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

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 p47 phox , and we systematically evaluated the signal relay mechanisms by which the ␣PDGF receptor (␣PDGFR) induces ROS liberation. Stimulation of the ␣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. ␣PDGFR activation is rapidly followed by the translocation of p47 phox 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 PDGFdependent Nox activation and ROS liberation. To evaluate the signaling pathway mediating PDGF-AA-dependent ROS production, we investigated Ph cells expressing mutant ␣PDGFRs that lack specific binding sites for ␣PDGFR-associated signaling molecules (Src, phosphatidylinositol 3-kinase (PI3K), phospholipase C␥, and SHP-2). Lack of PI3K signaling (but not Src, phospholipase C␥, or SHP-2) completely abolished PDGF-dependent p47 phox and Rac-1 translocation, increase of Nox activity, and ROS production. Conversely, a mutant ␣PDGFR able to activate only PI3K was sufficient to mediate these subcellular events. Furthermore, the catalytic PI3K subunit p110␣ (but not p110␤) was identified as the crucial isoform that elicits ␣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␣ mediates growth factor-dependent ROS production by recruiting p47 phox and Rac-1 to the cell membrane, thereby assembling the active Nox complex. ROS are required for PDGF-AA-dependent chemotaxis but not proliferation.

Selective Enhancement of Insulin Sensitivity in the Endothelium In Vivo Reveals a Novel Proatherosclerotic Signaling Loop

“…27 Here, we demonstrate that endothelial cell over-expression of IR increases basal and insulin stimulated activation of Akt, which increases the activity and expression of the superoxide generating enzyme Nox2 NADPH oxidase. While a link between PI3-K/Akt and NADPH oxidase has been proposed, 38,39 this is the first study to unequivocally demonstrate a direct pathway between enhanced insulin sensitivity, PI3-K, Akt activation and Nox2 NADPH oxidase activity. During normal physiology, the NADPH oxidases generate low levels of reactive oxygen species in a highly regulated fashion for use in redox-dependent signalling.…”

Rac1 Protein Regulates Glycogen Phosphorylase Activation and Controls Interleukin (IL)-2-dependent T Cell Proliferation

“…Gene expression levels were normalized with a housekeeping gene (YWHAZ) whose expression remains unchanged in Kit 225 cells regardless of the activation status. 5 Quantitative reverse transcription-PCR analysis showed that Kit 225 cells express PYGM in addition to PYGL. In fact, the expression of PYGM is substantially higher than that of PYGL.…”

“…Signals emanating from a large variety of membrane receptors positively regulate Rho GTPase activities. Examples include receptors with intrinsic tyrosine kinase activity such as EGF receptor (2)(3)(4) and PDGF receptor (5,6) or other types such as G protein-coupled receptors (7,8). In their active configuration, GTPases interact with downstream effector molecules to promote a variety of biological responses (9).…”

Intracellular reactive oxygen species are essential for PI3K/Akt/mTOR-dependent IL-7-mediated viability of T-cell acute lymphoblastic leukemia cells

“…35 Moreover, receptor tyrosine kinasemediated activation of PI3K results in the recruitment of p47 phox to the plasma membrane and consequent production of reactive oxygen species. 36 Mitochondrial respiration is the major cellular source of ROS. Under normal conditions, it is estimated that approximately 1-2% of the electrons leak out of mitochondrial electron transport chain, forming ROS, 37 and increased ROS levels associated with BCR-ABL transformation were linked to mitochondrial respiration.…”