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

Our recent study indicates that lysophosphatidylcholine (LPC) enhances Spl binding and Spl-dependent endothelial nitric oxide synthase (eNOS) promoter activity via the mitogen-activated protein kinase/extracellular signal-reg ulated kinase kinase 1 (MEK-1) signaling pathway (Cieslik, K,, Lee, C.-M, T ang, J,-L., and Wu, K, K, (1999) J, Biol, Chem. 274, 84669-94675). To ident ify upstream signaling molecules, we transfected human endothelial cells wi th dominant negative and active mutants of Bas 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 phosphat idylinositol 3-kinase gamma (PI-3K gamma) blocked the promoter activity ind uced by LPC, Wortmannin and LY 294002 had a similar effect. AG-490, a selec tive inhibitor of Janus kinase 2 (Jak2), also reduced the LPC-induced Spl b inding and eNOS promoter activity to the basal level. LPC induced Jak2 phos phorylation, which was abolished by LY 294002 and the dominant negative mut ant of PI-3K gamma. LY 294002 and AG-490 abrogated MEK-1 phosphorylation in duced by LPC but had no effect on Raf-l. These results indicate that PI-SKy and Jak2 are essential for LPC-induced eNOS promoter activity. This signal ing pathway was sensitive to pertussis toxin, suggesting the involvement of a Gi protein in PI-SK gamma activation. These results indicate that LPC en hances Sp1-dependent eNOS promoter activity by a pertussis toxin-sensitive, Res-independent novel pathway, PI-3K gamma /Jak2/MEK-1/ERK1/2.