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.

Methods and Results— Transfection of bovine aortic endothelial cells with small interfering RNA (siRNA) targeting either PKC-α, δ, or ε caused a reduction in the cognate PKC protein by 76% to 89% without changing expression of nontargeted isoforms. Downregulation of PKC-ε 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-α knockdown increased Akt and eNOS phosphorylation, whereas PKCδ knockdown had no significant effect. PKC-ε 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-ε, but increased during PKC-α knockdown.

Conclusions— By regulating VEGFR2 expression and activation, PKC-ε expression is critical for activation of Akt and eNOS by VEGF and contributes to VEGF-stimulated Erk activation, whereas PKC-α has opposite effects.