Intracellular TGF-β receptor blockade abrogates smad-dependent fibroblast activation in vitro and in vivo

Abstract

Fibrosis, the hallmark of scleroderma, is characterized by excessive synthesis of collagen and extracellular matrix proteins and accumulation of myofibroblasts. Transforming growth factor-β (TGF-β), a potent inducer of collagen synthesis, cytokine production, and myofibroblast transdifferentiation, is implicated in fibrosis. Profibrotic TGF-β responses are induced primarily via the type I activin-like receptor kinase 5 (ALK5) TGF-β receptor coupled to Smad signal transducers. Here, we investigated the effect of blocking ALK5 function with SM305, a novel small-molecule kinase inhibitor, on fibrotic TGF-β responses. In normal dermal fibroblasts, SM305 abrogated the ligand-induced phosphorylation, nuclear import, and DNA-binding activity of Smad2/3 and Smad4, and inhibited Smad2/3-dependent transcriptional responses. Furthermore, SM305 blocked TGF-β-induced extracellular matrix gene expression, cytokine production, and myofibroblast transdifferentiation. In unstimulated scleroderma fibroblasts, SM305 caused a variable and modest reduction in type I collagen levels, and failed to abrogate constitutive nuclear accumulation of Smad2/3, or alter the proportion of smooth muscle actin stress fiber-positive fibroblasts. In vivo, SM305 prevented TGF-β-induced Smad2/3 phosphorylation type I collagen (COL1)A2 promoter activation in dermal fibroblasts. Taken together, these results indicate that SM305 inhibits intracellular TGF-β signaling through selective interference with ALK5-mediated Smad activation, resulting in marked suppression of profibrotic responses induced by TGF-β in vivo and in vitro. © 2006 The Society for Investigative Dermatology.