Up-regulation of endothelial nitric-oxide synthase promoter by the phosphatidylinositol 3-kinase γ/Janus kinase 2/MEK-1-dependent pathway

Abstract

Our recent study indicates that lysophosphatidylcholine (LPC) enhances Sp1 binding and Sp1-dependent endothelial nitric oxide synthase (eNOS) promoter activity via the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 (MEK-1) signaling pathway (Cieslik, K., Lee, C.-M., Tang, J.-L., and Wu, K. K. (1999) J. Biol. Chem. 274, 34669-34675). To identify upstream signaling molecules, we transfected human endothelial cells with dominant negative and active mutants of Ras and evaluated their effects on eNOS promoter activity. Neither mutant altered the basal or LPC-induced eNOS promoter function. By contrast, a dominant negative mutant of phosphatidylinositol 3-ki-nase γ (PI-3Kγ) blocked the promoter activity induced by LPC. Wortmannin and LY 294002 had a similar effect. AG-490, a selective inhibitor of Janus kinase 2 (Jak2), also reduced the LPC-induced Sp1 binding and eNOS promoter activity to the basal level. LPC induced Jak2 phosphorylation, which was abolished by LY 294002 and the dominant negative mutant of PI-3Kγ. LY 294002 and AG-490 abrogated MEK-1 phosphorylation induced by LPC but had no effect on Raf-1. These results indicate that PI-3Kγ and Jak2 are essential for LPC-induced eNOS promoter activity. This signaling pathway was sensitive to pertussis toxin, suggesting the involvement of a Gi protein in PI-3Kγ activation. These results indicate that LPC enhances Sp1-dependent eNOS promoter activity by a pertussis toxin-sensitive, Ras-independent novel pathway, PI-3Kγ/Jak2/MEK-1/ERK1/2.