our results show that Tax has at least two distinct activities on T cells, and suggest that Tax plays a crucial role in IL-2-independent T-cell transformation induced by HTLV-1, in addition to its well-known IL-2-dependent cell transformation. 


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Soluble tumor necrosis factor receptors inhibit phorbol myristate acetate and cytokine-induced HIV-1 expression chronically infected U1 cells. 
Recombinant human tumor necrosis factor (TNF) binding protein-1 (r-h TBP-1) and recombinant human soluble dimeric TNF receptor (rhu TNFR:Fc) were used to determine the relative contributions of TNF to phorbol myristate acetate (PMA) and cytokine-induced human immunodeficiency virus type 1 (HIV-1) replication in chronically infected cell lines. Treatment of HIV-1-infected promonocytic U1 cells with r-h-TBP-1 or rhu TNFR:Fc reduced PMA-induced HIV-1 p24 antigen production in a concentration-dependent manner, with a maximal inhibition of approximately 90%. Maximal inhibition of p24 antigen production in T-lymphocytic ACH-2 cells was 47% with r-hTBP-1 and 42% with rhu TNFR:Fc. r-hTBP-1 and rhu TNFR:Fc also decreased p24 antigen synthesized by U1 cells in response to other stimuli, including phytohemagglutinin (PHA)-induced supernatant, granulocyte-macrophage colony-stimulating factor, interleukin-6, and TNF. Addition of r-hTBP-1 to U1 cells during the last 4 h of a 24 h incubation with PMA still inhibited p24 antigen production by 15%. U1 cells stimulated with 10(-7) M PMA released approximately 1 ng/ml endogenous TBP-1 with an initial peak observed at 1 h and a second peak at 24 h after PMA stimulation. r-hTBP-1 also partially reversed inhibition of U1 cellular proliferation caused by PMA. Both r-hTBP-1 and rhu TNFR:Fc blocked PMA induction of nuclear factor (NK)- kappa B DNA-binding activity in U1 cells in association with decreases in HIV-1 replication. We conclude that soluble TNF receptors can inhibit stimuli-induced HIV-1 expression and NK- kappa B DNA-binding activity in chronically infected U1 cells. 


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Transcriptional basis for hyporesponsiveness of the human inducible nitric oxide synthase gene to lipopolysaccharide/interferon-gamma. 
The work reported here resolves, at the level of gene regulation, the controversy as to whether or not human monocytes/macrophages can produce nitric oxide (NO) when stimulated with lipopolysaccharide (LPS), with or without co-stimulation by interferon-gamma (IFN-gamma). Studies included structural comparison of the promoters for human and mouse inducible NO synthase (iNOS) genes, transfection and assay of human and mouse iNOS promoter regions in response to LPS +/- IFN-gamma, and electrophoretic mobility shift assays of kappa B response elements. Two explanations for hyporesponsiveness of the human iNOS promoter to LPS +/- IFN-gamma were found: (1) multiple inactivating nucleotide substitutions in the human counterpart of the enhancer element that has been shown to regulate LPS/IFN-gamma induced expression of the mouse iNOS gene; and (2) and absence of one or more nuclear factors in human macrophages (e.g., an LPS-inducible nuclear factor-kappa B/Rel complex), that is (are) required for maximal expression of the gene. The importance of resolution of this controversy is that future research in this area should be directed toward the understanding of alternative mechanisms that can result in the successful production of NO. 


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Transcriptional regulation during T-cell development: the alpha TCR gene as a molecular model. 
The regulation of gene expression during lymphocyte differentiation is a complex process involving interactions between multiple positive and negative transcriptional regulatory elements. In this article, transcriptional regulation of the archetypal T-cell-specific gene, alpha TCR, is discussed. Major recent developments, including the identification of novel families of transcription factors that regulate multiple T-cell genes during thymocyte ontogeny and T-cell activation, are described. 


