Abstract
BackgroundPlasminogen 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 FindingsReactive oxygen species are generated within 5 minutes after addition of TGF-β1 to quiescent vascular smooth muscle cells (VSMCs) resulting in pp60c-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-1Y14 phosphoryation and initiation of downstream signaling.ConclusionsTGF-β1-initiated Src-dependent caveolin-1Y14 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.
Highlights
Plasminogen activator inhibitor type-1 (PAI-1, SERPINE1) is a major causative factor of arterial thrombosis and perivascular fibrosis [1,2,3,4] as well as a biomarker and prognostic indicator of cardiovascular disease-related death [5]
TGF-b1-initiated Src-dependent caveolin-1Y14 phosphorylation is a critical event in Rho-ROCK-mediated suppression of nuclear PPM1A levels maintaining, thereby, SMAD2/3-dependent transcription of the Plasminogen activator inhibitor-1 (PAI-1) gene
This paper provides novel evidence that TGF-b1 stimulation of vascular smooth muscle cells (VSMCs) leads to a reduction in nuclear levels of PPM1A, a recently identified C-terminal SMAD2/3 phosphatase capable of attenuating TGF-b1-mediated transcriptional responses including PAI-1 expression [19]
Summary
Plasminogen activator inhibitor type-1 (PAI-1, SERPINE1) is a major causative factor of arterial thrombosis and perivascular fibrosis [1,2,3,4] as well as a biomarker and prognostic indicator of cardiovascular disease-related death [5]. TGF-b1stimulated Rho-ROCK activation, for example, impacts the duration (but not the initiation) of SMAD2/3 activity but the underlying molecular basis and relationship to TGF-b1 target gene transcription is unknown. Caveolin-1 is required for TGF-b1mediated fibronectin expression in mesangial cells [18], suggesting that caveolin-1 regulation of TGF-b1 signaling may be cell type-specific. 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, in the context of elevated tissue TGF-b1. Identification of molecular events underlying to PAI-1 induction in response to TGF-b1 may yield novel targets for the therapy of cardiovascular disease
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