Identification of multiple cis-acting elements mediating the induction of prostaglandin G/H synthase-2 by phorbol ester in murine osteoblastic cells.
The tumor promoter phorbol 13-myristate 12-acetate (PMA), the best characterized protein kinase C agonist, frequently regulates gene expression via activation of Fos/Jun (AP-1) complexes. PMA rapidly and transiently induces prostaglandin G/H synthase-2 (PGHS-2) expression in murine osteoblastic MC3T3-E1 cells, but no functional AP-1 binding motifs in the 5'-flanking region have been identified. In MC3T3-E1 cells transfected with -371/+70 bp of the PGHS-2 gene fused to a luciferase reporter gene (Pluc), PMA stimulates luciferase activity up to eightfold. Computer analysis of the sequence of the PGHS-2 promoter region identified three potential AP-1 elements in the -371/+70 bp region, and deletion analysis suggested that the sequence 5'-aGAGTCA-3' at -69/-63 bp was most likely to mediate stimulation by PMA. Mutation of the putative AP-1 sequence reduces the ability of PMA to stimulate Pluc activity by 65%. On electrophoretic mobility shift analysis (EMSA), PMA induces binding to a PGHS-2 probe spanning this sequence, binding is blocked by an unlabeled AP-1 canonical sequence, and antibodies specific for c-Jun and c-Fos inhibit binding. Mutation of this AP-1 site also causes a small (22%) but significant reduction in the serum stimulation of Pluc activity in transiently transfected MC3T3-E1 cells. On EMSA, serum induces binding to a PGHS-2 probe spanning the AP-1 site, binding is blocked by an unlabeled AP-1 canonical sequence, and antibodies specific for c-Jun and c-Fos inhibit binding. Joint mutation of this AP-1 site and the nearby CRE site at -56/-52 bp, previously shown to mediate serum, v-src and PDGF induction of PGHS-2 in NIH-3T3 cells, blocks both PMA and serum induction of Pluc activity in MC3T3-E1 cells. Hence, the AP-1 and CRE binding sites are jointly but differentially involved in both the PMA and serum stimulation of PGHS-2 promoter activity.
Okada
Y
Y
University of Connecticut Health Center, Farmington, Connecticut 06030, USA.
Voznesensky
O
O
Herschman
H
H
Harrison
J
J
Pilbeam
C
C
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D051546:Cyclooxygenase 2; D004247:DNA / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004790:Enzyme Induction / Q000187:drug effects; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D017930:Genes, Reporter; D007527:Isoenzymes / Q000096:biosynthesis* / Q000235:genetics*; D008156:Luciferases / Q000235:genetics; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010006:Osteoblasts / Q000187:drug effects* / Q000201:enzymology* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis* / Q000235:genetics*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D017384:Sequence Deletion; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014162:Transfection
D017931:DNA Primers; D007527:Isoenzymes; D012333:RNA, Messenger; D018808:Transcription Factor AP-1; D004247:DNA; D008156:Luciferases; D051546:Cyclooxygenase 2; D011451:Prostaglandin-Endoperoxide Synthases; D013755:Tetradecanoylphorbol Acetate