Phosphatidylinositol 3-kinase-dependent membrane recruitment of Rac-1 and p47phox is critical for α-platelet-derived growth factor receptor-induced production of reactive oxygen species

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 p47(phox)(-/-) 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 p110 beta) 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 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.