TRANSCRIPTIONAL REGULATION OF THE RAT PROSTAGLANDIN ENDOPEROXIDE SYNTHASE-2 GENE IN GRANULOSA-CELLS - EVIDENCE FOR THE ROLE OF A CIS-ACTINGC 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) inducibilit y when ligated to a CAT (chloramphenicol acetyltransferase) reporter g ene and transfected into primary cultures of differentiated granulosa cells. To delineate cis-acting elements and trans-activating factors m ediating 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 as says revealed that the DNA subregion -142/-120 inhibited protein/DNA b inding observed between granulosa cell nuclear extracts and the labele d PGS-2 fragment -192/-53. The subregion -142/-120 was shown to contai n a consensus sequence for the cis-acting element C/EBPbeta, 5'-TTATGC AAT-3'. Point mutations within the C/EBPbeta element abolished protein /DNA binding and resulted in a 50% loss of forskolin/LH/FSH inducibili ty of reporter gene activity. C/EBPbeta mRNA and protein were induced rapidly in granulosa cells in vivo by an ovulatory dose of human chori onic gonadotropin (hCG). Collectively, these results indicate that C/E BPbeta appears to play a key role in regulating induction of the PGS-2 gene in granulosa cells prior to ovulation.