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Prostaglandin G/H Synthase (PGHS)-2 Expression in Bovine Myometrium: Influence of Steroid Hormones and PGHS Inhibitors¹

^a Perinatal Research and Developmental Pharmacology Unit, Lady Davis Institute for Medical Research, McGill University, Montréal, Québec, Canada H3T 1E2 ^b Unité de Génétique, Pavillon Saint-François d'Assise, ^c Laboratoire de Rhumatologie et Immunologie, ^d Unité de Recherche en Ontogénie et Reproduction, Pavillon C.H.U.L., Laval University, Québec, Canada G1V 4G2

	ABSTRACT

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Prostaglandins (PGs) are important mediators regulating uterine functions during the reproductive process. The objective of this study was to examine, in myocytes from the circular and longitudinal layers of bovine myometrium, the relative levels of mRNA and proteins corresponding to the gene expression of key enzymes (phospholipase A₂; prostaglandin G/H synthase-1 [PGHS-1]; prostaglandin G/H synthase-2 [PGHS-2]; prostaglandin I₂ synthase) involved in PG biosynthesis.

We examined the influence of estradiol-17ß and progesterone on the expression and activity of these enzymes. Treatment of myocytes with progesterone (P₄: 10 nM, 24 h) in the absence or presence of estradiol-17ß (E₂: 1 nM, 72 h) suppressed PG biosynthesis by approximately 60% in both myometrial layers. No significant effect was observed after E₂ treatment. The combined effect of E₂ and P₄ on PG accumulation was correlated with the modulation of PGHS-2 protein and mRNA levels in the two myometrial layers without affecting other enzymes of the PG cascade. Selective or nonselective inhibition of PGHS activity with CGP 28238 (PGHS-2-specific; a product from Ciba-Geigy: 6-[2,4-difluorophenoxy]-5-methyl-sulfonylamino-1-indanone) or indomethacin (PGHS-1 and -2) reduced prostacyclin accumulation (measured as 6-keto-PGF₁ in the culture medium) in a dose-dependent manner in the two myometrial layers. A significant inhibitory effect was obtained at a low concentration of indomethacin (1 nM, p < 0.05) compared to CGP 28238 (10 nM, p < 0.05). In both myometrial layers, the maximal effect of indomethacin and/or CGP 28238 on PG accumulation was observed at 100 nM and represented 85% and 65% inhibition, respectively. In the presence of phorbol 12-myristate (100 nM), CGP 28238 (10 nM) significantly suppressed PGHS-2 mRNA level by 44.80 ± 7.67% (p < 0.01) and 27.83 ± 7.62% (p < 0.05) in the longitudinal and circular layer, respectively. In contrast, indomethacin did not have any significant effect. These data constitute the first quantitative analysis of key enzymes involved in PG biosynthesis in separated myometrial layers. Furthermore, the results provide interesting information on the CGP 28238 drug modulating both enzymatic activity and mRNA expression of PGHS-2.

	INTRODUCTION

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The uterus is a complex organ composed of several cell types forming the myometrium and the endometrium. The successful outcome of the reproductive process requires a close collaboration between the different cell layers forming the uterus. Besides sex steroids, prostaglandins (PGs) appear as primary regulators of uterine functions during the entire reproductive cycle [1, 2]. The complex transformations affecting myometrial physiology during pregnancy require the liberation of such locally acting factors. PGs may come from intrauterine tissues such as the placenta, but these structures may also metabolize PGs before they reach the myometrium [3, 4]. Because PGs primarily exhibit paracrine or autocrine actions, and because we have shown that the myometrium has its own profile of prostanoid production, we believe that this tissue may control its own functions through the production of specific PGs. Previous studies from our laboratory have demonstrated that cultured myocytes from the circular and longitudinal layers of bovine myometrium express distinctive properties [5–7], and more particularly, that there is a predominant production of prostacyclin (PGI₂) by the longitudinal layer of bovine myometrium [6].

