on February 23, 2006
Published online before print February 23, 2006, doi: 10.1161/01.ATV.0000215179.76144.39
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Submitted on September 11, 2005
Accepted on February 8, 2006
Competitive Binding of CREB and ATF2 to cAMP/ATF Responsive Element Regulates eNOS Gene Expression in Endothelial Cells
Kazuo Niwano ;
From the Department of Medicine and Biological Science, Gunma University Graduate School of Medicine, Japan.
* To whom correspondence should be addressed. 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)-1
in cultured endothelial cells.
Method and Results--Exposure of human and bovine endothelial cells to IL-1 decreased eNOS expression. Western blot analysis using phospho-specific antibodies showed that IL-1 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-1 responsiveness. BPS attenuated the IL-1-mediated decrease in eNOS promoter activity and the expression of eNOS gene through PKA pathway. Electrophoretic gel mobility shift assays showed that IL-1 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-1 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.
Key words: cAMP • cytokine • endothelium • prostacyclin • transcription factor
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