Abstract
Endothelial cell surface expression of VCAM-1 is one of the initial steps in the pathogenesis of atherosclerosis. The inflammatory response transcription factor nuclear factor (NF)-kappaB plays an important role in the regulation of VCAM-1 expression by various stimuli including tumor necrosis factor (TNF)-alpha. Other transcription factors may modulate this response through interaction with NF-kappaB factors. Since c-Fos/c-Jun (activating protein-1 (AP-1)) are expressed in vascular endothelium during proinflammatory conditions, we investigated the role of AP-1 proteins in the expression of VCAM-1 by TNF-alpha in SV40 immortalized human microvascular endothelial cells (HMEC). TNF-alpha induced expression of both early protooncogenes, c-fos and c-jun. The ability of TNF-alpha to activate the kappaB-motif (kappaL-kappaR)-dependent VCAM-1 promoter-chloramphenicol acetyltransferase (CAT) reporter gene lacking a consensus AP-1 element was markedly inhibited by co-transfection of the expression vector encoding c-fos ribozyme, which decreases the level of c-fos by degrading c-fos mRNA, or c-fos or c-jun oligonucleotides. Conversely, co-transfection of c-Fos and c-Jun encoding expression vectors potentiated the p65/NF-kappaB-mediated transactivation of the VCAM-1 promoter-CAT reporter gene. Furthermore the c-Fos encoding expression vector potentiated by 2-fold the transactivation activity of a chimeric transcriptional factor Gal/p65 (containing the transactivation domain of p65 and the DNA binding domain of the yeast transcriptional factor Gal-4). Consistent with the promoter studies, curcumin and NDGA, inhibitors of AP-1 activation, markedly inhibited the ability of TNF-alpha to activate the expression of VCAM-1 mRNA levels at concentrations that did not inhibit the activation of NF-kappaB. In gel mobility supershift assays, the antibodies to c-Fos or c-Jun inhibited the binding of TNF-alpha-activated nuclear NF-kappaB to the kappaL-kappaR, suggesting that both c-Fos and c-Jun interacted with NF-kappaB. These results suggest that AP-1 proteins may mediate the effect of TNF-alpha in the regulation of VCAM-1 expression through interaction with NF-kappaB factors in endothelial cells.
Highlights
(TNF-␣),1 interleukin-1, and lipopolysaccharide) induce VCAM-1 gene expression in endothelial cells through activating the redox-sensitive transcription factor nuclear factor-B (NF-B) [1,2,3]
tumor necrosis factor (TNF)-␣ Induces c-fos and c-jun in human microvascular endothelial cells (HMEC) Cells—Because TNF-␣ is known to induce c-fos and c-jun in various cell types [7, 35, 36], we tested whether similar responses occur in HMEC
Confluent HMEC cells were treated with TNF-␣ (100 units/ml) for 15–120 min and RNA was prepared for analysis of c-fos and c-jun expression
Summary
(TNF-␣), interleukin-1, and lipopolysaccharide) induce VCAM-1 gene expression in endothelial cells through activating the redox-sensitive transcription factor nuclear factor-B (NF-B) [1,2,3]. Activation protein-1 (AP-1, c-Fos/c-Jun) designates another class of transcription factors that mediate inflammatory responses by various cytokines and growth factors in different cell types [6, 7]. AP-1 proteins are regulated by several growth factors, cytokines, and by various agents that induce oxidative stress [6, 7] Since several of these agents and the conditions that activate AP-1 proteins are present in atherogenic conditions, it is likely that AP-1 proteins play a role in the regulation of this inflammatory disease of the vasculature. At least part of these effects of AP-1 is independent of direct binding of AP-1 to its cognate enhancer element and is likely effected through direct interactions with NF-B factors
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