Prostaglandin G/H synthase (PGHS) and phospholipase A₂ (PLA₂) are the key enzymes in the release and conversion of arachidonic acid (AA) to PGH₂ that is subsequently converted to PGD₂, PGE₂, PGF₂, PGI₂, and thromboxane A₂ by specific synthases. Two PGHS isoforms are encoded by two different genes; the constitutive form (PGHS-1) is assumed to be involved in producing prostanoids for cellular "housekeeping" functions whereas the newly described inducible form (PGHS-2) is expressed under specific conditions in cells [8–12] and strongly down-regulated by glucocorticoids [13]. Although PGHS-1 and PGHS-2 enzymes share a high degree of homology (60–70%), several studies using available nonsteroidal anti-inflammatory drugs have demonstrated that the two isoforms can be inhibited differentially [14, 15].

Evidence supporting the role of prostanoids in the control of uterine contractility has been reported [1, 2]. However, little is known about the fine mechanisms involved in the local changes affecting myometrial PG biosynthesis. The aim of the present study was to document the specific mechanisms regulating PG formation in bovine myometrium. We examined the differential expression of the PGHS (PGHS-1 and PGHS-2), cytosolic phospholipase A2 (cPLA₂), and prostaglandin I₂ synthase (PGI synthase) transcripts and/or their related gene products in the two myometrial layers. We concurrently investigated the effects of sex steroid hormones (estradiol-17ß ± progesterone) and a specific PGHS-2 antagonist, CGP 28238 (Ciba-Geigy Canada Ltd., Calgary, AB; 6-[2,4-difluorophenoxy]-5-methyl-sulfonylamino-1-indanone), on PG biosynthesis and PGHS-2 gene expression.

	MATERIALS AND METHODS

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Reagents

Tissue culture reagents and fetal calf serum were purchased from ICN Biochemicals Inc. (Costa Mesa, CA). Human PGHS-2 cDNA was donated by Dr. Gary O'Neill (Merck Frosst Canada Inc., Pointe-Claire, PQ, Canada). The polyclonal antibodies to cPLA₂, PGHS-1, and PGHS-2 were obtained from Dr. J. Grassi (CEA, Saclay, France). The monoclonal antibody to PGI synthase was from Oxford Biomedical Research Inc. (Oxford, MI); [-³²P]dCTP (> 3.000 Ci/mmol) was purchased from Dupont (Boston, MA). All other chemicals were supplied by Sigma Chemical Co. (St. Louis, MO).

Cell Culture and Treatment

Bovine uteri were collected at local slaughterhouses, and the stage of the estrous cycle at which the uteri were collected (midluteal phase) was estimated by examining the ovarian morphology [16]. Myocytes from bovine myometrium were cultured in RPMI 1640 supplemented with 10% fetal bovine serum as previously described [6, 7]. When cell cultures reached confluency (Days 7–10 after plating), the culture medium was replaced with fresh medium without serum. The cells were then cultured in the absence or presence of 100 nM phorbol 12-myristate (PMA) to stimulate production of PGs. In experiment 1, during the 24-h serum withdrawal period, 100 nM PMA was added at 0, 6, or 24 h before collection of culture medium and recovery of the cells. In experiment 2, 72 h before expected confluency, the culture medium was replaced by fresh RPMI supplemented with 10% dextran-coated charcoal-treated fetal calf serum. During this period, 4 sex steroid treatment groups were analyzed. In treatment group 1, cells were treated with estradiol-17ß (E₂, 1 nM) for 72 h. In treatment group 2, cells were treated with ethanol (0.1%) for 48 h, then with progesterone (P₄, 10 nM) the last 24 h. In treatment group 3, cells were treated with dexamethasone (DEX) 24 h after a 48 h pretreatment with ethanol (0.1%). In treatment group 4, cells were treated with E₂ (1 nM) for 72 h plus P₄ (10 nM) the last 24 h. The control group corresponded to cells treated with 0.1% ethanol (vehicle) alone (72 h). In all cases, 3 h (corresponding to the maximal mRNA expression induced by PMA, data not shown) or 6 h (maximal protein expression) before the end of the incubation, PMA (100 nM) was added to the cultures. The culture medium was recovered for analysis of PGs, and cells were recovered for Northern or Western blot analysis. In experiment 3, cells were grown to confluency and the medium was replaced by fresh medium without serum. The cells were then incubated in the presence of PMA (100 nM) and in the presence or absence of increasing doses (0.01–100 nM) of indomethacin to block all PGHS activity or with CGP 28238 to block PGHS-2. The culture supernatant was then recovered for measurement of PGI₂. In experiment 4, cells were grown to confluency, the medium was replaced as described above, and cells were treated or not treated with CGP 28238 or indomethacin for 24 h. During the last 3 h, PMA (100 nM) was added to the cultures. The cells were then recovered for analysis of expression of PGHS-2 mRNA by Northern blot.

PG Assays

Accumulation of PGE₂ and 6-keto-PGF₁ (the stable metabolite of PGI₂) in the incubation medium was determined by ELISA as described previously [6].

RNA Isolation and Northern Blot Analysis

Total RNA was prepared by the method of Chomczynski and Sacchi [17]. In summary, after myocytes were homogenized in 4 M guanidium thiocyanate, RNA was extracted twice with phenol/chloroform-isoamyl alcohol (24:1), precipitated with isopropanol, and then washed with 70% ethanol. Total RNA (10 µg) was loaded on a 1% agarose-2.2 M formaldehyde gel, transferred to nylon membranes (Magna; Micron Separations Inc., Westboro, MA), and then cross-linked by UV irradiation [18]. After 4 h of prehybridization in a 50% formamide solution at 42°C, membranes were sequentially hybridized overnight at 42°C in the same solution containing [-³²P]dCTP-labeled cDNA probes corresponding to human PGHS-2 mRNA and [-³²P]dCTP-labeled 18S ribosomal DNA. Membranes were washed and exposed to x-ray films with an intensifying screen at -80°C for 4–72 h. Membranes were stripped for 30 min in 50% formamide, 6-strength saline-sodium phosphate-EDTA at 65°C between hybridizations. Signals were quantitated by densitometry of the autoradiograms using NIH (Bethesda, MD) Image 1.57 software.

Western Blot Analysis

Cells were rinsed with PBS and then lysed in a homogenization buffer (50 mM Tris, pH 6.8, 1% SDS, 10% glycerol, 1% ß-mercaptoethanol). For immunoblot analysis, 100 µg of protein [19] from cell lysates was separated by SDS-PAGE. Proteins were transferred onto polyvinylidene fluoride membranes (Micron Separations Inc.). Membranes were blocked in 5% nonfat milk-Tris-buffered saline with 0.1% Tween 20 (TBST) before incubation with rabbit antibody to either human PGHS-1 (dilution 1:5000), PGHS-2 (dilution 1:2500), or cPLA₂ (dilution 1:500) or mouse monoclonal antibody to PGI synthase (dilution 1:5000). Blots were incubated with donkey anti-rabbit IgG (PGHS-1, PGHS-2, and cPLA₂) or sheep anti-mouse IgG (PGI synthase) peroxidase-linked secondary antibody in TBST. Chemiluminescent detection was performed using reagents from Dupont, and bands were visualized on Kodak (Eastman Kodak, Rochester, NY) films.

Statistical Analysis

All experimental data are presented as the mean ± SEM. Statview 4.51 (Abacus Concepts, Berkeley, CA) statistical package for the Macintosh was used for all analyses. For Northern blot data, the Student's t-test was used to determine the level of significance for differences between two treatment (steroid hormones or PGHS inhibitors versus control) groups. Differences in dose response for each treatment were analyzed by ANOVA followed by Fisher's least-significant-difference procedure. The level of statistical significance was set at p < 0.05.

	RESULTS

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PGHS and cPLA₂ Protein Expression in Response to PMA in Cultured Myocytes

Figure 1A shows the effect of PMA on the pattern of PGHS-1, PGHS-2, and cPLA₂ proteins in the two smooth muscle layers of the bovine myometrium. Under basal conditions, without PMA stimulation, two immunoreactive bands were detected after immunoblotting with antiserum directed against cPLA₂ (approximately 85 kDa). After 6 h of PMA stimulation, only a single band with reduced mobility on SDS-PAGE was detected. After 24-h stimulation with the phorbol ester, corresponding to the maximal production of PGs [6], the signals were more intense, and two cPLA₂ immunoreactive bands, probably corresponding to the phosphorylated and unphosphorylated enzyme, were observed.

