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Biology of Reproduction 64, 983-991 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Molecular Characterization of Bovine Prostaglandin G/H Synthase-2 and Regulation in Uterine Stromal Cells1

Jianmin Liua, Martine Antayaa, Alan K. Goffa, Derek Boerbooma, David W. Silversidesa, Jacques G. Lussiera, and Jean Sirois2,a

a Centre de recherche en reproduction animale and Département de biomédecine vétérinaire, Faculté de médecine vétérinaire, Université de Montréal, Saint-Hyacinthe, Québec, Canada J2S 7C6

ABSTRACT

Prostaglandin G/H synthase (PGHS) is a key rate-limiting enzyme in the prostaglandin biosynthetic pathway, and prostaglandins play a central role in the control of the reproductive cycle. The objectives of this study were to clone and characterize the primary structure of bovine PGHS-2 and to study its regulation in uterine stromal cells in vitro. The bovine PGHS-2 cDNA was cloned by a combination of reverse transcription-polymerase chain reaction and cDNA library screening. Results showed that the complete bovine PGHS-2 cDNA is composed of a 5'-untranslated region of 128 bp, an open reading frame of 1815 bp, and a 3'-untranslated region of 1565 bp containing multiple repeats (n = 11) of the Shaw-Kamen sequence 5'-ATTTA-3'. The open reading frame encodes a 604-amino acid protein that is 86–97% identical to other mammalian PGHS-2 homologs. The regulation of PGHS-2 mRNA and protein was studied in primary cultures of bovine uterine stromal cells stimulated with phorbol 12-myristate 13-acetate (PMA; 100 nM). Northern and Western blot analyses reveal a marked induction in PGHS-2 transcript (4.0 kilobases) and protein (Mr = 72 000) after 3–12 h of PMA stimulation (P < 0.05). However, this induction was transient in nature as levels of PGHS-2 mRNA and protein returned to basal levels after 24 h of PMA stimulation. In contrast, PMA had no effect on levels of PGHS-1 (P > 0.05). The PMA-dependent induction of PGHS-2 was associated with a significant increase in prostaglandin E2 secretion in the culture media (P < 0.05). To study promoter activity of the 5'-flanking DNA region of the bovine PGHS-2 gene, the genomic fragment -1574/-2 (+1 = transcription start site), as well as a series of 5'-deletion mutants, were fused upstream of the firefly luciferase gene and transiently transfected into primary cultures of bovine uterine stromal cells. Results showed that a first promoter region located between -1574 and -492 and a second region between -88 and -39 appear to play important roles in PMA-dependent regulation of PGHS-2 promoter activity in bovine uterine cells. Thus, this study characterizes for the first time the structure of the bovine PGHS-2 transcript and the deduced amino acid sequence of its encoded protein and establishes an in vitro model to study the regulation of PGHS-2 gene expression in bovine uterine tissue.

gene regulation, uterus

INTRODUCTION

Prostaglandins (PGs) are potent mediators of a wide variety of physiological and pathological processes, including vascular homeostasis, reproductive and gastrointestinal functions, bone metabolism, glomerular filtration, inflammation, osteoarthritis, and oncogenesis [1–4]. Prostaglandin G/H synthase (PGHS) is the first rate-limiting enzyme in the biosynthetic pathway of PGs from arachidonic acid and is the target for a large group of nonsteroidal anti-inflammatory drugs (NSAIDs) [5]. The enzyme, a membrane-bound glycoprotein purified 20 yr ago from ovine and bovine seminal vesicles, is a homodimer composed of two subunits of about 70 000 Mr and one heme group [6–10]. Two sequential enzymatic functions are associated with PGHS, a cyclooxygenase reaction responsible for the conversion of arachidonic acid to PGG2 and a peroxidase reaction involved in the conversion of PGG2 to PGH2 [1]. The common precursor for the synthesis of all PGs, prostacyclins, and thromboxanes is PGH2.

Two isoforms of PGHS, referred to as PGHS-1 and PGHS-2 (or COX-1 and COX-2), have been identified, with PGHS-1 corresponding to the enzyme purified in the late 1970s and PGHS-2 to the isoform isolated in the early 1990s [1–4]. The two isoforms share important similarities at the protein level; they have a comparable size (70 000–72 000 Mr), their amino acid sequences are 60% identical, and all structural and functional domains are highly conserved [10–16]. However, the two isoforms are derived from distinct genes located on different chromosomes and encoding different-size mRNAs (2.8 vs. 4.0 kilobases [kb] for PGHS-1 and -2, respectively) [17–19]. Most importantly, the two isoforms differ markedly in their expression and regulation. For example, PGHS-1 is present in a variety of tissues and is often referred to as the constitutive isoform involved in the synthesis of PGs necessary for normal biological processes [2, 20]. In contrast, PGHS-2 is undetectable or present at very low levels in most tissues but can be induced by several agonists and is generally referred to as the inducible form [3, 20, 21]. Gene-targeting experiments have further underscored the distinct physiological roles of each isoform [22–24].

