Transcriptional regulation of the rat prostaglandin endoperoxide synthase 2 gene in granulosa cells. Evidence for the role of a cis-acting C/EBP beta promoter element.
The promoter of the rat prostaglandin endoperoxide synthase 2 (PGS-2) gene has recently been shown to confer gonadotropic hormone (follicle-stimulating hormone (FSH), luteinizing hormone (LH), cAMP) inducibility when ligated to a CAT (chloramphenicol acetyltransferase) reporter gene and transfected into primary cultures of differentiated granulosa cells. To delineate cis-acting elements and trans-activating factors mediating this response, deletions of the active promoter region (-192/-53 base pairs upstream of the transcriptional start site) were tested for their ability to bind protein of granulosa cell nuclear extracts and activate reporter gene activity. Electrophoretic mobility shift assays revealed that the DNA subregion -142/-120 inhibited protein/DNA binding observed between granulosa cell nuclear extracts and the labeled PGS-2 fragment -192/-53. The subregion -142/-120 acting element C/EBP beta, 5'-TTATGCAAT-3'. Point mutations within the C/EBP beta element abolished protein/DNA binding and resulted in a 50% loss of forskolin/LH/FSH inducibility of reporter gene activity. C/EBP beta mRNA and protein were induced rapidly in granulosa cells in vivo by an ovulatory dose of human chorionic gonadotropin (hCG). Collectively, these results indicate that C/EPB beta appears to play a key role in regulating induction of the PGS-2 gene in granulosa cells prior to ovulation.
Sirois
J
J
Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030.
Richards
J S
JS
D000818:Animals; D001483:Base Sequence; D022762:CCAAT-Enhancer-Binding Proteins; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D016861:Cyclooxygenase Inhibitors / Q000378:metabolism*; D004247:DNA; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005260:Female; D005498:Follicular Phase; D015971:Gene Expression Regulation, Enzymologic; D006062:Gonadotropins / Q000502:physiology; D006107:Granulosa Cells / Q000201:enzymology*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D014158:Transcription, Genetic
D022762:CCAAT-Enhancer-Binding Proteins; D016861:Cyclooxygenase Inhibitors; D004268:DNA-Binding Proteins; D006062:Gonadotropins; D009687:Nuclear Proteins; D012333:RNA, Messenger; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase