Potential regulatory elements of the constitutive up-regulated alpha2(I) collagen gene in scleroderma dermal fibroblasts.
The promoter activity of the full-length alpha2(I) collagen gene is higher in scleroderma fibroblasts, when compared to normal fibroblasts. In this study, to investigate the molecular mechanisms up-regulating the expression of the alpha2(I) collagen gene in scleroderma dermal fibroblasts more clearly, we compared promoter activities of serial 5'-deletion mutants and the substitution mutants of the alpha2(I) collagen promoter constructs between normal and scleroderma fibroblasts. The transient transfection assays using a series of 5'-deletions of the promoter revealed that the up-regulated fold-increase in scleroderma fibroblasts relative to that in normal fibroblasts was significantly decreased by the removal of bp -353 to -264 fragment or bp -264 to -186 fragment. The substitution mutations introduced into binding sites of Sp1 (bp -303 and -271), Ets1 (bp -285 and -282), as well as Smad (bp -263 and -258) also abrogated the fold-increase in promoter activity in scleroderma fibroblasts synergistically. A DNA affinity precipitation assay showed that the binding activity of Ets1 as well as Smad3 to their binding sites was increased in scleroderma fibroblasts compared with normal cells. Taken together, our promoter analysis emphasized that Ets1 form a transcriptionally active complex with Smad and Sp1 by autocrine transforming growth factor (TGF)-beta signaling, leading to the intrinsic up-regulation of alpha2(I) collagen promoter activity in scleroderma fibroblasts. The blockade of autocrine TGF-beta signaling is thought to be one of the most reliable approaches in the treatment of scleroderma, and further study targeting Ets1, Smad or Sp1 may contribute to this blockade.
Jinnin
Masatoshi
M
Department of Dermatology, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan.
Ihn
Hironobu
H
Mimura
Yoshihiro
Y
Asano
Yoshihide
Y
Tamaki
Kunihiko
K
D003094:Collagen / Q000235:genetics*; D024042:Collagen Type I; D020405:Dermis / Q000166:cytology / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism*; D006801:Humans; D011401:Promoter Regions, Genetic; D012595:Scleroderma, Systemic / Q000235:genetics*; D014157:Transcription Factors / Q000378:metabolism; D015533:Transcriptional Activation; D015854:Up-Regulation
D024042:Collagen Type I; D014157:Transcription Factors; D003094:Collagen