CHARACTERIZATION OF NOVEL PHORBOL ESTER-RESPONSIVE AND SERUM-RESPONSIVE SEQUENCES OF THE RAT ORNITHINE DECARBOXYLASE GENE PROMOTER

Ornithine decarboxylase (ODC), the key regulatory enzyme in mammalian polyamine biosynthesis, is rapidly induced by mitogens and tumor promo ters. We used transient expression assays and DNA-protein binding stud ies to examine the regulation of ODC promoter activity by phorbol este rs and serum growth factors. A fragment of the ODC 5' flanking region (nt -1156 to +13) was sufficient to confer 12-O-tetradecanoylphorbol-1 3-acetate (TPA)-responsive expression to a luciferase reporter gene wh en transfected into H35 cells. However, induction by TPA was not obser ved in Rat2 fibroblasts, although refeeding of serum-starved Rat2 cell s with fresh serum-containing medium rapidly induced a fivefold to six fold increase in ODC promoter activity, maximal about 8 h after refeed ing. Deletion analysis demonstrated that several sequences contributed to basal ODC promoter activity but that nt -92 to +13 was sufficient for induction by TPA or by serum. This sequence lacked canonical TPA-r esponsive elements, and an activator protein-1 (AP-1) consensus oligon ucleotide failed to compete effectively for proteins binding to this r egion. Two of four protein complexes observed by gel-shift analysis of nt -92 to +13 were competitively inhibited by wild-type but not mutan t oligonucleotides encompassing a variant cyclic AMP-response element (CRE) (ODC nt -50 to -42); however, a consensus CRE did not compete. M utagenesis of this site demonstrated that it contributes to basal expr ession of the ODC promoter but not to TPA or serum responsiveness. Thu s, we conclude that the proximal ODC promoter (nt -92 to +13) responds to TPA and serum stimulation in a cell-type-specific manner that is n ot mediated by canonical AP-1 elements. (C) 1995 Wiley-Liss, Inc.