title: An AP-1 binding sequence is essential for regulation of the human alpha2(I) collagen (COL1A2) promoter activity by transforming growth factor-beta.
abstract: Previous studies have shown that transforming growth factor-beta (TGF-be ta) and tumor necrosis factor-alpha (TNF-alpha) modulate type I collagen gene expression in fibroblasts. To fine-map the corresponding response elements in the human alpha2(I) collagen (COL1A2) promoter, we have generated a series of 5' deletion promoter/chloramphenicol acetyltransferase (CAT) reporter gene constructs. Transient cell transfection assays using human dermal fibroblasts and stable transfection experiments using NIH 3T3 fibroblasts identified the region located between residues -265 and -241, as critical for TGF-beta response. Specifically, we demonstrate that this 25-base pair region mediates the up-regulatory effect of TGF-beta on COL1A2 promoter activity and allows antagonistic activity of TNF-alpha on the TGF-beta effect. Gel mobility shift assays indicate that nuclear factor binding to this 25-base pair region of COL1A2 promoter is competed by AP-1, but not NF-1 or NF-kappaB, oligonucleotides. Transient cell transfection experiments with plasmid constructs in which the potential AP-1-binding site located within this short region of promoter was modified by site-directed mutagenesis indicated that this element plays a significant role in the basal activity of the promoter. Furthermore, this sequence is essential for TGF-beta response and does not require the presence of the three Sp-1-binding sites located further upstream, between nucleotides -273 and -304. In addition, overexpression of c-jun in co-transfection experiments with COL1A2 promoter/CAT constructs blocks the TGF- beta response, further implicating AP-1 in the regulation of COL1A2 gene expression. Our results clarify the molecular mechanisms involved in the regulation of type I collagen gene expression and further emphasize the importance of AP-1 in mediating some of the TGF-beta effects on gene transcription.
authors.affiliation: Department of Dermatology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
authors.forename: K Y
authors.initials: KY
authors.lastname: Chung
authors.forename: A
authors.initials: A
authors.lastname: Agarwal
authors.forename: J
authors.initials: J
authors.lastname: Uitto
authors.forename: A
authors.initials: A
authors.lastname: Mauviel
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D003094:Collagen / Q000096:biosynthesis* / Q000235:genetics*; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D017931:DNA Primers; D005347:Fibroblasts; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D007231:Infant, Newborn; D008297:Male; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000378:metabolism; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000187:drug effects; D011994:Recombinant Proteins / Q000096:biosynthesis; D012867:Skin / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D017931:DNA Primers; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D018808:Transcription Factor AP-1; D016212:Transforming Growth Factor beta; D014409:Tumor Necrosis Factor-alpha; D003094:Collagen; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase