Oxidative stress induces tyrosine phosphorylation of PDGF alpha-and beta-receptors and pp60c-src in mesangial cells

Kidney Int. 1996 Jul;50(1):164-73. doi: 10.1038/ki.1996.299.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Division / drug effects
  • Glomerular Mesangium / metabolism*
  • Hydrogen Peroxide / pharmacology
  • Oxidative Stress*
  • Phosphorylation
  • Proto-Oncogene Proteins pp60(c-src) / antagonists & inhibitors
  • Proto-Oncogene Proteins pp60(c-src) / metabolism*
  • Rats
  • Receptors, Platelet-Derived Growth Factor / metabolism*
  • Tyrosine / metabolism*

Substances

  • Tyrosine
  • Hydrogen Peroxide
  • Receptors, Platelet-Derived Growth Factor
  • Proto-Oncogene Proteins pp60(c-src)