Prostaglandins are involved in several uterine functions, and their regulation is important for successful pregnancy and parturition [25–27]. The endometrium consists of two basic cell types; epithelial cells that secrete predominantly PGF2{alpha}, and stromal cells that produce predominantly PGE2 [28]. In several species, PGF2{alpha} secretion from the endometrium is an important regulator of the estrous cycle as it controls the regression of the corpus luteum [29–31]. Its secretion is stimulated by oxytocin and involves the expression of PGHS-2 [32–35]. In contrast, PGE2 secretion is thought to have a luteotrophic effect during the time of maternal recognition of pregnancy in ruminants [36] and is known to be necessary for implantation in rodents [37]. Stimulation of PG synthesis by the endometrium appears to involve, at least in part, the phosphatidylinositol pathway [38]. Activation of protein kinase C with phorbol ester increases PG secretion from slices of bovine endometrium [39] and by both stromal and epithelial cell types [40], whereas activation of protein kinase A with cyclic AMP has no effect [39]. Although important advances have been achieved in recent years on the role and regulation of uterine PG synthesis, its molecular control remains largely uncharacterized. Thus, the objectives of this study were to clone and characterize the primary structure of the bovine PGHS-2 cDNA and to study its regulation in uterine stromal cells in vitro.

MATERIALS AND METHODS

Materials

Biotrans nylon membranes (0.2 µm) were obtained from ICN Pharmaceuticals (Montreal, PQ, Canada). [{alpha}-32P]dCTP and [35S]ATP were obtained from Mandel Scientific NEN Life Science Products (Mississauga, ON, Canada). QuikHyb hybridization solution, Poly(A) Quick mRNA purification kit and ZAP-cDNA/Gigapack cloning kit were purchased from Stratagene Cloning Systems (La Jolla, CA). TRIzol total RNA isolation reagent, RNA ladder (0.24–9.5 kb), 1 kb DNA ladder, synthetic oligonucleotides, lipofectamine, culture media, and bovine fetal calf serum were purchased from Life Technologies Inc (Gaithersburg, MD). Tissue culture plates were obtained from Corning-Costar (Fisher Scientific, Montreal, PQ, Canada). RNAsin, Prime-a-Gene labeling system, avian myeloblastosis virus (AMV) reverse transcriptase, Dual-Luciferase Reporter Assay, and plasmids pGEM3Zf(-), pRL-SV40, pGL3-basic, and pGL3-control were purchased from Promega (Madison, WI). Kodak film X-OMAT AR was obtained from Eastman Kodak Company (Rochester, NY). Electrophoretic reagents were purchased from Bio-Rad Laboratories (Richmond, CA). Vent DNA polymerase was obtained from New England Biolabs (Beverly, MA), while T4 polynucleotide kinase and all sequencing reagents were purchased from Pharmacia LKB Biotechnology (Piscataway, NJ).

Isolation and Characterization of the Bovine PGHS-2 cDNA

To clone the bovine PGHS-2 cDNA, we first used a reverse transcription/nested-polymerase chain reaction (RT-PCR) approach, as previously described [41]. Two RT reactions were performed with primers corresponding to the 3'-end and the midportion of the transcript (antisense primers 1 and 6, Fig. 1). The RT reactions were performed on 5 µg of total RNA extracted (TRIzol; Gibco BRL, Life Technologies, Gaithersburg, MD) from bovine preovulatory follicles obtained 24 h after i.v. administration of hCG [42] and using 20 µM of primers and 10 U of AMV reverse transcriptase (Promega). Nested PCR reactions were performed using Vent DNA polymerase (New England Biolabs), primers (20 µM) shown in Figure 1, and the following cycling conditions: 40 cycles of 45 sec denaturation at 95°C, 45 sec at the oligonucleotide-specific annealing temperature, and 2 min elongation at 72°C. Bovine PGHS-2 sense- and antisense-specific primers (oligonucleotides 4–9; Fig. 1) were designed from the coding sequence of bovine genomic clones [43].



