Journal of Biological Chemistry
Volume 271, Issue 6, 9 February 1996, Pages 3272-3278
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Nucleic Acids, Protein Synthesis, and Molecular Genetics
An AP-1 Binding Sequence Is Essential for Regulation of the Human α2(I) Collagen (COL1A2) Promoter Activity by Transforming Growth Factor-β(∗)

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Previous studies have shown that transforming growth factor-β (TGF-β) and tumor necrosis factor-α (TNF-α) modulate type I collagen gene expression in fibroblasts. To fine-map the corresponding response elements in the human α2(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-β response. Specifically, we demonstrate that this 25-base pair region mediates the up-regulatory effect of TGF-β on COL1A2 promoter activity and allows antagonistic activity of TNF-α on the TGF-β 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-κB, 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 sitedirected mutagenesis indicated that this element plays a significant role in the basal activity of the promoter. Furthermore, this sequence is essential for TGF-β 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-β 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-β effects on gene transcription.

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This research was supported in part by National Institutes of Health Grants R01-AR41439 and T32-AR07651 (to J. U.) and by a Research Career Development Award from the Dermatology Foundation (to A. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.