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Isolation of cDNA clones for 42 different Kruppel-related zinc finger proteins expressed in the human monoblast cell line U-937. 
To study the complexity and structural characteristics of zinc finger proteins expressed during human hematopoiesis and to isolate novel regulators of blood cell development, a degenerate oligonucleotide probe specific for a consensus zinc finger peptide domain was used to isolate 63 cDNA clones for Kruppel-related zinc finger genes from the human monoblast cell line U-937. By extensive nucleotide sequence and Northern blot analysis, these cDNA clones were found to originate from approximately 42 different genes (HZF 1-42) of which only 8 have previously been described. Northern blot analysis showed that a majority of these genes were expressed at comparable levels in U-937 and HeLa cells. The large number of individual genes represented among the 63 clones and their apparent non-cell-type-specific expression suggest that the majority of the Kruppel-related zinc finger genes are likely to be expressed in most human tissues. In contrast, some of the genes displayed a restricted expression pattern, indicating that they represent potential regulators of monocyte differentiation or proliferation. Detailed structural analysis of the first 12 cDNAs (HZF 1-10) and a partial characterization of HZF 11-42 revealed that a common feature of human Kruppel-related zinc finger proteins is the presence of tandem arrays of zinc fingers ranging in number from 3 to over 20 that are preferentially located in the carboxy-terminal regions of the proteins. In addition, several novel KRAB-containing zinc finger genes and a novel conserved sequence element were identified. 


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Contribution of NF-kappa B and Sp1 binding motifs to the replicative capacity of human immunodeficiency virus type 1: distinct patterns of viral growth are determined by T-cell types. 
Starting with a replication-incompetent molecular clone of human immunodeficiency virus type 1, lacking all the NF-kappa B and Sp1 binding sites present in the native long terminal repeat (LTR), proviruses containing reconstructed LTRs with individual or combinations of NF-kappa B and Sp1 elements were generated and evaluated for their capacity to produce virus progeny following transfection-cocultivation. Virus stocks obtained from these experiments exhibited a continuum of replicative capacities in different human T-cell types depending on which element(s) was present in the LTR. For example, in experiments involving proviral clones with LTRs containing one or two NF-kappa B elements (and no Sp1 binding sites), a hierarchy of cellular permissivity to virus replication (peripheral blood lymphocytes = MT4 greater than H9 greater than CEM greater than Jurkat) was observed. Of note was the associated emergence of second-site LTR revertants which involved an alteration of the TATA box. These results suggest that the human immunodeficiency virus type 1 LTR possesses functional redundancy which ensures virus replication in different T-cell types and is capable of changing depending on the particular combination of transcriptional factors present. 


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Oxidant-regulation of gene expression in the chronically inflamed intestine. 
It is becoming increasingly apparent that the chronic gut inflammation observed in the idiopathic inflammatory bowel diseases (e.g. ulcerative colitis, Crohn's disease) is associated with enhanced production of leukocyte-derived oxidants. Oxidants such as hydrogen peroxide are known to activate certain transcription factors such as nuclear transcription factor kappa beta. Nuclear transcription factor kB (NF-kappa B) is a ubiquitous transcription factor and pleiotropic regulator of numerous genes involved in the immune and inflammatory responses. This transcription factor is activated via the selective phosphorylation, ubiquination and degradation of its inhibitor protein I-kB thereby allowing translocation of NF-kappa B into the nucleus where it upregulates the transcription of a variety of adhesion molecules (e.g. ICAM-1, VCAM-1), cytokines (TNF, IL-1, IL-6) and enzymes (iNOS). The proteolytic degradation of the post-translationally modified I-kappa B is known to be mediated by the 26S proteasome complex. Based upon work from our laboratory, we propose that inhibition of NF-kappa B activation produces significant anti inflammatory activity which may be mediated by the inhibition of transcription of certain pro-inflammatory mediators and adhesion molecules. 