View larger version (29K): [in this window] [in a new window]   FIG. 1. Effect of PMA on steady state levels of cPLA2, PGHS-1, and PGHS-2 proteins in myocytes from the circular (C) and longitudinal (L) layers of bovine myometrium. When myocytes reached confluency, the culture medium was replaced by RPMI without serum, and incubation was performed for 24 h in the absence or presence of 100 nM PMA for the indicated time periods. Cells were lysed in homogenization buffer, and immunoblot analysis was performed in the presence of the respective antibodies as described in Materials and Methods. PLA2 and PGHS Western blots were run successively on the same membranes. Extracts from human platelet or interleukin-1-treated osteoblasts were used as PGHS-1 or PGHS-2 positive control, respectively (M). PG accumulation in the incubation media was measured by ELISA as described in Materials and Methods. Results are representative of one of 3 experiments.

A marked induction of immunoreactive PGHS-2 could be detected within 3 h after PMA treatment (data not shown), but it reached maximal levels after 6 h of incubation in the presence of phorbol ester. At that time point, the 69-kDa PGHS-2 protein appeared more abundant in the longitudinal than in the circular layer.

The polyclonal antibody used against PGHS-1 recognized a band of approximately 72 kDa corresponding to the migration point of the platelet PGHS-1 preparation that we used as a control. No significant induction of immunoreactive PGHS-1 was observed in response to PMA.

PMA (100 nM) induced a time-dependent stimulation of PGE₂ and PGI₂ (6-keto-PGF₁) synthesis (Fig. 1B). The maximal phorbol ester effect was obtained after 24-h stimulation, with a predominance of PGI₂ accumulation in the longitudinal layer (7200 ± 1800 pg/ml) in comparison to the circular layer (1600 ± 150 pg/ml), as previously described [6].

Effect of Exogenous Sex Steroid Hormones on Distinct PG Synthesis Pathways in Bovine Myometrium

The effects of steroid hormone E₂ alone or in combination with P₄ on PGHS-2 gene expression were assessed (Fig. 2). Myocytes from the circular and the longitudinal layers of bovine myometrium were treated with 1 nM E₂ (72 h) and/or 10 nM P₄ (24 h). The level of PGHS-2 mRNA was determined after incubation of the cells for 3 h in the presence of 100 nM PMA (Fig. 2A). Messenger RNA levels were higher in the longitudinal layer (0.57 ± 0.08; PGHS-2:18S relative optical density [ROD] units, n = 3) than in the circular layer (0.29 ± 0.07; PGHS-2:18S ROD units, n = 3) and were dramatically inhibited by 10 nM P₄ + E₂, but not by E₂ alone, in both layers (p < 0.05, n = 3). No effect of steroid hormone on PGHS-2 mRNA level was detected in the absence of PMA treatment (data not shown).

View larger version (51K): [in this window] [in a new window]   FIG. 2. Expression of PGHS-2 mRNA and protein in PMA-treated bovine myometrial cells; effect of steroid hormones. Confluent myometrial cells, from the circular (C) and the longitudinal (L) layers, were pretreated with 1 nM E2 (72 h), 1 nM E2 (72 h) with 10 nM P4 (for the last 24 h), or 100 nM DEX (24 h); and 100 nM PMA was added for the last 3 h (mRNA) or 6 h (protein) of incubation. A) Total RNA was isolated and hybridized with PGHS-2 cDNA probe. The 18S rRNA was used to normalize data. Results represent the mean ± SEM of 3 separate experiments, *p < 0.05. B) Western blotting was carried out as described in Materials and Methods. Analysis with PGHS-1, PGHS-2, and PGI synthase antibodies was performed successively on the same membranes.

