An E-box Region within the Prostaglandin Endoperoxide Synthase-2 (PGS-2) Promoter Is Required for Transcription in Rat Ovarian Granulosa Cells

• Published in: The Journal of biological chemistry Vol. 271; no. 28; pp. 16633 - 16643
• Main Authors: Morris, Jacqueline K., Richards, JoAnne S.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 12-07-1996; American Society for Biochemistry and Molecular Biology
• Subjects: Animals; Base Sequence; CCAAT-Enhancer-Binding Proteins; Cells, Cultured; Chloramphenicol O-Acetyltransferase - genetics; Cyclic AMP Response Element-Binding Protein - metabolism; DNA; DNA-Binding Proteins - genetics; Female; Granulosa Cells - enzymology; Humans; Mice; Molecular Sequence Data; Nuclear Proteins - genetics; Promoter Regions, Genetic; Prostaglandin-Endoperoxide Synthases - genetics; Prostaglandin-Endoperoxide Synthases - metabolism; Protein Binding; Rats; Sequence Homology, Nucleic Acid; Transcription Factors - metabolism; Transcription, Genetic; Upstream Stimulatory Factors
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.271.28.16633

The prostaglandin endoperoxide synthase-2 (PGS-2) gene encodes an isoform of prostaglandin synthase that is transiently induced by protein kinase 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 protein 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-activation 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), or neither region (−52 PGS-2·CAT) were transiently transfected into rat granulosa cell cultures. CAT activity was induced in both the −192 and −110 PGS-2·CAT vectors by luteinizing hormone (10-fold) and gonadotropin-releasing hormone (6-fold), whereas CAT activity of the −52 PGS-2·CAT construct did not differ from the promoterless vector (pCAT-Basic). Deletion of 1 base pair from the E-box within the −110 PGS-2·CAT construct, as well as point mutations within the CAAT box, E-box, or both regions of the −192 PGS-2·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-2 gene. An oligonucleotide spanning the rat PGS-2 E-box bound two specific protein complexes which were supershifted in the presence of antibody specific for the upstream stimulatory factor. Thus, in rat granulosa cells, the PGS-2 E-box region appears to interact with upstream cis-acting elements other than the CAAT box to confer hormonal regulation of the gene. The E-box region of the rat PGS-2 promoter does not contain ATF/CRE activity found in the human and mouse PGS-2 promoters, but is critical for basal transcription of the PGS-2 gene in rat granulosa cells and binds the upstream stimulatory factor (as do E-box regions of other genes regulated in the ovary).