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Coexpression of the interleukin-13 and interleukin-4 genes correlates with their physical linkage in the cytokine gene cluster on human chromosome 5q23-31. 
Interleukin-13 (IL-13) and IL-4 are cytokines produced by T cells that are encoded by the q23-31 region of human chromosome 5. To investigate the regulation of IL-13 gene expression by T cells, we isolated and sequenced the human IL-13 gene, analyzed its 5'-flanking region for potential transcriptional activation elements, and examined its expression in nontransformed T-lineage cell populations. The human IL-13 gene was located 12.5-kb upstream of the IL-4 gene and 2-kb downstream of a CpG island. The IL-13 gene 5' flank region included a segment with sequence homology to P elements of the IL-4 promoter involved in transcriptional activation in T cells. Mutation of the IL-13 P element site significantly reduced IL-13 promoter activity in response to T-cell activation. Oligonucleotides containing the IL-13 or IL-4 P element sites specifically bound the transcriptional activator protein, nuclear factor-activated T cells, preformed (NF-ATp), when incubated with nuclear protein extracts from activated T cells. Similar to IL-4, IL-13 mRNA expression was highest in T-cell populations enriched for cells that had previously been primed in vivo or in vitro, indicating that priming increases the expression of the IL-13 and IL-4 genes in a coordinate manner. Because the primed T cells contain higher levels of nuclear NF-ATp, capable of binding to P elements of the IL-4 and IL-13 promoters, than do freshly-isolated T cells, the NF-AT-binding P elements are attractive candidates to mediate the coordinate expression of these two cytokine genes. 


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Isolation of a rel-related human cDNA that potentially encodes the 65-kD subunit of NF-kappa B [published erratum appears in Science 1991 Oct 4;254(5028):11] 
A DNA probe that spanned a domain conserved among the proto-oncogene c-rel, the Drosophila morphogen dorsal, and the p50 DNA binding subunit of NF-kappa B was generated from Jurkat T cell complementary DNA with the polymerase chain reaction (PCR) and degenerate oligonucleotides. This probe was used to identify a rel-related complementary DNA that hybridized to a 2.6-kilobase messenger RNA present in human T and B lymphocytes. In vitro transcription and translation of the complementary DNA resulted in the synthesis of a protein with an apparent molecular size of 65 kilodaltons (kD). The translated protein showed weak DNA binding with a specificity for the kappa B binding motif. This protein-DNA complex comigrated with the complex obtained with the purified human p65 NF-kappa B subunit and binding was inhibited by I kappa B-alpha and -beta proteins. In addition, the 65-kD protein associated with the p50 subunit of NF-kappa B and the kappa B probe to form a complex with the same electrophoretic mobility as the NF-kappa B-DNA complex. Therefore the rel-related 65-kD protein may represent the p65 subunit of the active NF-kappa B transcription factor complex. 


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Induction of interleukin-12 p40 transcript by CD40 ligation via activation of nuclear factor-kappaB. 
Interleukin-12 is produced in response to infection with bacteria or parasites or to bacterial constituents such as LPS in monocytes/macrophages and dendritic cells, and also generated by the interaction between activated T cells and antigen-presenting cells via CD40-CD40 ligand (CD40L). So far, transcriptional analyses of p40 have been carried out only using bacterial constituents such as LPS as stimuli. In the present study, we have characterized the transcriptional induction of p40 by CD40 ligation in a human B lymphoblastoid cell line, Daudi, and a human acute monocytic leukemia cell line, THP-1. These cells, stimulated by an agonistic monoclonal antibody against CD40 or by transfection with a CD40L expression vector, secreted p40 and showed enhanced p40 mRNA expression. Sequence analysis of the p40 promoter region identified two potential nuclear factor (NF)-kappaB binding sites conserved between mouse and human. Electrophoretic mobility shift assay revealed that the potential NF-kappaB binding sequence which is located around 120 bp upstream of the transcription initiation site in murine and human p40 genes formed an NF-kappaB complex with nuclear extract from Daudi cells stimulated by CD40 ligation. Moreover, transfection of Daudi cells with the polymerized NF-kappaB binding sequence ligated to a thymidine kinase/chloramphenicol acetyltransferase (CAT) reporter plasmid greatly induced CAT activity, but transfection with the polymerized mutated NF-kappaB binding sequence did not. These results suggest that the NF-kappaB binding site located around 120 bp upstream of the transcription initiation site in murine and human p40 promoter regions could be important for the p40 induction by CD40 ligation via activation of NF-kappaB. 


