UI - 95080245 TI - Activation of JAK kinases and STAT proteins by interleukin-2 and interferon alpha, but not the T cell antigen receptor, in human T lymphocytes. AB - The activation of Janus protein tyrosine kinases (JAKs) and signal transducer and activator of transcription (STAT) proteins by interleukin (IL)-2, the T cell antigen receptor (TCR) and interferon (IFN) alpha was explored in human peripheral blood-derived T cells and the leukemic T cell line Kit225. An IL-2-induced increase in JAK1 and JAK3, but not JAK2 or Tyk2, tyrosine phosphorylation was observed. In contrast, no induction of tyrosine phosphorylation of JAKs was detected upon stimulation of the TCR. IFN alpha induced the tyrosine phosphorylation of JAK1 and Tyk2, but not JAK2 or JAK3. IFN alpha activated STAT1, STAT2 and STAT3 in T cells, but no detectable activation of these STATs was induced by IL-2. However, IL-2 regulates the DNA binding and tyrosine phosphorylation of two STAT-like protein complexes which do not include STAT1, STAT2 or STAT3. STAT4 is not activated by IL-2. The activation of STAT5 cannot be excluded, so the IL-2-activated complexes most probably include at least one novel STAT. No STAT activity was detected in TCR-stimulated lymphocytes, indicating that the JAK/STAT pathway defined in this study constitutes an IL-2R- mediated signaling event which is not shared by the TCR. Finally, in other cell types the correlation between JAK1 activation and the induction of STAT1 has suggested that JAK1 may activate STAT1. The observation that IL-2 and IFN alpha activate JAK1 to a comparable degree, but only IFN alpha activates STAT1, indicates that JAK1 activation is not the only determining factor for STAT1 activation. Moreover, the data show that JAK1 stimulation is also not sufficient for STAT3 activation. UI - 95009961 TI - Differential interactions of Rel-NF-kappa B complexes with I kappa B alpha determine pools of constitutive and inducible NF-kappa B activity. AB - The Rel-NF-kappa B family of transcription factors plays a crucial role in the regulation of genes involved in inflammatory and immune responses. We demonstrate that in vivo, in contrast to the other members of the family, RelB associates efficiently only with NF-kappa B1 (p105-p50) and NF-kappa B2 (p100-p52), but not with cRel or p65. The RelB-p52 heterodimers display a much lower affinity for I kappa B alpha than RelB-p50 heterodimers or p65 complexes. However, similarly to the other Rel-NF-kappa B complexes, RelB-p52 can upregulate the synthesis of I kappa B alpha leading to the cytoplasmic trapping of dimers which have a higher affinity for the inhibitor. We suggest that a hierarchy of interactions between I kappa B alpha and the different Rel-NF-kappa B complexes governs their cellular distribution. This results in the presence of two distinct pools of NF-kappa B activity which differ in their composition: one a constitutive nuclear and the other an inducible cytoplasmic activity. UI - 95009960 TI - Activation of NF-kappa B in vivo is regulated by multiple phosphorylations. AB - The activation of nuclear factor kappa B (NF-kappa B) in intact cells is mechanistically not well understood. Therefore we investigated the modifications imposed on NF-kappa B/I kappa B components following stimulation and show that the final step of NF-kappa B induction in vivo involves phosphorylation of several members of the NF-kappa B/I kappa B protein families. In HeLa cells as well as in B cells, TNF- alpha rapidly induced nuclear translocation primarily of p50-p65, but not of c-rel. Both NF-kappa B precursors and I kappa B alpha became strongly phosphorylated with the same kinetics. In addition to the inducible phosphorylation after stimulation, B lymphocytes containing constitutive nuclear NF-kappa B revealed constitutively phosphorylated p65 and I kappa B alpha. Phosphorylation was accompanied by induced processing of the precursors p100 and p105 and by degradation of I kappa B alpha. As an in vitro model we show that phosphorylation of p105 impedes its ability to interact with NF-kappa B, as has been shown before for I kappa B alpha. Surprisingly, even p65, but not c-rel, was phosphorylated after induction in vivo, suggesting that TNF-alpha selectively activates only specific NF-kappa B heteromers and that modifications regulate not only I kappa B molecules but also NF-kappa B molecules. In fact, cellular NF-kappa B activity was phosphorylation- dependent and the DNA binding activity of p65-containing NF-kappa B was enhanced by phosphorylation in vitro. Furthermore, we found that the induction by hydrogen peroxide of NF-kappa B translocation to the nucleus, which is assumed to be triggered by reactive oxygen intermediates, also coincided with incorporation of phosphate into the same subunits that were modified after stimulation by TNF-alpha. Thus, phosphorylation appears to be a general mechanism for activation of NF- kappa B in vivo. UI - 94155850 TI - Calcineurin acts in synergy with PMA to inactivate I kappa B/MAD3, an inhibitor of NF-kappa B. AB - The interleukin-2 (IL-2) promoter consists of several independent T cell receptor (TcR) responsive elements. The induction of promoters dependent on these elements is inhibitable by the immunosuppressants cyclosporin A (CsA) and tacrolimus (FK-506). Calcineurin, a Ca2+/calmodulin-dependent protein phosphatase, is the FK-506- and CsA- sensitive enzyme required for TcR mediated activation of the IL-2 promoter. We report that a constitutively active form of calcineurin partially substitutes for the Ca2+ co-stimulus required to activate the IL-2 promoter elements IL-2A (which binds the factors OAP and Oct-1) and IL-2E (which binds NF-AT), and completely substitutes for the Ca2+ co-stimulus required to stimulate an NF-kappa B-dependent element. Calcineurin stimulates the NF-kappa B element by enhancing inactivation of I kappa B/MAD3, an inhibitor of NF-kappa B, thereby increasing the amount of nuclear NF-kappa B DNA binding activity. These data provide the first demonstration in vivo that activation of a protein phosphatase can inactivate I kappa B, and suggest one possible explanation for mechanism-based toxicities associated with FK-506 and CsA by demonstrating that these drugs can inhibit the calcineurin- dependent activation of a virtually ubiquitous transcription factor. UI - 94038952 TI - In vivo control of NF-kappa B activation by I kappa B alpha. AB - The transcription factor NF-kappa B is stored in the cytoplasm in complexes with the inhibitor protein I kappa B alpha. It has been shown in vitro that dissociation of I kappa B alpha from these complexes results in active NF-kappa B. In this report we show that lipopolysaccharide (LPS)-induced activation of B or pre-B cells results in loss of I kappa B alpha from NF-kappa B complexes in vivo. Many liberated NF-kappa B dimers reached the nucleus, where increased c-rel, p65 and p50 were detected by immunoblotting and by DNA binding assays. Some liberated dimers were retained in the cytoplasm, however, through binding to newly synthesized I kappa B alpha, a finding which strongly suggests (i) that the LPS-induced signal causes dissociation of complexes rather than preventing their association and (ii) that dissociation results from modification of I kappa B alpha and not of c- rel or p65. No effect of LPS treatment was detected on p105 or p100, which also retain rel family members in the cytoplasm. Quite unexpectedly, we also found that in unstimulated cells there is a constant ongoing process of degradation and replacement of complexed I kappa B alpha. We propose that this turnover results in the low level of active NF-kappa B presumably necessary even in the unstimulated cell, and that the high rate of synthesis of I kappa B alpha provides the ability to turn off NF-kappa B activity rapidly as soon as the activating signal ceases. UI - 94008992 TI - Sox-4, an Sry-like HMG box protein, is a transcriptional activator in lymphocytes. AB - Previous studies in lymphocytes have described two DNA-binding HMG box proteins, TCF-1 and LEF-1, with affinity for the A/TA/TCAAAG motif found in several T cell-specific enhancers. Evaluation of cotransfection experiments in non-T cells and the observed inactivity of an AACAAAG concatamer in the TCF-1/LEF-1-expressing T cell line BW5147, led us to conclude that these two proteins did not mediate the observed enhancer effect. We therefore searched for additional HMG box proteins. By a PCR-aided strategy, we cloned Sox-4, a gene with homology to the HMG box region of the sex determining gene SRY. Sox-4 was expressed in T and pre-B lymphocyte lines and in the murine thymus. Significantly, BW5147 T cells did not express Sox-4. Recombinant Sox-4 bound with high affinity (Kd 3 x 10(-11) M) to the minor groove of the AACAAAG motif, most likely contacting all seven base pairs. In contrast with observations on TCF-1 and LEF-1, cotransfection with Sox-4 unveiled a transactivating capacity, which mapped to its serine-rich C terminus. This region remained functional upon grafting onto a GAL4 DNA- binding domain. Sox-4 is thus the first 'classical' transcription factor in the Sox gene family with separable DNA-binding and transactivation domains. Our observations indicate that a detailed understanding of T cell-specific gene control must integrate the concerted activity of at least three tissue-specific HMG box genes. UI - 92371447 TI - TAR-independent transactivation by Tat in cells derived from the CNS: a novel mechanism of HIV-1 gene regulation. AB - 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) UI - 92331609 TI - Transcription factor AP-2 activates gene expression of HTLV-I. AB - The HTLV-I LTR contains three conserved regulatory elements known as 21 base pair repeats which are required for stimulation of gene expression by the transactivator protein tax. Mutagenesis indicates that the 21 bp repeats can be subdivided into three motifs, A, B and C, each of which influences the level of tax activation. The A site in the 21 bp repeat has strong homology with previously described binding sites for the transcription factor AP-2. We demonstrated that AP-2 mRNA was present in T-lymphocytes and that cellular factors from both non-transformed and transformed T-lymphocytes specifically bound to the consensus motif for AP-2 in each 21 bp. To determine the role of AP-2 in the regulation of the HTLV-I LTR gene expression, we used an AP-2 cDNA in DNA binding and transient expression assays. Gel retardation and methylation interference studies revealed that bacterially produced AP-2 bound specifically and with high affinity to all three 21 bp repeats, and that it required the core sequence AGGC for specific binding. Binding of AP-2 prevented the subsequent binding of members of the CREB/ATF family to an adjacent regulatory motif in the 21 bp repeat. Transfection of an AP-2 expression construct into T-lymphocytes activated gene expression from the HTLV-I LTR. At least two 21 bp repeats were required for high levels of AP-2 activation and mutagenesis of the AP-2 consensus binding sequences in the 21 bp repeats eliminate this activation.(ABSTRACT TRUNCATED AT 250 WORDS) UI - 91293101 TI - Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-kappa B transcription factor and HIV-1. AB - 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. UI - 91266919 TI - Human erythroid 5-aminolevulinate synthase: promoter analysis and identification of an iron-responsive element in the mRNA. AB - 5-Aminolevulinate synthase (ALAS) catalyzes the first step of the heme biosynthetic pathway. cDNA clones for the human erythroid ALAS isozyme were isolated from a fetal liver library. It can be deduced that the erythroid ALAS precursor protein has a molecular weight of 64.6 kd, and is similar in size to the previously isolated human housekeeping ALAS precursor of molecular weight 70.6 kd. The mature mitochondrial forms of the erythroid and housekeeping ALAS isozymes are predicted to have molecular weights of 59.5 kd and 64.6 kd, respectively. The two isozymes show little amino acid identity in their N-terminal signal sequences but have considerable sequence identity in the C-terminal two- thirds of their proteins. An analysis of the immediate promoter of the human erythroid ALAS gene revealed several putative erythroid-specific cis-acting elements including both a GATA-1 and an NF-E2 binding site. An iron-responsive element (IRE) motif has been identified in the 5'- untranslated region of the human erythroid ALAS mRNA, but is not present in the housekeeping ALAS mRNA. Gel retardation experiments established that this IRE motif formed a protein - RNA complex with cytosolic extracts from human K562 cells and this binding was strongly competed with IRE transcripts from ferritin or transferrin receptor mRNAs. A transcript of the ALAS IRE, mutated in the conserved loop of the IRE, did not readily form this protein - RNA complex. These results suggest that the IRE motif in the ALAS mRNA is functional and imply that translation of the mRNA is controlled by cellular iron availability during erythropoiesis. UI - 91266912 TI - Inhibition of transcription factors belonging to the rel/NF-kappa B family by a transdominant negative mutant. AB - 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. UI - 91114695 TI - Identification and cloning of TCF-1, a T lymphocyte-specific transcription factor containing a sequence-specific HMG box. AB - CD3-epsilon expression is controlled by a downstream T lymphocyte- specific enhancer element. We report the identification of a T cell- specific transcription factor, TCF-1, binding to this element. The multimerized recognition motif of TCF-1 constituted a T cell-specific enhancer. Subsequent cloning of TCF-1 identified three splice alternatives. TCF-1 contained a single DNA-binding HMG box most closely related to similar boxes in the putative mammalian sex-determining gene SRY and in the Schizosaccharomyces pombe Mc mating type gene. TCF-1 mRNA was expressed uniquely in T lymphocytes. Upon cotransfection into non-T cells, TCF-1 could transactivate through its cognate motif. These results identify TCF-1 as a T cell-specific transcription factor, which might play a role in the establishment of the mature T cell phenotype. UI - 91092268 TI - Lymphoid specific gene expression of the adenovirus early region 3 promoter is mediated by NF-kappa B binding motifs. AB - 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) UI - 91092266 TI - TAR independent activation of the human immunodeficiency virus in phorbol ester stimulated T lymphocytes. AB - Multiple regulatory elements in the human immunodeficiency virus long terminal repeat (HIV LTR) are required for activation of HIV gene expression. Previous transfection studies of HIV LTR constructs linked to the chloramphenicol acetyltransferase gene indicated that multiple regulatory regions including the enhancer, SP1, TATA and TAR regions were important for HIV gene expression. To characterize these regulatory elements further, mutations in these regions were inserted into both the 5' and 3' HIV LTRs and infectious proviral constructs were assembled. These constructs were transfected into either HeLa cells, Jurkat cells or U937 cells in both the presence and absence of phorbol esters which have previously been demonstrated to activate HIV gene expression. Viral gene expression was assayed by the level of p24 gag protein released from cultures transfected with the proviral constructs. Results in all cell lines indicated that mutations of the SP1, TATA and the TAR loop and stem secondary structure resulted in marked decreases in gene expression while mutations of the enhancer motif or TAR primary sequence resulted in only slight decreases. However, viruses containing mutations in either the TAR loop sequences or stem secondary structure which were very defective for gene expression in untreated Jurkat cells, gave nearly wild-type levels of gene expression in phorbol ester-treated Jurkat cells but not in phorbol ester-treated HeLa or U937 cells. High level gene expression of these TAR mutant constructs in phorbol ester-treated Jurkat cells was eliminated by second site mutations in the enhancer region or by disruption of the tat gene.