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

Jacqueline K Morris,Joanne S Richards

doi:10.1074/jbc.271.28.16633

Abstract

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).

Highlights

Two isoforms of the Prostaglandin endoperoxide synthase (PGS) enzyme are present in the rat ovary [14, 15]
An E-box cis-Acting DNA Element Is Required for Transcriptional Activation of rPGS-2 Promoter in Rat Ovarian Granulosa Cells—Previous functional analyses of rat PGS-21⁄7CAT promoter expression vectors in primary ovarian granulosa cells demonstrated that Ϫ195 base pairs of the proximal promoter were sufficient to confer luteinizing hormone (LH), follicle-stimulating hormone (FSH), and forskolin inducibility to the PGS-21⁄7CAT vectors [38]
Induction of prostaglandin endoperoxide synthase-2 (PGS-2) mRNA and protein by protein kinase A (LH) and protein kinase C (GnRH) pathways occurs only in granulosa cells that have differentiated to a preovulatory phenotype [14, 24, 25, 26]

Summary

Introduction

Two isoforms of the PGS enzyme are present in the rat ovary [14, 15]. Each enzyme is the product of a distinct gene as evidenced by the cloning of cDNAs for PGS-1 (16 –18) and PGS-2 (19 –21). Transient transfection of reporter constructs in rat granulosa cells demonstrated that the E-box element is critical for basal activation of the PGS-2 gene in granulosa cells and that one transcription factor which binds to this region is an upstream stimulatory factor (USF).

Results

Conclusion