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(Received for publication, February 13, 1996, and in revised form, April 15, 1996)
From the Department of Cell Biology, Baylor College of Medicine,
Houston, Texas 77030
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 (
Volume 271, Number 28,
Issue of July 12, 1996
pp. 16633-16643
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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).
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