Redox-induced Src kinase and caveolin-1 signaling in TGF-β1-initiated SMAD2/3 activation and PAI-1 expression

PLoS One. 2011;6(7):e22896. doi: 10.1371/journal.pone.0022896. Epub 2011 Jul 28.


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.

Principal findings: 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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Aorta / cytology
  • Aorta / metabolism
  • Blotting, Western
  • Caveolin 1 / metabolism*
  • Cells, Cultured
  • Embryo, Mammalian / cytology
  • Embryo, Mammalian / metabolism
  • Fibroblasts / cytology
  • Fibroblasts / metabolism
  • Humans
  • Immunoenzyme Techniques
  • Mice
  • Mice, Knockout
  • Muscle, Smooth, Vascular / cytology
  • Muscle, Smooth, Vascular / metabolism*
  • Oxidation-Reduction
  • Phosphoprotein Phosphatases / antagonists & inhibitors
  • Phosphoprotein Phosphatases / genetics
  • Phosphoprotein Phosphatases / metabolism
  • Phosphorylation
  • Plasminogen Activator Inhibitor 1 / metabolism*
  • Protein Phosphatase 2C
  • Proto-Oncogene Proteins pp60(c-src) / metabolism*
  • RNA, Small Interfering / genetics
  • Rats
  • Reactive Oxygen Species
  • Signal Transduction
  • Smad2 Protein / genetics
  • Smad2 Protein / metabolism*
  • Smad3 Protein / antagonists & inhibitors
  • Smad3 Protein / genetics
  • Smad3 Protein / metabolism*
  • Transforming Growth Factor beta1 / pharmacology*


  • Caveolin 1
  • Plasminogen Activator Inhibitor 1
  • RNA, Small Interfering
  • Reactive Oxygen Species
  • Smad2 Protein
  • Smad3 Protein
  • Transforming Growth Factor beta1
  • Proto-Oncogene Proteins pp60(c-src)
  • PPM1A protein, human
  • Phosphoprotein Phosphatases
  • Ppm1a protein, mouse
  • Protein Phosphatase 2C