(ABSTRACT TRUNCATED AT 250 WORDS) UI - 91092267 TI - The actions of cyclosporin A and FK506 suggest a novel step in the activation of T lymphocytes. AB - Cyclosporin A and FK506 are immunosuppressive compounds that have similar inhibitory effects on the expression of several lymphokines produced by T lymphocytes. Despite their similar effects the drugs bind to two different cytosolic protein, cyclophilin and FKBP respectively, which raises the possibility that they have different modes of action. Using constructs in which mRNA production controlled by a specific transcription factor could be readily measured we found that both cyclosporin A and FK506 completely inhibited transcription activated by NF-AT, NFIL2 A, NFIL2 B and partially inhibited transcription activated by NF kappa B. Cyclosporin A and FK506 inhibited only transcriptional activation that was dependent on Ca2+ mobilization. However, cyclosporin A and FK506 did not inhibit Ca2+ mobilization dependent expression of c-fos mRNA indicating that only a subset of signalling pathways regulated by Ca2+ is sensitive to these drugs. Furthermore, we did not observe any qualitative differences between the effect of cyclosporin A and FK506 on six different transcription factors which suggests that these drugs may interfere with the activity of a novel Ca2+ dependent step that regulates several transcription factors. UI - 98315059 TI - Regulation of eosinophil-specific gene expression by a C/EBP-Ets complex and GATA-1. AB - The EOS47 antigen is an early and specific marker of eosinophil differentiation in the chicken haematopoietic system. To elucidate the transciptional events controlling commitment to the eosinophil lineage, we studied the regulation of the eosinophil-specific EOS47 promoter. This promoter is TATA-less, and binds trancription factors of the Ets, C/EBP, GATA and Myb families. These sites are contained within a 309 bp promoter fragment which is sufficient for specific high level transcription in an eosinophil cell line. Co-transfection experiments in Q2bn fibroblasts showed cooperative activation of the EOS47 proximal promoter by c-Myb, Ets-1/Fli-1, GATA-1 and C/EBPalpha. The Ets-1/Fli-1 and C/EBPalpha proteins were the most potent activators, and acted with high synergy through juxtaposed binding sites located approximately 60 bp upstream of the transcription start site. The Ets-1 and C/EBPalpha proteins were found to associate physically via their DNA-binding domains and to bind their combined binding site cooperatively. GATA-1 showed biphasic regulation of the EOS47 promoter, activating at low and repressing at high protein concentrations. These results demonstrate combinatorial activation of an eosinophil-specific promoter by ubiquitous and lineage-restricted haematopoietic transcription factors. They also indicate that direct interactions between C/EBPs and specific Ets family members, together with GATA-1, are important for eosinophil lineage determination. UI - 98211942 TI - Erythroid Kruppel-like factor (EKLF) is active in primitive and definitive erythroid cells and is required for the function of 5'HS3 of the beta-globin locus control region. AB - Disruption of the gene for transcription factor EKLF (erythroid Kruppel- like factor) results in fatal anaemia caused by severely reduced expression of the adult beta-globin gene, while other erythroid- specific genes, including the embryonic epsilon- and fetal gamma-globin genes, are expressed normally. Thus, EKLF is thought to be a stage- specific factor acting through the CACC box in the beta-gene promoter, even though it is already present in embryonic red cells. Here, we show that a beta-globin gene linked directly to the locus control region (LCR) is expressed at embryonic stages, and that this is only modestly reduced in EKLF-/- embryos. Thus, embryonic beta-globin expression is not intrinsically dependent on EKLF. To investigate whether EKLF functions in the locus control region, we analysed the expression of LCR-driven lacZ reporters. This shows that EKLF is not required for reporter activation by the complete LCR. However, embryonic expression of reporters driven by 5'HS3 of the LCR requires EKLF. This suggests that EKLF interacts directly with the CACC motifs in 5'HS3 and demonstrates that EKLF is also a transcriptional activator in embryonic erythropoiesis. Finally, we show that overexpression of EKLF results in an earlier switch from gamma- to beta-globin expression. Adult mice with the EKLF transgene have reduced platelet counts, suggesting that EKLF levels affect the balance between the megakaryocytic and erythroid lineages. Interestingly, the EKLF transgene rescues the lethal phenotype of EKLF null mice, setting the stage for future studies aimed at the analysis of the EKLF protein and its role in beta-globin gene activation. UI - 98211941 TI - Identification of BSAP (Pax-5) target genes in early B-cell development by loss- and gain-of-function experiments. AB - The Pax-5 gene codes for the transcription factor BSAP which is essential for the progression of adult B lymphopoiesis beyond an early progenitor (pre-BI) cell stage. Although several genes have been proposed to be regulated by BSAP, CD19 is to date the only target gene which has been genetically confirmed to depend on this transcription factor for its expression. We have now taken advantage of cultured pre- BI cells of wild-type and Pax-5 mutant bone marrow to screen a large panel of B lymphoid genes for additional BSAP target genes. Four differentially expressed genes were shown to be under the direct control of BSAP, as their expression was rapidly regulated in Pax-5- deficient pre-BI cells by a hormone-inducible BSAP-estrogen receptor fusion protein. The genes coding for the B-cell receptor component Ig- alpha (mb-1) and the transcription factors N-myc and LEF-1 are positively regulated by BSAP, while the gene coding for the cell surface protein PD-1 is efficiently repressed. Distinct regulatory mechanisms of BSAP were revealed by reconstituting Pax-5-deficient pre- BI cells with full-length BSAP or a truncated form containing only the paired domain. IL-7 signalling was able to efficiently induce the N-myc gene only in the presence of full-length BSAP, while complete restoration of CD19 synthesis was critically dependent on the BSAP protein concentration. In contrast, the expression of the mb-1 and LEF- 1 genes was already reconstituted by the paired domain polypeptide lacking any transactivation function, suggesting that the DNA-binding domain of BSAP is sufficient to recruit other transcription factors to the regulatory regions of these two genes. In conclusion, these loss- and gain-of-function experiments demonstrate that BSAP regulates four newly identified target genes as a transcriptional activator, repressor or docking protein depending on the specific regulatory sequence context. UI - 98169389 TI - Cooperative interaction of ets-1 with USF-1 required for HIV-1 enhancer activity in T cells. AB - The distal enhancer region of the human immunodeficiency virus 1 (HIV- 1) long terminal repeat (LTR) is known to be essential for HIV replication and to contain immediately adjacent E-box and Ets binding sites. Based on a yeast one-hybrid screen we have identified the E-box binding protein USF-1 as a direct interaction partner of Ets-1 and found that the complex acts on this enhancer element. The binding surfaces of USF-1 and Ets-1 map to their DNA-binding domains and although these domains are highly conserved, the interaction is very selective within the respective protein family. USF-1 and Ets-1 synergize in specific DNA binding as well as in the transactivation of reporter constructs containing the enhancer element, and mutations of the individual binding sites dramatically reduce reporter activity in T cells. In addition, a dominant negative Ets-1 mutant inhibits both USF- 1-mediated transactivation and the activity of the HIV-1 LTR in T cells. The inhibition is independent of Ets DNA-binding sites but requires the Ets binding surface on USF-1, highlighting the importance of the direct protein-protein interaction. Together these results indicate that the interaction between Ets-1 and USF-1 is required for full transcriptional activity of the HIV-1 LTR in T cells. UI - 98169386 TI - Epstein-Barr virus-mediated B-cell proliferation is dependent upon latent membrane protein 1, which simulates an activated CD40 receptor. AB - The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) is essential for the immortalization of human B cells and is linked etiologically to several human tumors. LMP1 is an integral membrane protein which acts like a constitutively active receptor. It binds tumor necrosis factor (TNF)-receptor-associated factors (TRAFs), activates NF-kappaB and triggers the transcription factor AP-1 via the c-Jun N-terminal kinase (JNK) cascade, but its specific contribution to B-cell immortalization has not been elucidated fully. To address the function of LMP1, we established B cell lines with a novel mini-EBV plasmid in which the LMP1 gene can be regulated at will without affecting the expression of other latent EBV genes. We demonstrate here that continuous expression of LMP1 is essential for the proliferation of EBV-immortalized B cells in vitro. Re-induction of LMP1 expression or activation of the cellular CD40 receptor both induce the JNK signaling cascade, activate the transcription factor NF-kappaB and stimulate proliferation of these B cells. Our findings strongly suggest that LMP1 mimics B-cell activation processes which are physiologically triggered by CD40-CD40 ligand signals. Since LMP1 acts in a ligand- independent manner, it replaces the T cell-derived activation signal to sustain indefinite B-cell proliferation. UI - 98026889 TI - Latent membrane protein 1 of Epstein-Barr virus mimics a constitutively active receptor molecule. AB - Latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) is an integral membrane protein which has transforming potential and is necessary but not sufficient for B-cell immortalization by EBV. LMP1 molecules aggregate in the plasma membrane and recruit tumour necrosis factor receptor (TNF-R) -associated factors (TRAFs) which are presumably involved in the signalling cascade leading to NF-kappaB activation by LMP1. Comparable activities are mediated by CD40 and other members of the TNF-R family, which implies that LMP1 could function as a receptor. LMP1 lacks extended extracellular domains similar to beta-adrenergic receptors but, in contrast, it also lacks any motifs involved in ligand binding. By using LMP1 mutants which can be oligomerized at will, we show that the function of LMP1 in 293 cells and B cells is solely dependent on oligomerization of its carboxy- terminus. Biochemically, oligomerization is an intrinsic property of the transmembrane domain of wild-type LMP1 and causes a constitutive phenotype which can be conferred to the signalling domains of CD40 or the TNF-2 receptor. In EBV, immortalized B cells cross-linking in conjunction with membrane targeting of the carboxy-terminal signalling domain of LMP1 is sufficient for its biological activities. Thus, LMP1 acts like a constitutively activated receptor whose biological activities are ligand-independent. UI - 98062995 TI - Epstein-Barr virus latent membrane protein-1 triggers AP-1 activity via the c-Jun N-terminal kinase cascade. AB - The Epstein-Barr virus latent membrane protein-1 (LMP-1) is an integral membrane protein which transforms fibroblasts and is essential for EBV- mediated B-cell immortalization. LMP-1 has been shown to trigger cellular NF-kappa B activity which, however, cannot fully explain the oncogenic potential of LMP-1. Here we show that LMP-1 induces the activity of the AP-1 transcription factor, a dimer of Jun/Jun or Jun/Fos proteins. LMP-1 effects on AP-1 are mediated through activation of the c-Jun N-terminal kinase (JNK) cascade, but not the extracellular signal-regulated kinase (Erk) pathway. Consequently, LMP-1 triggers the activity of the c-Jun N-terminal transactivation domain which is known to be activated upon JNK-mediated phosphorylation. Deletion analysis indicates that the 55 C-terminal amino acids of the LMP-1 molecule, but not its TRAF interaction domain, are essential for AP-1 activation. JNK- mediated transcriptional activation of AP-1 is the direct output of LMP- 1-triggered signaling, as shown by an inducible LMP-1 mutant. Using a tetracycline-regulated LMP-1 allele, we demonstrate that JNK is also an effector of non-cytotoxic LMP-1 signaling in B cells, the physiological target cells of EBV. In summary, our data reveal a novel effector of LMP-1, the SEK/JNK/c-Jun/AP-1 pathway, which contributes to our understanding of the immortalizing and transforming potential of LMP-1. UI - 97459950 TI - Cooperation of Spi-1/PU.1 with an activated erythropoietin receptor inhibits apoptosis and Epo-dependent differentiation in primary erythroblasts and induces their Kit ligand-dependent proliferation. AB - Spi-1/PU.1 is a myeloid- and B-cell specific transcription factor which is also involved in Friend virus-induced murine erythroleukemia. The pre-leukemic phase of Friend erythroleukemia results from activation of the erythropoietin receptor (EpoR) by the spleen focus forming virus (SFFV) envelope glycoprotein, followed by the emergence of leukemic clones characterized by overexpression of Spi-1 and mutation of the p53 tumor suppressor gene. We developed a heterologous system to analyze the contribution of these alterations to the induction of primary erythroblast transformation. Avian erythroblasts expressing the activated mouse EpoR(R129C) differentiated into erythrocytes in response to hEpo. Expression of Spi-1 in these cells inhibited this ability to differentiate and rescued the cells from the apoptotic cell death program normally induced upon hEpo withdrawal. Although devoid of any effect by itself, a mutant p53 cooperated with Spi-1 and EpoR(R129C) to reinforce both phenotypes. Analysis of erythroblasts co- expressing Spi-1 and the wild-type mouse EpoR showed that differentiation arrest and inhibition of apoptosis depended on specific cooperation between Spi-1 and EpoR(R129C). This cooperation was also required to induce the sustained proliferation of differentiation- blocked erythroblasts in response to ligand activation of the endogenous tyrosine kinase receptor c-Kit. These results show that Spi- 1/PU.1 requires signals emanating from specific cytokine and growth factor receptors to affect the survival, proliferation and differentiation control of primary erythroblasts. They also suggest that the function of Spi-1/PU.1 in the late phase of Friend leukemia requires specific signaling from the gp55-modified EpoR generated during the early phase of the disease. UI - 97377031 TI - c-Myb and Ets proteins synergize to overcome transcriptional repression by ZEB. AB - The Zfh family of zinc finger/homeodomain proteins was first identified in Drosophila where it is required for differentiation of tissues such as the central nervous system and muscle. ZEB, a vertebrate homolog of Zfh-1, binds a subset of E boxes and blocks myogenesis through transcriptional repression of muscle genes. We present evidence here that ZEB also has an important role in controlling hematopoietic gene transcription. Two families of transcription factors that are required for normal hematopoiesis are c-Myb and Ets. These factors act synergistically to activate transcription, and this synergy is required for transcription of at least several important hematopoietic genes. ZEB blocks the activity of c-Myb and Ets individually, but together the factors synergize to resist this repression. Such repression imposes a requirement for both c-Myb and Ets for transcriptional activity, providing one explanation for why synergy between these factors is important. The balance between repression by ZEB and transcriptional activation by c-Myb/Ets provides a flexible regulatory mechanism for controlling gene expression in hematopoietic cells. We demonstrate that one target of this positive/negative regulation in vivo is the alpha4 integrin, which play a key role in normal hematopoiesis and function of mature leukocytes. UI - 97180928 TI - Rapid Ca2+-mediated activation of Rap1 in human platelets. AB - Rap1 is a small, Ras-like GTPase whose function and regulation are still largely unknown. We have developed a novel assay to monitor the active, GTP-bound form of Rap1 based on the differential affinity of Rap1GTP and Rap1GDP for the Rap binding domain of RalGDS (RBD). Stimulation of blood platelets with alpha-thrombin or other platelet activators caused a rapid and strong induction of Rap1 that associated with RBD in vitro. Binding to RBD increased from undetectable levels in resting platelets to 50% of total Rap1 within 30 s after stimulation. An increase in the intracellular Ca2+ concentration is both necessary and sufficient for Rap1 activation since it was induced by agents that increase intracellular Ca2+ and inhibited by a Ca2+-chelating agent. Neither inhibition of translocation of Rap1 to the cytoskeleton nor inhibition of platelet aggregation affected thrombin-induced activation of Rap1. In contrast, prostaglandin I2 (PGI2), a strong negative regulator of platelet function, inhibited agonist-induced as well as Ca2+-induced activation of Rap1. From our results, we conclude that Rap1 activation in platelets is an important common event in early agonist-induced signalling, and that this activation is mediated by an increased intracellular Ca2+ concentration. UI - 97051821 TI - 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. AB - 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. UI - 96324400 TI - Multiple p21ras effector pathways regulate nuclear factor of activated T cells. AB - The transcription factor, Nuclear Factor of Activated T cells (NFAT) is a major target for p21ras and calcium signalling pathways in the IL-2 gene and is induced by p21ras signals acting in synergy with calcium/calcineurin signals. One p21ras effector pathway involves the MAP kinase ERK-2, and we have examined its role in NFAT regulation. Expression of dominant negative MAPKK-1 prevents NFAT induction. Constitutively active MAPKK-1 fully activates ERK-2 and the transcription factor Elk-1, but does not substitute for activated p21ras and synergize with calcium/calcineurin signals to induce NFAT. Expression of dominant negative N17Rac also prevents TCR and p21ras activation of NFAT, but without interfering with the ERK-2 pathway. The transcriptional activity of the NFAT binding site is mediated by a complex comprising a member of the NFAT group and AP-1 family proteins. The induction of AP-1 by p21ras also requires Rac-1 function. Activated Rac-1 could mimic activated p21ras to induce AP-1 but not to induce NFAT. Moreover, the combination of activated MAPKK-1 and Rac-1 could not substitute for activated p21ras and synergize with calcium signals to induce NFAT. Thus, p21ras regulation of NFAT in T cells requires the activity of multiple effector pathways including those regulated by MAPKK-1/ERK-2 and Rac-1. UI - 96256295 TI - The B cell coactivator Bob1 shows DNA sequence-dependent complex formation with Oct-1/Oct-2 factors, leading to differential promoter activation. AB - We have shown previously that both octamer binding transcription factors, namely the ubiquitous Oct-1 and the B cell-specific Oct-2A protein, can be enhanced in transcriptional activity by their association with the B cell-specific coactivator protein Bob1, also called OBF-1 or OCA-B. Here we study the structural requirements for ternary complex formation of DNA-Oct-Bob1 and coactivation function of Bob1. In analogy to DNA-bound transcription factors, Bob1 has a modular structure that includes an interaction domain (amino acids 1-65) and a C-terminal domain (amino acids 65-256), both important for transcriptional activation. A mutational analysis has resolved a region of seven amino acids (amino acids 26-32) in the N-terminus of Bob1 that are important for contacting the DNA binding POU domain of Oct-1 or Oct- 2. In contrast to the viral coactivator VP16 (vmw65), which interacts with Oct-1 via the POU homeosubdomain, Bob1 association with Oct factors requires residues located in the POU-specific subdomain. Because the same residues are also involved in DNA recognition, we surmised that this association would affect the DNA binding specificity of the Oct-Bob1 complex compared with free Oct factors. While Oct-1 or Oct-2 bind to a large variety of octamer sequences, Bob1 ternary complex formation is indeed highly selective and occurs only in a subset of these sequences, leading to the differential coactivation of octamer-containing promoters. The results uncover a new level in selectivity that furthers our understanding in the regulation of cell type-specific gene expression. UI - 96203119 TI - C-terminal activating and inhibitory domains determine the transactivation potential of BSAP (Pax-5), Pax-2 and Pax-8. AB - Pax-5 encodes the transcription factor BSAP which plays an essential role in early B cell development and midbrain patterning. In this study we have analysed the structural requirements for transcriptional activation by BSAP. In vitro mutagenesis and transient transfection experiments indicate that the C-terminal serine/threonine/proline-rich region of BSAP contains a potent transactivation domain of 55 amino acids which is active from promoter and enhancer positions. This transactivation domain was found to be inactivated by a naturally occurring frameshift mutation in one PAX-5 allele of the acute lymphoblastic leukemia cell line REH. The function of the transactivation domain is negatively regulated by adjacent sequences from the extreme C-terminus. The activating and inhibitory domains function together as an independent regulatory module in different cell types as shown by fusion to the GAL4 DNA binding domain. The same arrangement of positively and negatively acting sequences has been conserved in the mammalian Pax-2 and Pax-8, the zebrafish Pax-b as well as the sea urchin Pax-258 proteins. These data demonstrate that the transcriptional competence of a subfamily of Pax proteins is determined by a C-terminal regulatory module composed of activating and inhibitory sequences. UI - 96178227 TI - GATA transcription factors associate with a novel class of nuclear bodies in erythroblasts and megakaryocytes. AB - The nuclear distribution of GATA transcription factors in murine haemopoietic cells was examined by indirect immunofluorescence. Specific bright foci of GATA-1 fluorescence were observed in erythroleukaemia cells and primary murine erythroblasts and megakaryocytes, in addition to diffuse nucleoplasmic localization. These foci, which were preferentially found adjacent to nucleoli or at the nuclear periphery, did not represent sites of active transcription or binding of GATA-1 to consensus sites in the beta-globin loci. Immunoelectron microscopy demonstrated the presence of intensely labelled structures likely to represent the GATA-1 foci seen by immunofluorescence. The GATA-1 nuclear bodies differed from previously described nuclear structures and there was no co-localization with nuclear antigens involved in RNA processing or other ubiquitous (Sp1, c- Jun and TBP) or haemopoietic (NF-E2) transcription factors. Interestingly, GATA-2 and GATA-3 proteins also localized to the same nuclear bodies in cell lines co-expressing GATA-1 and -2 or GATA-1 and - 3 gene products. This pattern of distribution is, thus far, unique to the GATA transcription factors and suggests a protein-protein interaction with other components of the nuclear bodies via the GATA zinc finger domain. UI - 96176764 TI - Cross-linking CD40 on B cells preferentially induces stress-activated protein kinases rather than mitogen-activated protein kinases. AB - The B cell-associated surface molecule CD40 plays a key role in T cell- dependent B cell maturation, as individuals with defects in either CD40 or its ligand are impaired in immunoglobulin isotype class switching and germinal center formation. CD40 signaling activates downstream effectors, including the tyrosine protein kinase, Lyn, the phosphatidylinositol-3-kinase (PI-3 kinase), and the transcription factor, NF-kappa B. In this study, we demonstrate that stress-activated protein kinases (SAPK) are activated after CD40 cross-linking on various B cell lines or human tonsillar B cells. The activation is rapid and transient and is mediated through a 2cyclosporin A-insensitive pathway. Furthermore, this signaling pathway appears not to rely on protein kinase C. While CD40 ligation strongly activates the SAPKs (up to 25-fold), it does not affect members of the mitogen-activated protein kinase family (MAPK; ERK1 and ERK2). Consistent with these data, CD40 signals up-regulate c-jun but not c-fos mRNA and alter the transcription factor ATF2 but not the Raf-1 protein. In summary, CD40 signaling preferentially induces SAPK but not MAPK. UI - 96134911 TI - NF-M (chicken C/EBP beta) induces eosinophilic differentiation and apoptosis in a hematopoietic progenitor cell line. AB - CAAT/enhancer binding proteins (C/EBPs) are transcriptional activators implicated in the differentiation processes of various cell lineages. We have shown earlier that NF-M, the chicken homolog of C/EBP beta, is specifically expressed in myelomonocytic and eosinophilic cells of the hematopoietic system. To investigate the role of NF-M in hematopoietic cell lineage commitment, we constructed a conditional form of the protein by fusing it to the hormone binding domain of the human estrogen receptor. This construct was stably expressed in a multipotent progenitor cell line transformed by the Myb-Ets oncoprotein. We report here that both NF-M-dependent promoter constructs and resident genes could be activated by addition of beta-estradiol to the NF-M-estrogen receptor expressing progenitors. At the same time, we observed a down- regulation of progenitor-specific surface markers and the up-regulation of differentiation markers restricted to the eosinophil and myeloid lineages. In addition to the onset of differentiation, cell death was induced with typical apoptotic features. Our results suggest that NF-M plays an important role in commitment along the eosinophil lineage and in the induction of apoptosis. UI - 96067134 TI - In vivo regulation of interleukin-2 receptor alpha gene transcription by the coordinated binding of constitutive and inducible factors in human primary T cells. AB - IL-2R alpha transcription is developmentally restricted to T cells and physiologically dependent on specific stimuli such as antigen recognition. To analyse the mechanisms used to activate IL-2R alpha transcription as well as those used to block it in non-expressing cells, we determined the protein-DNA interactions at the IL-2R alpha locus in three different cell types using the DMS/LMPCR genomic footprinting method. CD25/IL-2R alpha can be efficiently induced in primary human T cells since approximately 100% express this gene when receiving an appropriate combination of mitogenic stimuli. To understand why IL-2R alpha is not expressed in other haematopoietic cell types, we analysed BJAB B lymphoma cells which do not express the IL-2R alpha gene and contain constitutively active nuclear NF-kappa B. Primary fibroblasts from embryo and adult skin were selected to examine the mechanisms that may be used to keep the IL-2R alpha gene inactive in non-haematopoietic cells. The three main results are: (i) the stable in vivo occupancy of IL-2R alpha kappa B element in resting T cells, most probably by constitutive NF-kappa B p50 homodimer that could impair SRF binding to the flanking SRE/CArG box; (ii) its inducible occupancy by NF-kappa B p50-p65 associated with the binding of an SRE/CArG box DNA-binding factor upon mitogenic stimulation; and (iii) a correlation between the precommitment of T cells to activation and the presence of stable preassembled protein-DNA complexes in contrast with the bare IL-2R alpha locus in non-T cells. UI - 95369245 TI - IL-2 gene expression and NF-kappa B activation through CD28 requires reactive oxygen production by 5-lipoxygenase. AB - Activation of the CD28 surface receptor provides a major costimulatory signal for T cell activation resulting in enhanced production of interleukin-2 (IL-2) and cell proliferation. In primary T lymphocytes we show that CD28 ligation leads to the rapid intracellular formation of reactive oxygen intermediates (ROIs) which are required for CD28- mediated activation of the NF-kappa B/CD28-responsive complex and IL-2 expression. Delineation of the CD28 signaling cascade was found to involve protein tyrosine kinase activity, followed by the activation of phospholipase A2 and 5-lipoxygenase. Our data suggest that lipoxygenase metabolites activate ROI formation which then induce IL-24 expression via NF-kappa B activation. These findings should be useful for therapeutic strategies and the development of immunosuppressants targeting the CD28 costimulatory pathway. UI - 95262639 TI - RelA/p65 is a molecular target for the immunosuppressive action of protein kinase A. AB - Stimulation of the protein kinase A (PKA) signalling pathway exerts an inhibitory effect on the proliferation of numerous cells, including T lymphocytes. In CD4+ T helper cells, stimulation of PKA leads to suppression of interleukin 2 (IL-2) induction, while induction of the genes coding for the lymphokines IL-4 and IL-5 is enhanced. We show that the differential effect of PKA activity on induction of the IL-2 and IL-4 genes is mediated through their promoters. One major target of the suppressive effect of PKA is the kappa B site in the IL-2 promoter. A kappa B site is missing in the IL-4 promoter. Mutations preventing factor binding to the IL-2 kappa B site result in a loss of PKA- mediated suppression of IL-2 promoter activity. Furthermore, activation of the PKA signalling pathway impairs the inducible activity of multiple kappa B sites of the IL-2 promoter, but not of other factor binding sites. The reduction in activity of kappa B sites in activated and PKA-stimulated T cells is accompanied by changes in the concentration and DNA binding of Rel/NF-kappa B factors. Stimulation of the PKA pathway in Jurkat T cells with the PKA activator forskolin leads to an increase in synthesis of c-Rel and p105/p50, while synthesis of p65/RelA remains unchanged. However, nuclear translocation and DNA binding of p65 is distinctly impaired, probably due to a retarded degradation of I kappa B-alpha. In a similar way, stimulation of the PKA signalling pathway inhibits nuclear translocation of p65 and generation of nuclear kappa B complexes in peripheral T lymphocytes from murine lymph nodes. These results indicate that PKA-mediated suppression of NF-kappa B activity plays an important role in the control of activation of peripheral T lymphocytes. UI - 95246748 TI - Absolute dependence on kappa B responsive elements for initiation and Tat-mediated amplification of HIV transcription in blood CD4 T lymphocytes. AB - The role of NF-kappa B-dependent signals in activating the transcriptional activity of the HIV regulatory region (LTR) was analyzed by systematic comparison of HIV LTR activity in human CD4 T cells purified from peripheral blood and a transformed lymphoblastoid T cell line. In normal CD4 T cells we also analyzed the role played by the viral kappa B responsive elements in HIV replication. Analysis of nuclear extracts of resting, normal T lymphocytes revealed the presence of the p50, but not the p65, NF-kappa B subunit and the induction by phorbol esters of bona fide (p50-p65) NF-kappa B complexes. In parallel, we observed clear enhancer-dependent HIV LTR transactivation comparable in intensity with that observed in lymphoblastoid cells. We show that unstimulated CD4 T lymphocytes offer a cellular environment of very low permissivity to HIV LTR functioning. This was in sharp contrast to the high spontaneous LTR activity observed in lymphoblastoid T cells, where LTR activity was essentially independent of kappa B-responsive elements. Due to the low basal LTR activity in resting T lymphocytes, NF-kappa B-dependent transactivation was a sine qua non event for induction of the HIV LTR. Surprisingly, even the function of HIV Tat in resting CD4 T lymphocytes was found to be absolutely dependent on LTR kappa B responsive elements. The relevance of these observations obtained in transient transfections was confirmed by the incapacity of blood CD4 T lymphocytes infected with an HIV infectious provirus carrying critical point mutations in the kappa B responsive elements to show any detectable transcriptional activity upon cell activation and prolonged culture in vitro.(ABSTRACT TRUNCATED AT 250 WORDS) UI - 95163587 TI - HIV-1 Tat potentiates TNF-induced NF-kappa B activation and cytotoxicity by altering the cellular redox state. AB - This study demonstrates that human immunodeficiency virus type 1 (HIV- 1) Tat protein amplifies the activity of tumor necrosis factor (TNF), a cytokine that stimulates HIV-1 replication through activation of NF- kappa B. In HeLa cells stably transfected with the HIV-1 tat gene (HeLa- tat cells), expression of the Tat protein enhanced both TNF-induced activation of NF-kappa B and TNF-mediated cytotoxicity. A similar potentiation of TNF effects was observed in Jurkat T cells and HeLa cells treated with soluble Tat protein. TNF-mediated activation of NF- kappa B and cytotoxicity involves the intracellular formation of reactive oxygen intermediates. Therefore, Tat-mediated effects on the cellular redox state were analyzed. In both T cells and HeLa cells HIV- 1 Tat suppressed the expression of Mn-dependent superoxide dismutase (Mn-SOD), a mitochondrial enzyme that is part of the cellular defense system against oxidative stress. Thus, Mn-SOD RNA protein levels and activity were markedly reduced in the presence of Tat. Decreased Mn-SOD expression was associated with decreased levels of glutathione and a lower ratio of reduced:oxidized glutathione. A truncated Tat protein (Tat1-72), known to transactivate the HIV-1 long terminal repeat (LTR), no longer affected Mn-SOD expression, the cellular redox state or TNF- mediated cytotoxicity. Thus, our experiments demonstrate that the C- terminal region of HIV-1 Tat is required to suppress Mn-SOD expression and to induce pro-oxidative conditions reflected by a drop in reduced glutathione (GSH) and the GSH:oxidized GSH (GSSG) ratio.(ABSTRACT TRUNCATED AT 250 WORDS) UI - 95163578 TI - Glucocorticoid-induced apoptosis of human leukemic cells is caused by the repressive function of the glucocorticoid receptor AB - Induction of apoptosis in lymphocytes, which may account for the therapeutic effects of glucocorticoids in various diseases including leukemia, depends on the glucocorticoid receptor. However, the events leading from the activated receptor to cell lysis are not understood. A prevailing hypothesis postulates induction of so-called 'lysis genes' by the activated receptor. In this study, we show that an activation- deficient glucocorticoid receptor mutant is as effective as the wild- type receptor in repression of AP-1 activity, inhibition of interleukin- 2 production, inhibition of c-myc expression and induction of apoptosis. Furthermore, we show that retinoic acid can also induce apoptosis in these cells through the retinoic acid receptor, whose repressive functions but not target site specificity, are similar to those of the glucocorticoid receptor. Therefore, the primary effect of the receptor in glucocorticoid-mediated apoptosis correlates with transcriptional repression rather than activation and could be mediated by interference with other transcription factors required for cell survival. UI - 95129555 TI - B-cell proliferation and induction of early G1-regulating proteins by Epstein-Barr virus mutants conditional for EBNA2. AB - Infection of primary B-lymphocytes by Epstein-Barr virus (EBV) leads to growth transformation of these B-cells in vitro. EBV nuclear antigen 2 (EBNA2), one of the first genes expressed after EBV infection of B- cells, is a transcriptional activator of viral and cellular genes and is essential for the transforming potential of the virus. We generated conditional EBV mutants by expressing EBNA2 as chimeric fusion protein with the hormone binding domain of the estrogen receptor on the genetic background of the virus. Growth transformation of primary normal B- cells by mutant virus resulted in estrogen-dependent lymphoblastoid cell lines expressing the chimeric EBNA2 protein. In the absence of estrogen about half of the cells enter a quiescent non-proliferative state whereas the others die by apoptosis. EBNA2 is thus required not only for initiation but also for maintenance of transformation. Growth arrest occurred at G1 and G2 stages of the cell cycle, indicating that functional EBNA2 is required at different restriction points of the cell cycle. Growth arrest is reversible for G1/G0 cells as indicated by the sequential accumulation and modification of cell cycle regulating proteins. EBV induces the same cell cycle regulating proteins as polyclonal stimuli in primary B-cells. These data suggest that EBV is using a common pathway for B-cell activation bypassing the requirement for antigen, T-cell signals and growth factors. UI - 99147079 TI - The hematopoietic-specific adaptor protein gads functions in T-cell signaling via interactions with the SLP-76 and LAT adaptors. AB - BACKGROUND: The adaptor protein Gads is a Grb2-related protein originally identified on the basis of its interaction with the tyrosine- phosphorylated form of the docking protein Shc. Gads protein expression is restricted to hematopoietic tissues and cell lines. Gads contains a Src homology 2 (SH2) domain, which has previously been shown to have a similar binding specificity to that of Grb2. Gads also possesses two SH3 domains, but these have a distinct binding specificity to those of Grb2, as Gads does not bind to known Grb2 SH3 domain targets. Here, we investigated whether Gads is involved in T-cell signaling. RESULTS: We found that Gads is highly expressed in T cells and that the SLP-76 adaptor protein is a major Gads-associated protein in vivo. The constitutive interaction between Gads and SLP-76 was mediated by the carboxy-terminal SH3 domain of Gads and a 20 amino-acid proline-rich region in SLP-76. Gads also coimmunoprecipitated the tyrosine- phosphorylated form of the linker for activated T cells (LAT) adaptor protein following cross-linking of the T-cell receptor; this interaction was mediated by the Gads SH2 domain. Overexpression of Gads and SLP-76 resulted in a synergistic augmentation of T-cell signaling, as measured by activation of nuclear factor of activated T cells (NFAT), and this cooperation required a functional Gads SH2 domain. CONCLUSIONS: These results demonstrate that Gads plays an important role in T-cell signaling via its association with SLP-76 and LAT. Gads may promote cross-talk between the LAT and SLP-76 signaling complexes, thereby coupling membrane-proximal events to downstream signaling pathways. UI - 99141160 TI - Mycobacterium avium complex activates nuclear factor kappaB via induction of inflammatory cytokines. AB - A variety of microorganisms has been reported to directly induce NF- kappaB, a critical step in the regulation of genes involved in the cellular immune response. In this study, we demonstrate that proinflammatory cytokines such as tumor necrosis factor alpha (TNFalpha) produced upon activation by the Mycobacterium avium complex (MAC) preceed NF-kappaB activity in U937, a human monocytoid cell line. MAC induction of TNFalpha mRNA expression was detected within 15 min after MAC infection, whereas enhanced NF-kappaB binding activity was not detected until 90 to 120 min postinfection. Supershift analysis revealed increased p50 in the MAC-induced NF-kappaB binding complexes. Consistent with an autocrine mechanism, anti-TNFalpha antibody and dexamethasone, a known cytokine inhibitor, both completely suppressed the effect of MAC on the induction of NF-kappaB. Taken together, these findings suggest that exposure of monocyte cell membranes to MAC induces endogenous TNFalpha, which in turn enhances NF-kappaB binding activity. The rapid induction of TNFalpha may be important in the initial host response to MAC infection. UI - 99101490 TI - IL-2 induces STAT4 activation in primary NK cells and NK cell lines, but not in T cells. AB - IL-2 exerts potent but distinct functional effects on two critical cell populations of the immune system, T cells and NK cells. Whereas IL-2 leads to proliferation in both cell types, it enhances cytotoxicity primarily in NK cells. In both T cells and NK cells, IL-2 induces the activation of STAT1, STAT3, and STAT5. Given this similarity in intracellular signaling, the mechanism underlying the distinct response to IL-2 in T cells and NK cells is not clear. In this study, we show that in primary NK cells and NK cell lines, in addition to the activation of STAT1 and STAT5, IL-2 induces tyrosine phosphorylation of STAT4, a STAT previously reported to be activated only in response to IL-12 and IFN-alpha. This activation of STAT4 in response to IL-2 is not due to the autocrine production of IL-12 or IFN-alpha. STAT4 activated in response to IL-2 is able to bind to a STAT-binding DNA sequence, suggesting that in NK cells IL-2 is capable of activating target genes through phosphorylation of STAT4. IL-2 induces the activation of Jak2 uniquely in NK cells, which may underlie the ability of IL-2 to activate STAT4 only in these cells. Although the activation of STAT4 in response to IL-2 occurs in primary resting and activated NK cells, it does not occur in primary resting T cells or mitogen- activated T cells. The unique activation of the STAT4-signaling pathway in NK cells may underlie the distinct functional effect of IL-2 on this cell population. UI - 99085032 TI - Lipopolysaccharide induction of tissue factor in THP-1 cells involves Jun protein phosphorylation and nuclear factor kappaB nuclear translocation. AB - Tissue Factor (TF) gene expression is transiently induced in human monocytic THP-1 cells by lipopolysaccharide (LPS). We characterized the transcription factor complexes binding to the TF gene promoter LPS response element (LRE) (-220 to -172), which contains binding sites for nuclear factor kappaB (NFkappaB) and activator protein 1 (AP1) transcription factors, and examined the nature of the activation of these factors during a 24-h time course of LPS stimulation. We found proteolysis of the cytoplasmic inhibitory protein IkappaBalpha and nuclear translocation of the NFkappaB/Rel family proteins p65 and c- Rel, corresponding to the transient binding of a p65/c-Rel heterodimer to the kappaB-like site of the LRE. AP1 binding to the LRE was found to be constitutive, with the majority of the AP1 complexes being JunD/Fra- 2 heterodimers. A change in the activation state of the AP1 complexes was, however, found to be transient, as determined by JunD phosphorylation of AP1 bound to the proximal binding site. This directly correlates to the transient activation of Jun N-terminal kinase (SAPK/JNK). These data indicate that LPS induction of TF gene expression in monocytic THP-1 cells is regulated by both the transient phosphorylation of Jun-family proteins and the nuclear translocation and transient binding of NFkappaB/Rel proteins. UI - 99084994 TI - Characterization of calcineurin in human neutrophils. Inhibitory effect of hydrogen peroxide on its enzyme activity and on NF-kappaB DNA binding. AB - We describe here a specific calcineurin activity in neutrophil lysates, which is dependent on Ca2+, inhibited by trifluoroperazine, and insensitive to okadaic acid. Immunoblotting experiments using a specific antiserum recognized both the A and B chains of calcineurin. Neutrophils treated with cyclosporin A or FK 506 showed a dose- dependent inhibition of calcineurin activity. The effect of oxidant compounds on calcineurin activity was also investigated. Neutrophils treated with hydrogen peroxide (H2O2), where catalase was inhibited with aminotriazole, exhibited a specific inhibition of calcineurin activity. However, the addition of reducing agents to neutrophil extracts partially reversed the inhibition caused by H2O2. A similar inhibitory effect of H2O2 on calcineurin activity was observed to occur in isolated lymphocytes. This is the first demonstration that redox agents modulate calcineurin activity in a cellular system. In addition, electrophoretic mobility shift assays revealed that lipopolysaccharide- induced activation of NF-kappaB in human neutrophils is inhibited by cell pretreatment with H2O2 in a dose-dependent manner. These data indicate that calcineurin activity regulates the functional activity of lipopolysaccharide-induced NF-kappaB/Rel proteins in human neutrophils. These data indicate a role of peroxides in the modulation of calcineurin activity and that the H2O2-dependent NF-kappaB inactivation in neutrophils occurs in concert with inhibition of calcineurin. UI - 99074247 TI - The roles of nuclear factor of activated T cells and ying-yang 1 in activation-induced expression of the interferon-gamma promoter in T cells. AB - Nuclear factor of activated T cells (NFAT) plays an important role in expression of many cytokine genes including interleukin-2 and interleukin-4. However, its role in interferon-gamma (IFN-gamma) expression is not well understood. In the current studies, two strong NFAT-binding sites in the IFN-gamma promoter were identified by DNase I footprint analysis at positions -280 to -270 and -163 to -155. NFATp bound independently to both sites and was required for the formation of a composite element with AP-1 spanning position -163 to -147. In Jurkat T cells and primary lymphocytes, activation-induced expression of IFN- gamma reporter constructs containing point mutations in either NFAT site or the AP-1 component of the composite site was decreased by approximately 40-65%. Despite elimination of both strong NFAT-binding sites, the IFN-gamma promoter remained completely sensitive to inhibition by cyclosporin. This suggests that other elements in the IFN- gamma promoter, such as the IFN-gamma proximal element, are sufficient for cyclosporin sensitivity of this gene. Ying-Yang 1 (YY1), a potential inhibitor of IFN-gamma expression, binds to sites located between the two NFAT sites. Mutation of the YY1 sites alone had little effect on IFN-gamma promoter activity. However, mutation of both the NFAT and YY1-binding sites abolished activation-induced expression in primary murine splenocytes but not in Jurkat T cells. This suggests that under some conditions, YY1 may play a positive role in activation- induced transcription of IFN-gamma. UI - 99064095 TI - [Novel function of Calcineurin--multipotential factor as protein a phosphatase] AB - Calcineurin, serine/threonine phosphatase2B, is well known as a target of immunophilin-immunosuppressant complex such as cyclophilin- cyclosporinA and FKBP -FK506. It has been disclosed that Calcineurin is involved in interleukin 2 gene activation pathway lead to T lymphocyte proliferation, however, its functions as a multipotential factor still remains unknown. Here we mention about a new aspect of Calcineurin- involved pathway through its direct interaction to Bcl-2, an apoptosis suppressor. This direct binding of Calcineurin to Bcl-2 results in blockage of KFAT4 nuclear import by the prevention of Calcineurin- targetted dephosphorylation of NFAT4. Moreover, the tight binding between Calcineurin and Bcl-2 facilitate Bcl-2 activation as a apoptosis inhibitor through dephosphorylation of phosphorylated form of Bcl-2 serving to apoptosis regulation. UI - 99061801 TI - Interleukin-15 (IL-15) induces NF-kappaB activation and IL-8 production in human neutrophils. AB - Interleukin-2 (IL-2) and IL-15 exert similar biological actions, which largely reflect the fact that their receptors share common beta and gamma subunits; in contrast, distinct subunits are required for high- affinity binding of either cytokine to a heterotrimeric receptor complex. Human neutrophils are known to express both the beta and gamma subunits of the IL-2/IL-15 receptor complex, and we now report that they also constitutively express messenger RNA transcripts encoding the IL-15 receptor chain, suggesting that they possess functional, heterotrimeric IL-15 receptors. Accordingly, we show that in neutrophils, IL-15 elicits several functional responses. In particular, neutrophils synthesize and release IL-8 in response to IL-15, but not to IL-2. Moreover, a nuclear factor-kappaB (NF-kappaB) DNA-binding activity was enhanced in nuclear extracts of IL-15-treated neutrophils, which could be supershifted by antibodies to p50 or RelA. Again, no detectable effect of IL-2 was observed on this response. In peripheral blood lymphocytes (PBL), however, both IL-2 and IL-15 were potent inducers of NF-kappaB activation. Conversely, neither IL-15 nor IL-2 elicited the formation of activator protein-1 (AP-1) DNA-binding complexes in neutrophils, even though both cytokines were found to activate these DNA-binding activities in PBL. Collectively, these observations establish neutrophils as a useful cellular model to discriminate between the actions of IL-15 and IL-2. More importantly, this is the first demonstration that IL-15 has the ability to induce NF- kappaB and AP-1 activation, which further emphasizes the potential relevance of this newly discovered cytokine to immune and inflammatory processes. UI - 99061782 TI - Thrombopoietin induces association of Crkl with STAT5 but not STAT3 in human platelets. AB - Crkl, a 39-kD SH2, SH3 domain-containing adapter protein, is constitutively tyrosine phosphorylated in hematopoietic cells from chronic myelogenous leukemia (CML) patients. We recently reported that thrombopoietin induces tyrosine phosphorylation of Crkl in normal platelets. In this study, we demonstrate that thrombopoietin induces association of Crkl with a tyrosine phosphorylated 95- to 100-kD protein in platelets and in UT7/TPO cells, a thrombopoietin-dependent megakaryocytic cell line. With specific antibodies against STAT5, we demonstrate that the 95- to 100-kD protein in Crkl immunoprecipitates is STAT5. This coimmunoprecipitation was specific in that Crkl immunoprecipitates do not contain STAT3, although STAT3 becomes tyrosine phosphorylated in thrombopoietin-stimulated platelets. The coimmunoprecipitaion of Crkl with STAT5 was inhibited by the immunizing peptide for Crkl antisera or phenyl phosphate (20 mmol/L). After denaturing of Crkl immunoprecipitates, Crkl was still immunoprecipitated by Crkl antisera. However, coimmunoprecipitation of STAT5 was not observed. Coincident with STAT5 tyrosine phosphorylation, thrombopoietin induces activation of STAT5 DNA-binding activity as demonstrated by electrophoretic mobility shift assays (EMSA). Using a beta-casein promoter STAT5 binding site as a probe, we have also demonstrated that Crkl antisera supershift the STAT5-DNA complex, suggesting that Crkl is a component of the complex in the nucleus. Furthermore, interleukin-3 (IL-3), granulocyte-macrophage colony- stimulating factor (GM-CSF), and erythropoietin also induce Crkl-STAT5 complex formation in responding cells in a stimulation-dependent manner. In vitro, glutathione S-transferase (GST)-Crkl bound to STAT5 inducibly through its SH2 domain. These results indicate that thrombopoietin, IL-3, GM-CSF, and erythropoietin commonly induce association of STAT5 and Crkl and that the complex translocates to the nucleus and binds to DNA. Interestingly, such association between STAT5 and Crkl was not observed in cytokine-stimulated murine cells, suggesting an intriguing possibility that components of the human STAT5- DNA complex may be different from those of the murine counterpart. UI - 99045307 TI - Activation of EGF receptor family members suppresses the cytotoxic effects of tumor necrosis factor-alpha. AB - Tumor necrosis factor (TNF)-alpha has a broad range of biological activities, which depend heavily on cell type and physiological condition. In a panel of human tumor cell lines we analyzed expression of the receptor tyrosine kinases EGFR, ErbB2 and ErbB3, and the response to TNF-alpha. Among the cell lines tested those resistant to TNF-alpha were found to express high levels of either EGFR, or ErbB2 and ErbB3. In TNF-sensitive breast and cervical carcinoma cells activation of EGFR or ErbB2 by the exogenous growth factors EGF and heregulin beta1 resulted in a significant increase in the number of cells surviving TNF-alpha treatment. In contrast, inhibition of EGFR activation in TNF-resistant breast carcinoma cells by the novel antagonistic anti-EGFR antibody 14E1 sensitized the cells to the cytotoxic effects of TNF-alpha. A bacterially expressed fusion protein consisting of a 14E1 single-chain (sc) Fv antibody fragment linked to human TNF-alpha retained TNF-alpha activity. This scFv(14E1)-TNF-alpha molecule localized specifically to EGFR on the surface of tumor cells and activated the NF-kappaB pathway in co-cultured T cells, as demonstrated by electrophoretic mobility shift assays. UI - 99030442 TI - Mast cell-/basophil-specific transcriptional regulation of human L- histidine decarboxylase gene by CpG methylation in the promoter region. AB - L-Histidine decarboxylase (HDC) catalyzes the formation of histamine from L-histidine, and in hematopoietic cell lineages the gene is expressed only in mast cells and basophils. We attempted here to discover how HDC gene expression is restricted in these cells. In the cultured cell lines tested, only the mast cells and basophils strongly transcribed the HDC gene. However, in transient transfection analysis, the reporter constructs with the HDC promoter were active not only in expressing cells but also in nonexpressing cells. Detailed analyses of the HDC promoter region revealed that the GC box is essential for transactivation. Also, the promoter region of the HDC gene proved to be sensitive to DNase I and restriction endonucleases exclusively in HDC- expressing cells, suggesting that the promoter region is readily accessible to trans-acting factor(s). Furthermore, the promoter region in HDC-expressing cell lines was found to be selectively unmethylated. The correlation between HDC expression and hypomethylation was also found in primary human mast cells. Methylation of the HDC promoter in vitro reduced the luciferase reporter activity in transient expression analysis, suggesting that methylation of the promoter region is functionally important for HDC gene expression. These results imply that alteration of DNA methylation is one of the mechanisms regulating cell-specific expression of the HDC gene. UI - 99030394 TI - The interleukin-4 receptor activates STAT5 by a mechanism that relies upon common gamma-chain. AB - Interleukin (IL)-4 signaling proceeds via cytoplasmic activation of the Janus kinases JAK1 and JAK3 and the signal transducer and activator of transcription STAT6. We show that the IL-4 receptor, like other cytokine receptor systems utilizing the common receptor gamma-chain (gammac), is also connected to a signaling pathway that involves STAT5. Both STAT5a and STAT5b become tyrosine-phosphorylated and acquire specific DNA-binding properties in response to IL-4 receptor stimulation in the murine pro-B cell line Ba/F3. In preactivated human T cells, STAT5 became activated in an IL-4-dependent fashion as assayed by IL-4-induced STAT5 translocation from the cytoplasm to the cell nucleus and by binding to cognate DNA. Moreover, stimulation of preactivated human T cells by IL-4 led to specific transcriptional up- regulation of STAT5 target genes. IL-4 receptor-mediated STAT5 activation is dependent on the presence of gammac and JAK3 within the receptor complex. In COS-7 cells, the JAK/STAT pathway leading from the IL-4 receptor to STAT5-dependent regulation of a reporter gene relied largely on coexpression of JAK3. In Ba/F3 cells, studies on signal transduction evoked by directed specific receptor homo- or heterodimerization revealed that STAT5 activation can be triggered exclusively by IL-4R heterodimers containing gammac. UI - 98440523 TI - Immediate-early transactivator Rta of Epstein-Barr virus (EBV) shows multiple epitopes recognized by EBV-specific cytotoxic T lymphocytes. AB - We analyzed the immediate-early transactivator Rta of Epstein-Barr virus (EBV) for its role as a target for specific cytotoxic T lymphocytes (CTL). Panels of overlapping peptides covering the entire amino acid sequence of Rta were synthesized and used to induce and analyze specific CTL responses in EBV-positive donors. Using peptide- pulsed target cells, we found nine different CTL epitopes that are distributed over the entire protein sequence. One epitope restricted by HLA-A24 could be mapped to the decameric sequence DYCNVLNKEF between amino acid positions 28 and 37 of the Rta protein. A second epitope could be assigned to the same region of Rta (residues 25 to 39) and was shown to be restricted by HLA-B18. Another, minimal epitope could be mapped to the nonameric sequence ATIGTAMYK between amino acid positions 134 and 142; this peptide was restricted by HLA-A11. Another four epitopes were proven to be restricted by HLA-A2, -A3, -B61, and -Cw4 and were located between Rta residues 225 and 239, 145 and 159, 529 and 543, and 393 and 407, respectively. For two other epitopes, only the location within the Rta protein is known so far (residues 121 to 135 and 441 to 455); their exact HLA restriction patterns have not yet been identified. Using target cells infected with recombinant vaccinia virus containing the gene for Rta, we showed that six of eight Rta-specific CTL lines recognized the corresponding peptides also after endogenous processing. These data suggest that Rta comprises an important target for EBV-specific cellular cytotoxicity. Together with recent findings of other immediate-early and early proteins also acting as CTL targets, they reveal the role of proteins of the lytic cycle in the immune recognition of EBV-infected cells. UI - 98438532 TI - Herpesvirus entry mediator ligand (HVEM-L), a novel ligand for HVEM/TR2, stimulates proliferation of T cells and inhibits HT29 cell growth. AB - Herpesvirus entry mediator (HVEM), a member of the tumor necrosis factor (TNF) receptor family, mediates herpesvirus entry into cells during infection. Upon overexpression, HVEM activates NF-kappaB and AP- 1 through a TNF receptor-associated factor (TRAF)-mediated mechanism. Using an HVEM-Fc fusion protein, we