Bcl-2 and Bcl-XL serve an anti-inflammatory function in endothelial cells through inhibition of NF-kappaB.
(1/2441)
To maintain the integrity of the vascular barrier, endothelial cells (EC) are resistant to cell death. The molecular basis of this resistance may be explained by the function of antiapoptotic genes such as bcl family members. Overexpression of Bcl-2 or Bcl-XL protects EC from tumor necrosis factor (TNF)-mediated apoptosis. In addition, Bcl-2 or Bcl-XL inhibits activation of NF-kappaB and thus upregulation of proinflammatory genes. Bcl-2-mediated inhibition of NF-kappaB in EC occurs upstream of IkappaBalpha degradation without affecting p65-mediated transactivation. Overexpression of bcl genes in EC does not affect other transcription factors. Using deletion mutants of Bcl-2, the NF-kappaB inhibitory function of Bcl-2 was mapped to bcl homology domains BH2 and BH4, whereas all BH domains were required for the antiapoptotic function. These data suggest that Bcl-2 and Bcl-XL belong to a cytoprotective response that counteracts proapoptotic and proinflammatory insults and restores the physiological anti-inflammatory phenotype to the EC. By inhibiting NF-kappaB without sensitizing the cells (as with IkappaBalpha) to TNF-mediated apoptosis, Bcl-2 and Bcl-XL are prime candidates for genetic engineering of EC in pathological conditions where EC loss and unfettered activation are undesirable. (+info)
B-MYB transactivates its own promoter through SP1-binding sites.
(2/2441)
B-MYB is an ubiquitous protein required for mammalian cell growth. In this report we show that B-MYB transactivates its own promoter through a 120 bp segment proximal to the transcription start site. The B-MYB-responsive element does not contain myb-binding sites and gel-shift analysis shows that SP1, but not B-MYB, protein contained in SAOS2 cell extracts binds to the 120 bp B-myb promoter fragment. B-MYB-dependent transactivation is cooperatively increased in the presence of SP1, but not SP3 overexpression. When the SP1 elements of the B-myb promoter are transferred in front of a heterologous promoter, an increased response to B-MYB results. In contrast, c-MYB, the prototype member of the Myb family, is not able to activate the luciferase construct containing the SP1 elements. With the use of an SP1-GAL4 fusion protein, we have determined that the cooperative activation occurs through the domain A of SP1. These observations suggest that B-MYB functions as a coactivator of SP1, and that diverse combinations of myb and SP1 sites may dictate the responsiveness of myb-target genes to the various members of the myb family. (+info)
Neu differentiation factor stimulates phosphorylation and activation of the Sp1 transcription factor.
(3/2441)
Neu differentiation factors (NDFs), or neuregulins, are epidermal growth factor-like growth factors which bind to two tyrosine kinase receptors, ErbB-3 and ErbB-4. The transcription of several genes is regulated by neuregulins, including genes encoding specific subunits of the acetylcholine receptor at the neuromuscular junction. Here, we have examined the promoter of the acetylcholine receptor epsilon subunit and delineated a minimal CA-rich sequence which mediates transcriptional activation by NDF (NDF-response element [NRE]). Using gel mobility shift analysis with an NRE oligonucleotide, we detected two complexes that are induced by treatment with neuregulin and other growth factors and identified Sp1, a constitutively expressed zinc finger phosphoprotein, as a component of one of these complexes. Phosphatase treatment, two-dimensional gel electrophoresis, and an in-gel kinase assay indicated that Sp1 is phosphorylated by a 60-kDa kinase in response to NDF-induced signals. Moreover, Sp1 seems to act downstream of all members of the ErbB family and thus may funnel the signaling of the ErbB network into the nucleus. (+info)
Activation-dependent transcriptional regulation of the human Fas promoter requires NF-kappaB p50-p65 recruitment.
(4/2441)
Fas (CD95) and Fas ligand (CD95L) are an interacting receptor-ligand pair required for immune homeostasis. Lymphocyte activation results in the upregulation of Fas expression and the acquisition of sensitivity to FasL-mediated apoptosis. Although Fas upregulation is central to the preservation of immunologic tolerance, little is known about the molecular machinery underlying this process. To investigate the events involved in activation-induced Fas upregulation, we have examined mRNA accumulation, fas promoter activity, and protein expression in the Jurkat T-cell line treated with phorbol myristate acetate and ionomycin (P/I), pharmacological mimics of T-cell receptor activation. Although resting Jurkat cells express Fas, Fas mRNA was induced approximately 10-fold in 2 h upon P/I stimulation. Using sequential deletion mutants of the human fas promoter in transient transfection assays, we identified a 47-bp sequence (positions -306 to -260 relative to the ATG) required for activation-driven fas upregulation. Sequence analysis revealed the presence of a previously unrecognized composite binding site for both the Sp1 and NF-kappaB transcription factors at positions -295 to -286. Electrophoretic mobility shift assay (EMSA) and supershift analyses of this region documented constitutive binding of Sp1 in unactivated nuclear extracts and inducible binding of p50-p65 NF-kappaB heterodimers after P/I activation. Sp1 and NF-kappaB transcription factor binding was shown to be mutually exclusive by EMSA displacement studies with purified recombinant Sp1 and recombinant p50. The functional contribution of the kappaB-Sp1 composite site in P/I-inducible fas promoter activation was verified by using kappaB-Sp1 concatamers (-295 to -286) in a thymidine kinase promoter-driven reporter construct and native promoter constructs in Jurkat cells overexpressing IkappaB-alpha. Site-directed mutagenesis of the critical guanine nucleotides in the kappaB-Sp1 element documented the essential role of this site in activation-dependent fas promoter induction. (+info)
Functional studies by site-directed mutagenesis on the role of Sp1 in the expression of the pyruvate kinase M and aldolase A genes.
