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Interaction of smad3 with a proximal smad-binding element of the human alpha2(I) procollagen gene promoter required for transcriptional activation by TGF-beta.
J Cell Physiol. 2000 Jun; 183(3):381-92.JC

Abstract

Transcription of the alpha2(I) collagen gene (COL1A2) in fibroblasts is potently induced by transforming growth factor-beta (TGF-beta). Smad family proteins function as intracellular signal transducers for TGF-beta 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-beta-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-beta 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-beta or overexpressed Smad3 in transient transfection assays. The insertion of tandem repeats of CAGACA conferred TGF-beta 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-beta 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-beta, and demonstrate that interaction of this Smad-binding element with endogenous Smads is required for the full TGF-beta response in fibroblasts.

Authors+Show Affiliations

Section of Rheumatology, University of Illinois College of Medicine at Chicago, Chicago, Illinois 60607-7171, USA.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

10797313

Citation

Chen, S J., et al. "Interaction of Smad3 With a Proximal Smad-binding Element of the Human alpha2(I) Procollagen Gene Promoter Required for Transcriptional Activation By TGF-beta." Journal of Cellular Physiology, vol. 183, no. 3, 2000, pp. 381-92.
Chen SJ, Yuan W, Lo S, et al. Interaction of smad3 with a proximal smad-binding element of the human alpha2(I) procollagen gene promoter required for transcriptional activation by TGF-beta. J Cell Physiol. 2000;183(3):381-92.
Chen, S. J., Yuan, W., Lo, S., Trojanowska, M., & Varga, J. (2000). Interaction of smad3 with a proximal smad-binding element of the human alpha2(I) procollagen gene promoter required for transcriptional activation by TGF-beta. Journal of Cellular Physiology, 183(3), 381-92.
Chen SJ, et al. Interaction of Smad3 With a Proximal Smad-binding Element of the Human alpha2(I) Procollagen Gene Promoter Required for Transcriptional Activation By TGF-beta. J Cell Physiol. 2000;183(3):381-92. PubMed PMID: 10797313.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Interaction of smad3 with a proximal smad-binding element of the human alpha2(I) procollagen gene promoter required for transcriptional activation by TGF-beta. AU - Chen,S J, AU - Yuan,W, AU - Lo,S, AU - Trojanowska,M, AU - Varga,J, PY - 2000/5/8/pubmed PY - 2000/6/8/medline PY - 2000/5/8/entrez SP - 381 EP - 92 JF - Journal of cellular physiology JO - J Cell Physiol VL - 183 IS - 3 N2 - Transcription of the alpha2(I) collagen gene (COL1A2) in fibroblasts is potently induced by transforming growth factor-beta (TGF-beta). Smad family proteins function as intracellular signal transducers for TGF-beta 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-beta-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-beta 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-beta or overexpressed Smad3 in transient transfection assays. The insertion of tandem repeats of CAGACA conferred TGF-beta 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-beta 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-beta, and demonstrate that interaction of this Smad-binding element with endogenous Smads is required for the full TGF-beta response in fibroblasts. SN - 0021-9541 UR - https://www.unboundmedicine.com/medline/citation/10797313/Interaction_of_smad3_with_a_proximal_smad_binding_element_of_the_human_alpha2_I__procollagen_gene_promoter_required_for_transcriptional_activation_by_TGF_beta_ L2 - https://doi.org/10.1002/(SICI)1097-4652(200006)183:3<381::AID-JCP11>3.0.CO;2-O DB - PRIME DP - Unbound Medicine ER -