VEGF regulation of endothelial nitric oxide synthase in glomerular endothelial cells.
BACKGROUND
Vascular endothelial growth factor (VEGF) regulation of endothelial nitric oxide synthase (eNOS) and signaling pathways involved have not been well studied in glomerular endothelial cells (GENCs).


METHODS
GENCs grown from tsA58 Immortomice were used. Immunoblotting and in-cell Western blot analysis were employed to assess changes in VEGF receptor signaling pathway and eNOS phosphorylation of ser1177. Immunokinase assay and immunoblotting with phosphospecific antibodies were performed to assess activity of kinases.


RESULTS
VEGF rapidly induced tyrosine phosphorylation of type 1 and type 2 VEGF receptors. Physical association between VEGF-receptor 2 (VEGF-R2) and insulin receptor substrate (IRS-1) and phosphatidylinositol 3'-kinase (PI3K) was induced by VEGF, which augmented PI3K activity in VEGF-R2 immunoprecipitates. VEGF stimulated Akt phosphorylation in a PI3K-dependent manner. VEGF increased eNOS phosphorylation on Ser1177. Activation of eNOS was associated with nitric oxide generation as measured by medium nitrite content. Signaling mechanisms involved in VEGF stimulation of eNOS were explored. VEGF-induced eNOS phosphorylation was abolished by SU1498, a VEGF-R2 inhibitor, LY294002, a PI3K inhibitor, and infection of cells with an adenovirus carrying a dominant negative-mutant of Akt, demonstrating the requirement of the VEGF-R2/IRS-1/PI3K/Akt axis for activation of eNOS. VEGF also activated extracellular signal-regulated protein kinase (ERK) in a time-dependent manner; and VEGF-stimulated eNOS phosphorylation on Ser1177 was prevented by PD098059, an upstream inhibitor of ERK, demonstrating that ERK was involved in VEGF regulation of eNOS. ERK phosphorylation was abolished by LY294002, suggesting ERK was downstream of PI3K in VEGF-treated GENC.


CONCLUSIONS
Our data demonstrate that in GENC, VEGF stimulates VEGF-R2/IRS-1/PI3K/Akt axis to regulate eNOS phosphorylation on Ser1177 in conjunction with the ERK signaling pathway.
Feliers
Denis
D
South Texas Veterans Healthcare System and O'Brien Kidney Research Center, University of Texas Health Science Center, San Antonio, Texas 78229, USA.
Chen
Xiaoyan
X
Akis
Nese
N
Choudhury
Goutam Ghosh
GG
Madaio
Michael
M
Kasinath
Balakuntalam S
BS
D000818:Animals; D002461:Cell Line, Transformed; D055504:Insulin Receptor Substrate Proteins; D007678:Kidney Glomerulus / Q000166:cytology / Q000201:enzymology*; D020935:MAP Kinase Signaling System / Q000502:physiology*; D051379:Mice; D008822:Mice, Transgenic; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*; D040281:Vascular Endothelial Growth Factor Receptor-1 / Q000378:metabolism; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism
D055504:Insulin Receptor Substrate Proteins; C526295:Irs1 protein, mouse; D010750:Phosphoproteins; D042461:Vascular Endothelial Growth Factor A; C467480:vascular endothelial growth factor A, mouse; D014443:Tyrosine; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; C496315:Nos3 protein, mouse; D019869:Phosphatidylinositol 3-Kinases; D040281:Vascular Endothelial Growth Factor Receptor-1; D040301:Vascular Endothelial Growth Factor Receptor-2; D051057:Proto-Oncogene Proteins c-akt