Phosphatidylinositol 3-Kinase-dependent Membrane Recruitment of Rac-1 and p47phox Is Critical for {alpha}-Platelet-derived Growth Factor Receptor-induced Production of Reactive Oxygen Species

Abstract

Platelet-derived growth factor (PDGF) plays a critical role in the pathogenesis of proliferative diseases. NAD(P)H oxidase (Nox)-derived reactive oxygen species (ROS) are essential for signal transduction by growth factor receptors. Here we investigated the dependence of PDGF-AA-induced ROS production on the cytosolic Nox subunits Rac-1 and p47phox, and we systematically evaluated the signal relay mechanisms by which the {alpha}PDGF receptor ({alpha}PDGFR) induces ROS liberation. Stimulation of the {alpha}PDGFR led to a time-dependent increase of intracellular ROS levels in fibroblasts. Pharmacological inhibitor experiments and enzyme activity assays disclosed Nox as the source of ROS. {alpha}PDGFR activation is rapidly followed by the translocation of p47phox and Rac-1 from the cytosol to the cell membrane. Experiments performed in p47phox(-/-) cells and inhibition of Rac-1 or overexpression of dominant-negative Rac revealed that these Nox subunits are required for PDGF-dependent Nox activation and ROS liberation. To evaluate the signaling pathway mediating PDGF-AA-dependent ROS production, we investigated Ph cells expressing mutant {alpha}PDGFRs that lack specific binding sites for {alpha}PDGFR-associated signaling molecules (Src, phosphatidylinositol 3-kinase (PI3K), phospholipase C{gamma}, and SHP-2). Lack of PI3K signaling (but not Src, phospholipase C{gamma}, or SHP-2) completely abolished PDGF-dependent p47phox and Rac-1 translocation, increase of Nox activity, and ROS production. Conversely, a mutant {alpha}PDGFR able to activate only PI3K was sufficient to mediate these subcellular events. Furthermore, the catalytic PI3K subunit p110{alpha} (but not p110β) was identified as the crucial isoform that elicits {alpha}PDGFR-mediated production of ROS. Finally, bromodeoxyuridine incorporation and chemotaxis assays revealed that the lack of ROS liberation blunted PDGF-AA-dependent chemotaxis but not cell cycle progression. We conclude that PI3K/p110{alpha} mediates growth factor-dependent ROS production by recruiting p47phox and Rac-1 to the cell membrane, thereby assembling the active Nox complex. ROS are required for PDGF-AA-dependent chemotaxis but not proliferation.