Competitive Binding of CREB and ATF2 to cAMP/ATF Responsive Element Regulates eNOS Gene Expression in Endothelial Cells

Abstract

Expression of endothelial nitric oxide synthase (eNOS) is a critical determinant for vascular homeostasis. We examined the effects of Beraprost sodium (BPS), a stable analogue of prostacyclin, on the eNOS gene expression in the presence of inflammatory cytokine interleukin (IL)-1beta in cultured endothelial cells. Exposure of human and bovine endothelial cells to IL-1beta decreased eNOS expression. Western blot analysis using phospho-specific antibodies showed that IL-1beta stimulated p38 MAP kinase and phosphorylated ATF2. BPS inhibited these effects via protein kinase A (PKA)/cAMP-responsive element binding protein (CREB) activation. Transfection assays using site-specific mutation constructs showed that CRE/ATF elements located at -733 and -603 within the human eNOS promoter are necessary for full IL-1beta responsiveness. BPS attenuated the IL-1beta-mediated decrease in eNOS promoter activity and the expression of eNOS gene through PKA pathway. Electrophoretic gel mobility shift assays showed that IL-1beta increased the binding of phosphorylated ATF2 to CRE/ATF. On treatment with BPS, phosphorylated CREB predominantly bound to CRE/ATF. These results indicate that IL-1beta and BPS antagonistically regulates the eNOS expression through the activation of p38 and PKA, respectively. Furthermore, the ability to bind both CREB and ATF2 implicates the CRE/ATF sequence as a potential target for multiple signaling pathways in the regulation of the eNOS gene transcription.