Differential regulation of VEGF signaling by PKC-alpha and PKC-epsilon in endothelial cells.
OBJECTIVE
Vascular endothelial growth factor (VEGF) stimulates proangiogenic signal transduction and cell function in part through activation of protein kinase C (PKC). Our aim was to examine how individual isoforms of PKC affect VEGF action.


RESULTS
Transfection of bovine aortic endothelial cells with small interfering RNA (siRNA) targeting either PKC-alpha, delta, or epsilon caused a reduction in the cognate PKC protein by 76% to 89% without changing expression of nontargeted isoforms. Downregulation of PKC-epsilon abrogated VEGF-stimulated phosphorylation of Akt at Ser473 and eNOS at Ser1179 and decreased VEGF-stimulated NO synthase activity in intact cells. In contrast, PKC-alpha knockdown increased Akt and eNOS phosphorylation, whereas PKCdelta knockdown had no significant effect. PKC-epsilon knockdown also decreased VEGF-stimulated Erk1/2 phosphorylation and abolished VEGF-stimulated DNA synthesis. Consistent with an effect on several pathways of VEGF signaling, VEGF receptor-2 (VEGFR2) tyrosine phosphorylation and expression of VEGFR2 protein and mRNA was decreased by 81, 90, and 84%, respectively, during knockdown of PKC-epsilon, but increased during PKC-alpha knockdown.


CONCLUSIONS
By regulating VEGFR2 expression and activation, PKC-epsilon expression is critical for activation of Akt and eNOS by VEGF and contributes to VEGF-stimulated Erk activation, whereas PKC-alpha has opposite effects.
Rask-Madsen
Christian
C
Research Division, Joslin Diabetes Center, Harvard Medical School, Boston, Mass. 02215, USA.
King
George L
GL
D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D051571:Protein Kinase C-alpha / Q000378:metabolism*; D051745:Protein Kinase C-delta / Q000187:drug effects / Q000378:metabolism; D051744:Protein Kinase C-epsilon / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism
D034741:RNA, Small Interfering; D042461:Vascular Endothelial Growth Factor A; D052250:Nitric Oxide Synthase Type III; D040301:Vascular Endothelial Growth Factor Receptor-2; D051057:Proto-Oncogene Proteins c-akt; D051571:Protein Kinase C-alpha; D051745:Protein Kinase C-delta; D051744:Protein Kinase C-epsilon; D048049:Extracellular Signal-Regulated MAP Kinases