Competitive binding of CREB and ATF2 to cAMP/ATF responsive element regulates eNOS gene expression in endothelial cells.
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)-1beta in cultured endothelial cells.


RESULTS
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.


CONCLUSIONS
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.
Niwano
Kazuo
K
Department of Medicine and Biological Science, Gunma University Graduate School of Medicine, Japan.
Arai
Masashi
M
Koitabashi
Norimichi
N
Hara
Shiro
S
Watanabe
Atai
A
Sekiguchi
Kenichi
K
Tanaka
Toru
T
Iso
Tatsuya
T
Kurabayashi
Masahiko
M
D051698:Activating Transcription Factor 2 / Q000378:metabolism*; D000818:Animals; D001667:Binding, Competitive; D002417:Cattle; D002478:Cells, Cultured; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology; D042783:Endothelial Cells / Q000201:enzymology*; D011464:Epoprostenol / Q000031:analogs & derivatives / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology; D052250:Nitric Oxide Synthase Type III / Q000235:genetics*; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D020218:Response Elements / Q000502:physiology*; D048051:p38 Mitogen-Activated Protein Kinases / Q000502:physiology
D051698:Activating Transcription Factor 2; D017362:Cyclic AMP Response Element-Binding Protein; D007375:Interleukin-1; C048081:beraprost; D011464:Epoprostenol; D052250:Nitric Oxide Synthase Type III; D017868:Cyclic AMP-Dependent Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases