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.
Grotendorst
G R
GR
Department of Cell Biology and Anatomy, University of Miami School of Medicine, FL 33136, USA.
Okochi
H
H
Hayashi
N
N
D016475:3T3 Cells; D000818:Animals; D000841:Anisomycin / Q000494:pharmacology; D001483:Base Sequence; D001665:Binding Sites; D015152:Blotting, Northern; D055513:Connective Tissue Growth Factor; D003513:Cycloheximide / Q000494:pharmacology; D005347:Fibroblasts; D017353:Gene Deletion; D005786:Gene Expression Regulation; D006133:Growth Substances / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; D008156:Luciferases; D051379:Mice; D008934:Mitogens / Q000235:genetics*; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000235:genetics; D011485:Protein Binding; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011691:Puromycin / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000494:pharmacology*
C526317:CCN2 protein, human; C526318:CCN2 protein, mouse; D006133:Growth Substances; D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; D008934:Mitogens; D009841:Oligonucleotides; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta; D055513:Connective Tissue Growth Factor; D011691:Puromycin; D000841:Anisomycin; D003513:Cycloheximide; D008156:Luciferases