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 11

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The effect of the transient overexpression of Smurf1/Smurf2 on the level...
The effect of the transient overexpression of Smurf1/Smurf2 on the levels of TβRI protein in normal and scleroderma fibroblasts. Cells were transfected with 1 μg each of the Smurf1 and Smurf2 expression vectors or the corresponding empty constructs. After a 48-hour incubation, the levels of TβRI or exogenous overexpressed Smurf1/Smurf2 were determined by Western blot analysis using anti-TβRI Ab or anti-Flag Ab, respectively. In some experiments, cells were treated with TGF-β1 (2 μg/ml) for the last 24 hours. One representative of five independent experiments is shown (a). The protein levels quantitated by scanning densitometry are shown relative to those in untreated normal fibroblasts transfected with empty vector (100 AU) (b). Data are expressed as the mean ± SD of five independent experiments.