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Inactivation of IkappaBbeta by the tax protein of human T-cell leukemia virus type 1: a potential mechanism for constitutive induction of NF-kappaB. 
In resting T lymphocytes, the transcription factor NF-kappaB is sequestered in the cytoplasm via interactions with members of the I kappa B family of inhibitors, including IkappaBalpha and IkappaBbeta. During normal T-cell activation, IkappaBalpha is rapidly phosphorylated, ubiquitinated, and degraded by the 26S proteasome, thus permitting the release of functional NF-kappaB. In contrast to its transient pattern of nuclear induction during an immune response, NF-kappaB is constitutively activated in cells expressing the Tax transforming protein of human T-cell leukemia virus type I (HTLV-1). Recent studies indicate that HTLV-1 Tax targets IkappaBalpha to the ubiquitin-proteasome pathway. However, it remains unclear how this viral protein induces a persistent rather than transient NF-kappaB response. In this report, we provide evidence that in addition to acting on IkappaBalpha, Tax stimulates the turnover Of IkappaBbeta via a related targeting mechanism. Like IkappaBalpha, Tax-mediated breakdown of IkappaBbeta in transfected T lymphocytes is blocked either by cell-permeable proteasome inhibitors or by mutation Of IkappaBbeta at two serine residues present within its N-terminal region. Despite the dual specificity of HTLV-1 Tax for IkappaBalpha and IkappaBbeta at the protein level, Tax selectively stimulates NF-kappaB-directed transcription of the IkappaBalpha gene. Consequently, IkappaBbeta protein expression is chronically downregulated in HTLV-1-infected T lymphocytes. These findings with IkappaBbeta provide a potential mechanism for the constitutive activation of NF-kappaB in Tax-expressing cells. 


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The role of nuclear factor-kappa B in cytokine gene regulation. 
Transcription factors are DNA-binding proteins that regulate gene expression. Nuclear factor-kappa B (NF-kappa B) is a critical transcription factor for maximal expression of many cytokines that are involved in the pathogenesis of inflammatory diseases, such as adult respiratory distress syndrome (ARDS) and sepsis syndrome. Activation and regulation of NF-kappa B are tightly controlled by a group of inhibitory proteins (I kappa B) that sequester NF-kappa B in the cytoplasm of immune/inflammatory effector cells. NF-kappa B activation involves signaled phosphorylation, ubiquitination, and proteolysis of I kappa B. Liberated NF-kappa B migrates to the nucleus, where it binds to specific promoter sites and activates gene transcription. The activation of NF-kappa B initiates both extracellular and intracellular regulatory events that result in autoregulation of the inflammatory cascade through modulation of NF-kappa B activation. Recently, activation of NF-kappa B has been linked to ARDS and has been shown to be a critical proximal step in the initiation of neutrophilic inflammation in animal models. Activation of NF-kappa B can be inhibited in vivo by treatment with antioxidants, corticosteroids, and the induction of endotoxin tolerance. Identification of more specific and efficacious inhibitors of NF-kappa B activation might prove beneficial for the treatment of cytokine-mediated inflammatory diseases. 


