Protein kinase G mediates vascular endothelial growth factor-induced Raf-1 activation and proliferation in human endothelial cells.
Vascular endothelial growth factor (VEGF) is an endothelium-specific, secreted protein that acts as a vasodilator, angiogenic peptide, and hyperpermeability factor. Recent reports have shown that nitric oxide synthase inhibitors block proliferation and microvascular hyperpermeability induced by VEGF. This study examined the mechanisms by which nitric oxide and its downstream signals mediate the VEGF-induced proliferative response in human umbilical vein endothelial cells (HUVECs). Nitric oxide synthase blockade by Nomega-nitro-L-arginine methyl ester prevented both the proliferative effect of VEGF and Raf-1 activation by VEGF as measured by cell counting and the capacity of immunoprecipitated Raf-1 to phosphorylate syntide 2, a Raf-1-specific synthetic substrate. VEGF-induced proliferation and Raf-1 kinase activity were also inhibited by Rp-8-pCPT-cGMPs and KT5823, inhibitors of the regulatory and catalytic subunits of cGMP-dependent protein kinase (PKG), respectively. The ability of PKG to stimulate proliferation was verified by the observation that the PKG activator, 8-pCPT-cGMPs, stimulated both Raf-1 kinase activity and endothelial proliferation in a dose-dependent manner. Furthermore, recombinant catalytically active PKG phosphorylated and activated Raf-1 in a reconstituted system. Finally, Raf-1 immunoprecipitated from VEGF-stimulated endothelial cells coprecipitated with PKG, indicating a direct protein-protein interaction in activated cells. We conclude that VEGF induces increases in both proliferation and Raf-1 kinase activity in HUVECs and these activities are dependent on NO and its downstream effector, PKG.
Microcirculation Research Institute and Department of Medical Physiology, Texas A&M University System Health Science Center, College Station, Texas 77843-1114, USA.
J
J
Hood
H J
HJ
Granger
D000470:Alkaloids / Q000494:pharmacology; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D002227:Carbazoles; D002455:Cell Division; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000031:analogs & derivatives; D016228:Endothelial Growth Factors / Q000494:pharmacology*; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D004789:Enzyme Activation; D006801:Humans; D007211:Indoles; D036341:Intercellular Signaling Peptides and Proteins; D008222:Lymphokines / Q000494:pharmacology*; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D015398:Signal Transduction; D014471:Umbilical Veins / Q000166:cytology; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D000470:Alkaloids; D002227:Carbazoles; D016228:Endothelial Growth Factors; D007211:Indoles; D036341:Intercellular Signaling Peptides and Proteins; D008222:Lymphokines; D010455:Peptides; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; C063391:syntide-2; C073601:KT 5823; D019001:Nitric Oxide Synthase; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D006152:Cyclic GMP; D019331:NG-Nitroarginine Methyl Ester