Vascular Biology |
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-1ßmediated 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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