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OBF-1, a novel B cell-specific coactivator that stimulates immunoglobulin promoter activity through association with octamer-binding proteins. 
Recent biochemical and genetic studies indicate that in addition to the octamer-binding proteins Oct-1 and Oct-2, other B cell components are required for lymphoid-restricted, octamer site-mediated immunoglobulin gene promoter activity. Using a genetic screen in yeast, we have isolated B cell-derived cDNAs encoding Oct-binding factor 1 (OBF-1), a novel protein that specifically associates with Oct-1 and Oct-2. Biochemical studies demonstrate that OBF-1 has no intrinsic DNA-binding activity and recognizes the POU domains of Oct-1 and Oct-2, but not those of Oct-4 and Oct-6. The OBF-1 mRNA is expressed in a highly cell-specific manner, being most abundant in B cells and essentially absent in most of the other cells or tissues tested. Furthermore, expression of OBF-1 in HeLa cells selectively stimulates the activity of a natural immunoglobulin promoter in an octamer site-dependent manner. Thus, OBF-1 has all the properties expected for a B cell-specific transcriptional coactivator protein. 


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T cells from renal cell carcinoma patients exhibit an abnormal pattern of kappa B-specific DNA-binding activity: a preliminary report. 
Recent data suggest that the poor induction of a T-cell response to human renal cell carcinoma (RCC) may be related to alterations in signal transduction pathways. We report that T cells from RCC patients have two alterations in kappa B motif-specific DNA-binding activity. The first alteration involves the constitutive expression of substantial kappa B-binding activity in nuclear extracts, which was observed in the electrophoretic mobility shift assay. The magnitude of kappa B activity in unstimulated patient T cells was similar to that observed in T cells from normal individuals that had been activated in vitro. On the basis of Western blotting experiments using antibodies to kappa B/Rel family proteins, the kappa B-binding activity constitutively expressed in T cells from RCC patients is composed mostly of the NF-kappa B1 (p50) subunit. The second abnormality in kappa B-binding activity in T cells from these patients is that RelA, a member of the Rel homology family which is part of the normal NF-kappa B complex, was not induced in the nucleus following activation. Western blotting analysis did not detect any RelA in nuclear extracts either before or after stimulation of T cells. The altered kappa B-binding activity in T cells from RCC patients may impair their capacity to respond normally to various stimuli. 


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Effects of prostaglandin E2 on Th0-type human T cell clones: modulation of functions of nuclear proteins involved in cytokine production. 
The effects of prostaglandin E2 (PGE2) on cytokine production and proliferation of the CD4+ human helper T cell clone SP-B21 were investigated. In cells stimulated with anti-CD3 mAb, PGE2 inhibited cell proliferation and the production of all the cytokines examined. Addition of rIL-2 fully restored the proliferative response and partially restored the production of IL-4 and IL-5, but not that of other cytokines. In contrast, in cells stimulated with phorbol myristate acetate (PMA)/A23187, PGE2 enhanced the production of IL-4 and IL-5, and only partially inhibited the production of other cytokines. Therefore, the effects of PGE2 vary depending on the mode of T cell activation, and the IL-4 and IL-5 are regulated differently from other cytokines. In a mobility shift assay, only the NF-kappa B (p50/p50) homodimer was observed in a complex formed with the kappa B sequence in unstimulated SP-B21 cells. When cells were stimulated with anti-CD3 mAb or PMA/A23187, a complex formation of NF-kappa B (p50/p65) heterodimer with the kappa B sequence was induced. Interestingly, PGE2 or di-butyryl (Bt2)cAMP abolished the binding of NF-kappa B (p50/p65) heterodimer to the kappa B sequence in cells stimulated with anti-CD3 mAb but not with PMA/A23187. Our results suggest that the target of PGE2 action is a component in the signal transduction pathway leading to the activation of protein kinase C. However, the inhibition of the T cell activation signals by PGE2 is selective. PGE2 enhanced the complex formation with NF-AT, AP-1 and CLE0 sequences when the cells were activated by either anti-CD3 mAb or PMA/A23187 stimulation. It seems therefore that PGE2, by elevating cAMP levels, interferes with the activation pathway for NF-kappa B but not for NF-AT, AP-1 or CLE0 binding protein. 


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