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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2006;26:1036.)
© 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

Kazuo Niwano; Masashi Arai; Norimichi Koitabashi; Shiro Hara; Atai Watanabe; Kenichi Sekiguchi; Toru Tanaka; Tatsuya Iso; Masahiko Kurabayashi

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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