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 7

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Constitutive complex formation of Smad7 with TβRI in scleroderma fibrobl...
Constitutive complex formation of Smad7 with TβRI in scleroderma fibroblasts. (a) In the left panels, confluent quiescent normal fibroblasts were incubated in the absence or presence of TGF-β1 (2 ng/ml) for 24 hours. In the right panels, normal fibroblasts were transfected with the Smad7 expression vector (2 μg) and incubated for 48 hours prior to cell extraction. Some transfectants were stimulated with TGF-β1 (2 ng/ml) for the last 3 hours. Cell lysates (1 mg of protein/sample) were subjected to immunoprecipitation (IP) using anti-TβRI Ab. Immunoprecipitates were subjected to immunoblotting (Blot) using anti-Smad7 Ab. After stripping, total TβRI levels were determined by immunoblotting. Total Smad7 levels were determined by immunoblotting using whole-cell lysates (20 μg of protein/sample). (b) Cell lysates prepared from confluent quiescent fibroblasts were subjected to immunoprecipitation and immunoblotting as described above (upper panels). Reverse immunoprecipitation using anti-Smad7 Ab was also performed (lower panels). Each figure shows one representative of five independent experiments.