AN AP-1 BINDING SEQUENCE IS ESSENTIAL FOR REGULATION OF THE HUMAN ALPHA-2(I) COLLAGEN (COL1A2) PROMOTER ACTIVITY BY TRANSFORMING GROWTH-FACTOR-BETA

Previous studies have shown that transforming growth factor-beta (TGF- beta) and tumor necrosis factor-alpha (TNF-alpha) modulate type I coll agen gene expression in fibroblasts. To fine-map the corresponding res ponse elements in the human alpha 2(I) collagen (COL1A2) promoter, we have generated a series of 5' deletion promoter/ chloramphenicol acety ltransferase (CAT) reporter gene constructs, Transient cell transfecti on assays using human dermal fibroblasts and stable transfection exper iments using NIH 3T3 fibroblasts identified the region located between residues -265 and -241, as critical for TGF-beta response, Specifical ly, we demonstrate that this 25-base pair region mediates the up-regul atory effect of TGF-beta on COL1A2 promoter activity and allows antago nistic activity of TNF-alpha on the TGF-beta effect. Gel mobility shif t assays indicate that nuclear factor binding to this 25-base pair reg ion of COL1A2 promoter is competed by AP-1, but not NF-1 or NF-kappa B , oligonucleotides. Transient cell transfection experiments with plasm id constructs in which the potential AP-1-binding site located within this short region of promoter was modified by site-directed mutagenesi s indicated that this element plays a significant role in the basal ac tivity of the promoter. Furthermore, this sequence is essential for TG F-beta response and does not require the presence of the three Sp-1-bi nding sites located further upstream, between nucleotides -273 and -30 4. In addition, overexpression of c-jun in co-transfection experiments with COL1A2 promoter/CAT constructs blocks the TGF-beta response, fur ther implicating AP-1 in the regulation of COL1A2 gene expression. Our results clarify the molecular mechanisms involved in the regulation o f type I collagen gene expression and further emphasize the importance of AP-1 in mediating some of the TGF-beta effects on gene transcripti on.