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Platelet-derived growth factor activates production of reactive oxygen species by NAD(P)H oxidase in smooth muscle cells through G_i1,2¹

J. KREUZER, C. VIEDT, R. P. BRANDES, F. SEEGER, A. S. ROSENKRANZ, H. SAUER^||, A. BABICH, B. NÜRNBERG, H. KATHER^* and H. I. KRIEGER-BRAUER^*2

Innere Medizin III,
^* Innere Medizin I, Universität Heidelberg, Heidelberg, Germany;
Institut für Kardiovaskuläre Physiologie, Universität Frankfurt, Frankfurt, Germany;
Abteilung Pharmakologie und Toxikologie, Universität Ulm, Ulm, Germany;
Klinik III für Innere Medizin, Universität zu Köln;
^|| Institut für Neurophysiologie, Universität zu Köln, Köln, Germany; and
Instituts für Physiologische Chemie II, Heinrich-Heine-Universität, Düsseldorf, Germany

²Correspondence: Innere Medizin I, Universität Heidelberg, Bergheimer Str. 58, D-69115 Heidelberg, Germany. E-mail: joerg_kreuzer{at}med.uni-heidelberg.de

SPECIFIC AIMS

Induction of reactive oxygen species in smooth muscle cells (SMC) by platelet-derived growth factor (PDGF) has been proposed to contribute to lesion progression in arteriosclerosis. The aim of the study was to identify the source and signal transduction pathway of PDGF-dependent reactive oxygen species (ROS) formation in SMC.

PRINCIPAL FINDINGS

1. PDGF-induced ROS release depends on G-proteins
Assessment of ROS production demonstrated that PDGF AA caused a marked dose-dependent threefold increase in ROS levels in the presence but not in the absence of 10 µM GTPS. PDGF AA-induced ROS generation was mediated through the PDGF receptor (R).

2. The p22phox NAD(P)H oxidase subunit is essential for ROS production by PDGF
Preincubation of plasma membranes with diphenyleneiodonium, an inhibitor of flavoproteins, completely prevented PDGF AA-stimulated ROS generation. Accordingly, a p22phox antibody blocked PDGF AA-stimulated ROS generation back to basal levels. These data identify p22phox and/or a spatially proximate molecule as a critical component of the membrane-bound ROS generating system stimulated by PDGF.

3. Gi2 couples to the PDGF receptor and is crucial for ROS release
The functional importance of G-proteins in up-regulation of NADPH oxidase is supported further by the effects of isolated G-proteins. Only Gi2 induced ROS production in SMC plasma membranes whereas Gi3, G0, Gs and Gß subunits did not. GDPßS, which irreversibly inactivates G-protein-coupled events, did not yield in any PDGF AA-dependent ROS formation. Pretreatment of membranes with pertussis toxin (PTX) had no effect on basal ROS generation but inhibited PDGF AA-stimulated ROS production. ROS production could be reconstituted after PTX treatment by addition of Gi2-GTPS but not by Gi2-GDPßS.

A functional interaction between the PDGF R and Gi proteins was examined using PTX-mediated ADP ribosylation in the presence of PDGF AA (Fig. 1 a). A dose-dependent reduction in the PTX-mediated ADP ribosylation of Gi was seen when SMC membranes were exposed to PDGF AA in the presence of GTP.

View larger version (33K): [in this window] [in a new window]   Figure 1. a) Effect of PDGF AA on PTX-mediated ADP ribosylation of Gi. Plasma membranes were treated with PDGF AA and PTX A protomer (2 µg/mL) and subjected to SDS-PAGE, followed by autoradiography and immunoblotting. Left: representative autoradiograph of pertussis toxin catalyzed ADP ribosylation in the absence and presence of PDGF AA (10 ng/mL) together with the corresponding immunoblot. Right: densitometric analysis of PTX labeling of Gi and the corresponding Gi1,2 immunoblot. Data are mean ± SD, n = 5. b) PDGF AA-stimulated association of Gi1,2 protein with the PDGF receptor. Plasma membranes were incubated with PDGF AA in the presence or absence of GTPS. Pretreatment with PTX A was carried out for ROS generation. Membranes were subjected to an anti-PDGF R immunoaffinity column. Proteins were eluted and subjected to SDS-PAGE and immunoblotting with either anti-PDGF R or anti-Gi1,2. Shown is densitometric quantitation of the bands that were normalized to the amount of PDGF R eluted from the column. Results are mean ± SD, n = 3. *P < 0.05 compared to control, ¶P < 0.05 compared to PDGF 100 ng/mL in the absence of PTX. c) PDGF AA-stimulated association of Gi1,2 protein with the PDGF type R. Plasma membranes were incubated with PDGF AA (5 ng/mL) in the presence or absence of GTPS (10 µM) and subjected to an anti-PDGF R immunoaffinity column. After washing, proteins were eluted and subjected to SDS-PAGE and immunoblotting with either PDGF receptor (PDGF R), Gi1,2, or Gß antibody. Left: representative immunoblot of immunoaffinity eluates. Right: densitometric quantitation of the bands that were normalized to the amount of PDGF R. Results are mean ± SD, n = 3. *P < 0.05.

The ability of PDGF AA to induce association of Gi1,2 with the R could be demonstrated by immunoadsorption of the receptor-Gi1,2 complex to an R agarose antibody affinity resin. A dose-dependent stimulation of Gi in association with the PDGF receptor was seen with different concentrations of PDGF AA in the absence of GTPS (Fig. 1b ). The association of Gi1,2 was reduced by pretreatment of the membranes with PDGF AA and GTPS, but the dissociation of the Gß subunit was not affected by GTPS (Fig. 1c ).

CONCLUSION AND SIGNIFICANCE

In the present study, strong evidence is provided that the PDGF R in SMC is coupled through Gi1,2 to membrane-bound NAD(P)H oxidase. The PDGF R can form a complex with Gi1,2 on binding PDGF AA and the Gi1,2 protein appears to be an important component in activating NAD(P)H oxidase.

Previous studies have shown that agonist binding to the receptor in the absence of guanine nucleotides results in the increase of G-protein association, whereas guanine nucleotides induced dissociation of the ternary complex. Our data on the modulation of Gi binding to the R after ligand interaction fully fit these observations. By analogy with hepta-helical receptors, PDGF AA promoted formation of a PDGF R–G-protein ternary complex and addition of GTPS caused a dissociation of the Gi subunit from the receptor. Our findings expand previous data describing a role of G-proteins for intracellular signaling via tyrosine kinase receptors such as IGF I, EGF, FGF, insulin, and PDGF.

In the past, only a few studies investigated the role of PDGF for NAD(P)H oxidase-dependent ROS release in nonphagocytic cells but failed to identify p22phox as an essential component for PDGF AA-mediated ROS release.

Recent work showed that ROS release through ligands such as fMLP requires G-protein activation and inhibition of NAD(P)H oxidase by Gß. In light of our present data, it is conceivable that activation of G-proteins is the common pathway leading to regulation of NAD(P)H oxidase.

View larger version (22K): [in this window] [in a new window]   Figure 2. Schematic. Stimulation of the PDGF receptor leads to dissociation of Gi1,2, which in turn activates NAD(P)H oxidase leading to release of reactive oxygen species. The NAD(P)H oxidase subunit p22phox is crucial for the activation of ROS release by PDGF AA.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-1036fje; to cite this article, use FASEB J. (November 1, 2002) 10.1096/fj.01-1036fje

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