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  		FIG. 1. Cloning strategy for isolating the bovine PGHS-2 cDNA. A) To obtain a 1.6-kb fragment corresponding to the 3'-end of the bovine PGHS-2 cDNA (clone 1.6), RNA was reverse transcribed with a polydeoxythymidine adapter-primer (primer 1), and the product was subjected to a first PCR with primers 2 and 4, and a second nested PCR with primers 3 and 5. A 1.1-kb internal fragment of bovine PGHS-2 (clone 1.1) was obtained by RT of bovine RNA with primer 6 followed by a first PCR with primers 6 and 8, and a second nested PCR with primers 7 and 9. B) List of primers used. Specific sense (4, 5, 8, 9) and anti-sense (6, 7, 10) oligonucleotide primers were designed from bovine PGHS-2 genomic clones [43]. Primers 1, 2, and 3 were not specific to bovine PGHS-2

Library screening was used as a second approach to characterize the 5'-end of the bovine PGHS-2 cDNA. A bovine expression library was prepared with RNA extracted from preovulatory follicles isolated 24 h post-hCG [42]. Poly(A)+ RNA was purified with the Poly(A) Quick mRNA purification kit (Stratagene), and the library was constructed using a ZAP-cDNA/Gigapack cloning kit (Stratagene) and following the manufacturer's protocol. One round of 150 000 plaques was screened with a 5' 1.2-kb EcoRI fragment of the mouse PGHS-2 cDNA [44]. The probe was labeled with [{alpha}-32P]deoxy-CTP using the Prime-a-Gene labeling system (Promega) to a final specific activity greater than 1 x 108 cpm/µg DNA, and hybridization was performed at 55°C with QuikHyb hybridization solution (Stratagene). Three positive clones were plaque purified through secondary and tertiary screening, and pBluescript phagemids containing the cloned DNA insert were excised with the Ex-Assist/SOLR system (Stratagene). The RT-PCR fragments and cDNA clones obtained through the library screening were sequenced by the Sanger dideoxy nucleotide chain termination method [45] using a T7 Sequencing Kit (Pharmacia).

Isolation and Culture of Uterine Stromal Cells

Uteri from cows at Days 1–3 of the estrous cycle were collected at the slaughterhouse and transported on ice to the laboratory. The endometrial stromal cells were separated as described previously [46]. Briefly, the two horns of each uterus were washed with sterile Hanks balanced salt solution (HBSS) containing 100 units penicillin, 100 µg streptomycin, and 0.25 µg amphotericin per ml. The myometrial layers were dissected away and the horns were then inverted to expose the epithelium. The everted horns were digested for 2 h in HBSS with 0.3% trypsin at room temperature to remove the epithelial cells. At the end of incubation, the digested horns were scraped gently with forceps and then washed twice in HBSS. The horns were further digested to obtain stromal cells by incubating in HBSS with 0.064% (w/v) trypsin III, 0.064% (w/v) collagenase II, and 0.032% (w/v) DNase I for 45 min at 37°C. Immediately after the cell suspension was collected, 10% newborn calf serum (NBCS) was added to inhibit trypsin. The cell suspension was initially centrifuged at 60 x g for 5 min to remove clumps of cells, and then the supernatant was centrifuged at 1000 x g for 10 min. The pelleted stromal cells were washed twice with HBSS. The stromal cells were plated onto dishes at a concentration of 1 x 107 cells per dish, and after a 3-h incubation, the floating cells were washed away by gentle pipetting. The attached stromal cells were then cultured in RPMI-1640 medium containing 10% NBCS that was depleted of steroids by dextran-charcoal (DC) extraction, at 37°C in humidified air (5% CO2) for 4–8 days. Medium was changed every 2 days.

Northern Blot Analysis

Total RNA was extracted from uterine cells using TRIzol (Lifetechnologies Inc.) following the manufacturer's protocol. For Northern analysis, RNA samples (10 µg) were denatured at 55°C for 15 min in denaturing buffer, electrophoresed on a 1.2% agarose gel, and transferred by capillarity to a nylon membrane, as previously described [42]. A ladder of RNA standards was run with each gel, and ethidium bromide (10 µg) was added to each sample prior to electrophoresis to compare RNA loading and determine migration of standards. The membrane was first hybridized to a 5' 1.6-kb PstI fragment of the bovine PGHS-2 cDNA using QuikHyb solution (Stratagene). After stripping the radioactivity with 0.1% saline-sodium citrate (0.15 M NaCl and 0.015 M sodium citrate)-0.1% SDS for 30 min at 100°C, the same blot was subsequently hybridized with a rat elongation factor Tu cDNA as a control gene for RNA loading and transfer [47]. Probes were labeled with [{alpha}-32P]deoxy-CTP using the Prime-a-Gene labeling system (Promega) to a final specific activity greater than 1 x 108 cpm/µg DNA.

