Impaired Smad7-Smurf–mediated negative regulation of TGF-β signaling in scleroderma fibroblasts
Yoshihide Asano, … , Masahide Kubo, Kunihiko Tamaki
Yoshihide Asano, … , Masahide Kubo, Kunihiko Tamaki
Published January 15, 2004
Citation Information: J Clin Invest. 2004;113(2):253-264. https://doi.org/10.1172/JCI16269.
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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 3

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The phosphorylation levels of Smad2 in normal and scleroderma fibroblast...
The phosphorylation levels of Smad2 in normal and scleroderma fibroblasts either treated or untreated [TGF-β1 (–)] with TGF-β1. Cell extracts were prepared from confluent quiescent fibroblasts either treated or untreated with 2 ng/ml of TGF-β1 for 1 hour, electrophoresed through a 10% polyacrylamide gel, transferred to a nitrocellulose membrane, and probed with anti–phospho-Smad2 Ab (pSmad2). After stripping, total levels of Smad2 were determined using anti-Smad2/Smad3 Ab. One representative of five independent experiments is shown.