The effect of steroid hormones on PGHS-2, PGHS-1, and PGI synthase protein levels was also assessed (Fig. 2B). Protein expression was analyzed after incubation of the cells for 6 h in the presence of 100 nM PMA. DEX, which is recognized as inhibiting PG production and PGHS-2 induction [13], was used as an inhibitory control. PGHS-2 protein level was higher in the longitudinal layer than in the circular layer and was dramatically inhibited by 10 nM P₄ with or without E₂ or 100 nM DEX but not by E₂ alone. By contrast, PGHS-1 and PGI synthase protein levels were not affected by steroid hormone treatments.

It is interesting to note that the effect of steroid hormones on PGHS-2 protein or mRNA level correlates well with PG accumulation in both myometrial layers (Table 1). Indeed, compared to those in the control group (6-h treatment with PMA in the absence of steroid), PG levels were not affected by E₂ treatment whereas P₄ or P₄ + E₂ significantly decreased the PG level in both layers.

Effect of Indomethacin and CGP 28238 on PGI₂ Accumulation

The accumulation of PGI₂ in myocytes cultured in the presence of PMA and increasing concentrations (0, 0.01, 0.1, 1, 10, 100 nM) of the PGHS inhibitors, indomethacin and CGP 28238, is illustrated in Figure 3. Both inhibitors induced a dose-dependent inhibition of PGI₂ accumulation. The inhibitory effect was observed at low concentrations of indomethacin (1 nM, p < 0.05) in comparison to the specific PGHS-2 inhibitor, CGP 28238, which was effective only at concentrations above 10 nM (p < 0.05). When the two inhibitors were combined, their effect was additive for concentrations lower than 1 nM, after which it was equal to the effect with indomethacin alone.

View larger version (23K): [in this window] [in a new window]   FIG. 3. Effect of PGHS inhibitors on PGI2 production in cultured myocytes. Myocytes from the longitudinal layers of bovine myometrium were incubated with CGP 28238 (solid circles), indomethacin (open circles), or CGP 28238 plus indomethacin (open squares) at the indicated concentrations, in the presence of 100 nM PMA. PGI2 production was measured after 24 h. Results represent the mean ± SEM of 3 separate experiments run in quadruplicate. Similar results were obtained in the circular layer. *p < 0.05 and **p < 0.01 when compared to untreated cells.

Effect of CGP 28238 and Indomethacin on PGHS-2 mRNA Levels

The pattern of PGHS-2 mRNA expression in bovine myometrium was evaluated after treatment with 10 nM CGP 28238 or indomethacin in the presence of PMA (Fig. 4). By analogy with previous Western blot analysis, the PGHS-2 transcript (4.1 kilobases) was more abundant in the longitudinal layer, confirming preferential expression of this enzyme in this smooth muscle layer. The level of PGHS-2 mRNA in PMA-stimulated myocytes was decreased by 44.80 ± 7.67% (p < 0.01, n = 5) in the longitudinal layer as compared to 27.83 ± 7.62% (p < 0.05, n = 6) in the circular layer after treatment with 10 nM CGP 28238. Treatment with 10 nM indomethacin affected the PGHS-2 mRNA level slightly but not significantly (p > 0.05, n = 3).

View larger version (29K): [in this window] [in a new window]   FIG. 4. Effect of CGP 28238 and indomethacin on PGHS-2 mRNA accumulation in bovine myometrium. Confluent myocytes from the circular (C) and longitudinal (L) myometrial layers were incubated for 24 h in the absence or presence of 10 nM CGP 28238 (open columns) or indomethacin (filled columns). During the last 3 h of incubation, 100 nM PMA was added. RNA extraction and Northern blot analysis were performed as described in Materials and Methods. Ten micrograms of total RNA was loaded in each lane. Each Northern blot was hybridized successively with PGHS-2 and 18S rRNA. Data are expressed as mean ± SEM from 5 or 6 separate experiments. *p < 0.05 and **p < 0.01 when compared to untreated cells.

