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

Anselm T Bäumer,Henrik Ten Freyhaus,Heinrich Sauer,Maria Wartenberg,Kai Kappert,Petra Schnabel,Christian Konkol,Jürgen Hescheler,Marius Vantler,Stephan Rosenkranz

doi:10.1074/jbc.m704997200

Anselm T Bäumer, Henrik Ten Freyhaus + Show 8 more

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https://doi.org/10.1074/jbc.m704997200

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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 alphaPDGF receptor (alphaPDGFR) induces ROS liberation. Stimulation of the alphaPDGFR 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. alphaPDGFR 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 PDGF-dependent Nox activation and ROS liberation. To evaluate the signaling pathway mediating PDGF-AA-dependent ROS production, we investigated Ph cells expressing mutant alphaPDGFRs that lack specific binding sites for alphaPDGFR-associated signaling molecules (Src, phosphatidylinositol 3-kinase (PI3K), phospholipase Cgamma, and SHP-2). Lack of PI3K signaling (but not Src, phospholipase Cgamma, or SHP-2) completely abolished PDGF-dependent p47(phox) and Rac-1 translocation, increase of Nox activity, and ROS production. Conversely, a mutant alphaPDGFR able to activate only PI3K was sufficient to mediate these subcellular events. Furthermore, the catalytic PI3K subunit p110alpha (but not p110beta) was identified as the crucial isoform that elicits alphaPDGFR-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/p110alpha 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.

Highlights

Several growth factors and cytokines induce the production of reactive oxygen species (ROS)3 in nonphagocytic cells such as endothelial cells, vascular smooth muscle cells (VSMC), and fibroblasts [1, 2]
The ␣PDGF receptor (PDGFR) Induces Intracellular ROS Production via NAD(P)H Oxidase—To investigate whether ␣PDGFR activation induces the liberation of ROS in fibroblasts, intracellular
Selective stimulation of the ␣PDGFR with platelet-derived growth factor (PDGF)-AA (50 ng/ml) in wild type (WT) ␣PDGFR expressing Ph cells led to a time-dependent increase of intracellular ROS, which was maximally 189 Ϯ 24% at 4 h (Fig. 1, A–C)

Summary

Introduction

Several growth factors and cytokines induce the production of reactive oxygen species (ROS) in nonphagocytic cells such as endothelial cells, vascular smooth muscle cells (VSMC), and fibroblasts [1, 2]. A similar enzyme with distinct characteristics has been described in a wide variety of nonphagocytic cells, e.g. the vascular system [6], thyroid gland [12], and cartilage [13] It generally consists of two membranous components, the flavocytochrome b protein Nox (nonphagocytes) or gp91phox (phagocytes) and p22phox, and the two cytosolic subunits. This study focuses on the signal relay mechanisms by which the ␣PDGFR mediates NAD(P)H oxidase activation and superoxide production in nonphagocytic cells. To systematically evaluate the role of each of the signaling enzymes that are recruited to the activated ␣PDGFR, we stably expressed tyrosine to phenylalanine ␣PDGFR mutants that fail to associate with one or more of the signaling enzymes (Src, PI3K, PLC␥, and SHP-2) in an ␣PDGFR-deficient cell line (Ph cells) [19] and compared their ability to mediate PDGF-dependent NAD(P)H oxidase activation and ROS production to that of the wild type (WT) ␣PDGFR. We sought to investigate the role of specific class IA PI3K isoforms and to evaluate the importance of ROS for PDGF-dependent cellular functions

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