A novel transforming growth factor beta response element controls the expression of the connective tissue growth factor gene.

We reported previously that transforming growth factor beta (TGF-beta) selectively induced high levels of connective tissue growth factor (CTGF) mRNA and protein in human skin fibroblasts. In this study, we investigated the molecular mechanism for TGF-beta regulation of CTGF gene expression. Northern blot and run-on transcription assays indicate that TGF-beta directly activates transcription of the CTGF gene. Fragments of the 5'flanking region of the human CTGF gene were linked to luciferase reporter constructs. TGF-beta induced a 25-30 fold increase in luciferase activity in NIH/3T3 fibroblasts that had been transfected with this construct compared with nontreated cells after 24 h incubation. Other growth factors, such as platelet derived growth factor or fibroblast growth factor, caused only a 2-3-fold induction. This response to TGF-beta occurred only in human skin fibroblasts, fetal bovine aortic smooth muscle cells, and NIH/3T3 fibroblasts but not in the epithelial cell lines tested. Analysis of deletion mutants indicated that an important TGF-beta regulatory element is located between positions -162 and -128 of the CTGF promoter sequence. A fragment of the promoter containing this region conferred TGF-beta induction to a SV40 enhanceriess promoter. Methylation interference and competition gel shift assays mapped a unique 13-nucleotide sequence delineating a novel TGF-beta cis-regulatory element. Point mutations in this region result in a complete loss of the TGF-beta induction, identifying this sequence as a new TGF-beta response element.

[1]  F. Sanger,et al.  DNA sequencing with chain-terminating inhibitors. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[2]  K. Münger,et al.  Factor-binding element in the human c-myc promoter involved in transcriptional regulation by transforming growth factor beta 1 and by the retinoblastoma gene product. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[3]  A. Brunner,et al.  Identification of a gene family regulated by transforming growth factor-beta. , 1991, DNA and cell biology.

[4]  M. Sporn,et al.  Transforming growth factor-beta in human platelets. Identification of a major storage site, purification, and characterization. , 1983, The Journal of biological chemistry.

[5]  P. Chomczyński,et al.  Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. , 1987, Analytical biochemistry.

[6]  R. Ross,et al.  TGF-beta induces bimodal proliferation of connective tissue cells via complex control of an autocrine PDGF loop. , 1990, Cell.

[7]  Joseph Schlessinger,et al.  Signal transduction by receptors with tyrosine kinase activity , 1990, Cell.

[8]  K. Anderson,et al.  decapentaplegic acts as a morphogen to organize dorsal-ventral pattern in the Drosophila embryo , 1992, Cell.

[9]  D. Edwards,et al.  Protein synthesis inhibitors differentially superinduce c‐fos and c‐jun by three distinct mechanisms: lack of evidence for labile repressors. , 1992, The EMBO journal.

[10]  P. Bork.  The modular architecture of a new family of growth regulators related to connective tissue growth factor , 1993, FEBS letters.

[11]  C. Nüsslein-Volhard,et al.  The origin of pattern and polarity in the Drosophila embryo , 1992, Cell.

[12]  W. Gelbart,et al.  Human BMP sequences can confer normal dorsal-ventral patterning in the Drosophila embryo. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[13]  Gary R. Grotendorst,et al.  TGF‐β stimulates primary human skin fibroblast DNA synthesis via an autocrine production of PDGF‐related peptides , 1989 .

[14]  S. Coughlin,et al.  Signal transduction by the platelet-derived growth factor receptor. , 1988, Cold Spring Harbor symposia on quantitative biology.

[15]  R. Coffey,et al.  Selective inhibition of growth-related gene expression in murine keratinocytes by transforming growth factor beta , 1988, Molecular and cellular biology.

[16]  D. Levy,et al.  Identification of a phorbol ester-repressible v-src-inducible gene. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[17]  M. Greenberg,et al.  Effect of protein synthesis inhibitors on growth factor activation of c-fos, c-myc, and actin gene transcription , 1986, Molecular and cellular biology.

[18]  S. Schwartz,et al.  Inhibition of endothelial regeneration by type-beta transforming growth factor from platelets. , 1986, Science.

[19]  J. Oikarinen,et al.  Separate binding sites for nuclear factor 1 and a CCAAT DNA binding factor in the mouse alpha 2(I) collagen promoter. , 1987, The Journal of biological chemistry.

[20]  K. Anderson,et al.  Dorsal-ventral pattern formation in the Drosophila embryo: the role of zygotically active genes. , 1991, Current topics in developmental biology.

[21]  Gary R. Grotendorst,et al.  Significant correlation between connective tissue growth factor gene expression and skin sclerosis in tissue sections from patients with systemic sclerosis. , 1995, The Journal of investigative dermatology.

