Characterization of novel phorbol ester- 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 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.
Mar
P K
PK
University of Texas M. D. Anderson Cancer Center, Science Park-Research Division, Smithville 78957, USA.
Kumar
A P
AP
Kang
D C
DC
Zhao
B
B
Martinez
L A
LA
Montgomery
R L
RL
Anderson
L
L
Butler
A P
AP
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics; D004247:DNA / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects; D004790:Enzyme Induction; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology / Q000502:physiology; D017353:Gene Deletion; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006133:Growth Substances / Q000494:pharmacology; D008114:Liver Neoplasms, Experimental / Q000188:drug therapy / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000378:metabolism; D009955:Ornithine Decarboxylase / Q000096:biosynthesis / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000187:drug effects*; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D012680:Sensitivity and Specificity; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D017362:Cyclic AMP Response Element-Binding Protein; D006133:Growth Substances; D009687:Nuclear Proteins; D004247:DNA; D011493:Protein Kinase C; D009955:Ornithine Decarboxylase; D013755:Tetradecanoylphorbol Acetate