screened soluble forms of novel TNF- related proteins derived from an expressed sequence tag data base. One of these, which we designated HVEM-L, specifically bound to HVEM-Fc with an affinity of 44 nM. This association was confirmed with soluble and membrane forms of both receptor and ligand. HVEM-L mRNA is expressed in spleen, lymph nodes, macrophages, and T cells and encodes a 240-amino acid protein. A soluble, secreted form of the protein stimulates proliferation of T lymphocytes during allogeneic responses, inhibits HT-29 cell growth, and weakly stimulates NF-kappaB-dependent transcription. UI - 98438350 TI - Alterations in protein-DNA interactions in the gamma-globin gene promoter in response to butyrate therapy. AB - The mechanisms by which pharmacologic agents stimulate gamma-globin gene expression in beta-globin disorders has not been fully established at the molecular level. In studies described here, nucleated erythroblasts were isolated from patients with beta-globin disorders before and with butyrate therapy, and globin biosynthesis, mRNA, and protein-DNA interactions were examined. Expression of gamma-globin mRNA increased twofold to sixfold above baseline with butyrate therapy in 7 of 8 patients studied. A 15% to 50% increase in gamma-globin protein synthetic levels above baseline gamma globin ratios and a relative decrease in beta-globin biosynthesis were observed in responsive patients. Extensive new in vivo footprints were detected in erythroblasts of responsive patients in four regions of the gamma-globin gene promoter, designated butyrate-response elements gamma 1-4 (BRE-G1-4). Electrophoretic mobility shift assays using BRE-G1 sequences as a probe demonstrated that new binding of two erythroid- specific proteins and one ubiquitous protein, alphaCP2, occurred with treatment in the responsive patients and did not occur in the nonresponder. The BRE-G1 sequence conferred butyrate inducibility in reporter gene assays. These in vivo protein-DNA interactions in human erythroblasts in which gamma-globin gene expression is being altered strongly suggest that nuclear protein binding, including alphaCP2, to the BRE-G1 region of the gamma-globin gene promoter mediates butyrate activity on gamma-globin gene expression. UI - 98434392 TI - Analysis of the TPA regulatory element in the genomic poly(ADP-ribose) synthetase gene in human leukemia U937 cells. AB - The human leukemia U937 cells differentiate into monocyte/macrophage- like cells when treated with 12-O-tetradecanoylphorbol-13-acetate (TPA). We observed that during this process, both protein and mRNA levels for PARS markedly decreased in U937 cells. Through deletion analysis of the PARS regulatory gene, we found that the sequence within the first intron region was responsible for the TPA-dependent repression. Electrophoretic mobility shift assays (EMSAs) and Southwestern blot analysis indicate that this element bound specifically to a nuclear protein. TPA treatment abolished the binding of the protein in U937 cells but not in HeLa cells. DNase I footprinting data show that the cis regulatory element is located between residues 328 and 383. We further examined the function of this cis element (BS207) in a basal promoter regulatory reporter construct and found that this cis element (BS207) functions as an enhancer via the binding of an unknown trans-acting factor. TPA treatment diminished the binding activity of the factor in U937 cells, resulting in a decrease in the enhanced activity to the basal level. These results suggest that abolishment of the binding of a special nuclear protein to the first intron of the PARS gene is related to the TPA-responsive downregulation of PARS in U937 cells. UI - 98430673 TI - Structural basis of specificity and degeneracy of T cell recognition: pluriallelic restriction of T cell responses to a peptide antigen involves both specific and promiscuous interactions between the T cell receptor, peptide, and HLA-DR. AB - TCR engagement of peptide-MHC class II ligands involves specific contacts between the TCR and residues on both the MHC and peptide molecules. We have used molecular modeling and assays of peptide binding and T cell function to characterize these interactions for a CD4+ Th1 cell clone, ESL4.34, which recognizes a peptide epitope of the herpes simplex type 2 virus virion protein, VP16 393-405, in the context of several HLA-DR alleles. This clone responded to VP16 393-405 in proliferation and cytotoxicity assays when presented by DRB1*0402, DRB1*1102, and DRB1*1301, which share a common amino acid sequence, ILEDE, at residues 67-71 in the alpha-helical portion of the DRbeta polypeptide, but not when presented by other DR4, DR11, and DR13 alleles that are negative for this sequence. Using a panel of APCs expressing DR4 molecules that were mutagenized in vitro at individual residues within this shared epitope and using peptide analogues with single amino acid substitutions of predicted MHC and TCR contact residues, a unit of recognition was identified dependent on DRbeta residues 67-71 and relative position 4 (P4) of the VP16 393-405 peptide. The interactions of this portion of the peptide-DR ligand with the ESL4.34 TCR support a structural model for MHC-biased recognition in some Ag-specific and alloreactive T cell responses and suggest a possible mechanism for autoreactive T cell selection in rheumatoid arthritis. UI - 98430636 TI - p38 MAPK is required for CD40-induced gene expression and proliferation in B lymphocytes. AB - We have investigated the activation of the p38 MAPK pathway in response to CD40 engagement in multiple B cell lines and in human tonsillar B cells to define the role of p38 MAPK in proliferation, NF-kappaB activation and gene expression. Cross-linking CD40 rapidly stimulates both p38 MAPK and its downstream effector, MAPKAPK-2. Inhibition of p38 MAPK activity in vivo with the specific cell-permeable inhibitor, SB203580, under conditions that completely prevented MAPKAPK-2 activation, strongly perturbed CD40-induced tonsillar B cell proliferation while potentiating the B cell receptor (BCR)-driven proliferative response. SB203580 also significantly reduced expression of a reporter gene driven by a minimal promoter containing four NF- kappaB elements, indicating a requirement for the p38 MAPK pathway in CD40-induced NF-kappaB activation. However, CD40-mediated NF-kappaB binding was not affected by SB203580, suggesting that NF-kappaB may not be a direct target for the CD40-induced p38 MAPK pathway. In addition, SB203580 selectively reduced CD40-induced CD54/ICAM-1 expression, whereas CD40-dependent expression of CD40 and CD95/Fas and four newly defined CD40-responsive genes cIAP2, TRAF1, TRAF4/CART and DR3 were unaffected. Our observations show that the p38 MAPK pathway is required for CD40-induced proliferation and that CD40 induces gene expression via both p38 MAPK-dependent and -independent pathways. UI - 98434612 TI - The early growth response protein (EGR-1) regulates interleukin-2 transcription by synergistic interaction with the nuclear factor of activated T cells. AB - The early growth response-1 gene (EGR-1) is induced by a wide range of stimuli in diverse cell types; however, EGR-1-regulated genes display a highly restricted pattern of expression. Recently, an overlapping Sp1.EGR-1 binding site has been identified within the interleukin-2 (IL- 2) gene promoter directly upstream of the binding site for the nuclear factor of activated T cells (NFAT). We used transfection assays to study how the abundantly and constitutively expressed Sp1 protein and the immediate early EGR-1 zinc finger protein regulate IL-2 gene expression. Here, we identify EGR-1 as an important activator of the IL- 2 gene. In Jurkat T cells, EGR-1 but not Sp1 acts as a potent coactivator for IL-2 transcription, and in combination with NFATc, EGR- 1 increases transcription of an IL-2 reporter construct 200-fold. Electrophoretic mobility shift assays reveal that recombinant EGR-1 and NFATc bind independently to their target sites within the IL-2 promoter, and the presence of both sites on the same DNA molecule is required for EGR-1.NFATc.DNA complex formation. The transcriptional synergy observed here for EGR-1 and NFATc explains how the abundant nuclear factor EGR-1 contributes to the expression of restrictively expressed genes. UI - 98413865 TI - An AP-1 site in the promoter of the human IL-5R alpha gene is necessary for promoter activity in eosinophilic HL60 cells. AB - Interleukin-5 (IL-5) plays a crucial role in the proliferation, differentiation and activation of eosinophils. The IL-5 receptor is composed of an IL-5-specific alpha subunit, which is expressed by eosinophils and basophils, and a beta c-subunit shared with the receptors for IL-3 and GM-CSF. We identified an AP-1 element which is important for IL-5R alpha promoter activity in eosinophilic HL60 cells. The AP-1 site and the previously identified EOS1 site cooperate, since single mutation of either of the sites decreased promoter activity. We show that the AP-1 site of the IL-5R alpha promoter binds multiple proteins, including cJun, CREB, and CREM. UI - 98401159 TI - Association of Cdk2/cyclin E and NF-kappa B complexes at G1/S phase. AB - NF-kappa B/Rel family plays a pivotal role in a wide variety of cellular functions including growth, development, apoptosis and stress responses. Recent studies indicated that NF-kappa B is also involved in the cell cycle regulation, and high expression of c-Rel results in a cell cycle arrest at the G1/S-phase transition (Bash, J., Zong, W,-X., and Gelinas, C.(1997) Mol.Cell.Biol.17, 6526-6536). Here we report the detection of Cdk2, a critical kinase responsible for the G1/S-phase transition, in immune complexes precipitated by the NF-kappa B antisera. Cdk2 and NF-kappa B association was detected by co- precipitation in the nuclear lysates of the G1/S-phase cells, and was found in cultured cell lines and in T cells purified from human peripheral blood. Using an affinity column containing the C-terminal peptide of human c-Rel, we isolated cyclin E, the regulatory subunit of the Cdk2 complex, as a c-Rel-binding protein. These findings support and provide physical basis for the involvement of NF-kappa B in the G1/S-phase cell cycle control, and suggest an important role played by the C-terminal sequence of c-Rel. UI - 98389747 TI - The alpha-chemokine, stromal cell-derived factor-1alpha, binds to the transmembrane G-protein-coupled CXCR-4 receptor and activates multiple signal transduction pathways. AB - The alpha-chemokine stromal cell-derived factor (SDF)-1alpha binds to the seven transmembrane G-protein-coupled CXCR-4 receptor and acts to modulate cell migration and proliferation. The signaling pathways that mediate the effects of SDF-1alpha are not well characterized. We studied events following SDF-1alpha binding to CXCR-4 in a model murine pre-B cell line transfected with human CXCR-4. There was enhanced tyrosine phosphorylation and association of components of focal adhesion complexes such as the related adhesion focal tyrosine kinase, paxillin, and Crk. We also observed activation of phosphatidylinositol 3-kinase. Wortmannin, a selective inhibitor of phosphatidylinositol 3- kinase, partially inhibited the SDF-1alpha-induced migration and tyrosine phosphorylation of paxillin. SDF-1alpha treatment selectively activated p44/42 mitogen-activated protein kinase (Erk 1 and Erk 2) and its upstream kinase mitogen-activated protein kinase kinase but not p38 mitogen-activated protein kinase, c-Jun amino-terminal kinase or mitogen activated protein kinase kinase. We also observed that SDF- 1alpha treatment increased NF-kappaB activity in nuclear extracts from the CXCR-4 transfectants. Taken together, these studies revealed that SDF-1alpha activates distinct signaling pathways that may mediate cell growth, migration, and transcriptional activation. UI - 98375856 TI - Requirement for dual signals by anti-CD40 and IL-4 for the induction of nuclear factor-kappa B, IL-6, and IgE in human B lymphocytes. AB - Stimulation of human peripheral B cells via the CD40 receptor and IL-4R together lead to IgE synthesis and secretion, but the intracellular signaling mechanisms by which these signals lead to IgE production are unclear. Roles for the transcription factor NF-kappa B and IL-6 have been postulated in the induction of IgE synthesis by IL-4/CD40. We found that neither anti-CD40 Ab nor IL-4 alone was able to induce significant proliferation of human B cells. However, the combination of anti-CD40 and IL-4 was a potent inducer of B cell proliferation in addition to IgE production from purified human B cells. Furthermore, IL- 4 and anti-CD40 synergized for the production of IL-6. While neither IL- 4 alone nor anti-CD40 alone was able to induce significant NF-kappa B DNA binding activity, the combination of IL-4 and anti-CD40 induced a strong activation of NF-kappa B, a transcription factor that regulates IL-6 production. These data indicate that both IL-4 and anti-CD40 are required to induce NF-kappa B activation and IL-6 transcription and production, and implicate these events in a signaling pathway augmenting IgE production in human B lymphocytes. UI - 98374021 TI - Activation of NF-kappaB in human neutrophils during phagocytosis of bacteria independently of oxidant generation. AB - We have exposed human neutrophils to opsonized Staphylococcus aureus and used an electrophoretic mobility shift assay to show activation of the transcription factor NF-kappaB above basal levels. Activation was evident within 10 min and was increased with higher bacteria:neutrophil ratios. The neutrophil NADPH oxidase inhibitor diphenylene iodonium, catalase, and other oxidant scavengers did not inhibit NF-kappaB activation, and no activation was seen with added hydrogen peroxide. Oxidants produced during phagocytosis, therefore, are not involved in the activation mechanism. UI - 98370990 TI - Molecular cloning and characterization of a transcription regulator with homology to GC-binding factor. AB - GC-binding factor (GCF) represses transcription of certain genes and is encoded by a 3.0-kilobase mRNA (Kageyama, R., and Pastan, I.(1989) Cell 59, 815-825). The GCF cDNA hybridizes to two additional mRNA species, 4.2 and 1.2 kilobases. We have used differential hybridization to identify a cDNA clone (termed GCF2) for the 4. 2-kilobase mRNA and find that it is highly expressed in HUT-102 cells. The open reading frame consists of 2256 nucleotides and encodes a protein of 752 amino acids with a calculated molecular mass of 83 kilodaltons. GCF2 expressed in vitro using reticulocyte lysates and Escherichia coli migrates as a 160-kilodalton protein in SDS-polyacrylamide gel electrophoresis but has a molecular mass of 83 kilodaltons as determined by mass spectrum analysis. GCF2 binds to epidermal growth factor receptor promoter fragments, and the major binding site is located between nucleotides -249 and -233. Cotransfection assays show that GCF2 acts to repress transcription from the epidermal growth factor receptor promoter in constructs containing the major GCF2 binding site and not when the site had been mutated. Thus, GCF2 is a newly identified transcriptional repressor with aberrant electrophoretic mobility. UI - 98361995 TI - Complex NF-kappaB interactions at the distal tumor necrosis factor promoter region in human monocytes. AB - We describe a dense cluster of DNA-protein interactions located 600 nucleotides upstream of the transcriptional start site of the human tumor necrosis factor (TNF) gene. This area was identified as being of potential importance for lipopolysaccharide-inducible TNF expression in the human monocyte cell line Mono Mac 6, based on reporter gene analysis of point mutations at a number of nuclear factor kappaB (NF- kappaB)-like motifs within the human TNF promoter region. The area contains two NF-kappaB sites, which are here shown by DNase I and methylation interference footprinting to flank a novel binding site. UV cross-linking studies reveal that the novel site can also bind NF- kappaB as well as an unknown protein(s) of approximately 40 kDa. We show that these three adjacent kappaB-binding sites differ markedly in their relative affinities for p50/p50, p65/p65, and p65/p50, yet this 39-nucleotide segment of DNA appears capable of binding up to three NF- kappaB heterodimers simultaneously. Reporter gene studies indicate that each element of the cluster contributes to lipopolysaccharide-induced transcriptional activation in Mono Mac 6 cells. These findings suggest that NF-kappaB acts in a complex manner to activate TNF transcription in human monocytes. UI - 98361782 TI - Differential binding activity of the transcription factor LIL-STAT in immature and differentiated normal and leukemic myeloid cells. AB - Cytokines and growth factors induce activation of the family of signal transducers and activators of transcription (Stats) that directly activate gene expression. Recently, constitutively activated Stat1, Stat3, and Stat5 were identified in nuclear extracts of acute myeloid leukemia (AML) patients, suggesting involvement of constitutive Stat activity in the events of leukemogenesis. In the present study, blasts of nine AML cases were investigated for the constitutive binding activity of the recently identified transcription factor LIL-Stat (LPS- and IL-1-inducible Stat). Band-shift assays were performed using the LPS-and IL-1-responsive element (LILRE) oligonucleotide, a gamma interferon activation site-like site that is present in the human IL- 1beta promoter. Constitutive LIL-Stat binding activity was observed in three leukemic cell lines and in seven out of nine AML cases. Transient transfection studies with a reporter plasmid containing three sequential LIL-Stat binding sites showed distinct transcriptional activity of LIL-Stat only in those AML blasts that constitutively expressed LIL-Stat. In CD34+ cells LIL-Stat also constitutively bound to its consensus sequence. However, when these cells were cultured in the presence of macrophage-colony stimulating factor (M-CSF) and stem cell factor (SCF) for differentiation along the monocytic lineage, the LIL-Stat binding activity disappeared totally. In agreement with these findings neither mature monocytes nor granulocytes showed constitutive or inducible LIL-Stat binding activity. We conclude that the LIL-Stat transcription factor is constitutively activated in undifferentiated and leukemic hematopoietic cells, but not in mature cells. This may suggest a role for this transcription factor in the process of differentiation. UI - 98361779 TI - Impaired activation of NFkappaB in T cells from a subset of renal cell carcinoma patients is mediated by inhibition of