title: Activation of integrin β1-focal adhesion kinase-RasGTP pathway plays a critical role in TGF beta1-induced podocyte injury.
abstract: The depletion of glomerular podocytes is the key mechanism of glomerulosclerosis and progressive renal failure. Transforming growth factor-β (TGFβ) is a central mediator of signaling networks that control a diverse set of cellular processes, such as cell proliferation, differentiation, and apoptosis. Though many key events in TGFβ1 signaling have been documented at cellular and molecular level in podocytes, the complete effects of TGFβ1 on podocyte integrity are still elusive. In this study, the function of adhesion protein integrin β1, focal adhesion kinase (FAK), and a small GTPase Ras was explored in TGFβ1-induced podocyte injury. In cultured mouse podocyte, caspase 3-positive cells were counted by flow cytometry to evaluate podocyte damage at different time points after TGFβ1 treatment. Immunoblotting assay showed that integrin β1, FAK, Src kinase, and an adaptor protein Grb2 were activated rapidly after TGFβ1 stimulation. Active Ras Pull-Down assay revealed that the active Ras (GTP-bound Ras) level was upregulated in TGFβ1-treated cell. Immunoprecipitation results displayed that TGFβ1 enhanced the complex formation of integrin β1, FAK and Src kinase, as well as FAK, Grb2 and Ras. The FAK inhibitor TAE226 and the specific knockdown of Grb2 remarkably alleviated TGFβ1-induced podocyte apoptosis. The activation of p38MAPK and Erk1/2, and the nuclear translocation of NFκB(p65) were increased evidently in TGFβ1-treated cell, which could be dramatically prohibited by the application of the p38MAPK inhibitor SB202190 and the Ras inhibitor FPT Inhibitor III. The Src kinase inhibitor PP2 obviously prevented the activation of FAK and Ras, as well as the translocation of NFκB(p65) from cytoplasm to nuclei. The PP2, FPT Inhibitor III, and SB202190 significantly decreased TGFβ1-induced podocyte apoptosis. Taken together, these data demonstrated that the activation of integrin β1/Src/FAK and Grb2/RasGTP should be responsible for TGFβ1-induced podocyte damage through the p38MAPK and Erk1/2-mediated nuclear translocation of NFκB(p65).
authors.affiliation: Department of Pediatrics, the Second Affiliate Hospital of Harbin Medical University, Harbin, Heilongjiang Province 150086, PR China.
authors.forename: Yu-Jing
authors.initials: YJ
authors.lastname: Zhang
authors.forename: Zhi-Liang
authors.initials: ZL
authors.lastname: Tian
authors.forename: Xin-Yan
authors.initials: XY
authors.lastname: Yu
authors.forename: Xia-Xia
authors.initials: XX
authors.lastname: Zhao
authors.forename: Li
authors.initials: L
authors.lastname: Yao
mesh_terms: D000818:Animals; D017209:Apoptosis; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000276:immunology*; D051380:GRB2 Adaptor Protein / Q000276:immunology; D019012:Integrin beta1 / Q000276:immunology*; D020935:MAP Kinase Signaling System; D051379:Mice; D050199:Podocytes / Q000166:cytology / Q000276:immunology* / Q000473:pathology; D015398:Signal Transduction; D053773:Transforming Growth Factor beta1 / Q000276:immunology*; D048051:p38 Mitogen-Activated Protein Kinases / Q000276:immunology; D018631:ras Proteins / Q000276:immunology*; D019061:src-Family Kinases / Q000276:immunology
chemical_list: D051380:GRB2 Adaptor Protein; C495949:Grb2 protein, mouse; D019012:Integrin beta1; D053773:Transforming Growth Factor beta1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D019061:src-Family Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D018631:ras Proteins
keywords: FAK; GTP-Ras; Grb2; Integrin; Podocyte apoptosis; TGFβ1