The delayed activation of the prostaglandin G/H synthase-2 promoter in bovine granulosa cells is associated with down-regulation of truncated upstream stimulatory factor-2.
To elucidate the molecular mechanisms involved in the delayed induction of PGHS-2 in species with a long ovulatory process, a 1. 6-kilobase fragment of the bovine PGHS-2 promoter was isolated, and its activity was characterized in primary cultures of bovine granulosa cells. Promoter activity assays performed with a series of deletion mutants revealed that the promoter region from -149 to -2 (+1 = transcription start site) confers full-length promoter activity in response to forskolin (10 microM). Four consensus cis-elements were identified within this region, including an E-box, ATF/CRE, C/EBP, and AP2 site. Site-directed mutagenesis showed that the E-box was required for PGHS-2 promoter activity, that disruption of the C/EBP element decreased forskolin inducible activity by 29%, whereas point mutation within the ATF/CRE and AP2 element had no inhibitory effect. Electrophoretic mobility shift assays (EMSAs) performed with the -149/-2 fragment and granulosa cell nuclear extracts obtained before (0 h) and after (18 and 20 h) human chorionic gonadotropin (hCG) revealed the regulation of multiple DNA-protein complexes. The 0-h extract generated four complexes at the E-box, whereas only one complex was produced at this site with the 18-h extract. Supershift EMSAs identified that upstream stimulatory factor-1 and -2 (USF-1 and -2) were part of these complexes. Interestingly, the presence of the amino-terminal truncated USF-2, which lacks the transcription activation domain, was detected in the 0-h extract, but not in extracts prepared post-hCG. Supershift EMSAs also indicated high levels of C/EBPbeta binding to its cis-element in the 0-h extract, which contrasts with results previously reported in rats. Thus, high levels of amino-terminal truncated USF-2 and C/EBPbeta in bovine granulosa cells prior to hCG treatment could repress gene expression, and be involved in the delayed induction of PGHS-2 in species with a long ovulatory process.
Liu
J
J
Centre de Recherche en Reproduction Animale, Faculté de Médecine Vétérinaire, Université de Montréal, C.P. 5000, Saint-Hyacinthe, Québec J2S 7C6, Canada.
Antaya
M
M
Boerboom
D
D
Lussier
J G
JG
Silversides
D W
DW
Sirois
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D051546:Cyclooxygenase 2; D004268:DNA-Binding Proteins; D015536:Down-Regulation; D005260:Female; D006062:Gonadotropins / Q000378:metabolism; D006107:Granulosa Cells / Q000187:drug effects / Q000378:metabolism*; D015151:Immunoblotting; D007527:Isoenzymes / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009841:Oligonucleotides / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012689:Sequence Homology, Nucleic Acid; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism*; D051720:Upstream Stimulatory Factors
D004268:DNA-Binding Proteins; D006062:Gonadotropins; D007527:Isoenzymes; D009841:Oligonucleotides; D014157:Transcription Factors; D051720:Upstream Stimulatory Factors; D005576:Colforsin; D051546:Cyclooxygenase 2; D011451:Prostaglandin-Endoperoxide Synthases