Oxidative stress induces tyrosine phosphorylation of PDGF alpha-and beta-receptors and pp60c-src in mesangial cells.
Reactive oxygen species are autocrine and paracrine modulators of cell behavior. Hydrogen peroxide, a cellular oxidant, has been shown to stimulate mesangial cell proliferation. In the present study we analyzed the H2O2-induced early signaling events. Immunofluorescence analysis revealed a H2O2 induced dose-dependent increase in tyrosine phosphorylation. Short treatment (2 or 5 min) with 5 mM H2O2 induced a mitogenic response and a significant (P < 0.01) increase in the number of cells compared to non-treated controls. Proteins extracted from H2O2 (0.1 to 10 mM) treated cells were separated on SDS-PAGE and subjected to immunoblot analysis with anti-phosphotyrosine. A dose-dependent induction of tyrosine phosphorylation of 180 kDa, 120 kDa and 60 kDa proteins was observed within 1 to 10 minutes. By sequentially using immunoprecipitation and immunoblotting the 180 kDa tyrosine phosphorylated band was shown to represent both PDGF alpha- and beta-receptors. The tyrosine phosphorylated 60 kDa protein was identified as the cytoplasmic protein tyrosine kinase pp60c-src. The c-src phosphorylation was associated with an inhibition of c-src kinase activity, suggesting phosphorylation of tyrosine 527 in the c-src regulatory domain. Pretreatment with catalase completely abrogated the H2O2-induced PDGF receptor and c-src tyrosine phosphorylation. These data support the notion that the activation of a signaling pathway involving the PDGF receptors and c-src contributes to the mitogenic effects of reactive oxygen species.
González-Rubio
M
M
Medizinische Klinik IV, Universität Erlangen-Nümberg, Germany.
Voit
S
S
Rodríguez-Puyol
D
D
Weber
M
M
Marx
M
M
D000818:Animals; D002455:Cell Division / Q000187:drug effects; D005920:Glomerular Mesangium / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000494:pharmacology; D018384:Oxidative Stress; D010766:Phosphorylation; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D017479:Receptors, Platelet-Derived Growth Factor / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*
D014443:Tyrosine; D006861:Hydrogen Peroxide; D017479:Receptors, Platelet-Derived Growth Factor; D016392:Proto-Oncogene Proteins pp60(c-src)