Effect of TGF-β/Smad signaling pathway on lung myofibroblast differentiation1

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Abstract

Aim

Myofibroblasts play important roles in the pathogenesis of lung fibrosis. Transforming growth factor (TGF)-β1 has been widely recognized as a key fibrogenic cytokine. The major signaling pathway of TGF-β1 is through cytoplasmic Smad proteins. Our study investigated the role of individual TGF-β1/Smad signal proteins in mediating α-smooth muscle actin (α-SMA) gene expression, which is a well-known key marker of myofibroblast differentiation.

Methods

We transiently cotransfected α-SMA promoter-luciferase fusion plasmid (p895-Luc) and Smad expression plasmids and measured Luc activity in TGF-β1-treated human fetal lung fibroblasts. We induced Smad3 knockout mice lung fibrosis by bleomycin. α-SMA protein expression was assessed by Western blotting. Collagen protein was analyzed by measuring hydroxyprolin. Myofibroblast morphology was assessed by immunohistochemistry.

Results

We found that the overexpression of Smad3, not Smad2 markedly increased TGF-β1-induced α-SMA promoter activity and α-SMA protein expression in vitro, whereas the over-expression of dominant negative mutant Smad3 and Smad7 repressed TGF-β1-induced α-SMA gene expression. Compared to wild-type mice, Smad3 knockout mice showed attenuated lung fibrosis after bleomycin treatment, manifested by lower collagen production and myofibroblast differentiation.

Conclusion

Our study suggested TGF-β1/Smad3 is a major pathway which regulated the myofibroblast differentiation. This result indicates a potential significance for future attempts of attenuating the progression of human lung fibrosis by the inhibition of the Smad3 cascade.

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