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Interaction of Smad3 with a proximal smad‐binding element of the human α2(I) procollagen gene promoter required for transcriptional activation by TGF‐β
Author(s) -
Chen ShuJen,
Yuan Weihua,
Lo Sientay,
Trojanowska Maria,
Varga John
Publication year - 2000
Publication title -
journal of cellular physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.529
H-Index - 174
eISSN - 1097-4652
pISSN - 0021-9541
DOI - 10.1002/(sici)1097-4652(200006)183:3<381::aid-jcp11>3.0.co;2-o
Subject(s) - smad , microbiology and biotechnology , biology , transfection , transcription (linguistics) , promoter , reporter gene , transcription factor , gene , gene expression , transforming growth factor , genetics , linguistics , philosophy
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. J. Cell. Physiol. 183:381–392, 2000. © 2000 Wiley‐Liss, Inc.