Differential Regulation of VEGF Signaling by PKC-α and PKC-ε in Endothelial Cells

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Abstract
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 exp... Downregulation of PKCε with siRNA decreased Akt, eNOS, and Erk1/2 phosphorylation, NO synthase activity, and DNA synthesis stimulated by VEGF, likely through a dramatic decrease in VEGF receptor-2 (VEGFR2) tyrosine phosphorylation, and VEGFR2 protein and mRNA expression. In contrast, PKC-α knockdown increased VEGFR2 phosphorylation, VEGFR2 expression, and downstream signaling.

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