Characterization of novel phorbol ester‐ and serum‐responsive sequences of the rat ornithine decarboxylase gene promoter

Abstract

Ornithine decarboxylase (ODC), the key regulatory enzyme in mammalian polyamine biosynthesis, is rapidly induced by mitogens and tumor promoters. We used transient expression assays and DNA-protein binding studies to examine the regulation of ODC promoter activity by phorbol esters and serum growth factors. A fragment of the ODC 5' flanking region (nt-1156 to +13) was sufficient to confer 12-O-tetradecanoylphorbol-13-acetate (TPA)-responsive expression to a luciferase reporter gene when transfected into H35 cells. However, induction by TPA was not observed in Rat2 fibroblasts, although refeeding of serum-starved Rat2 cells with fresh serum-containing medium rapidly induced a fivefold to sixfold increase in ODC promoter activity, maximal about 8 h after refeeding. 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-responsive elements, and an activator protein-1 (AP-1) consensus oligonucleotide failed to compete effectively for proteins binding to this region. Two of four protein complexes observed by gel-shift analysis of nt -92 to +13 were competitively inhibited by wild-type but not mutant oligonucleotides encompassing a variant cyclic AMP-response element (CRE) (ODC nt -50 to -42); however, a consensus CRE did not compete. Mutagenesis of this site demonstrated that it contributes to basal expression of the ODC promoter but not to TPA or serum responsiveness. Thus, 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 not mediated by canonical AP-1 elements.

A negative regulatory element within the proximal promoter region of the rat ornithine decarboxylase gene

“…In contrast, a ®fth Sp1-binding site at nt À108 contributes little to ODC promoter activity [19]. Nevertheless, Sp1 is not required for induction of ODC by TPA because ODC promoter fragments lacking the Sp1-binding sites remain responsive to TPA [16]. However, Sp1 may play a role in overexpression of ODC during carcinogenesis because we found that Sp1 expression and DNAbinding activity are elevated in papillomas, carcinomas, and cell lines derived from epidermal tumors [21].…”

“…An ATF/CRE site is located at nt À50 to À43 of the ODC promoter and contributes to regulation of ODC transcription by protein kinase A [22]. However, mutation of this site does not signi®cantly affect the ratio of activity in TPAtreated cells relative to that in untreated cells (the induction ratio) [16]. Two conserved myc-binding sites in intron 1 of mammalian ODC genes have been shown to mediate induction of ODC by myc [23].…”

“…Two conserved myc-binding sites in intron 1 of mammalian ODC genes have been shown to mediate induction of ODC by myc [23]. However, these intronic myc sites are not required for ODC induction by TPA because ODC promoter fragments lacking these sites are fully responsive to induction by TPA and by serum growth factors [16,17].…”

Microarray Analysis Supports a Role for CCAAT/Enhancer-binding Protein-β in Brain Injury

“…To test whether the C/EBP␤ protein regulates transcription of the 24p3 and ODC genes, we next performed transient transfection experiments with the reporter plasmids pODClux1m (20) and p24p3luc, containing 1.2 and 0.8 kb, respectively, of the promoter regions of the ODC and 24p3 genes. As shown in Fig.…”

“…The entire promoter fragment (Ϫ719 to ϩ18, p24p3) was subcloned in the promoterless luciferase reporter vector pXP1. The construct pODClux1m (containing 1.2 kb of the rat ODC promoter) was a gift of Dr. A. P. Butler (Anderson Cancer Center, University of Texas) (20).…”