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Ornithine decarboxylase (ODC) is the rate-limiting enzyme of polyamine biosynthesis. We investigated the transcriptional regulation of the rat ODC gene using transient expression assays. The 5'-flanking region (-1156 to +13) of the ODC gene was sufficient to mediate strong basal expression of a luciferase reporter. Sequences between -345 and -93 contributed to basal promoter activity. This region, containing five potential Sp1 binding sites, was analyzed by electrophoretic mobility shift assays. Three specific DNA-protein complexes were identified using H35 nuclear extracts and the -345/-93 ODC probe. Binding to all three was eliminated by competition with an oligonucleotide containing an Sp1 binding site, but not by a mutant Sp1 oligonucleotide. Preincubation with an antibody against Sp1 supershifted complexes associated with one or more of Sp1 binding sites 1-4 as well as with site 5. DNase I footprinting revealed two protected regions: PR-I (-92 to -130) and PR-II (-304 to -332). PR-I contains a putative binding site for Sp1 that was protected by recombinant Sp1 protein. Transfection studies in Schneider SL2 cells demonstrated that the ODC promoter is transactivated up to 350-fold by Sp1 and that this transactivation is dependent on the presence of Sp1 binding sites 1-4. Thus, although the ODC promoter binds multiple nuclear proteins, Sp1 or a related protein appears to be a critical determinant of ODC transcription, possibly through cooperative interactions between Sp1 and additional transcription factors.

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.

Tumor-promoting phorbol esters and insulin produce similar effects in Reuber H35 rat hepatoma cell proliferation, including increased ornithine decarboxylase (ODC) enzyme activity, DNA synthesis, and mitogenesis. We investigated ODC mRNA accumulation in cells treated with either insulin or 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Both agents caused rapid accumulation of ODC mRNA: for TPA, it was maximal 3 hr after treatment (4-6-fold greater than control cells) and returned quickly to control levels; for insulin, it was significantly longer, continuing to increase for at least 6 hr. Simultaneous treatment with TPA and insulin led to additive effects on ODC mRNA. Induction of ODC by TPA was blocked by down-regulation or inhibition of protein kinase C (PKC), consistent with a PKC-mediated mechanism. In contrast, PKC down-regulation had little effect on ODC induction by insulin. Furthermore, although both agents stimulated ribosomal S6 protein phosphorylation in cells containing normal amounts of PKC, the response to TPA was abolished in PKC-depleted cells; the effect of insulin was only slightly inhibited. TPA caused a rapid redistribution of essentially all of the PKC activity from the cytosolic to the membrane fraction of the cells, whereas insulin had no effect on PKC distribution. These results suggest that although insulin and TPA share some common cytoplasmic signalling pathways, their effects on phosphorylation of nuclear proteins and transcription of ODC may be mediated by distinct factors.

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