The adult kidney is derived from the interaction between the metanephric blastema and the ureteric bud. Platelet-derived growth factor (PDGF) receptor β is essential for the development of the mature glomerular tuft, as mice deficient for this receptor lack mesangial cells. This study investigated the role of Src tyrosine kinase in PDGF-mediated reactive oxygen species (ROS) generation and migration of metanephric mesenchymal cells (MMCs). Cultured embryonic MMCs from wildtype and PDGF receptor-deficient embryos were established. Migration was determined via wound-healing assay. Unlike PDGF AA, PDGF BB-induced greater migration in MMCs with respect to control. This was abrogated by neutralizing an antibody to PDGF BB. Phosphatidylinositol 3-kinase (PI3K) inhibitors suppressed PDGF BB-induced migration. Conversely, mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) inhibitors had no effect. Src inhibitors inhibited PDGF-induced cell migration, PI3K activity, and Akt phosphorylation. Adenoviral dominant negative Src (AD DN Src) abrogated PDGF BB-induced Akt phosphorylation. Hydrogen peroxide stimulated cell migration. PDGF BB-induced wound closure was inhibited by the antioxidants N-acetyl-L-cysteine, tiron, and the flavoprotein inhibitor diphenyleneiodonium. These cells express the NADPH oxidase homolog Nox4. Inhibiting Nox4 with antisense oligonucleotides or small interfering RNA (siRNA) suppressed PDGF-induced wound closure. Inhibition of Src with siRNA reduced PDGF BB-induced ROS generation as assessed by 2',7'-dichlorodihydrofluorescein diacetate fluorescence. Furthermore, PDGF BB-stimulated ROS generation and migration were similarly suppressed by Ad DN Src. In MMCs, PDGF BB-induced migration is mediated by PI3K and Src in a redoxdependent manner involving Nox4. Src may be upstream to PI3K and Nox4.
- Platelet-derived growth factor
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