Immunoblot Analysis

Bovine uterine stromal cells were collected after 0, 1, 3, 6, 12, and 24 h of culture in the presence of 100 nM phorbol 12-myristate 13-acetate (PMA; diluted from a 1.62 mM PMA stock solution prepared in dimethyl sulfoxide), and solubilized cell extracts were prepared as previously described [42]. Protein concentration was determined by the method of Bradford (Bio-Rad Protein Assay). Samples (50 µg protein) were resolved by one-dimensional SDS-PAGE, and electrophoretically transferred to nitrocellulose filters [42]. Membranes were incubated with polyclonal antibodies selective for PGHS-1 (8223) [42] or PGHS-2 (MF243) [48], and 125I-labeled protein A was used to visualize immunoreactive proteins, as described [42]. Filters were exposed to film at -70°C.

Radioimmunoassay of PGE2

Concentrations of PGE2 were measured directly in 10- or 100-µl aliquots of culture medium, as previously described [35]. The antiserum was purchased from Assay Designs Inc. (Ann Arbor, MI); its cross-reactivities against PGE1, PGF1{alpha}, PGF2{alpha}, and 6-keto PGF1{alpha} were 70%, 1.4%, 0.7%, and 0.6%, respectively. The sensitivity of the assay was 40 pg/ml, and the intra- and interassay coefficients of variation were 6.3% and 8.6%, respectively.

Prostaglandin G/H Synthase-2 Promoter Constructs, Transient Transfections, and Promoter Activity Assays

The production of bovine PGHS-2 promoter/firefly luciferase reporter gene constructs has been previously described [43]. Briefly, bovine PGHS-2 promoter fragments, including -1574/-2, -492/-2, -325/-2, -149/-2, -88/-2, and -39/-2 (where +1 = transcription initiation site) were inserted upstream of the firefly luciferase reporter gene in the vector pGL3.Basic (Promega). The resulting PGHS-2 promoter/firefly luciferase chimeric constructs were referred to as -1574/-2PGHS.LUC, -492/-2PGHS.LUC, -325/-2PGHS.LUC, -149/-2PGHS.LUC, -88/-2PGHS.LUC, and -39/-2PGHS.LUC.

Bovine uterine stromal cells were seeded in 24-well plates at a density of 2–5 x 105 cells per 0.5 ml RPMI supplemented with 5% DC-NBCS. Cultures were incubated at 37°C in a humidified incubator gassed with 95% air and 5% CO2 until they became 75–80% confluent. Cells were transiently transfected with 90 fmol/well of various PGHS.LUC constructs using 2 µl lipofectamine (Life Technologies) in 0.3 ml of serum/antibiotic-free RPMI, and following the manufacturer's protocol. Cotransfection with the SV40 Renilla luciferase control vector (pRL.SV40; Promega) was performed to normalize results. A ratio of experimental (PGHS.LUC) to control vector (pRL.SV40) of 10:1 was used. After 3 h of transfection, cells were incubated in 0.5 ml fresh RPMI in the absence or presence of PMA (100 nM) for variable intervals of time. At the end of the culture period, cell lysates were prepared, and firefly and Renilla luciferase activities were determined using the Promega Dual Luciferase Assay System.

Statistical Analysis

Each experiment was carried out using the cells from one uterus and was repeated with three different uteri. Effects of treatment on PGE2 production and PGHS-1 and PGHS-2 expression in uterine cells were evaluated by least-squares analysis of variance. For PGE2 production, the effect of treatment was analyzed using the two-way factorial design that included the main effects of PMA treatment and incubation time, and the treatment x time interaction. Because uterus was nested within an experiment, it was included as a random variable in the F-test for the effect of experiment. Difference between individual means were determined by orthogonal contrasts. For PGHS expression, an effect of time was analyzed by one-way ANOVA followed by Dunnett test to determine differences from time zero. A probability of P < 0.05 was considered to be significant statistically. Autoradiographic bands were scanned by Foto/analyst (Fotodyne Inc., New Berlin, WI), and the intensity of the bands was quantified by the National Institutes of Health (NIH)-Image program. The data were analyzed using the computer program JMP (SAS Institute Inc., Cary, NC).

RESULTS

Cloning and Characterization of Bovine PGHS-2 cDNA

Two strategies were used to clone the complete bovine PGHS-2 cDNA. First, an RT-PCR approach allowed the isolation of two contiguous fragments corresponding to an internal region and the 3'-end of the cDNA (Fig. 1). The internal fragment consisted of 1.1 kb corresponding to a PGHS-2 genomic region extending from exon 5 to the end of the coding region of exon 10, whereas the 3'-end fragment consisted of 1.6 kb corresponding to the 3'-untranslated region of exon 10 [43]. Despite numerous attempts, the 5'-end of the bovine PGHS-2 cDNA could not be isolated by RT-PCR. Therefore, an expression library was prepared with bovine follicle mRNA and was screened with a 5' fragment of the mouse PGHS-2 cDNA. Three nearly full-length clones were isolated and shown to differ only by the length of their 5'-ends, with a corresponding 5'-untranslated region of either 102, 106, or 109 bp. The bovine PGHS-2 cDNA contains an open reading frame of 1815 bp (including the stop codon) and a long 3'-UTR of 1565 bp containing multiple repeats of the Shaw-Kamen sequence 5'-ATTTA-3' (n = 11, Fig. 2).



