Protein Kinase C-Mediated Phosphorylation of p47^phox modulates platelet-derived growth factor-induced H₂ O₂ generation and cell proliferation in human umbilical vein endothelial cells

Substantial evidence indicate that growth factors such as platelet-derived growth factor (PDGF) exert their effect, at least in part, through reactive oxygen species (ROS) generated via NAD(P)H oxidase. In this work, the role of p47^phox, a key component of the phagocytic NAD(P)H oxidase in cell proliferation, was addressed. The authors show that diphenylene iodonium (DPI) and apocynin, but not N^G-nitro-L-arginine methyl esterL-NAME, reduced PDGF-induced ROS generation and proliferation in human umbilical vein endothelial cells (HUVECs). Pharmacological inhibition of protein kinase C (PKC) as well as dominant-negative mutants of p47^phox directed to PKC-dependent phosphorylation targets inhibited PDGF-stimulated ROS production and cell proliferation. Hydrogen peroxide restored PDGF-stimulated proliferation in cells that was inhibited by apocynin, DPI, or by the dominant-negative mutants. PDGF-induced proliferation was reduced in the HUVEC-derived cell line E.A.hy926 overexpressing catalase. On the contrary, cells overexpressing superoxide dismutase 1 exhibited increased proliferation. These results demonstrate that PKC-dependent phosphorylation of p47^phox is essential for PDGF-stimulated ROS generation and proliferation in HUVECs. More relevant, H₂O₂ is identified as the key molecule that signals proliferation in the systems studied.