Vascular endothelial growth factor governs endothelial nitric-oxide synthase expression via a KDR/Flk-1 receptor and a protein kinase C signaling pathway.
The mechanism by which vascular endothelial growth factor (VEGF) regulates endothelial nitric-oxide synthase (eNOS) expression is presently unclear. Here we report that VEGF treatment of bovine adrenal cortex endothelial cells resulted in a 5-fold increase in both eNOS protein and activity. Endothelial NOS expression was maximal following 2 days of constant VEGF exposure (500 pM) and declined to base-line levels by day 5. The elevated eNOS protein level was sustained over the time course if VEGF was co-incubated with L-N(G)-nitroarginine methyl ester, a competitive eNOS inhibitor. Addition of S-nitroso-N-acetylpenicillamine, a nitric oxide donor, prevented VEGF-induced eNOS up-regulation. These data suggest that nitric oxide participates in a negative feedback mechanism regulating eNOS expression. Various approaches were used to investigate the role of the two high affinity VEGF receptors in eNOS up-regulation. A KDR receptor-selective mutant increased eNOS expression, whereas an Flt-1 receptor-selective mutant did not. Furthermore, VEGF treatment increased eNOS expression in a KDR but not in an Flt-1 receptor-transfected porcine aorta endothelial cell line. SU1498, a selective inhibitor of the KDR receptor tyrosine kinase, blocked eNOS up-regulation, thus providing further evidence that the KDR receptor signals for eNOS up-regulation. Finally, treatment of adrenal cortex endothelial cells with VEGF or phorbol ester resulted in protein kinase C activation and elevated eNOS expression, whereas inhibition of protein kinase C with isoform-specific inhibitors abolished VEGF-induced eNOS up-regulation. Taken together, these data demonstrate that VEGF increases eNOS expression via activation of the KDR receptor tyrosine kinase and a downstream protein kinase C signaling pathway.
Shen
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Department of Pharmacokinetics, Genentech, Inc., South San Francisco, California 94080, USA.
Lee
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Zioncheck
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D000302:Adrenal Cortex; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D016228:Endothelial Growth Factors / Q000494:pharmacology*; D004730:Endothelium, Vascular / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D006133:Growth Substances / Q000494:pharmacology; D008222:Lymphokines / Q000494:pharmacology*; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D010396:Penicillamine / Q000031:analogs & derivatives / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D017978:Receptors, Growth Factor / Q000378:metabolism*; D040262:Receptors, Vascular Endothelial Growth Factor; D011994:Recombinant Proteins; D015398:Signal Transduction; D015854:Up-Regulation / Q000187:drug effects; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D016228:Endothelial Growth Factors; D004791:Enzyme Inhibitors; D006133:Growth Substances; D008222:Lymphokines; D017978:Receptors, Growth Factor; D011994:Recombinant Proteins; C110881:S-nitro-N-acetylpenicillamine; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D020794:Receptor Protein-Tyrosine Kinases; D040262:Receptors, Vascular Endothelial Growth Factor; D011493:Protein Kinase C; D010396:Penicillamine; D019331:NG-Nitroarginine Methyl Ester