Molecular Characterization and Role of Bovine Upstream Stimulatory Factor 1 and 2 in the Regulation of the Prostaglandin G/H Synthase-2 Promoter in Granulosa Cells

Abstract

The transcriptional activation of the prostaglandin G/H synthase-2 (PGHS-2) gene in granulosa cells is required for ovulation. To directly study the ability of upstream stimulatory factor 1 (USF1) and USF2 to trans-activate the bovine PGHS-2 promoter in granulosa cells, USF1 or USF2 expression vectors were cotransfected with the PGHS-2/luciferase (LUC) chimeric construct, ؊149/؊2PGHS-2.LUC. Results revealed that overexpression of USF1 or USF2 caused a marked and significant increase in basal and forskolin-inducible promoter activities (p < 0.05), and these effects were dependent on the presence of a consensus E-box ciselement within the promoter fragment. Co-transfections with different N-and C-terminal truncated USF mutants led to significant reductions in promoter activation, as compared with full-length constructs (p < 0.05), thus allowing identification of putative bovine USF functional domains. Overexpression of a USF2 truncated mutant lacking the first 220 residues (U2⌬1-220) acted as a dominant negative mutant and blocked endogenous and USF-stimulated PGHS-2 promoter activation. Interestingly, transfections with U2⌬1-220 blocked the forskolin-dependent induction of PGHS-2 mRNA in granulosa cells, whereas transfections with full-length USF2 increased PGHS-2 transcript levels. Immunoblot analyses confirmed overexpression of full-length and truncated USF proteins, and electrophoretic mobility shift assays (EMSAs) and supershift EMSAs established that the observed effects were dependent on specific interactions between USF proteins and the consensus E-box cis-element. Stimulation of cells with forskolin increased, whereas treatment of extracts with phosphatase decreased USF binding activities to the E-box. Thus, this study presents for the first time direct evidence for a role of USF proteins in the regulation of the PGHS-2 promoter in preovulatory granulosa cells.Prostaglandins are key mediators of inflammation, and the process of ovulation shares numerous signs of an acute inflammatory reaction, including hyperemia, edema, emigration of leukocytes, and induction of proteolytic and collagenolytic activities (1, 2). Likewise, there is a marked increase in intrafollicular levels of prostaglandin E 2 (PGE 2 ) 1 and PGF 2␣ just prior to ovulation, and inhibitors of prostaglandin synthesis were shown to block ovulation in several species (3-6). Prostaglandin G/H synthase (PGHS, also known as COX) is the first rate-limiting enzyme in the biosynthesis of all prostaglandins from arachidonic acid, and two PGHS isoforms derived from distinct genes located on separate chromosomes have been characterized, and are referred to as PGHS-1 and PGHS-2 (7-9). A third PGHS isoform produced as an alternate splice variant of the PGHS-1 gene was recently identified as PGHS-3 (10). Several studies have established that the increase in follicular prostaglandin synthesis prior to ovulation is caused by a selective, gonadotropin-dependent induction of . Within the preovulatory follicle, the enzyme was shown to be induced exclusively i...

Cyclooxygenase-2 Gene Transcription in a Macrophage Model of Inflammation

“…The overlapping CRE-1 and E-box element of the human promoter have been studied in transfected LPS-stimulated RAW264.7 macrophages (14) and shown to be involved in stimulation of COX-2 transcription. The E-box element has also been shown to act as a positive regulatory element for COX-2 expression in bovine granulosa cells (15) and in PMA-treated human gastric epithelial cells (16). Although previous studies have suggested that the CRE-1, C/EBP, and NF-B sites are involved in regulating COX-2 formation in RAW264.7 cells, no other cis-acting elements have been shown to be functional.…”

Gonadotropin-dependent regulation of bovine pituitary adenylate cyclase-activating polypeptide in ovarian follicles prior to ovulation

“…Furthermore, previous studies have shown that PACAP increases the production of progesterone and PGE2 in the ovary of crested newt, Triturus carnifex (Zhong & Kasson 1994, Gobbetti et al 1997, Gras et al 1999, Ko et al 1999. In the present work, we used primary granulosa cell cultures that have been previously established as a valuable in vitro model to study the regulation of several bovine genes, including PGHS-2 and cPLA2, and the production of PGE2 recapitulating results observed in vivo (Liu et al 1999, Sayasith et al 2004, Diouf et al 2006. A similar in vitro model was employed in rodents to study the gonadotropin-dependent regulation of PACAP and its role in the suppression of follicle apoptosis (Lee et al 1999.…”

“…Indeed, it has been reported that incubation of preovulatory follicles or granulosa cells with PACAP markedly stimulates cAMP production and amplifies FSH stimulation of cAMP, suggesting that PACAP may act as a mediator for LH during ovulation (Tornell et al 1988, Heindel et al 1996, Cecconi et al 2004. It is known that the preovulatory LH surge induces follicular cAMP production, causing a transient increase of several enzymes and receptors, such as PGHS-2, PR and ADAMTS-1, and production of PGE2 (Richards et al 2002, Sirois et al 2004. Furthermore, previous studies have shown that PACAP increases the production of progesterone and PGE2 in the ovary of crested newt, Triturus carnifex (Zhong & Kasson 1994, Gobbetti et al 1997, Gras et al 1999, Ko et al 1999.…”

“…The present study also indicated that high levels of PACAP mRNA were found in the uterus, intestine, pituitary and testis, suggesting that PACAP may play important roles in these tissues. Ovulation is a complex process triggered by LH surge, during which there is increased expression of several genes thought to be involved in a rupture of the follicle and the release of the oocyte, including PGHS-2, mPGES, membrane type 1-metalloproteinase, early growth factor-1, and PACAP (Richards et al 2002, Sirois et al 2004, Sayasith et al 2006. Many studies have suggested the role of PACAP as a regulator of ovarian functions in the rodent ovary (Lee et al 1999, Cecconi et al 2004.…”

“…Primary cultures of bovine granulosa cells were prepared as previously described (Liu et al 1999, Sayasith et al 2004. Briefly, pairs of bovine ovaries bearing a newly formed corpus luteum and a follicle of 8-12 mm in diameter (dominant follicle of the first wave of the estrous cycle) were obtained from a slaughterhouse.…”