c-Src Induces Phosphorylation and Translocation of p47phox: Role in Superoxide Generation by Angiotensin II in Human Vascular Smooth Muscle Cells

Abstract

Objectives— The aim of this study was to determine molecular mechanisms whereby c-Src regulates angiotensin II (Ang II)-mediated NAD(P)H oxidase-derived ·O 2 − in human vascular smooth muscle cells (VSMCs). Methods and Results— VSMCs from human small arteries were studied. Ang II increased NAD(P)H oxidase-mediated generation of ·O 2 − and H 2 O 2 ( P <0.01). PP2, c-Src inhibitor, attenuated these effects by 70% to 80%. Immunoprecipitation of p47phox, followed by immunoblotting with antiphosphoserine antibody, demonstrated a rapid increase (1.5- to 2-fold) in p47phox phosphorylation in Ang II-stimulated cells. This was associated with p47phox translocation from cytosol to membrane, as assessed by immunoblotting and immunofluorescence. PP2 abrogated these effects. Long-term Ang II stimulation (6 to 24 hours) increased NAD(P)H oxidase subunit expression. c-Src inhibition decreased abundance of gp91phox, p22phox, and p47phox. Confirmation of c-Src-dependent regulation of NAD(P)H oxidase was tested in VSMCs from c-Src −/− mice. Ang II-induced ·O 2 − generation was lower in c-Src −/− than c-Src +/+ counterparts. This was associated with decreased p47phox phosphorylation, blunted Ang II-stimulated NAD(P)H oxidase activation, and failure of Ang II to increase subunit expression. Conclusions— c-Src regulates NAD(P)H oxidase-derived ·O 2 − generation acutely by stimulating p47phox phosphorylation and translocation and chronically by increasing protein content of gp91phox, p22phox, and p47phox in Ang II-stimulated cells. These novel findings identify NAD(P)H oxidase subunits, particularly p47phox, as downstream targets of c-Src.