[22]  Gary R. Grotendorst,et al.  Production of transforming growth factor beta by human peripheral blood monocytes and neutrophils , 1989, Journal of cellular physiology.

[23]  V. Joliot,et al.  Proviral rearrangements and overexpression of a new cellular gene (nov) in myeloblastosis-associated virus type 1-induced nephroblastomas , 1992, Molecular and cellular biology.

[24]  Gary R. Grotendorst,et al.  Regulation of connective tissue growth factor gene expression in human skin fibroblasts and during wound repair. , 1993, Molecular biology of the cell.

[25]  H. Moses,et al.  Growth inhibitor from BSC-1 cells closely related to platelet type beta transforming growth factor. , 1984, Science.

[26]  Gary R. Grotendorst,et al.  Connective tissue growth factor: a cysteine-rich mitogen secreted by human vascular endothelial cells is related to the SRC-induced immediate early gene product CEF-10 , 1991, The Journal of cell biology.

[27]  Gary R. Grotendorst,et al.  TGF-β inhibition of endothelial cell proliferation: Alteration of EGF binding and EGF-induced growth-regulatory (competence) gene expression , 1987, Cell.

[28]  J. Heath,et al.  CHAPTER 16 - Regulation of transcription by transforming growth factor-β , 1991 .

[29]  H. Olsen,et al.  Transforming growth factor beta 1-responsive element: closely associated binding sites for USF and CCAAT-binding transcription factor-nuclear factor I in the type 1 plasminogen activator inhibitor gene , 1992, Molecular and cellular biology.

[30]  J. Marsh,et al.  Dorsal midline fate in Drosophila embryos requires twisted gastrulation, a gene encoding a secreted protein related to human connective tissue growth factor. , 1994, Genes & development.

[31]  M. Sporn,et al.  Expression and secretion of type beta transforming growth factor by activated human macrophages. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[32]  Brent H. Cochran,et al.  Molecular cloning of gene sequences regulated by platelet-derived growth factor , 1983, Cell.

[33]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[34]  B. Rollins,et al.  Cloning and expression of JE, a gene inducible by platelet-derived growth factor and whose product has cytokine-like properties. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[35]  R. de Groot,et al.  Transcriptional activation by TGF beta 1 mediated by the dyad symmetry element (DSE) and the TPA responsive element (TRE). , 1990, Biochemical and biophysical research communications.

[36]  M. Mattei,et al.  Structure, mapping, and expression of fisp-12, a growth factor-inducible gene encoding a secreted cysteine-rich protein. , 1991, Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research.

[37]  M. Rosner,et al.  Induction of epidermal growth factor receptor gene transcription by transforming growth factor beta 1: association with loss of protein binding to a negative regulatory element. , 1994, Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research.

[38]  L. Matrisian,et al.  TGF-β1 inhibition of transin/stromelysin gene expression is mediated through a fos binding sequence , 1990, Cell.

[39]  M. Sporn,et al.  Autoinduction of transforming growth factor beta 1 is mediated by the AP-1 complex , 1990, Molecular and cellular biology.

[40]  Michael E. Greenberg,et al.  Stimulation of 3T3 cells induces transcription of the c-fos proto-oncogene , 1984, Nature.

[41]  L. Lau,et al.  Expression of cyr61, a growth factor-inducible immediate-early gene , 1990, Molecular and cellular biology.

[42]  G. Todaro,et al.  Growth factors from murine sarcoma virus-transformed cells. , 1978, Proceedings of the National Academy of Sciences of the United States of America.

[43]  Gary R. Grotendorst,et al.  Connective Tissue Growth Factor , 1992, The Journal of dermatology.

[44]  M. Yaar,et al.  Dissociation of proto‐oncogene induction from growth response in normal human fibroblasts , 1990, Journal of cellular physiology.

[45]  C. B. Childs,et al.  Serum contains a platelet-derived transforming growth factor. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[46]  M. Majesky,et al.  Role of PDGF-A expression in the control of vascular smooth muscle cell growth by transforming growth factor-beta , 1990, The Journal of cell biology.

[47]  M. Levine,et al.  The screw gene encodes a ubiquitously expressed member of the TGF-beta family required for specification of dorsal cell fates in the Drosophila embryo. , 1994, Genes & development.

[48]  T. Guadagno,et al.  G1/S control of anchorage-independent growth in the fibroblast cell cycle , 1991, The Journal of cell biology.

[49]  V. Rosen,et al.  Identification of transforming growth factor beta family members present in bone-inductive protein purified from bovine bone. , 1990, Proceedings of the National Academy of Sciences of the United States of America.