phosphorylation and degradation of the inhibitor, IkappaBalpha. AB - Activation of the transcription factor NFkappaB in peripheral blood T cells from patients with renal cell carcinoma (RCC) is compromised. This impaired signaling function results from a failure of RelA and c- Rel to translocate to the nucleus though normal levels of Rel proteins are present in the cytoplasm. We demonstrate here in a subset of RCC patients that the defect in NFkappaB activation is attributable to the absence of phosphorylation and degradation of the inhibitor IkappaBalpha. In patient T cells there was no stimulus dependent decrease in the cytoplasmic level of IkappaBalpha. Coimmunoprecipitation studies showed that RelA was in complex with IkappaBalpha and was not released after stimulation. Moreover, the phosphorylated form of IkappaBalpha detected in normal T cells after activation is absent in patient T cells. Additional experiments showed that soluble products from RCCs (RCC-S) can reproduce the same phenotype in T cells from healthy individuals. Supernatant fluid from cultured explants of RCC, but not normal kidney, inhibited the stimulus dependent nuclear translocation of NFkappaB without altering the cytoplasmic levels of RelA, c-Rel, and NFkappaB1. Phosphorylation and degradation of IkappaBalpha was also blocked by RCC-S. The mechanistic similarities between patient-derived T cells and normal T cells cultured with RCC-S suggest that the tumor-derived products may be the primary mediators of impaired T-cell function in this tumor system. UI - 98349390 TI - Th2-specific protein/DNA interactions at the proximal nuclear factor-AT site contribute to the functional activity of the human IL-4 promoter. AB - IL-4 is a pleiotropic immunoregulatory cytokine secreted by activated Th2, but not Th1, cells. The proximal IL-4 promoter contains MARE, C/EBP, P0, octamer-like, P1, and activating protein-1 elements. The half c-Maf binding site (MARE), P0, and P1 sites were previously shown to be involved in Th2-specific transcriptional activity. Except the MARE and P1 site, the molecular basis for Th2 specificity of the P0 site has not been analyzed. Here, we provide the first detailed analysis of the P0 binding factors and show that in Th2, but not in Th1, cells, NF-AT and proteins of the activating protein-1 family are involved in cooperative binding to the P0 and the adjacent octamer-like site. In the mouse Th2 D10 cells, Oct-1/Oct-2 are also found to participate in formation of the P0-binding complexes. Mutation, deletion, and methylation interference analysis demonstrate that both the P0 and the octamer-like sequence are required for inducible binding. Furthermore, we provide the first report of the functional relevance of each site in the human IL-4 promoter by mutagenesis/transfection analysis and demonstrate that the octamer- like, P0 and P1 sites are important for the biologic function of the IL- 4 promoter. The MARE site, although it was shown to be critical for the function of the murine IL-4 promoter, does not appear essential for human IL-4 promoter activity in Jurkat T cells. These findings suggest that besides c-Maf, another Th2-specific factor(s) may be involved in tissue-specific expression of the IL-4 gene. UI - 98349353 TI - A newly identified response element in the CD95 ligand promoter contributes to optimal inducibility in activated T lymphocytes. AB - Inducible expression of CD95 ligand on activated T lymphocytes contributes to both cytotoxic effector mechanisms and peripheral T cell homeostasis. To understand better the transcriptional events that regulate this expression, we have examined the CD95 ligand promoter to determine which regions are required for its induced activity following T cell stimulation. We report here the identification of a new response element within the promoter that is required for its optimal function in activated Jurkat T cells. This region is bound by proteins contained in nuclear extracts of activated, but not resting, T cells. Multimerization of this sequence independently drives transcription in response to T cell activation, while mutation of it substantially decreases inducible promoter activity. Finally, we provide evidence that T cell activation-induced transcription of the CD95 ligand gene is regulated coordinately by this response element together with two previously defined sites for nuclear factor of activated T cells (NFAT). UI - 98352032 TI - Glyceraldehyde-3-phosphate dehydrogenase and Nm23-H1/nucleoside diphosphate kinase A. Two old enzymes combine for the novel Nm23 protein phosphotransferase function. AB - We have recently discovered an alternative function of the putative metastasis suppressor protein Nm23, which is identical to nucleoside diphosphate kinase, as a protein phosphotransferase in vitro. While purified native Nm23 protein did not phosphorylate other proteins, we could purify a Nm23-associated protein that activates the protein phosphotransferase function; it was identified as a glyceraldehyde-3- phosphate dehydrogenase (GAPDH) isoenzyme. Co-expression and purification of (His)6-tagged GAPDH in combination with either Nm23-H1 or Nm23-H2 in baculovirus-infected Sf9 cells showed that only Nm23-H1, but not Nm23-H2, forms a stable complex with GAPDH. Protein phosphotransferase activity was confirmed for the recombinant GAPDH. Nm23-H1 complex but not for either of the enzymes alone, nor was this activity observed after simple mixing of the purified proteins in vitro. The molecular mass of the highly purified recombinant GAPDH. Nm23- H1 complex suggests that a dimer of GAPDH interacts with a dimer of Nm23-H1. In contrast to the complex with GAPDH, co-expression of Nm23- H1 with antioxidant protein (MER-5) or creatine kinase did not activate the protein phosphotransferase function, indicating that this activation may specifically require GAPDH as a binding partner. UI - 98343919 TI - The level of the tissue-specific factor GATA-1 affects the cell-cycle machinery. AB - GATA-1 is a tissue-specific DNA-binding protein containing two zinc- finger-like domains. It is expressed predominantly in erythrocytes. Consensus binding sites for GATA-1 have been found in the regulatory elements of all erythroid-specific genes examined. GATA-1 protein is required for erythroid differentiation beyond the proerythroblast stage. In this paper, we demonstrate that the overexpression of GATA-1 in murine erythroleukaemia (MEL) cells alleviates DMSO-induced terminal erythroid differentiation. Hence, there is no induction of globin gene transcription and the cells do not arrest in the G1 phase of the cell cycle. Furthermore, we demonstrate that expression of GATA-1 in non- transformed erythroid precursors also affects their proliferative capacity and terminal differentiation, as assayed by adult globin gene transcription. To gain insight into the mechanism of this effect, we studied the levels and activities of regulators of cell-cycle progression during DMSO-induced differentiation. A decrease in cyclin D- dependent kinase activity was observed during the induction of both control and GATA-1-overexpressing MEL cells. However, cyclin E- dependent kinase activity decreased more than 20-fold in control but less than 2-fold in GATA-1-overexpressing MEL cells upon induction. Thus GATA-1 may exert its effects by regulating cyclin E-dependent kinase activity. We also show that GATA-1 binds to the retinoblastoma protein in vitro, but not to the related protein p107, which may indicate that GATA-1 interacts directly with specific members of the cell-cycle machinery in vivo. We conclude that GATA-1 regulates cell fate, in terms of differentiation or proliferation, by affecting the cell-cycle apparatus. UI - 98344097 TI - Association of p59(fyn) with the T lymphocyte costimulatory receptor CD2. Binding of the Fyn Src homology (SH) 3 domain is regulated by the Fyn SH2 domain. AB - Human CD2 is a 50-55-kDa cell surface receptor specifically expressed on the surface of T lymphocytes and NK cells. Stimulation of human peripheral blood T cells with mitogenic pairs of anti-CD2 monoclonal antibodies (mAbs) is sufficient to induce interleukin-2 production and T cell proliferation in the absence of an antigen-specific signal through the T cell receptor. CD2 has been shown previously to associate physically with the Src family protein-tyrosine kinases p56(lck) and p59(fyn). We now report that stimulation of T cells with mitogenic pairs of anti-CD2 mAbs enhanced the association of the Fyn polypeptide with the CD2 complex, whereas stimulation with single anti-CD2 mAb had minimal effect. Using glutathione S-transferase (GST) fusion proteins, we found that CD2 bound to the Src homology (SH) 3 domain of Fyn. Interestingly, the CD2-Fyn association was negatively regulated by the Fyn SH2 domain; CD2 bound poorly to GST fusion proteins expressing both the SH2 and SH3 domains of Fyn. However, the inhibitory effect of the Fyn SH2 domain on binding of the Fyn SH3 domain to CD2 was relieved by peptides containing a phosphorylated YEEI sequence that bound directly to the Fyn SH2 domain. In addition, we found that the ability of the Fyn SH2 domain to precipitate tyrosine-phosphorylated proteins, including the CD3zeta chain, was enhanced after T cell stimulation with mitogenic pairs of CD2 mAbs. Finally, overexpression of a mutated Fyn molecule, in which the ability of the Fyn SH2 domain to bind phosphotyrosine-containing proteins was abrogated, inhibited CD2- induced transcriptional activation of the nuclear factor of activated T cells (NFAT), suggesting a functional involvement of the Fyn SH2 domain in CD2-induced T cell signaling. We thus propose that stimulation through the CD2 receptor leads to the tyrosine phosphorylation of intracellular proteins, including CD3zeta itself, which in turn bind to the Fyn-SH2 domain, allowing the direct association of the Fyn SH3 domain with CD2 and the initiation of downstream signaling events. UI - 98309441 TI - V7 (CD101) ligation inhibits TCR/CD3-induced IL-2 production by blocking Ca2+ flux and nuclear factor of activated T cell nuclear translocation. AB - Ligation of the V7 (CD101) molecule on T cells with anti-V7 mAb blocks TCR/CD3-induced proliferation by inhibiting IL-2 transcription. To explore the basis for this observation, we analyzed the effects of V7 ligation on CD3/TCR-induced changes in intracellular free Ca2+ and Ca2+- dependent nuclear factor of activated T cells (NF-AT) translocation to the nucleus, which is required for IL-2 transcription. T cells exposed to anti-V7 mAb fluxed Ca2+ transiently, but did not flux Ca2+ in response to subsequent treatment with anti-CD3; however, they recovered the capacity to flux Ca2+ after treatment with pervanadate, indicating that tyrosine dephosphorylation of a critical V7-related substrate is required in the desensitization process. One such substrate, phospholipase C (PLC)-gamma1, becomes tyrosine phosphorylated on CD3/TCR activation and mediates inositol triphosphate-dependent Ca2+ flux. Co-cross-linking of T cells with anti-CD3 and anti-V7 resulted in selective inhibition of PLC-gamma1 tyrosine phosphorylation, which may explain V7-mediated blockade of anti-CD3-induced Ca2+ flux. Moreover, anti-CD3-induced binding of transcription factors to a consensus NF-AT- binding oligonucleotide, which is dependent on Ca2+, was blocked completely by treatment of the cells with anti-V7, whereas binding to a consensus-activating protein-1 oligonucleotide was unaffected. Western blot analysis of cytoplasmic and nuclear extracts confirmed that anti- V7 prevented nuclear translocation of NF-ATc induced by anti-CD3. We conclude that V7 ligation interferes with T cell activation and IL-2 secretion through a Ca2+ and tyrosine kinase-dependent pathway that inhibits PLC-gamma1 phosphorylation and prevents NF-AT translocation to the nucleus. UI - 98307914 TI - Identification and characterization of a conserved erythroid-specific enhancer located in intron 8 of the human 5-aminolevulinate synthase 2 gene. AB - Thirty five kilobases of sequence encompassing the human erythroid 5- aminolevulinate synthase (ALAS2) gene have been determined. Analysis revealed a very low GC content, few repetitive elements, and evidence for the insertion of a reverse-transcribed mRNA sequence and a neighboring gene. We have investigated whether introns 1, 3, and 8, which correspond to DNase I-hypersensitivity sites in the structurally related mouse ALAS2 gene, affect expression of the human ALAS2 promoter in transient expression assays. Whereas intron 3 was marginally inhibitory, introns 1 and 8 of the human gene stimulated promoter activity. Intron 8 harbored a strong erythroid-specific enhancer activity which was orientation-dependent. Deletion analysis of this region localized enhancer activity to a fragment of 239 base pairs. Transcription factor binding sites clustered within this region include GATA motifs and CACCC boxes, critical regulatory sequences of many erythroid cell-expressed genes. These sites were also identified in the corresponding intron of both the murine and canine ALAS2 genes. Mutagenesis of these conserved sites in the human intron 8 sequence and transient expression analysis in erythroid cells established the functional importance of one GATA motif and two CACCC boxes. The GATA motif bound GATA-1 in vitro. The two functional CACCC boxes each bound Sp1 or a related protein in vitro, but binding of the erythroid Kruppel- like factor and the basic Kruppel-like factor could not be detected. The intron 8 enhancer region was not activated by GATA-1 together with Sp1 in transactivation experiments in COS-1 cells indicating the involvement of a related Sp1 protein or of another unidentified erythroid factor. Overall, these results demonstrate that a GATA-1- binding site and CACCC boxes located within the human ALAS2 intron 8 are critical for the erythroid-specific enhancer activity in transfected erythroid cells, and due to the conserved nature of these binding sites across species, it seems likely that these sites play a functional role in the tissue-restricted expression of the gene in vivo. UI - 98300945 TI - Sp1 and related factors fail to interact with the NF-kappaB-proximal G/C box in the LTR of a replication competent, brain-derived strain of HIV-1 (YU-2). AB - The HIV-1 LTR promoter proximal G/C box array has been demonstrated to function by interacting with the Sp1 transcription factor family whose members can act as either activators or repressors of transcription. In this regard, we have examined the interaction of the HIV-1 Sp binding sites with nuclear factors that are present in cell types that support HIV-1 replication, including those of lymphocytic, monocytic, and astrocytic origin. As determined by electrophoretic mobility shift (EMS) competition analyses using oligonucleotides containing the sequences of each of the Sp1 sites of HIV-1 strain LAI, the NF-kappaB- proximal Sp site (site III) displayed the highest binding activity compared to sites I and II with regard to Sp1 and related factors present in lymphocytic (Jurkat) and astrocytic (U-373 MG) nuclear extracts. Sp1 and two Sp3 isoforms were detected as the primary cellular constituents of DNA-protein complexes formed with the NF- kappaB-proximal site. Only modest differences in Sp1:Sp3 binding ratios were observed when this site was reacted with either astrocytic or lymphocytic nuclear extract. However, when nuclear extracts derived from two monocytic cell types that differ in the degree of differentiation were reacted with the HIV-1 LAI Sp site III, a large difference in Sp1 and Sp3 binding was observed. To determine if naturally occurring and replication-competent strains of HIV-1 contain base pair alterations within the Sp elements that affect the ability of the site to interact with Sp1 and related factors, a series of Sp site III variants were constructed and examined by EMS analyses. One of these sites, obtained from the published sequence of the YU-2 strain (a brain-derived macrophage tropic strain of HIV-1), displayed almost no Sp1 or Sp3 binding activity as a result of a single base pair alteration in Sp site III. This base-pair alteration, when placed in the context of an HIV-1 LAI LTR-luciferase construct, resulted in a 40- 50% reduction in LTR activity in transiently transfected Jurkat and U- 373 MG cells. Overall, these results suggest that specific G/C box sequence alterations present in the brain-derived HIV-1 variant YU-2, or possibly other brain-derived variants, may exhibit altered replication properties as a result of the low affinity of the NF-kappaB- proximal G/C box for members of the Sp transcription factor family. UI - 98289963 TI - Folding requirements of the ligand-binding domain of the human mineralocorticoid receptor. AB - The effects of aldosterone are mediated by the mineralocorticoid receptor (MR), a ligand-dependent transcription factor. We investigated the structural determinants for ligand binding to the receptor using a series of human MR (hMR) deletion mutants. These proteins were produced in vitro in rabbit reticulocyte lysate and analyzed for their ability to bind agonists, antagonists, and the heat shock protein hsp90, which is a prerequisite for ligand binding to hMR. Studies on N terminus- truncated hMRs showed that the ligand-binding domain (LBD: amino acids 734-984) has a lower affinity for aldosterone than the entire receptor [dissociation constant (Kd) 2.9 vs. 0.47 nM] and does not interact with hsp90. Addition of the five-amino acid sequence (729-733) upstream from the LBD is necessary for interaction with hsp90, but a larger region is needed for high aldosterone affinity. Deletions at the C-terminal end of the hMR greatly reduced both agonist and antagonist binding: deletion of the last three amino acids reduced the affinity for aldosterone to 1/20 that of the entire protein, and deletion of the last four amino acids completely abolished binding, although the interaction with hsp90 was not affected. These effects can be explained by misfolding of the receptor, since limited proteolysis assays showed that deletions at the C-terminal end of hMR affect the accessibility of the cleavage sites within the DNA-binding domain and the N-terminal part of the hinge region to trypsin. Thus, our results support the idea that a short sequence upstream of the LBD is essential for the interaction of hMR with hsp90 and that the C terminus of hMR and hsp90 are both essential for folding of the receptor in a high-affinity hormone-binding state. UI - 98282522 TI - Interleukin 2 up-regulates glucocorticoid receptor number in human peripheral blood mononuclear cells and the osteosarcoma cell line Saos- 2 in vitro. AB - Glucocorticoids are well-recognized