Published online before print February 23, 2006, doi: 10.1161/01.ATV.0000215179.76144.39
© 2006 American Heart Association, Inc.
Vascular Biology |
Competitive Binding of CREB and ATF2 to cAMP/ATF Responsive Element Regulates eNOS Gene Expression in Endothelial Cells
From the Department of Medicine and Biological Science, Gunma University Graduate School of Medicine, Japan.
Correspondence to Masahiko Kurabayashi, MD, PhD, 3-39-15 Showa-machi, Maebashi, Gunma, 371-8511, Japan. E-mail mkuraba{at}med.gunma-u.ac.jp
Objective— 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)-1ß in cultured endothelial cells.
Method and Results— Exposure of human and bovine endothelial cells to IL-1ß decreased eNOS expression. Western blot analysis using phospho-specific antibodies showed that IL-1ß 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-1ß responsiveness. BPS attenuated the IL-1ß–mediated decrease in eNOS promoter activity and the expression of eNOS gene through PKA pathway. Electrophoretic gel mobility shift assays showed that IL-1ß increased the binding of phosphorylated ATF2 to CRE/ATF. On treatment with BPS, phosphorylated CREB predominantly bound to CRE/ATF.
Conclusions— These results indicate that IL-1ß 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.
The expression level of the eNOS gene is a critical determinant of endothelial integrity. Beraprost sodium (BPS) inhibited the downregulation of eNOS gene expression by IL-1ß through competitive binding of ATF2 and CREB to ATF/CREB sequences. The data indicate a potential role of ATF/CREB family of transcription factors in atherosclerosis.
Key Words: cAMP • cytokine • endothelium • prostacyclin • transcription factor
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