Transcription of the α2(I) collagen gene (COL1A2) in fibroblasts is potently induced by transforming growth factor-β (TGF-β). Smad family proteins function as intracellular signal transducers for TGF-β that convey information from the cell membrane to the nucleus. In the present study, we establish the functional requirement for endogenous Smad3 and Smad4 in TGF- β-stimulated COL1A2 transcription in human skin fibroblasts in vitro. Furthermore, using transfections with a series of 5' deletions of the human COL1A2 promoter, we identify a proximal region between -353 and -148 bp, which is required for full stimulation of transcription by a constitutively active TGF-β type I receptor. This region of the COL1A2 promoter contains a CAGA motif also found in the promoter of the plasminogen activator inhibitor- 1. Substitutions disrupting this sequence decreased the binding of nuclear extracts or recombinant Smad3 to the CAGACA oligonucleotide, and markedly reduced the transcriptional response to TGF-β or overexpressed Smad3 in transient transfection assays. The insertion of tandem repeats of CAGACA conferred TGF-β stimulation to a heterologous minimal promoter-reporter construct. Inhibition of endogenous Smad expression in fibroblasts by antisense oligonucleotides or cDNA against Smad3 or Smad4, and transfection of COL1A2 promoter constructs into Smad4-deficient breast adenocarcinoma cells, indicated the critical role of Smads for the full TGF-β response. The importance of Smad binding to the CAGACA box of COL1A2 was further established by transcriptional decoy oligonucleotide competition. Taken together, the results identify a functional Smad-binding element of the COL1A2 promoter harboring a CAGACA consensus sequence that is both necessary and sufficient for stimulation by TGF-β, and demonstrate that interaction of this Smad-binding element with endogenous Smads is required for the full TGF- β response in fibroblasts. (C) 2000 Wiley-Liss, Inc.
|Original language||English (US)|
|Number of pages||12|
|Journal||Journal of Cellular Physiology|
|State||Published - 2000|
All Science Journal Classification (ASJC) codes
- Clinical Biochemistry
- Cell Biology