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  		FIG. 2. Primary structure of the bovine PGHS-2 cDNA. The bovine PGHS-2 cDNA is composed of a 5'-untranslated region of 128 bp (lowercase letters), an open reading frame of 1815 bp (uppercase letters), and a 3'-untranslated region of 1565 bp (lowercase letters). The nucleotide sequence was derived from clones isolated by RT-PCR and cDNA library screening as described in Materials and Methods; the first 19 nucleotides were obtained from a genomic clone [43]. The translation initiation (ATG) and stop (TAG) codons are shown in bold type, Shawn-Kamen motifs (ATTTA) are underlined, and numbers appearing on the left refer to the first nucleotide on that line. The nucleotide sequence was submitted to GenBank (accession number AF031698)

The amino acid sequence of bovine PGHS-2 was deduced from the coding region of the cDNA, and comparisons were made with other known mammalian homologs (Fig. 3). The coding region of the bovine PGHS-2 cDNA encodes a 604-amino acid protein as reported for other species except for ovine PGHS-2 that consists of 603 residues (Fig. 3). The enzyme appears to share all the important structural and functional domains implicated in PGHS function (Fig. 3). Overall comparisons between bovine PGHS-2 and other mammalian homologs revealed more than 85% identity at the amino acid and nucleic acid level. Interestingly, comparative analyses of aligned PGHS-2 amino acid sequences reveal that the region located between residues 275 and 375 is remarkably conserved across all mammalian species (91–100% identity; Fig. 3), suggesting an important role of this region in PGHS-2 function.



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  		FIG. 3. Predicted amino acid sequence of bovine PGHS-2 and comparison with other mammalian homologs. The deduced amino acid sequence of the bovine (bov) PGHS-2 is aligned with the human (hum), rat, mouse (mou), ovine (ovi), equine (equ), rabbit (rab), guinea pig (gpg), and mink (min) homologs. Identical residues are marked with a printed period; the putative signal peptide cleavage site is indicated by an inverse triangle; putative N-glycosylation sites are marked with an asterisk; the putative transmembrane domain is doubled underlined; proximal and distal heme coordination residues are overlined; the tyrosine associated with the cyclooxygenase active site is underlined and the aspirin-acetylation site is indicated by a number sign. Numbers appearing on the right refer to the last amino acid residue on that line

Induction of PGHS-2 mRNA in Bovine Uterine Stromal Cells

Northern blot analysis was used to study the effect of PMA on steady-state levels of PGHS-2 mRNA in bovine uterine stromal cells in vitro. Results showed a marked regulation of PGHS-2 transcript in uterine cells after agonist treatment (Fig. 4). Whereas only low or undetectable levels of PGHS-2 mRNA (4.0 kb) were present at 0 h, a marked induction was observed within 6 h of culture in the presence of PMA (100 nM). However, this induction was transient in nature as levels of PGHS-2 transcript decreased progressively after 12 and 24 h of culture (Fig. 4A). When results from three independent experiments were quantified by densitometric analyses and corrected with the control gene EFTu, a significant increase in levels of PGHS-2 mRNA was observed at 3 and 6 h after PMA treatment (P < 0.05) (Fig. 4B).



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  		FIG. 4. Induction of PGHS-2 mRNA by PMA bovine uterine stromal cells in vitro. Confluent cultures of stromal cells were incubated in the presence of PMA (100 nM) for 0, 1, 3, 6, 12, and 24 h, as described in Materials and Methods. A) Samples of total RNA (10 µg/lane) were analyzed by Northern blotting using a bovine PGHS-2 cDNA probe and the elongation factor Tu (EFTu) as a control gene for RNA loading. Filters were exposed to film at -70°C for 15 h (PGHS-2) and 3.5 h (EFTu). Markers on the right indicate migration of intact PGHS-2 (4.0 kb) and EFTu (1.8 kb) transcripts. B) Northern blots from three independent experiments were quantified by densitometric analysis, and data from PGHS-2 mRNA were normalized with the control gene EFTu. Results are presented as a ratio PGHS-2 to EFTu ([PGHS-2/EFTu] x 10) (mean ± SEM). Columns marked with an asterisk are significantly different (P < 0.05) from 0 h

