AN E-BOX REGION WITHIN THE PROSTAGLANDIN ENDOPEROXIDE SYNTHASE-2 (PGS-2) PROMOTER IS REQUIRED FOR TRANSCRIPTION IN RAT OVARIAN GRANULOSA-CELLS

The prostaglandin endoperoxide synthase-2 (PGS-2) gene encodes an isof orm of prostaglandin synthase that is transiently induced by protein k inase A (luteinizing hormone/cAMP) and protein kinase C (gonadotropin- releasing hormone) agonists in granulosa cells of ovulating follicles. The promoter of the rat PGS-2 gene contains a CAAT enhancer-binding p rotein consensus site (CAAT box) which can confer hormone inducibility to a PGS-2-CAT reporter gene, as well as a putative E-box region. To determine if the E-box region was involved in hormone induced trans-ac tivation of the rat PGS-2 gene, constructs with the CAAT box and E-box regions (-192 PGS-2-CAT), only the putative E-box (-110 PGS-2 CAT), o r neither region (-52 PGS-2-CAT) were transiently transfected into rat granulosa cell cultures. CAT activity was induced in both the -192 an d -110 PGS-S-CAT vectors by luteinizing hormone (10-fold) and gonadotr opin releasing hormone (6-fold), whereas CAT activity of the -52 PGS-S -CAT construct did not differ from the promoterless vector (pCAT-Basic ). Deletion of 1 base pair from the E-box within the -110 PGS-S CAT co nstruct, as well as point mutations within the CAAT box, E-box, or bot h regions of the -192 PGS-S CAT construct, demonstrated that the E-box is critical for basal transcription, and that regions, in addition to the CAAT box, are involved in hormone induction of the PGS-S gene. An oligonucleotide spanning the rat PGS-2 E-box bound two specific prote in complexes which were supershifted in the presence of antibody speci fic for the upstream stimulatory factor. Thus, in rat granulosa cells, the PGS-2 E-box region appears to interact with upstream cis-acting e lements other than the CAAT box to confer hormonal regulation of the g ene. The E-box region of the rat PGS-2 promoter does not contain ATF/C RE activity found in the human and mouse PGS-2 promoters, but is criti cal for basal transcription of the PGS-2 gene in rat granulosa cells a nd binds the upstream stimulatory factor (as do E-box regions of other genes regulated in the ovary).