Stimulation of type I collagen transcription in human skin fibroblasts by TGF-beta: Involvement of Smad 3

Transforming growth factor-beta (TGF-beta) stimulates the transcription of the alpha 2(I) procollagen gene (COL1A2), The intracellular mediators invol ved in this response remain poorly understood. In this study, we demonstrat e that primary human skin fibroblasts express Smads, a novel family of sign aling molecules, in vitro in the absence of TGF-beta. The levels of Smad 7 mRNA was rapidly and transiently increased by TGF-beta. Transient overexpre ssion of Smad 3 and Smad 4, but not Smad 1 or Smad 2, caused trans-activati on of a CAT reporter gene driven by a 772 bp segment of the human COL1A2 pr omoter containing putative TGF-beta response elements. Smad stimulation of promoter activity was ligand independent, but was further enhanced by TGF-b eta, Overexpression of a phosphorylation-deficient Smad 3 mutant or wild-ty pe Smad 7, which lacks the carboxy-terminal phosphorylation motif, specific ally inhibited TGF-beta-induced activation of COL1A2 promoter. A CAGACA seq uence shown to be a functional Smad-binding element in the plasminogen acti vator inhibitor-1 gene promoter was found within the TGF-beta-response regi on of the proximal COL1A2 promoter. Gel mobility shift assays showed protei n phosphorylation-dependent binding activity in fibroblast nuclear extracts specific for this sequence; TGF-beta treatment strongly stimulated the for mation of this DNA-protein complex. Smad was identified as a component of t he CAGACA-binding transcription complex in TGF-beta-treated fibroblasts by antibody supershifting, These results demonstrate that (i) Smad 3 transmits TGF-beta signals from the receptor to the COL1A2 promoter in human fibrobl asts, and is Likely to play an important role in stimulation of COL1A2 prom oter activity elicited by TGF-beta; (ii) in fibroblasts, Smads appear to fu nction as inducible DNA-binding transcription factors; and (iii) Smad 7 may be involved in autocrine negative feedback in the regulation of COL1A2 pro moter activity by TGF-beta.