Although
hypoxia is known to induce upregulation of endothelial
NO synthase (eNOS) gene expression, the underlying mechanism is largely unclear. In this study, we show that
hypoxia increases eNOS gene expression through the binding of phosphorylated cAMP-responsive
element binding (
CREB) protein (pCREB) to the eNOS gene promoter.
Hypoxia (1% O2) increased both eNOS expression and NO production, peaking at 24 hours, in bovine aortic endothelial cells, and these increases were accompanied by increases in pCREB. Treatment with the
protein kinase A inhibitor
H-89 or transfection with dominant-negative inhibitor of CREB reversed the
hypoxia-induced increases in eNOS expression and NO production, with concomitant inhibition of the phosphorylation of CREB induced by
hypoxia, suggesting an involvement of
protein kinase A/pCREB-mediated pathway. To map the regulatory elements of the eNOS gene responsible for pCREB binding under
hypoxia, we constructed an eNOS gene promoter (-1600 to +22
nucleotides) fused with a
luciferase reporter gene [pGL2-eNOS(-1600)].
Hypoxia (for 24-hour incubation) increased the promoter activity by 2.36+/-0.18-fold in the bovine aortic endothelial cells transfected with pGL2-eNOS(-1600). However, progressive 5'-deletion from -1600 to -873 completely attenuated the
hypoxia-induced increase in promoter activity. Electrophoretic mobility shift, anti-pCREB antibody supershift, and site-specific mutation analyses showed that pCREB is bound to the Tax-responsive
element (TRE) site, a cAMP-responsive
element-like site, located at -924 to -921 of the eNOS promoter. Our data demonstrate that the interaction between pCREB and the Tax-responsive
element site within the eNOS promoter may represent a novel mechanism for the mediation of
hypoxia-stimulated eNOS gene expression.