Transcriptional Stimulation of the eNOS Gene by the Stable Prostacyclin Analogue Beraprost Is Mediated Through cAMP-Responsive Element in Vascular Endothelial Cells: Close Link Between PGI2 Signal and NO Pathways

ABSTRACT—Beraprost sodium (BPS), an orally active prostacyclin analogue, has been reported to be beneficial in the treatment of primary pulmonary hypertension and obstructive peripheral arterial disease. Although BPS was originally described for its effects on platelet aggregation and vasodilatory response, the effect on endothelial cells has been poorly understood. In this study, we examined the effects of BPS on the eNOS gene expression in mouse aorta and cultured human and bovine aortic endothelial cells. Treatment of these cells with BPS increased the eNOS expression as assessed by Northern blots, Western blots, and NO production by NO-specific fluorescence (DAF2-DA) and by the Griess method. Standard mRNA decay assays showed that BPS increases the stability of eNOS mRNA. In addition, BPS increased the promoter activity of the human eNOS gene, as determined by luciferase assays of the eNOS promoter gene. Progressive 5′-deletion and site-specific mutation analyses defined the BPS-responsive sequences as cAMP-responsive elements (CRE) located at −733 and −603. By using the oligonucleotide probe containing this CRE sequence in electrophoretic mobility shift assays, we showed that the phosphorylated form of CRE-binding protein is a major constituent of the complex in BPS-treated cells. Western blot analyses indicate that BPS but not endogenous prostacyclin phosphorylates CRE-binding protein. The presence of functional CRE sites within human eNOS promoter may represent a novel mechanism for regulating eNOS gene expression.