Acta Pharmacologica Sinica 2007 March; 28 (3): 382-391; doi: 10.1111/j.1745-7254.2007.00468.x

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.

Keywords: myofibroblasts; transforming growth factor b1; Smad