title: Src tyrosine kinase mediates platelet-derived growth factor BB-induced and redox-dependent migration in metanephric mesenchymal cells.
abstract: 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 wild-type 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 redox-dependent manner involving Nox4. Src may be upstream to PI3K and Nox4.
authors.affiliation: South Texas Veterans Health Care System, Div. of Nephrology MC 7882, 7703 Floyd Curl Dr., San Antonio, TX 78229-3900. wagnerb@uthscsa.edu.
authors.forename: Brent
authors.initials: B
authors.lastname: Wagner
authors.forename: Yves
authors.initials: Y
authors.lastname: Gorin
mesh_terms: D000818:Animals; D000077214:Becaplermin; D002465:Cell Movement; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D004622:Embryo, Mammalian; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018507:Gene Expression Regulation, Developmental / Q000502:physiology; D059630:Mesenchymal Stem Cells / Q000378:metabolism*; D051379:Mice; D009025:Morpholines / Q000494:pharmacology; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D058539:Phosphatidylinositol 3-Kinase / Q000235:genetics / Q000378:metabolism; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism; D020574:Proto-Oncogene Proteins c-sis / Q000494:pharmacology*; D019061:src-Family Kinases / Q000235:genetics / Q000378:metabolism*
chemical_list: D002867:Chromones; D004791:Enzyme Inhibitors; D009025:Morpholines; D010982:Platelet-Derived Growth Factor; D020574:Proto-Oncogene Proteins c-sis; C075278:platelet-derived growth factor A; D000077214:Becaplermin; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C422943:Nox4 protein, mouse; D058539:Phosphatidylinositol 3-Kinase; D019061:src-Family Kinases; D051057:Proto-Oncogene Proteins c-akt
keywords: Nox4; mesenchyme; metanephric; migration; platelet-derived growth factor