title: Redox-induced Src kinase and caveolin-1 signaling in TGF-β1-initiated SMAD2/3 activation and PAI-1 expression.
abstract: BACKGROUND
  Plasminogen activator inhibitor-1 (PAI-1), a major regulator of the plasmin-based pericellular proteolytic cascade, is significantly increased in human arterial plaques contributing to vessel fibrosis, arteriosclerosis and thrombosis, particularly in the context of elevated tissue TGF-β1. Identification of molecular events underlying to PAI-1 induction in response to TGF-β1 may yield novel targets for the therapy of cardiovascular disease.
  
  
  RESULTS
  Reactive oxygen species are generated within 5 minutes after addition of TGF-β1 to quiescent vascular smooth muscle cells (VSMCs) resulting in pp60(c-src) activation and PAI-1 expression. TGF-β1-stimulated Src kinase signaling sustained the duration (but not the initiation) of SMAD3 phosphorylation in VSMC by reducing the levels of PPM1A, a recently identified C-terminal SMAD2/3 phosphatase, thereby maintaining SMAD2/3 in an active state with retention of PAI-1 transcription. The markedly increased PPM1A levels in triple Src kinase (c-Src, Yes, Fyn)-null fibroblasts are consistent with reductions in both SMAD3 phosphorylation and PAI-1 expression in response to TGF-β1 compared to wild-type cells. Activation of the Rho-ROCK pathway was mediated by Src kinases and required for PAI-1 induction in TGF-β1-stimulated VSMCs. Inhibition of Rho-ROCK signaling blocked the TGF-β1-mediated decrease in nuclear PPM1A content and effectively attenuated PAI-1 expression. TGF-β1-induced PAI-1 expression was undetectable in caveolin-1-null cells, correlating with the reduced Rho-GTP loading and SMAD2/3 phosphorylation evident in TGF-β1-treated caveolin-1-deficient cells relative to their wild-type counterparts. Src kinases, moreover, were critical upstream effectors of caveolin-1(Y14) phosphoryation and initiation of downstream signaling.
  
  
  CONCLUSIONS
  TGF-β1-initiated Src-dependent caveolin-1(Y14) phosphorylation is a critical event in Rho-ROCK-mediated suppression of nuclear PPM1A levels maintaining, thereby, SMAD2/3-dependent transcription of the PAI-1 gene.
authors.affiliation: Center for Cell Biology and Cancer Research, Albany Medical College, Albany, New York, United States of America.
authors.forename: Rohan
authors.initials: R
authors.lastname: Samarakoon
authors.forename: Subhanir S
authors.initials: SS
authors.lastname: Chitnis
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Higgins
authors.forename: Craig E
authors.initials: CE
authors.lastname: Higgins
authors.forename: Joan C
authors.initials: JC
authors.lastname: Krepinsky
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Higgins
mesh_terms: D000818:Animals; D001011:Aorta / Q000166:cytology / Q000378:metabolism; D015153:Blotting, Western; D051242:Caveolin 1 / Q000378:metabolism*; D002478:Cells, Cultured; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D006801:Humans; D007124:Immunoenzyme Techniques; D051379:Mice; D018345:Mice, Knockout; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*; D010084:Oxidation-Reduction; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism*; D000071636:Protein Phosphatase 2C; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D017382:Reactive Oxygen Species; D015398:Signal Transduction; D051899:Smad2 Protein / Q000235:genetics / Q000378:metabolism*; D051900:Smad3 Protein / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology*
chemical_list: D051242:Caveolin 1; D017395:Plasminogen Activator Inhibitor 1; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D051899:Smad2 Protein; D051900:Smad3 Protein; D053773:Transforming Growth Factor beta1; D016392:Proto-Oncogene Proteins pp60(c-src); C000607456:PPM1A protein, human; D010749:Phosphoprotein Phosphatases; C000607466:Ppm1a protein, mouse; D000071636:Protein Phosphatase 2C