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 4

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Effects of TGF-β1 stimulation, anti–TGF-β Ab, or TGF-β1 antisense oligon...
Effects of TGF-β1 stimulation, anti–TGF-β Ab, or TGF-β1 antisense oligonucleotide on Smad7 mRNA expression in normal and scleroderma fibroblasts. (a) Smad7 mRNA levels in normal and scleroderma fibroblasts treated or untreated with TGF-β1 (2 ng/ml) for 3 hours were investigated by Northern blot analysis. Representative results are shown (upper panels). Smad7 mRNA levels quantitated by scanning densitometry and corrected for the level of GAPDH in the same samples are shown relative to those in normal fibroblasts without TGF-β1 stimulation (100 AU) (lower panel). Data are expressed as the mean ± SD of five independent experiments. (b) Cells were treated with the indicated reagent for 48 hours, and Smad7 mRNA levels were investigated by Northern blot analysis. Representative results are shown (upper panels). Smad7 mRNA levels quantitated by scanning densitometry and corrected for the level of GAPDH in the same samples are shown relative to those in normal fibroblasts (100 AU) (lower panels). Data are expressed as the mean ± SD of five independent experiments.