Inhibition of the hedgehog pathway leads to antifibrotic effects in dermal fibrosis

Dermal fibrosis is characterized by the activation of the matrix-producing 'positive' myofibroblasts, and the relentless production and deposition of extracellular matrix. The hedgehog pathway has recently been demonstrated to work in a pro-fibrotic manner in systemic sclerosis (SSc). A negative regulator of the hedgehog pathway (Hh), the suppressor of fused (Sufu), was shown to be involved in the activation of fibrotic diseases. However, the exact role of Sufu in fibrosis has not been investigated so far. In our study, we aimed to define the role of sufu in the process of fibrosis using dermal fibroblasts of healthy donors that were cultured in vitro. Cyclopamine, a Smo antagonist, and Sufu lentivector were used to treat or transfect cells. The expression of fibrosis markers and ERK1/2, Smad2, and GSK3β at the protein level was determined by Western blot. Fibroblast migration was measured by in vitro wound healing assay. Bleomycin-induced dermal fibrosis mouse model was introduced to assess the effect of cyclopamine on dermal fibrosis in vivo. We found that cyclopamine significantly upregulated the expression of Sufu. Both cyclopamine and Sufu lentivector reduced migration and myofibroblast differentiation of human dermal fibroblasts at a statistically significant level. Furthermore, cyclopamine reversed dermal fibrosis induced by TGF-β1. Cyclopamine and the overexpression of Sufu inhibited the phosphorylation of GSK-3β and restrained the migration of fibroblasts. Dermal fibrosis was inhibited by intraperitoneal injection of cyclopamine in a mouse model of scleroderma. Our findings suggest that cyclopamine and Sufu-overexpression may effectively inhibit the endogenous as well as the TGF-β1-induced activation of fibroblasts through subsequent activation of GSK-3β. Sufu agonists may be a promising approach in the development of antifibrotic medications for dermal fibrosis and systemic sclerosis.