Novel phorbol ester response region in the collagenase promoter binds Fos and Jun

Abstract

In rabbit fibroblasts the AP‐1 sequence (5′‐ATGAGTCAC‐3′) is necessary but not sufficient for induction of collagenase transcription by phorbol esters (PMA) (Auble and Brinckerhoff: Biochemistry 30(18):4629–4635, 1991). In this study we identified additional sequences involved in PMA‐induced transcription. Using fibroblasts transiently transfected with chimeric constructs containing fragments of the rabbit collagenase 5′‐flanking DNA linked to the chloramphenicol acetyl transferase (CAT) gene, we found that deletion of nucleotides −182 to −141 in a 380 bp promoter construct resulted in about a 7‐fold loss of induction by PMA. Mobility shift assays revealed that nuclear proteins from fibroblasts specifically bound to 20‐bp at −182 to −161. Binding was competed completely by self and only partially by the AP‐1 sequence, implying that proteins binding to the AP‐1 sequence could also bind to this region. In vitro transcribed and translated c‐Fos and c‐Jun bound to both the AP‐1 site and to the sequences from −182 to −141. DNAase I footprinting of the collagenase promoter with purified c‐Jun or c‐Fos/c‐Jun protected the AP‐1 sequence at −77 to −69 in addition to a region from −189 to −178 which overlaps a putative AP‐1‐like site, 5′‐ATTAATCAT‐3′. Finally, deletion of the −182 to −161 region in a 380‐bp CAT construct resulted in a substantial reduction of PMA responsiveness. Thus, we have identified a novel phorbol‐responsive region that binds c‐Fos and c‐Jun, and we suggest that these or similar proteins may regulate transcription of the collagenase gene by binding to sequences within and adjacent to the −182 to −161 region.