TY - JOUR
T1 - Stimulation of type I collagen transcription in human skin fibroblasts by TGF-β
T2 - Involvement of Smad 3
AU - Chen, Shu Jen
AU - Yuan, Weihua
AU - Mori, Yasuji
AU - Levenson, Anait
AU - Trojanowska, Maria
AU - Varga, John
N1 - Funding Information:
We thank Rik Derynck, Harvey Lodish, Howard Goldberg, Peter ten Dijke, Masa Kawabata, Masao Takase, Takeshi Watanabe, Liliana Attisano, and Carolyn Bruzdzinski for their generous gifts of reagents used in this study. The work was supported by the National Institutes of Health (AR-42309).
PY - 1999
Y1 - 1999
N2 - Transforming growth factor-β (TGF-β) stimulates the transcription of the α2(I) procollagen gene (COL1A2). The intracellular mediators involved in this response remain poorly understood. In this study, we demonstrate that primary human skin fibroblasts express Smads, a novel family of signaling molecules, in vitro in the absence of TGF-β. The levels of Smad 7 mRNA was rapidly and transiently increased by TGF-β. Transient overexpression of Smad 3 and Smad 4, but not Smad 1 or Smad 2, caused trans-activation of a CAT reporter gene driven by a 772 bp segment of the human COL1A2 promoter containing putative TGF-β response elements. Smad stimulation of promoter activity was ligand independent, but was further enhanced by TGF-β. Overexpression of a phosphorylation-deficient Smad 3 mutant or wild-type Smad 7, which lacks the carboxy-terminal phosphorylation motif, specifically inhibited TGF-β-induced activation of COL1A2 promoter. A CAGACA sequence shown to be a functional Smad-binding element in the plasminogen activator inhibitor-1 gene promoter was found within the TGF-β-response region of the proximal COL1A2 promoter. Gel mobility shift assays showed protein phosphorylation-dependent binding activity in fibroblast nuclear extracts specific for this sequence; TGF-β treatment strongly stimulated the formation of this DNA-protein complex. Smad was identified as a component of the CAGACA-binding transcription complex in TGF-β-treated fibroblasts by antibody supershifting. These results demonstrate that (i) Smad 3 transmits TGF-β signals from the receptor to the COL1A2 promoter in human fibroblasts, and is likely to play an important role in stimulation of COL1A2 promoter activity elicited by TGF-β; (ii) in fibroblasts, Smads appear to function as inducible DNA-binding transcription factors; and (iii) Smad 7 may be involved in autocrine negative feedback in the regulation of COL1A2 promoter activity by TGF-β.
AB - Transforming growth factor-β (TGF-β) stimulates the transcription of the α2(I) procollagen gene (COL1A2). The intracellular mediators involved in this response remain poorly understood. In this study, we demonstrate that primary human skin fibroblasts express Smads, a novel family of signaling molecules, in vitro in the absence of TGF-β. The levels of Smad 7 mRNA was rapidly and transiently increased by TGF-β. Transient overexpression of Smad 3 and Smad 4, but not Smad 1 or Smad 2, caused trans-activation of a CAT reporter gene driven by a 772 bp segment of the human COL1A2 promoter containing putative TGF-β response elements. Smad stimulation of promoter activity was ligand independent, but was further enhanced by TGF-β. Overexpression of a phosphorylation-deficient Smad 3 mutant or wild-type Smad 7, which lacks the carboxy-terminal phosphorylation motif, specifically inhibited TGF-β-induced activation of COL1A2 promoter. A CAGACA sequence shown to be a functional Smad-binding element in the plasminogen activator inhibitor-1 gene promoter was found within the TGF-β-response region of the proximal COL1A2 promoter. Gel mobility shift assays showed protein phosphorylation-dependent binding activity in fibroblast nuclear extracts specific for this sequence; TGF-β treatment strongly stimulated the formation of this DNA-protein complex. Smad was identified as a component of the CAGACA-binding transcription complex in TGF-β-treated fibroblasts by antibody supershifting. These results demonstrate that (i) Smad 3 transmits TGF-β signals from the receptor to the COL1A2 promoter in human fibroblasts, and is likely to play an important role in stimulation of COL1A2 promoter activity elicited by TGF-β; (ii) in fibroblasts, Smads appear to function as inducible DNA-binding transcription factors; and (iii) Smad 7 may be involved in autocrine negative feedback in the regulation of COL1A2 promoter activity by TGF-β.
KW - Smad signaling
UR - http://www.scopus.com/inward/record.url?scp=0032921867&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0032921867&partnerID=8YFLogxK
U2 - 10.1046/j.1523-1747.1999.00477.x
DO - 10.1046/j.1523-1747.1999.00477.x
M3 - Article
C2 - 9886263
AN - SCOPUS:0032921867
VL - 112
SP - 49
EP - 57
JO - Journal of Investigative Dermatology
JF - Journal of Investigative Dermatology
SN - 0022-202X
IS - 1
ER -