Abstract 970: Competitive binding of CREB and ATF2 to cAMP-responsive/Activating Transcription Factor Element Regulates Endothelial Nitric Oxide Synthase Gene Expression in Endothelial Cells
Introduction Expression of endothelial nitric oxide synthase (eNOS) is a critical determinant for vascular homeostasis. Inflammatory cytokines including interleukin-1β (IL-1β) impair the eNOS expression and initiate atherosclerosis. Our previous study has shown that beraprost sodium (BPS), a stable analogue of prostacyclin, increases eNOS gene expression through the sustained activation of cAMP-dependent protein kinase (PKA) pathway. In this study, we examined the effects of BPS on the eNOS gene expression in the presence of IL-1β.
Results Exposure of human and bovine aortic endothelial cells (BAEC) to IL-1β decreased eNOS expression. Western blot analysis using phospho-specific antibodies showed that IL-1β stimulated p38MAPK and phosphorylated ATF2. Treatment of BAEC with BPS inhibited these effects via PKA activation. Overexpression of ATF2 decreased eNOS promoter activity, and coexpression of CREB attenuated this effect. Transfection assays using site-specific mutation constructs showed that the cAMP-responsive element/activating transcription factor (CRE/ATF) element located at −733 and −603 are necessary for full IL-1β responsiveness; deletion of these elements abolished the IL-1β-mediated down-regulation of eNOS promoter activity. BPS attenuated the IL-1b-mediated decrease in eNOS promoter activity and the stability of eNOS mRNA through PKA pathway. Electrophoretic gel mobility shift assays showed that IL-1β increased the binding of phosphorylated ATF2 to CRE/ATF. Upon treatment with BPS, phosphorylated CREB predominantly bound to CRE/ATF.
Conclusion Taken together, our findings indicate that IL-1β and BPS antagonistically regulates the eNOS expression through the activation of p38 MAPK and PKA pathways, respectively. Further, the ability to bind both CREB and ATF2 implicates the CRE/ATF as a potential target for multiple signaling pathways in the regulation of the eNOS gene.