Regulation of PGHS-2 Protein and PGE2 Synthesis in Bovine Uterine Stromal Cells

To determine whether the regulation of PGHS-2 mRNA was associated with changes in levels of PGHS-2 protein, immunoblot analyses were performed on extracts of proteins prepared from cultures of bovine uterine stromal cells stimulated with PMA (100 nM). Results showed a significant time-dependent induction of PGHS-2, but not of PGHS-1 protein, after PMA treatment (Fig. 5, A and B). Levels of PGHS-2 were low or undetectable at 0 h but significantly increased thereafter to reach their maximal levels at 6 h of PMA stimulation. The PGHS-2 protein appeared more stable than the transcript as levels of the protein remained relatively high at 12 h of culture (Fig. 5A). However, levels of PGHS-2 returned to almost basal levels after 24 h of PMA stimulation (Fig. 5A). In contrast, levels of PGHS-1 protein were relatively high in bovine uterine cells prior to PMA treatment and did not vary significantly between 0–24 h of treatment (Fig. 5B).



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  		FIG. 5. Regulation of PGHS-2 and PGHS-1 proteins by PMA in bovine uterine stromal cells in vitro. Cell extracts were prepared from cultures of uterine stromal cells treated for 0–24 h with PMA (100 nM), and proteins (50 µg/lane) were analyzed by one-dimensional SDS-PAGE and immunoblotting techniques using PGHS-2-selective (A) and PGHS-1-selective (B) antibodies. After autoradiography, the band intensity was quantified by densitometric analysis. Results are presented as mean ± SEM of three independent experiments. Columns marked with an asterisk are significantly different (P < 0.05) from 0 h. Inserts in A and B show results from one experiment. Markers on the right indicate migration of intact PGHS-2 (Mr = 72 000) and PGHS-1 (Mr = 70 000) proteins. The lower band in B is thought to correspond to a PGHS-1 proteolytic fragment

To ascertain whether the induction of PGHS-2 mRNA and protein were associated with changes in PG synthetic activity, concentrations of PGE2 were determined in media of bovine stromal cells cultured in the absence or presence of PMA (100 nM). Results showed a significant effect of treatment between 3–24 h (Fig. 6). The concentration of PGE2 was significantly higher in cultures stimulated with PMA than in controls (P < 0.05). An effect of time of culture was also observed. Concentrations of PGE2 increased progressively between 1–24 h in PMA-stimulated cultures, whereas in control cultures they remained unchanged between 1 and 12 h and increased at 24 h (Fig. 6).



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  		FIG. 6. Effect of PMA on cumulative secretion of PGE2 by uterine stromal cells in vitro. Confluent cultures of stromal cells were incubated in the absence or presence of PMA (100 nM) for 0, 1, 3, 6, 12, and 24 h, as described in Materials and Methods. Concentrations of PGE2 in culture medium were determined by specific RIA. Results are presented as means ± SEM of duplicate cultures from three independent experiments. Bars marked with an asterisk are significantly different from their control at each time point (P < 0.05)

PMA-Dependent Regulation of the Bovine PGHS-2 Promoter in Uterine Stromal Cells

To study whether changes in PGHS-2 expression were related with the activation of bovine PGHS-2 promoter activity, the chimeric PGHS-2 promoter/luciferase construct -1574/-2PGHS.LUC was transiently transfected into primary cultures of bovine uterine stromal cells that were then stimulated with PMA (100 nM). Results from a time-course study showed that reporter gene activity was relatively low after 6 h and increased to reach maximal levels after 12 h of PMA treatment (Fig. 7A). Thus, a period of 12 h of PMA stimulation was selected for subsequent transient transfections.



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  		FIG. 7. Functional analysis of the bovine PGHS-2 promoter in primary cultures of uterine stromal cells. A) Time-dependent induction of the bovine PGHS-2 promoter in uterine stromal cells. Primary cultures of bovine uterine stromal cells were transiently transfected with the chimeric construct -1574/-2PGHS.LUC and incubated for 6, 12, and 24 h in the presence of PMA (100 nM), as described in Materials and Methods. Results are presented as luciferase activity normalized per µg of protein (mean ± SEM of triplicate cultures from three independent experiments). B) Deletion mutant analysis of the bovine PGHS-2 promoter. Bovine PGHS-2 promoter fragments -1574/-2, -492/-2, -325/-2, -149/-2, -88/-2, and -39/-2 subcloned upstream of the firefly luciferase reporter gene in the pGL3.Basic vector were transiently transfected into cultures of uterine stromal cells. Following transfection, cells were incubated for 12 h in the absence or presence of PMA (100 nM). All cultures were cotransfected with the SV40 Renilla luciferase vector (SV40.pRL) as an internal control to normalize experimental reporter gene activity. Results are presented as normalized relative luciferase activity (firefly/Renilla; mean ± SEM of triplicate cultures from three independent experiments)

