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 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-kinase gamma (PI-3Kgamma) 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-3Kgamma. LY 294002 and AG-490 abrogated MEK-1 phosphorylation induced by LPC but had no effect on Raf-1. These results indicate that PI-3Kgamma and Jak2 are essential for LPC-induced eNOS promoter activity. This signaling pathway was sensitive to pertussis toxin, suggesting the involvement of a G(i) protein in PI-3Kgamma activation. These results indicate that LPC enhances Sp1-dependent eNOS promoter activity by a pertussis toxin-sensitive, Ras-independent novel pathway, PI-3Kgamma/Jak2/MEK-1/ERK1/2.
Cieslik
K
K
Vascular Biology Research Center and Division of Hematology, University of Texas Medical School, Houston, Texas 77030, USA.
Abrams
C S
CS
Wu
K K
KK
D000906:Antibodies / Q000494:pharmacology; D002460:Cell Line; D002867:Chromones / Q000494:pharmacology; D058544:Class Ib Phosphatidylinositol 3-Kinase; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000502:physiology*; D011905:Genes, ras; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D053614:Janus Kinase 2; D008244:Lysophosphatidylcholines / Q000494:pharmacology; D048369:MAP Kinase Kinase 1; D020935:MAP Kinase Signaling System; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D009025:Morpholines / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000235:genetics*; D052250:Nitric Oxide Synthase Type III; D037342:Pertussis Toxin; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion; D016329:Sp1 Transcription Factor / Q000378:metabolism; D014162:Transfection; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D000906:Antibodies; D002867:Chromones; D058544:Class Ib Phosphatidylinositol 3-Kinase; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D053614:Janus Kinase 2; D008244:Lysophosphatidylcholines; D048369:MAP Kinase Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases; D009025:Morpholines; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D037342:Pertussis Toxin; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D016329:Sp1 Transcription Factor; D010566:Virulence Factors, Bordetella; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C496314:NOS3 protein, human; C546760:PIK3CG protein, human; C507924:JAK2 protein, human; C482125:MAP2K1 protein, human