(5/2441)
During the cell cycle of mitogen stimulated rat thymocytes, an 8-10-fold induction of glycolytic enzymes and a corresponding increase in the mRNA levels has been observed. This prompted us to study the transcriptional regulation of the rat aldolase A and pyruvate kinase M genes. cis-Regulatory elements of both promoters were evaluated by site-directed mutagenesis of promoter/luciferase constructs and transient transfections of rat hepatoma FTO2B cells. Furthermore, the binding proteins were identified by mobility shift assays in the presence of specific antibodies. In the aldolase AH1 promoter, five binding sites for Sp1 and Sp3 and a TPA responsive element were identified as essential for transcriptional regulation. Most of the promoter activity can be attributed to these regulatory elements. In the pyruvate kinase M promoter three out of five binding sites of Sp1 and Sp3 (B box and GC boxes 1 and 3) turned out to be functional in the transfection assays whereas the disruption of GC box 2 had no effect, and the disruption of the GC box 4 had only a minor effect on the promoter activity. Both promoters are stimulated by Sp1 as well as Sp3, as judged by cotransfection experiments of Drosophila SL2 cells. Therefore, the Sp1- and Sp3-directed transcription provides a means for common regulatory mechanism of the aldolase A and the pyruvate kinase M genes. (+info)
Differential regulation of the human nidogen gene promoter region by a novel cell-type-specific silencer element.
(6/2441)
Transfection analyses of the human nidogen promoter region in nidogen-producing fibroblasts from adult skin revealed multiple positive and negative cis-acting elements controlling nidogen gene expression. Characterization of the positive regulatory domains by gel mobility-shift assays and co-transfection studies in Drosophila SL2 cells unequivocally demonstrated that Sp1-like transcription factors are essential for a high expression of the human nidogen gene. Analysis of the negative regulatory domains identified a novel silencer element between nt -1333 and -1322, which is bound by a distinct nuclear factor, by using extracts from adult but not from embryonal fibroblasts. In embryonal fibroblasts, which express significantly higher amounts of nidogen mRNA as compared with adult fibroblasts, this inhibitory nidogen promoter region did not affect nidogen and SV40 promoter activities. The silencer element seems to be active only in nidogen-producing cells. Therefore this regulatory element might function in vivo to limit nidogen gene expression in response to external stimuli. However, none of the identified regulatory elements, including the silencer, contribute significantly to cell-specific expression of the human nidogen gene. Instead we provide evidence that gene expression in epidermal keratinocytes that are not producing nidogen is repressed by methylation-specific and chromatin-dependent mechanisms. (+info)
Evolutionary analysis of TATA-less proximal promoter function.
(7/2441)
Many molecular studies describe how components of the proximal promoter affect transcriptional processes. However, these studies do not account for the likely effects of distant enhancers or chromatin structure, and thus it is difficult to conclude that the sequence variation in proximal promoters acts to modulate transcription in the natural context of the whole genome. This problem, the biological importance of proximal promoter sequence variation, can be addressed using a combination of molecular and evolutionary analyses. Provided here are molecular and evolutionary analyses of the variation in promoter function and sequence within and between populations of Fundulus heteroclitus for the lactate dehydrogenase-B (Ldh-B) proximal promoter. Approximately one third of the Ldh-B proximal promoter contains interspersed regions that are functionally important: (1) they bind transcription factors in vivo, (2) they effect a change in transcription as assayed by transient transfection into two different fish cell lines, and (3) they bind purified transcription factors in vitro. Evolutionary analyses that compare sequence variation in these functional regions versus the nonfunctional regions indicate that the changes in the Ldh-B proximal promoter sequences are due to directional selection. Thus, the Ldh-B proximal promoter sequence variations that affect transcriptional processes constitute a phenotypic change that is subject to natural selection, suggesting that proximal promoter sequence variation affects transcription in the natural context of the whole genome. (+info)
Activation and repression of p21(WAF1/CIP1) transcription by RB binding proteins.
(8/2441)
The Cdk inhibitor p21(WAF1/CIP1) is a negative regulator of the cell cycle, although its expression is induced by a number of mitogens that promote cell proliferation. We have found that E2F1 and E2F3, transcription factors that activate genes required for cell cycle progression, are strong activators of the p21 promoter. In contrast, HBP1 (HMG-box protein-1), a novel retinoblastoma protein-binding protein, can repress the p21 promoter and inhibit induction of p21 expression by E2F. Both E2Fs and HBP1 regulate p21 transcription through cis-acting elements located between nucleotides -119 to +16 of the p21 promoter and the DNA binding domains of each of these proteins are required for activity. Sequences between -119 and -60 basepairs containing four Sp1 consensus elements and two noncanonical E2F binding sites are of major importance for E2F activation, although E2F1 and E2F3 differ in the extent of their ability to activate expression when this segment is deleted. The opposing effects of E2Fs and HBP1 on p21 promoter activity suggest that interplay between these factors may determine the level of p21 transcription in vivo. (+info)