To identify regions within the -1574/-2 fragment involved in PMA-regulation of PGHS-2 promoter activity, a series of 5'-deletion mutants fused upstream of the firefly luciferase reporter gene were transiently transfected into uterine stromal cells that were then cultured in the absence or presence of PMA (Fig. 7B). Results showed that the longest construct, -1574/-2PGHS.LUC, produced the maximal basal and PMA-stimulated reporter gene activity (Fig. 7B). Deletion of the promoter region between -1574 and -492 resulted in a 55% and 65% decrease in basal and PMA-stimulated promoter activities, respectively. Further deletions from promoter regions -492 to -325, -325 to -149, and -149 to -88 had no marked effect on basal and PMA-stimulated promoter activities (Fig. 7B). A second important loss in PGHS-2 promoter activity occurred with the deletion between -88 and -39, with 71% and 89% reduction in basal and PMA-stimulated promoter activities (Fig. 7B). The amount of reported gene activities observed with the -39/-2PGHS.LUC construct were very low and compared with those generated with the promoterless pGL3-Basic vector. Thus, these results suggest that a first region located between -1574 and -492, and a second region between -88 and -39, play an important role in the regulation of the PGHS-2 promoter in bovine uterine stromal cells.

DISCUSSION

This is the first study to document the cloning and characterization of the full-length bovine PGHS-2 cDNA. Although previous reports had documented the isolation of small cDNA fragments of 0.4 kb [33] and 0.5 kb [49], the complete structure of the bovine PGHS-2 transcript and its deduced protein had not been characterized. Comparative analysis revealed that the amino acid sequence of the bovine protein is very similar to that of other mammalian homologs, being 89%, 87%, 87%, 89%, 90%, 86%, and 86% identical to human [16], rat [14], mouse [13], horse [50], rabbit [51], guinea pig [52], and mink PGHS-2 [53], respectively. Not surprisingly, it is the comparison between species most phylogeneticaly related that showed the highest levels of similarity, with a 97% identity between the bovine and ovine PGHS-2 protein sequence [54]. All known structural and functional domains involved in PGHS-2 activity are conserved in the bovine enzyme, including a putative transmembrane region, heme-coordinating histidines 295 and 374, the cyclooxygenase active-site tyrosine 371, the aspirin acetylation-site serine 516, and four putative N-linked glycosylation sites [13, 14–16, 50–54].

The presence of multiple copies of the pentameric sequence 5'-ATTTA-3' in the 3'-untranslated region of the bovine PGHS-2 cDNA is a structural feature conserved in other species [13, 14–16, 50–54]. The position of these motifs within the 3'-untranslated regions could be of functional significance, as revealed by the highly conserved location of five AUUUA elements, of which three are overlapping, within 70 nucleotides immediately downstream of the translation stop codon. The 3'-untranslated region plays a key role in the regulation of mRNA stability, and the presence of AUUUA motifs has been described as an instability determinant of rapidly degraded transcripts, including various cytokine- and protooncogene-encoding mRNAs [55, 56]. In the present study, the rapid down-regulation of bovine PGHS-2 mRNA in uterine stromal cells following PMA stimulation further underscores the unstable nature of the transcript and compares well with results observed in other cell types [2, 21]. This characteristic of the PGHS-2 mRNA was elegantly shown in a recent study in which the insertion of the 3'-untranslated region of human PGHS-2 downstream of the ß-globin gene was sufficient to transform a stable wild-type ß-globin transcript into a rapidly degraded chimeric ß-globin/PGHS-2 mRNA [57].

Previous studies have shown that PGE2 is the predominant PG produced by uterine stromal cells [28, 58]. The present report establishes that PMA, an activator of the protein kinase C pathway, is a potent inducer of PGHS-2 expression in these cells. The PMA-dependent induction of PGHS-2 was transient in nature, although the PGHS-2 protein appeared slightly more stable that the transcript. The observed decrease in PMA stimulation of PGE2 synthesis at 24 h of culture appeared to be due, at least in part, to the loss of PGHS-2 expression at this time point. The close temporal association between induction of PGHS-2 mRNA, PGHS-2 protein, and PGE2 synthesis is clearly supportive of a causal relationship between PGHS-2 expression and increased PG synthesis. Such a finding is in keeping with the reported effect of PMA on fibroblasts of nonuterine origin [13, 59, 60]. Interestingly, although PMA had no effect on PGHS-1, the enzyme was constitutively expressed at high levels in bovine uterine stromal cells. The considerable amounts of PGE2 secreted by unstimulated cultures of stromal cells were likely derived from PGHS-1. Thus, both PGHS-1 and PGHS-2 are thought to contribute to the overall synthesis of PGE2 by stromal cells; constitutive synthesis of PGHS-1-derived prostaglandins could serve housekeeping uterine functions, whereas inducible synthesis of PGHS-2-derived prostaglandins could be necessary for transient physiological (and pathological) uterine processes.

