Impaired Smad7-Smurf–mediated negative regulation of TGF-β signaling in scleroderma fibroblasts
Yoshihide Asano, Hironobu Ihn, Kenichi Yamane, Masahide Kubo, Kunihiko Tamaki
Yoshihide Asano, Hironobu Ihn, Kenichi Yamane, Masahide Kubo, Kunihiko Tamaki
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Article Autoimmunity

Impaired Smad7-Smurf–mediated negative regulation of TGF-β signaling in scleroderma fibroblasts

Abstract

The principal effect of TGF-β1 on mesenchymal cells is its stimulation of ECM synthesis. Previous reports indicated the significance of the autocrine TGF-β loop in the pathogenesis of scleroderma. In this study, we focused on Smad7 and Smurfs, principal molecules in the negative regulation of TGF-β signaling, to further understand the autocrine TGF-β loop in scleroderma. Scleroderma fibroblasts exhibited increased Smad7 levels compared with normal fibroblasts in vivo and in vitro. Smad7 constitutively formed a complex with the TGF-β receptors, and the inhibitory effect of Smad7 on the promoter activity of human α2(I) collagen and 3TP-lux was completely impaired in scleroderma fibroblasts. Furthermore, the protein stability of TGF-β receptor type I was significantly increased in scleroderma fibroblasts compared with normal fibroblasts. There was no significant difference in Smurf1 and Smurf2 levels between normal and scleroderma fibroblasts, and the transiently overexpressed Smurf1 and/or Smurf2 did not affect TGF-β receptor type I protein levels in scleroderma fibroblasts. These results indicate that the impaired Smad7-Smurf–mediated inhibitory effect on TGF-β signaling might contribute to maintaining the autocrine TGF-β loop in scleroderma fibroblasts. To our knowledge, this is the first report of a disturbed negative regulation of TGF-β signaling in fibrotic disorders.

Authors

Yoshihide Asano, Hironobu Ihn, Kenichi Yamane, Masahide Kubo, Kunihiko Tamaki

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Figure 12

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Smad7 can interact with Smurfs in scleroderma fibroblasts. (a) Whole-cel...
Smad7 can interact with Smurfs in scleroderma fibroblasts. (a) Whole-cell lysates (1 mg of protein/sample) prepared from confluent quiescent normal fibroblasts or normal fibroblasts transfected with the Smad7 expression vector were subjected to immunoprecipitation using anti-Smad7 Ab. In some experiments, cells were stimulated with TGF-β1 (2 ng/ml) for 3 hours. Immunoprecipitates were subjected to immunoblotting using anti-Smurf1 Ab (left panels) or anti-Smurf2 Ab (right panels). After stripping, total Smad7 levels were determined by immunoblotting. Total Smurf1 or Smurf2 levels were determined by immunoblotting using whole-cell lysates (20 μg protein/sample). The same experiments were performed using whole-cell lysates prepared from normal and scleroderma fibroblasts (lower panels). (b) Reverse immunoprecipitation using anti-Smurf1 Ab (upper panels) or anti-Smurf2 Ab (lower panels) was performed.