An E-box Region within the Prostaglandin Endoperoxide Synthase-2 (PGS-2) Promoter Is Required for Transcription in Rat Ovarian Granulosa Cells*
- ‡ To whom correspondence and requests for reprints should be addressed: Dept. of Cell Biology, One Baylor Plaza, Baylor College of Medicine, Houston, TX 77030. Tel.: 713-798-6259; Fax: 713-790-1275; E-mail: joanner{at}bcm.tmc.edu
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).
Footnotes
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↵* This work was supported by National Institutes of Health Grant HD-16229 and the Reproductive Biology: Regulatory Mechanisms Training Grant HD-07165. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵1 The abbreviations used are:
- PGS
-
prostaglandin endoperoxide synthase
- LH
-
luteinizing hormone
- GnRH
-
gonadotropin-releasing hormone
- FSH
-
follicle-stimulating hormone
- FSH-R
-
follicle-stimulating hormone-receptor
- USF
-
upstream stimulatory factor
- bp
-
base pair(s)
- C/EBP
-
CAAT/enhancer binding protein
- ATF
-
activating transcription factor
- CRE
-
cAMP response element
- CREB
-
cAMP response element binding protein
- PBS
-
phosphate-buffered saline
- hCG
-
human chorionic gonadotropin
- SF-1
-
steroidogenic factor-1
- RIIβ
-
type II regulatory subunit of protein kinase A
- FSH
-
follicle-stimulating hormone
- EMSA
-
electrophoretic mobility shift assay
- PCR
-
polymerase chain reaction
- Mt
-
mutant
- CHO
-
Chinese hamster ovary
- CAT
-
chloramphenicol acetyltransferase
- rPGS-2
-
rat PGS-2.
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- Received February 13, 1996.
- Revision received April 15, 1996.
- © 1996 by The American Society for Biochemistry and Molecular Biology, Inc.