The pulsatile release of PGF2{alpha} by uterine epithelial cells has been identified as the uterine luteolytic factor that controls the lifespan of the corpus luteum [29, 32], but the precise roles of uterine stromal PGE2 secretion in ruminants have not been as convincingly established. Some of the putative roles of uterine PGE2 in ruminants include its luteotrophic effect on the corpus luteum [36], and its modulatory effect on the local immune response [61]. In contrast, results from studies in rodents [62, 63], minks [53], and humans [64] suggest that induction of PGHS-2 expression and PGE2 synthesis by uterine stromal cells is involved during the implantation process. Genetic deletion of PGHS-2 proved to impair implantation severely in mice [65]. However, in the sheep, a species with a much less invasive placenta, there is no evidence that stromal COX-2 expression is involved in the early interactions between the conceptus and the endometrium [66]. Further studies will be required to determine whether similar results are observed in cows, or whether stromal COX-2 expression and PGE2 synthesis play a role later in the development of the bovine placenta.

Lastly, this report is the first to investigate the regulation of the bovine PGHS-2 promoter in uterine stromal cells and to establish the promoter activity of the 5'-flanking DNA region of the bovine PGHS-2 gene in homologous uterine cells. The PMA-dependent induction of promoter activity observed with the -1574/-2 PGHS-2 construct compares with the induction observed in a recent study where human uterine stromal cells were transiently transfected with a human -891/+9 PGHS-2 promoter construct [67]. The moderate levels of PMA inducibility observed in human cells (2.6-fold induction) and bovine cells (2.7-fold induction) appeared attributed, at least in part, to the relatively high levels of basal activities observed in both studies [42]. However, in contrast to the human study in which only one PGHS-2 promoter construct was tested, the use of several deletion constructs in the present study allowed for identification of at least two putative promoter regions involved in the regulation of PGHS-2 gene expression in uterine stromal cells. The importance of the proximal promoter region located at -88/-39 is identical to that reported for forskolin-dependent induction of PGHS-2 promoter in bovine granulosa cells [43]. Further studies should reveal whether the E-box present within this proximal region is responsible for driving PGHS-2 promoter activity in uterine cells, as observed in granulosa cells [43]. However, one key difference observed in uterine cells is the presence of another important promoter region located at -1574/-492 that was not shown to play a role in granulosa cells [43]. The distinct nature of the two experimental paradigms, PMA stimulation of uterine stromal cells versus forskolin stimulation of granulosa cells, likely argues for the observed difference. Indeed, several studies have shown that the importance of specific cis-acting elements within the PGHS-2 promoter varies greatly depending on the agonist and cell type used [68–73].

In summary, this study characterizes the primary structure of the bovine PGHS-2 transcript and the deduced amino acid sequence of its encoded protein and establishes an in vitro model to study the regulation of PGHS-2 gene expression in bovine uterine tissue. The identification of two regions conferring PGHS-2 promoter activities in uterine stromal cells provides the basis for further efforts aimed at unraveling the precise molecular control of the PGHS-2 promoter in uterine cells. Future studies should also focus on the transcriptional mechanisms involved in the regulation of PGHS-2 gene expression in uterine epithelial cells.

ACKNOWLEDGMENTS

_We thank Dr. Daniel Simmons (Brigham Young University, Utah) for the mouse PGHS-2 cDNA, Dr. R. Levine (Cornell University, Ithaca, NY) for the rat EF-Tu cDNA, Drs. Jilly F. Evans and Stacia Kargman (Merck Frosst Center for Therapeutic Research, Québec, ON, Canada) for providing the MF243 antibody, and Danielle Rannou and Nadine Bouchard for technical assistance.

FOOTNOTES

First decision: 20 October 2000.

1 Supported in part by Canadian Institutes of Health Research (CIHR) Grant MT-13190 (J.S.), Natural Sciences and Engineering Research Council of Canada Grant 8161-98 (A.K.G), and Fonds pour la Formation de Chercheurs et l'Aide à la Recherche (FCAR) Grant 99-ER-3016 (J.G.L., D.W.S., and J.S.). J.L. is supported by an FCAR of Québec Doctoral Fellowship, D.B. is supported by a CIHR Doctoral Research Award, and J.S. is supported by a CIHR Investigator Award. Back

2 Correspondence: Jean Sirois, CRRA, Faculté de médecine vétérinaire, Université de Montréal, C.P. 5000, Saint-Hyacinthe, PQ, Canada J2S 7C6. FAX: 450 778 8103; siroisje{at}medvet.umontreal.ca Back

Accepted: November 2, 2000.

Received: October 4, 2000.

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