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Rheumatology Advance Access originally published online on November 30, 2005
Rheumatology 2006 45(2):157-165; doi:10.1093/rheumatology/kei124
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© The Author 2005. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Constitutively phosphorylated Smad3 interacts with Sp1 and p300 in scleroderma fibroblasts

H. Ihn, K. Yamane, Y. Asano, M. Jinnin and K. Tamaki

Department of Dermatology, Faculty of Medicine, University of Tokyo, Tokyo, Japan.

Correspondence to: H. Ihn, Department of Dermatology, Faculty of Medicine, University of Tokyo, 7–3–1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. E-mail: IN-DER{at}h.u-tokyo.ac.jp

Objective. To elucidate the role of transforming growth factor-ß (TGF-ß)/Smad signalling in the increased expression of the collagen gene in systemic sclerosis (SSc) fibroblasts.

Methods. Dermal fibroblasts from seven patients with diffuse SSc of recent onset and from seven healthy individuals were studied. The expression levels of Smad2, Smad3 and Smad4 proteins were determined by immunoblotting. Smad3 phosphorylation and the interaction of Smad3 with Sp1 or p300 were analysed using immunoprecipitation. The effects of overexpression of Smad proteins or Sp1 on the human {alpha}2(I) collagen gene transcription were investigated with chloramphenicol acetyltransferase (CAT) assays using the –772 COL1A2/CAT construct.

Results. Constitutive increased Smad3 phosphorylation was detected in SSc fibroblasts compared with normal fibroblasts. Increased interaction of Smad3 with Sp1 as well as p300 was also detected in SSc fibroblasts. The overexpression of Smad3 caused an increase of up to 5-fold in COL1A2 promoter activity in normal fibroblasts, while Smad3 caused a small increase in COL1A2 promoter activity in SSc fibroblasts. However, neither Smad2 nor Smad4 caused significant effects in COL1A2 promoter activity in normal fibroblasts or SSc fibroblasts. The overexpression of Sp1 caused further increase in COL1A2 promoter activity stimulated by TGF-ß in normal fibroblasts, but did not change COL1A2 promoter activity in the presence of TGF-ß in SSc fibroblasts. The combined overexpression of Smad3 and Sp1 significantly enhanced TGF-ß response in normal fibroblasts, but less markedly in SSc fibroblasts.

Conclusions. These results suggested that SSc fibroblasts are less sensitive to exogenous TGF-ß stimulation because they are already activated by the autocrine TGF-ß loop.

KEY WORDS: Collagen diseases, Connective tissue, Cytokines


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