modulators of immunocytes and osteoblasts via specific receptor-mediated mechanisms. We have evaluated the in vitro effect of interleukin 2 (IL-2) on the expression of glucocorticoid receptors (GRs) in peripheral blood mononuclear cells (PBMCs) obtained from healthy donors and osteoblast-like Saos-2 cells. Aliquots of PBMC or Saos-2 cells were incubated for 20 h in the presence or absence of recombinant human IL-2 (100 IU/mL) at 37 degrees C. After incubation, a [3H]dexamethasone radioligand-binding assay and Scatchard analysis were used to determine GR-binding parameters in both cell populations. Saos-2 cells basally express higher numbers of GR than PBMCs. After IL-2, a significant increase in GR number was found for both PBMCs and Saos-2 cells. The relative increase was higher in Saos-2 cells; in PBMCs, the apparent affinity fell to almost half. These data represent an additional piece of evidence that cytokine and steroid hormones may act in a complementary way to regulate specific cell functions. UI - 98289590 TI - Identification of the critical portions of the human IL-4 receptor alpha chain for activation of STAT6. AB - Interleukin-4 (IL-4) has been shown to activate Janus kinase (Jak)-1 and Jak-3, followed by activation of STAT (signal transducers and activators of transcription) 6. This Jak-STAT pathway is central to the initiation of IL-4 activities. In this study, we identified the essential region for the proliferation signal and activation of Jak-1, Jak-3, and STAT6 in the cytoplasmic domain of the human IL-4 receptor alpha chain (hIL-4R alpha) using a mouse T cell line CTLL-2. We found that the region between amino acid 353 and 393 is critical for the proliferation signal and activation of STAT6, but not for tyrosine phosphorylation of Jaks. These results suggest that in addition to the "Box-1" portion, which is known to be essential for Jak-1 activation, the more membrane-distal region of hIL-4R alpha is also necessary for activation of STAT6. UI - 98264614 TI - PMA/ionomycin induces Ig kappa 3' enhancer activity which is in part mediated by a unique NFAT transcription complex. AB - The Ig kappa 3' enhancer is required for high levels of Ig kappa gene expression. We now show that kappa 3' enhancer function increases five- to eightfold after stimulation of primary murine B cells with phorbol 12-myristate 13-acetate (PMA) and the calcium ionophore ionomycin. In the presence of cyclosporin A this induction is almost halved, suggesting that transcription factors of the NFAT family contribute to kappa 3' enhancer induction. Indeed, we identify a novel NFAT binding site which is required for full enhancer function. We find that this site is transcriptionally active in stimulated B cells, T cells and fibroblasts and that both PMA and ionomycin are required for maximal induction. Time course analysis of the components of the protein-DNA complex in primary lymphocytes reveals that both NFATp and NFATc are present in the complex after 15 min, while only NFATc is detectable after 4 h. This suggests that NFATc plays the dominant role in controlling long-term responses of this transcription factor family. Furthermore, JunB, JunD, FosB and cFos form part of the DNA-protein complex in Bal-17 B cells. Complex formation as well as transcriptional activity can also be induced by crosslinking of surface Ig. We have, thus, identified a unique NFAT complex in B cells that contributes to Ig kappa gene expression. UI - 98226553 TI - Tumor necrosis factor alpha gene regulation: enhancement of C/EBPbeta- induced activation by c-Jun. AB - Tumor necrosis factor alpha (TNF alpha) is a key regulatory cytokine whose expression is controlled by a complex set of stimuli in a variety of cell types. Previously, we found that the monocyte/macrophage- enriched nuclear transcription factor C/EBPbeta played an important role in the regulation of the TNF alpha gene in myelomonocytic cells. Abundant evidence suggests that other transcription factors participate as well. Here we have analyzed interactions between C/EBPbeta and c-Jun, a component of the ubiquitously expressed AP-1 complex. In phorbol myristate acetate (PMA)-treated Jurkat T cells, which did not possess endogenous C/EBPbeta, expression of c-Jun by itself had relatively little effect on TNF alpha promoter activity. However, the combination of C/EBPbeta and c-Jun was synergistic, resulting in greater than 130- fold activation. This effect required both the leucine zipper and DNA binding domains, but not the transactivation domain, of c-Jun, plus the AP-1 binding site centered 102/103 bp upstream of the transcription start site in the TNF alpha promoter. To determine if C/EBPbeta and c-Jun might cooperate to regulate the cellular TNF alpha gene in myelomonocytic cells, U937 cells that possess endogenous C/EBPbeta and were stably transfected with either wild-type c-Jun or the transactivation domain deletion mutant (TAM-67) were examined. U937 cells expressing ectopic wild-type c-Jun or TAM-67 secreted over threefold more TNF alpha than the control line in response to PMA plus lipopolysaccharide. Transient transfection of the U937 cells expressing TAM-67 suggested that TAM-67 binding to the -106/-99-bp AP-1 binding site cooperated with endogenous C/EBPbeta in the activation of the -120 TNF alpha promoter-reporter. DNA binding assays using oligonucleotides derived from the TNF alpha promoter suggested that C/EBPbeta and c-Jun interact in vitro and that the interaction may be DNA dependent. Our data demonstrate that the TNF alpha gene is regulated by the interaction of the ubiquitous AP-1 complex protein c-Jun and the monocyte/macrophage-enriched transcription factor C/EBPbeta and that this interaction contributes to the expression of the cellular TNF alpha gene in myelomonocytic cells. This interaction was unique in that it did not require the c-Jun transactivation domain, providing new insight into the cell-type-specific regulation of the TNF alpha gene. UI - 98226532 TI - Alternative splicing variants of IkappaB beta establish differential NF-kappaB signal responsiveness in human cells. AB - To release transcription factor NF-kappaB into the nucleus, the mammalian IkappaB molecules IkappaB alpha and IkappaB beta are inactivated by phosphorylation and proteolytic degradation. Both proteins contain conserved signal-responsive phosphorylation sites and have conserved ankyrin repeats. To confer specific physiological functions to members of the NF-kappaB/Rel family, the different IkappaB molecules could vary in their specific NF-kappaB/Rel factor binding activities and could respond differently to activation signals. We have demonstrated that both mechanisms apply to differential regulation of NF-kappaB function by IkappaB beta relative to IkappaB alpha. Via alternative RNA processing, human IkappaB beta gives rise to different protein isoforms. IkappaB beta1 and IkappaB beta2, the major forms in human cells, differ in their carboxy-terminal PEST sequences. IkappaB beta2 is the most abundant species in a number of human cell lines tested, whereas IkappaB beta1 is the only form detected in murine cells. These isoforms are indistinguishable in their binding preferences to cellular NF-kappaB/Rel homo- and heterodimers, which are distinct from those of IkappaB alpha, and both are constitutively phosphorylated. In unstimulated B cells, however, IkappaB beta1, but not IkappaB beta2, is found in the nucleus. Furthermore, the two forms differ markedly in their efficiency of proteolytic degradation after stimulation with several inducing agents tested. While IkappaB beta1 is nearly as responsive as IkappaB alpha, indicative of a shared activation mechanism, IkappaB beta2 is only weakly degraded and often not responsive at all. Alternative splicing of the IkappaB beta pre- mRNA may thus provide a means to selectively control the amount of IkappaB beta-bound NF-kappaB heteromers to be released under NF-kappaB stimulating conditions. UI - 98220845 TI - Co-stimulation of human peripheral blood mononuclear cells with IL-2 and anti-CD3 monoclonal antibodies induces phosphorylation of CREB. AB - Phosphorylation of the cAMP-response element binding protein CREB within 1 h of CD2 but not CD3 cross-linking of human PBMC was recently demonstrated. The absence of P-CREB following CD3 cross-linking was unexpected, as other laboratories reported increased phosphorylation of CREB following CD3 cross-linking of the Jurkat lymphocyte cell line. Due to Jurkat T-cells being IL-2-independent, it was postulated that IL-2 might provide a necessary co-stimulus for phosphorylation of CREB in primary lymphocytes. Therefore, P-CREB was evaluated following co- stimulation of human PBMC through the IL-2 and CD2 or CD3 receptors. IL-2 did not further augment phosphorylation of CREB following CD2 cross- linking. However, while neither IL-2 nor CD3 cross-linking alone induced P-CREB, a 4.5-fold increase in phosphorylation of CREB within 1 h of IL-2/CD3 co-stimulation was observed. Phosphorylation was not associated with the induction of cAMP, and inhibition of PKA signaling had no effect on P-CREB. Consistent with signal transduction through p56lck or p59fyn, inhibition of PTK signaling reduced phosphorylation 50%. Interestingly, inhibiting PKC signaling with calphostin C further increased P-CREB levels 3-fold over that observed in IL-2/CD3 co- stimulated cells not pretreated with a PKC inhibitor. In contrast to previous studies performed in the absence of exogenous IL-2, no increase in binding of CREB to a 32P-labeled oligonucleotide probe was observed by electrophoretic mobility shift assay. These data suggest that the IL-2 and CD3 signaling pathways provide a necessary and co- operative stimulus promoting phosphorylation of CREB following receptor cross-linking. UI - 98211655 TI - High affinity receptor for IgG (Fc gamma RI/CD64) gene and STAT protein binding to the IFN-gamma response region (GRR) are regulated differentially in human neutrophils and monocytes by IL-10. AB - Since IL-10 has been shown to up-regulate the expression of the high affinity receptor for IgG (FcgammaRI/CD64) in human monocytes, we examined whether the cytokine exerts a similar action toward polymorphonuclear neutrophils (PMN). Unexpectedly, we found that in neutrophils, IL-10 failed to induce either the mRNA accumulation or the surface expression of FcgammaRI. Consistent with these findings, stimulation of PMN with IFN-gamma, but not with IL-10, resulted in the induction of specific DNA-binding activities to the IFN-gamma response region (GRR), a regulatory element located in the FcgammaRI gene promoter, required for transcriptional activation. In electrophoretic mobility shift assays (EMSAs), we confirmed that in PBMC, IL-10 induces the binding to the GRR of both STAT1 and STAT3, two members of the STAT family. In neutrophils, however, these activators did not bind to the GRR in response to IL-10, despite the fact that both STAT1 and STAT3 are expressed in these cells. On the other hand, IFN-gamma was an efficient inducer of STAT1 binding to the GRR in both PMN and PBMC. The lack of inducible GRR-binding activity in IL-10-treated PMN could not be ascribed to a lack of IL-10R, and did not appear to reflect an inhibitory effect of the cytokine. Taken together, our data suggest that IL-10 is unable to induce FcgammaRI gene expression in neutrophils because the intracellular signaling pathway triggered by the cytokine is impaired at the level of, or upstream of, STAT1 and/or STAT3 activation. UI - 98211645 TI - Activation of STAT5 by lipopolysaccharide through granulocyte- macrophage colony-stimulating factor production in human monocytes. AB - LPS is a potent stimulator of monocytes, inducing many of their functions. Although the details of how LPS exerts such functions remain largely unknown, transcription factors such as nuclear factor-kappaB, nuclear factor-IL-6, and activator protein-1 have been shown to be involved in this process. However, to date it has been thought that no known STAT molecule plays a role in the activation of monocytes by LPS. In this study we examined whether some known STAT molecule is stimulated by LPS, based on the finding that a GAS motif sequence is conserved in the promoter regions of human, mouse, and rat cyclo- oxygenase-2 (COX-2) genes. Consequently, LPS induced activation of STAT5 in human monocytes, and this STAT5 activation occurred in an indirect way via granulocyte-macrophage CSF (GM-CSF) secreted by LPS- stimulated monocytes. Expression of COX-2 protein was partially reduced by treatment of anti-human GM-CSF Ab. Activation of STAT5 was inhibited by either IL-10 or dexamethasone (Dex), but not by aspirin. IL-10 blocked activation of STAT5 indirectly by suppressing GM-CSF production, while Dex inhibited this activation both directly and indirectly. Taken together, these results suggest that in addition to other transcription factors, STAT5 plays an important role in activation of monocytes by LPS, and that STAT5 is another target for IL-10 and Dex to inhibit COX-2 expression in activated monocytes. UI - 98165830 TI - Synergistic regulation of the human interleukin-12 p40 promoter by NFkappaB and Ets transcription factors in Epstein-Barr virus- transformed B cells and macrophages. AB - Monocytes/macrophages produce interleukin-12 (IL-12) in response to pathogenic stimulation, whereas most Epstein-Barr virus-transformed (EBV+) B cells constitutively secrete IL-12. The molecular mechanism regulating the constitutive IL-12 gene expression in EBV+ B cells has not been addressed. In this study, using the EBV+ B cell line RPMI-8866, we localized to the human IL-12 p40 promoter two essential cis elements, the NFkappaB site and the Ets site. The NFkappaB site was shown to interact with members of the NFkappaB family: p50 and c-Rel. The Ets site constitutively bound a multi-component Ets-2-containing complex. While the NFkappaB and Ets sites appear equally critical for inducible p40 promoter activity in macrophage cell lines, NFkappaB plays a more dominant role in the constitutive p40 promoter activity in EBV+ B cells. Transient expression of Ets-2 and c-Rel in B, T, and monocytic cell lines synergistically activated the IL-12 p40 promoter, apparently overcoming the requirement for cell type- or stimulant- specific transcription factors. These data provide new evidence that full activation of the human IL-12 p40 promoter may result primarily from the interplay between NFkappaB and Ets family members. UI - 98157993 TI - Association of activating transcription factor 2 (ATF2) with the ubiquitin-conjugating enzyme hUBC9. Implication of the ubiquitin/proteasome pathway in regulation of ATF2 in T cells. AB - Activating transcription factor 2 (ATF2) is regulated by phosphorylation via the Jun N-terminal kinase, and its binding activity is markedly induced at late stages of T and B lymphocyte activation (Feuerstein, N., Firestein, R., Aiyer, N., Xiao, H., Murasko, D., and Cristofalo, V.(1996) J.Immunol.156, 4582-4593). To identify proteins that interact specifically with ATF2 in lymphocytes, the yeast two- hybrid interaction system was employed using ATF2 cDNA as a "bait".] In two separate screenings, a clone was identified that revealed a novel sequence with homology to several members of the ubiquitin-conjugating enzyme family. An identical sequence was recently reported as the human homolog of the yeast UBC9, hUBC9. Northern blot analysis revealed a 1.3- kilobase RNA transcript, which showed differential levels of expression in various human tissues and a moderate induction after a 48-h stimulation of peripheral blood T lymphocytes. An antibody that was generated against the bacterially expressed glutathione S-transferase- hUBC9 detected a approximately 19-kDa protein, which localizes predominantly in the nuclei of T cells. Further quantitative assays using the yeast two-hybrid system confirmed a high and specific level of interaction of hUBC9 with ATF2 and lack of interaction with lamin or control vectors. Two other cyclic AMP-responsive element-binding transcription factors, CREB and ATF1, also showed significant levels of interaction with hUBC9. However, this interaction was severalfold lower as compared with ATF2. Far Western blot analysis confirmed the specific binding of ATF2 and hUBC9 also in vitro. Evidence is presented that indicates a physiological significance for the interaction of hUBC9 with ATF2. (a) We show that ATF2 is ubiquitinated in vivo and in vitro, and (b) ATF2 ubiquitination in vitro is facilitated by addition of purified hUBC9. (c) ATF2 is shown to undergo a proteolytic process, which is rapidly regulated upon T cell activation concomitant with induction of ATF2 phosphorylation. (d) A proteasome inhibitor delays the down-regulation of ATF2 phophorylation after T cell activation. Taken collectively, these results implicate a role for hUBC9 and the ubiquitin/proteasome pathway in regulation of ATF2 in T cells. UI - 98129351 TI - Involvement of extracellular signal-regulated kinase module in HIV- mediated CD4 signals controlling activation of nuclear factor-kappa B and AP-1 transcription factors. AB - Although the molecular mechanisms by which the HIV-1 triggers either T cell activation, anergy, or apoptosis remain poorly understood, it is well established that the interaction of HIV-1 envelope glycoproteins with cell surface CD4 delivers signals to the target cell, resulting in activation of transcription factors such as NF-kappa B and AP-1. In this study, we report the first evidence indicating that kinases MEK-1 (MAP kinase/Erk kinase) and ERK-1 (extracellular signal-regulated kinase) act as intermediates in the cascade of events that regulate NF-kappa B and AP-1 activation upon HIV-1 binding to cell surface CD4. We found that CEM cells transfected with dominant negative forms of MEK-1 or ERK-1 do not display NF-kappa B activation after HIV-1 binding to CD4. In contrast, NF-kappa B activation was observed in these cells after PMA stimulation. Although the different cell lines studied expressed similar amounts of CD4 and p56(lck), HIV-1 replication and HIV-1-induced apoptosis were slightly delayed in cells expressing dominant negative forms of MEK-1 or ERK-1 compared with parental CEM cells and cells expressing a constitutively active mutant form of MEK-1 or wild-type ERK-1. In light of recently published data, we propose that a positive signal initiated following oligomerization of CD4 by the virus is likely to involve a recruitment of active forms of p56(lck), Raf-1, MEK-1, and ERK-1, before AP-1 and NF-kappa B activation. UI - 98127791 TI - Transcriptional regulation in the immune system: all roads lead to AP-1. AB - The mechanisms regulating the development and function of the immune system are diverse and complicated. The signaling pathways and target genes that become activated upon cell-surface stimulation are currently being defined, and transcription factor activator protein 1 (AP-1) is proving to be an important regulator of nuclear gene expression in leukocytes. In vitro and in vivo studies have demonstrated that AP-1 expression is induced after a diverse range of stimuli and that AP-1 contributes to the regulation of a large number of genes. In this review we will examine the role of AP-1 during leukocyte activation and differentiation in the immune system. UI - 98125541 TI - Human 4-1BB (CD137) signals are mediated by TRAF2 and activate nuclear factor-kappa B. AB - Human 4-1BB (CD137), a member of the tumor necrosis factor receptor (TNFR) superfamily, costimulates T cell activation. No apparent intrinsic kinase activity is seen with 4-1BB, which suggests that 4-1BB- associated molecules may be involved in 4-1BB-mediated signal transduction. We found that tumor necrosis factor receptor-associated factor (TRAF) 1, TRAF2, and TRAF3, all interacted with the cytoplasmic domain of 4-1BB. Mutation analysis showed that TRAF1, TRAF2, and TRAF3 were associated with one of two runs of acidic residues found in the cytoplasmic domain of 4-1BB. In addition, 4-1BB cross-linking with TCR signal in Jurkat cells and overexpression of 4-1BB in 293 cells were able to induce activation of the nuclear factor-kappa B (NF-kappa B). 