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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2008;28:919.)
© 2008 American Heart Association, Inc.

Cell Biology and Signaling

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

Christian Rask-Madsen; George L. King

From the Research Division, Joslin Diabetes Center, Harvard Medical School, Boston, Mass.

Correspondence to George L. King, MD, Joslin Diabetes Center, Harvard Medical School, One Joslin Place, Room 4504, Boston, MA 02215. E-mail george.king{at}joslin.harvard.edu

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-, , or caused a reduction in the cognate PKC protein by 76% to 89% without changing expression of nontargeted isoforms. Downregulation of PKC- 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- knockdown increased Akt and eNOS phosphorylation, whereas PKC knockdown had no significant effect. PKC- 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-, but increased during PKC- knockdown.

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

Downregulation of PKC 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- knockdown increased VEGFR2 phosphorylation, VEGFR2 expression, and downstream signaling.

Key Words: endothelium vascular • vascular endothelial growth factor A • receptors vascular growth factor • nitric oxide synthase type III • protein kinase C

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