	DISCUSSION

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The relative levels of mRNA and proteins corresponding to the expression of key enzymes involved in the biosynthesis of prostanoids were measured in bovine myometrium. One important feature of our approach was the use of primary cultures of separated myometrial layers. We have shown that the two smooth muscle layers have distinct patterns of PG production [6], and exploratory studies in our laboratory suggested that a subculture of myocytes could alter PGHS gene regulation (data not shown). Our study shows that the modulation of PG synthesis in the myometrium parallels some changes in cPLA₂. The reduced mobility of the band suggests that the phosphorylated form, corresponding to the fully active enzyme [20, 21], is stimulated by PMA. Consequently, after PMA treatment, increased PG production would be favored by increased availability both of the AA substrate through cPLA₂ and of its PGHS-2-converting enzyme. No rapid induction of PGHS-2 mRNA comparable to that with 6-h PMA treatment was observed after oxytocin treatment (data not shown). Our results describe both differences and similarities in the regulation of PG synthesis in the two myometrial layers. While increased expression of PGHS-2 in response to PMA is more evident in the longitudinal layer, the effect of steroids is comparable in the two layers. It is interesting to note in this respect that DEX and P₄ have comparable inhibitory effects. DEX is recognized as a potent inhibitor of PGI₂ production in human myometrium [22]. Because the levels of PGI synthase are comparable in the two muscle layers and do not vary with steroid treatments, it is tempting to conclude that the regulation of PGI₂ production in the myometrium is regulated through the expression of PGHS-2. It is also interesting if not intriguing to see that the selective PGHS-2 inhibitor CGP 28238 not only inhibited the converting activity [14] but also reduced the overexpression of PGHS-2 mRNA in response to PMA. Selective stimulation of PGHS mRNA and protein levels by PGE₂ has been demonstrated in osteoclastic MC3T3-E1 [23] and bone cells [24].

High amounts of PGs are produced by the endometrium [25] and other intrauterine tissues during pregnancy and may influence myometrial contractility. However, present and previous data [6] from our laboratory indicate the presence of endogenous PG regulatory pathways within the myometrium. An imbalance in the production of PG, particularly PGI₂, may lead to the modification of uterine reactivity to exogenous regulators of uterine contractility. This concept is supported by our previous observation that the longitudinal layer, which produces large amounts of PGI₂, concurrently expresses less connexin-43 [7].

Although we have not observed any modulation of PGHS-1 gene expression in the cyclic bovine myometrium, the role of this enzyme in the regulation of other reproductive processes such as parturition remains to be elucidated. There is good evidence demonstrating the importance of PGHS-1 in the maintenance of gestation in mice [26].

The availability of drugs that will selectively inhibit PGHS-1, in addition to the available PGHS-2 inhibitors, may contribute to a better understanding of the relative contribution of PGHS-1 and PGHS-2 isoforms to myometrial physiology. The present data clearly demonstrate that among the key enzymes involved in the biosynthesis of PG production, only PGHS-2 was responsive to steroid hormones. More interestingly, we indicated a double function of a specific PGHS-2 inhibitor acting both on the enzyme activity and on the enzyme expression. Taken together, these results suggest that a local regulation of myometrial PG production (via an up-regulation of PGHS-2), analogous to the local differential distribution of sex steroids evidenced in pregnant myometrium [27], may be instrumental in the study of uterine contractility regulation.

	ACKNOWLEDGMENTS

 
We thank Drs. T.G. Kennedy and J.M. Moutquin for their generous contribution of the PGE₂ and PGI₂ antisera and Dr J.A. Zidichouski (Ciba-Geigy Canada Ltd.) for providing CGP 28238. We thank Sandy Fraiberg for reviewing the English text.

	FOOTNOTES

 
¹ This work was supported in part by grants #OGP0183695 (F.D-B.) and #OGPIN030 (M.A.F.) from the Natural Sciences and Engineering Research Council of Canada (NSERC).

² Correspondence: Florence Doualla-Bell, Perinatal Research and Developmental, Pharmacology Unit, Lady Davis Institute for Medical Research, Jewish General Hospital, 3755 chemin de la Côte-Ste-Catherine, Montréal, PQ, Canada H3T 1E2. FAX: 514 340 7573; fdoualla{at}ldi.jgh.mcgill.ca

Accepted: August 6, 1998.

Received: January 28, 1997.

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