4-1BB-mediated NF-kappa B activation was inhibited by a dominant negative- TRAF2 or -NF-kappa B-inducing kinase (NIK). These data suggest that 4-1BB functions may be mediated by NF-kappa B activation, which requires a TRAF2/NIK pathway. UI - 98058928 TI - Two NFAT transcription factor binding sites participate in the regulation of CD95 (Fas) ligand expression in activated human T cells. AB - Antigen receptor engagement on T lymphocytes activates transcription factors important for stimulating cytokine gene expression. This is critical for clonal expansion of antigen-specific T cells and propagation of immune responses. Additionally, under some conditions antigen receptor stimulation initiates apoptosis of T lymphocytes through the induced expression of CD95 ligand and its receptor. Here we demonstrate that the transcription factor, NFAT, which is critical for the inducible expression of many cytokine genes, also plays a critical role in the regulation of T cell receptor-mediated CD95 ligand expression. Two sites within the CD95 ligand promoter, identified through DNase I footprinting, bind NFAT proteins from nuclear extracts of activated T cells. Although both sites appear important for optimal expression of CD95 ligand in activated T cells, mutational analysis suggests that the distal NFAT site plays a more significant role. Furthermore, these sites do not appear to be required for constitutive CD95 ligand expression in Sertoli cells. UI - 98043747 TI - Interaction of NF-kappaB and NFAT with the interferon-gamma promoter. AB - Interferon-gamma (IFN-gamma) is a pleiotropic lymphokine whose production is restricted to activated T cells and NK cells. Along with other cytokines, IFN-gamma gene expression is inhibited by the immunosuppressant cyclosporin A. We have previously identified an intronic enhancer region (C3) of the IFN-gamma gene that binds the NF-kappaB protein c-Rel and that shows partial DNA sequence homology with the cyclosporin A-sensitive NFAT binding site and the 3'-half of the NF-kappaB consensus site. Sequence analysis of the IFN-gamma promoter revealed the presence of two additional C3-related elements (C3-1P and C3-3P). In addition, an NF-kappaB site (IFN-gamma kappaB) was identified within the promoter region. Based on this observation, we have analyzed the potential role of NF-kappaB and NFAT family members in regulating IFN-gamma transcription. Electrophoretic mobility shift assay analysis demonstrated that after T cell activation, the p50 and p65 NF-kappaB subunits bind specifically to the newly identified IFN-gamma kappaB and C3-related sites. In addition, we identified the NFAT proteins as a component of the inducible complexes that bind to the C3-3P site. Site-directed mutagenesis and transfection studies demonstrate that calcineurin-inducible transcriptional factors enhance the transcriptional activity of the IFN-gamma promoter through the cyclosporin-sensitive C3-3P site, whereas NF-kappaB proteins functionally interact with the C3-related sites. In addition, when located downstream to the beta-galactosidase gene driven by the IFN-gamma promoter, the intronic C3 site worked in concert with both the IFN-gamma kappaB and the C3-3P site to enhance gene transcription. These results demonstrate that the coordinate activities of NFAT and NF-kappaB proteins are involved in the molecular mechanisms controlling IFN-gamma gene transcription. UI - 98058742 TI - Inhibition of NF-kappaB DNA binding and nitric oxide induction in human T cells and lung adenocarcinoma cells by selenite treatment. AB - NF-kappaB is a major transcription factor consisting of 50(p50)- and 65(p65)-kDa proteins that controls the expression of various genes, among which are those encoding cytokines, cell adhesion molecules, and inducible NO synthase (iNOS). After initial activation of NF-kappaB, which involves release and proteolysis of a bound inhibitor, essential cysteine residues are maintained in the active reduced state through the action of thioredoxin and thioredoxin reductase. In the present study, activation of NF-kappaB in human T cells and lung adenocarcinoma cells was induced by recombinant human tumor necrosis factor alpha or bacterial lipopolysaccharide. After lipopolysaccharide activation, nuclear extracts were treated with increasing concentrations of selenite, and the effects on DNA-binding activity of NF-kappaB were examined. Binding of NF-kappaB to nuclear responsive elements was decreased progressively by increasing selenite levels and, at 7 microM selenite, DNA-binding activity was completely inhibited. Selenite inhibition was reversed by addition of a dithiol, DTT. Proportional inhibition of iNOS activity as measured by decreased NO products in the medium (NO2- and NO3-) resulted from selenite addition to cell suspensions. This loss of iNOS activity was due to decreased synthesis of NO synthase protein. Selenium at low essential levels (nM) is required for synthesis of redox active selenoenzymes such as glutathione peroxidases and thioredoxin reductase, but in higher toxic levels (5-10 microM) selenite can react with essential thiol groups on enzymes to form RS-Se-SR adducts with resultant inhibition of enzyme activity. Inhibition of NF-kappaB activity by selenite is presumed to be the result of adduct formation with the essential thiols of this transcription factor. UI - 98030791 TI - Early phosphorylation of the retinoblastoma gene product regulates protein binding to the c-fos retinoblastoma control element during T cell activation. AB - Function of the retinoblastoma tumor suppressor protein [pRb] is regulated by phosphorylation during the G1 and S phases of the cell cycle. pRb regulates transcription of several genes, including c-fos. However, since c-fos is regulated during exit from G0, it has remained unclear how pRb participates in c-fos regulation. We have identified a protein complex, the retinoblastoma control factor A [RCF-A] which binds to the c-fos retinoblastoma control element [RCE] and is regulated by pRb within 10 min after T cell activation. We demonstrate that pRb control of RCF-A is dependent upon the state of phosphorylation of pRb. pRb becomes hyperphosphorylated on specific peptides at 10 min after mitogenic stimulation and pRb is dephosphorylated by 30 min. This time course coincides with RCF-A DNA binding. RCF-A binds RCE DNA longer when cells are treated with okadaic acid, and okadaic acid prevents pRb dephosphorylation. Dephosphorylated pRb inhibits RCF-A binding in vitro but phosphorylated pRb does not. Thus, in addition to the described G1/S regulation of pRb, transient inactivation by phosphorylation of pRb in T cells may also be important as resting cells leave G0. UI - 97473984 TI - Novel flow cytometric method for quantifying nuclear binding of the transcription factor nuclear factor kappa B in unseparated human monocytes and polymorphonuclear cells. AB - The transcription factor nuclear factor kappa B (NF kappa B) regulates the production of a number of pro-inflammatory mediators involved in polymorphonuclear and mononuclear cell activation. Measurement of NF kappa B DNA binding is used as an estimate of cellular activation and is usually measured by the DNA mobility shift assay. Results from the mobility shift assay can be difficult to quantify and do not allow discrimination of activity in different populations of cells in whole blood without prior separation. This paper describes a new flow cytometric method which allows rapid detection and quantification of DNA-bound NF kappa B in white cell populations of whole blood. The technique is sensitive and allows discriminate analysis and quantification of bound NF kappa B in nuclei from polymorphonuclear and mononuclear cell populations in whole blood. UI - 97459719 TI - A new member of the I kappaB protein family, I kappaB epsilon, inhibits RelA (p65)-mediated NF-kappaB transcription. AB - A novel member of the I kappaB family has been identified as a protein that associated with the p50 subunit of NF-kappaB in a yeast two-hybrid screen. Similar to previously known I kappaB proteins, this member, I kappaB epsilon, has six consecutive ankyrin repeats. I kappaB epsilon mRNA is widely expressed in different human tissues, with highest levels in spleen, testis, and lung. I kappaB epsilon interacts with different NF-kappaB proteins, including p65 (RelA), c-Rel, p50, and p52, in vitro and in vivo and inhibits the DNA-binding activity of both p50-p65 and p50-c-Rel complexes effectively. Endogenous and transfected NF-kappaB (RelA-dependent) transcriptional activation is inhibited by I kappaB epsilon. I kappaB epsilon mRNA is expressed at different levels in specific cell types and is synthesized constitutively in transformed B-cell lines. It also displays differential induction in response to tumor necrosis factor alpha, interleukin-1, or phorbol ester stimulation compared to I kappaB alpha in non-B-cell lines. Therefore, I kappaB epsilon represents a novel I kappaB family member which provides an alternative mechanism for regulation of NF-kappaB-dependent transcription. UI - 97410329 TI - Kinetic mechanism for p38 MAP kinase. AB - p38 has been shown to be a critical enzyme in the pro-inflammatory cytokine pathway and is a member of the mitogen-activated protein (MAP) kinase family. While the details for p38 activation and subsequent signal transduction have begun to be elucidated, little is known about the kinetic mechanism for p38. In this study, we have determined the kinetic mechanism for p38 MAP kinase. Data from initial velocity patterns in the presence and absence of a dead-end inhibitor and two triarylimidazole p38 inhibitors were consistent with an ordered sequential mechanism for p38 with protein substrate, glutathione S- transferase-activating transcription factor 2 (GST-ATF2), binding before ATP. The ATP analog, adenylyl methylenediphosphonate (AMP-PCP), and two triarylimidazoles were competitive inhibitors versus ATP and uncompetitive inhibitors versus GST-ATF2. Equilibrium binding studies utilizing a tritiated ATP-competitive inhibitor were also consistent with this mechanism and suggest an inability of ATP to bind to p38 in the absence of protein substrate. Moreover, the Michaelis constant for GST-ATF2 was 12-fold greater than the dissociation constant, indicating that the binding of ATP affected the binding of GST-ATF2. An ordered sequential mechanism with protein substrate binding first is unique to p38 compared to cyclic AMP-dependent protein kinase (cAPK) and most tyrosine kinases and helps to explain the interaction between enzyme, substrates, and inhibitors. UI - 97407915 TI - The 90-kDa ribosomal S6 kinase (pp90rsk) phosphorylates the N-terminal regulatory domain of IkappaBalpha and stimulates its degradation in vitro. AB - Nuclear factor kappaB (NF-kappaB) is a eukaryotic member of the Rel family of transcription factors whose biological activity is post- translationally regulated by its assembly with various ankyrin-rich cytoplasmic inhibitors, including IkappaBalpha. Expression of NF-kappaB in the nucleus occurs after signal-induced phosphorylation, ubiquitination, and proteasome-mediated degradation of IkappaBalpha. The induced proteolysis of IkappaBalpha unmasks the nuclear localization signal within NF-kappaB, allowing its rapid migration into the nucleus, where it activates the transcription of many target genes. At present, the identity of the IkappaBalpha kinase(s) that triggers the first step in IkappaBalpha degradation remains unknown. We have investigated the potential function of the 90-kDa ribosomal S6 kinase, or pp90(rsk), as a signal-inducible IkappaBalpha kinase. pp90(rsk) lies downstream of mitogen-activated protein (MAP) kinase in the well characterized Ras-Raf-MEK-MAP kinase pathway that is induced by various growth factors and phorbol ester. We now show that pp90(rsk), but not pp70(S6K) or MAP kinase, phosphorylates the regulatory N terminus of IkappaBalpha principally on serine 32 and triggers effective IkappaBalpha degradation in vitro. When co-expressed in vivo in COS cells, IkappaBalpha and pp90(rsk) readily assemble into a complex that is immunoprecipitated with antibodies specific for either partner. While phorbol 12-myristate 13-acetate produced rapid activation of pp90(rsk), in vivo, other potent NF-kappaB inducers, including tumor necrosis factor alpha and the Tax transactivator of human T-cell lymphotrophic virus, type I, failed to activate pp90(rsk). These data suggest that more than a single IkappaBalpha kinase exists within the cell and that these IkappaBalpha kinases are differentially activated by different NF-kappaB inducers. UI - 97376993 TI - Inhibition of E2F-4/DP-1-stimulated transcription by p202. AB - The interferon (IFN)-inducible proteins mediate activities of the interferons including the cell growth-regulatory activity. We have shown that p202, an IFN-inducible 52kDa primarily nuclear phosphoprotein whose expression in transfected cells inhibits cell proliferation, interacts with the retinoblastoma tumor suppressor protein (pRb) and the transcription factor E2F (E2F-1/ DP-1) in vitro and in vivo. p202 was shown to inhibit E2F-1/DP-1-stimulated transcription of a reporter gene and of endogenous genes. Here we report that expression of p202 inhibited E2F-4/DP-1-stimulated transcription of a reporter gene in transfected cells. Furthermore, this inhibition was associated with the inhibition of the sequence- specific DNA-binding of E2F-4 both in complex with the pocket proteins p107 or p130 and in its 'free' form in vitro. p202 bound to p107 and p130 in vitro and in vivo and also associated with E2F-4, supporting the notion that complexes containing p107/E2F-4 or p130/E2F-4 and p202 exist in vivo. Moreover, cotransfection of E2F-4-encoding plasmid in AKR-2B cells overcame p202-mediated inhibition of cell growth, raising the possibility that p202 contributes to cell growth inhibition by the interferons, at least in part, by modulating E2F-4-mediated transcription. UI - 94222041 TI - Induction by interleukin-6 of interferon regulatory factor 1 (IRF-1) gene expression through the palindromic interferon response element pIRE and cell type-dependent control of IRF-1 binding to DNA. AB - The effects of interleukin-6 (IL-6) on interferon regulatory factor 1 (IRF-1) gene expression were studied in B-hybridoma B9 cells which are growth-stimulated by IL-6 and breast carcinoma T47D cells which are growth-inhibited. IL-6 induced the production of IRF-1 mRNA and protein in both cell types, but IRF-1 binding activity to its target DNA sequence was induced only in T47D cells. With B9 cells, there was no IRF-1 binding but instead strong constitutive binding of the IRF-2 repressor, indicating that binding of IRF-1 to DNA is an important regulatory step. The IRF-1 gene promoter element, palindromic IFN- response element (pIRE), was found to respond to IL-6 with high efficiency as compared with IFN-gamma or IFN-beta. On this palindromic TTC...GAA sequence, two protein complexes (pIRE-a and pIRE-b) were induced within minutes by IL-6. pIRE-b is similar to the main complex induced by IFN-gamma and contains the Stat91 protein. pIRE-a predominantly induced by IL-6 is a slowly migrating complex which does not contain Stat91 and has low affinity for IFN-gamma activated sequence (GAS)-type sequences. Comparison of the relative effects of IL- 6 and IFN-gamma shows that pIRE enhancers are differently regulated than GAS elements. Distinct transcription complexes, forming in ratios dependent on the inducer, help explain how various cytokines sharing effects through Stat91 on related enhancers can produce specific patterns of gene expression. Activation of the pIRE-a factors defines a novel transcriptional activity of IL-6 in epithelial and lymphoid cells. UI - 93154317 TI - The Epstein-Barr virus nuclear antigen 2 interacts with an EBNA2 responsive cis-element of the terminal protein 1 gene promoter. AB - The Epstein-Barr virus protein EBNA2 acts as a transcriptional activator of cellular and viral genes and plays a crucial role in the immortalization of human primary B-cells by EBV. We have shown previously that EBNA2 transactivates the promoters of the latent membrane antigens LMP, TP1 and TP2. The promoter of the TP1 gene was chosen as a model system to study the molecular mechanism of EBNA2 mediated transactivation. To identify an EBNA2 dependent cis-acting element, various TP1 promoter-reporter gene constructs were transfected in the absence and presence of an EBNA2 expression vector into the established B-cell line BL41-P3HR1. We were able to delineate an 81 bp EBNA2 responsive region between -258 and -177 relative to the TP1 RNA start site. The element worked in either orientation and could mediate EBNA2 dependent transactivation on a heterologous promoter. Electrophoretic mobility shift assays revealed three specific protein- DNA complexes formed with sequences of the EBNA2 responsive element. Two of these were not cell type specific, but the third was detected only in EBNA2 positive cell extracts. Gel-shift analysis in the presence of EBNA2 specific monoclonal antibodies revealed that EBNA2 is a component of the third complex. Thus, these experiments demonstrate that EBNA2 interacts with an EBNA2 responsive cis-element of the TP1 promoter.