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Endocrinology Vol. 141, No. 11 4136-4145
Copyright © 2000 by The Endocrine Society

ARTICLES

Complementary Mechanisms of Enhanced Oxytocin-Stimulated Prostaglandin E2 Synthesis in Rabbit Amnion at the End of Gestation1

Yow-Jiun Jeng, Daniel Liebenthal, Zuzana Strakova2, Kirk L. Ives, Mark R. Hellmich and Melvyn S. Soloff

Department of Obstetrics and Gynecology (Y.-J.J., D.L., Z.S., M.S.S.), Sealy Center for Molecular Science (M.S.S.), and the Department of Surgery (K.L.I., M.R.H.), University of Texas Medical Branch, Galveston, Texas 77555-1062

Address all correspondence and requests for reprints to: Dr. Melvyn S. Soloff, Department of Obstetrics and Gynecology, University of Texas Medical Branch, 301 University Boulevard, Galveston, Texas 77555-1062. E-mail: msoloff{at}utmb.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The up-regulation of oxytocin (OT) receptors in rabbit amnion at the end of gestation is associated with a large increase in the ability of OT to stimulate PGE2 synthesis. The purpose of these investigations was to determine what other factors contribute to this increase. OT enhanced PGE2 synthesis at several levels. The concentrations of cytosolic phospholipase A2, which generates arachidonic acid for PGE2 synthesis, and PGH endoperoxide synthases (types 1 and 2), which catalyze the conversion of arachidonic acid to prostanoids, rose substantially in rabbit amnion at term. OT stimulated translocation of cytosolic phospholipase A2 to the cell particulate fraction, presumably by a Ca2+-mediated process, and phosphorylation of cytosolic phospholipase A2 via the extracellular regulated protein kinase 2/1-mediated pathway. OT-stimulated increases in intracellular Ca2+ concentrations and extracellular regulated protein kinase 2/1 phosphorylation were both mediated by Gq/11 activation. OT also increased the expression of PGH endoperoxide synthase-2 after treatment of amnion cells in culture for 2 h; however, PGE2 release in response to OT was virtually immediate. These findings show that the rapid stimulation of PGE2 synthesis by OT occurs through cytosolic phospholipase A2 activation and PGH endoperoxide synthase-1 activity, both of which, along with OT receptor concentrations, are considerably up-regulated in the amnion at the end of gestation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THERE IS A substantial increase in oxytocin (OT)-stimulated PGE2 synthesis by rabbit amnion at the end of gestation (1), suggesting that both OT and PGE2 are important for processes associated with parturition or the termination of pregnancy in rabbits. Experiments using mice deficient in the ability to respond to PGF or to synthesize it have clearly illustrated the requirement of PGs in the initiation of labor in mice (2, 3). We have shown previously that the increased PGE2 response to OT by rabbit amnion cells is due in part to the marked up-regulation of OT receptor (OTR) ligand-binding sites and messenger RNA (mRNA) concentrations (1, 4). Epidermal growth factor (EGF) also stimulates PGE2 production, but only in amnion cells taken from rabbits several days before parturition and not at the end of gestation (5). As there was no marked reduction in the number of EGF receptor sites at term (5), the uncoupling of signal transduction pathways from EGF receptors appears to occur. OT therefore appears to be the major stimulus of PGE2 synthesis by amnion tissue at the end of pregnancy.

As pregnancy proceeds, the concentrations of cortisol and agents that stimulate cAMP synthesis increase in amniotic fluid (6). These factors are probably the physiological regulators of OTR concentrations in the amnion, as the addition of cortisol and/or forskolin to rabbit amnion cells in primary culture up-regulates OTR ligand-binding sites and the OTR mRNA concentration (4, 5). Both agents increase the rate of transcription of the OTR gene, as measured by either nuclear run-on synthesis or the incorporation of 4-thioruridine into nascent RNA (4). The addition of cortisol and forskolin to rabbit amnion cells in culture results in about a 200-fold increase in ligand-binding sites, but there is more than a 5000-fold increase in PGE2 synthesized in response to the addition of OT (5). One interpretation of these results is that agents that up-regulate OTR concentrations also up-regulate intracellular pathways associated with PGE2 synthesis. The two major enzyme activities involved in PGE2 production are cytosolic phospholipase A2 (cPLA2), which generates arachidonic acid from membrane glycerophospholipids, and PG endoperoxide H synthase (PGHS; cyclooxygenase), a bifunctional enzyme catalyzing both the oxidation of arachidonic acid to the PG endoperoxide (PGG2) and its subsequent reduction to PGH2. The latter is the precursor of all biologically active PGs and thromboxanes. Two PGHS isoforms have been characterized in considerable detail (7, 8, 9, 10, 11). PGHS-1 is constitutively expressed in most cell types, whereas PGHS-2 mRNA is rapidly and transiently induced. Normally, PGHS-2 is undetectable, but is rapidly induced by cytokines, growth factors, hormones, bacterial endotoxin, and phorbol ester (12). Depending on the cell type and stimulus, either cPLA2 or PGHS can be rate limiting with respect to PG synthesis. Our goal was to determine whether OT-stimulated production of PGE2 by amnion tissue at the end of pregnancy and by cultured amnion cells pretreated with cortisol and forskolin is due at least in part to up-regulation of PG- synthesizing enzymes in the rabbit amnion at term. Both mRNA and protein levels of cPLA2, PGHS-1, and PGHS-2 were measured in amnions taken from rabbits in the latter part of pregnancy and in cultured amnion cells.

Cytosolic PLA2 catalysis involves its translocation from the cytosol to membrane binding sites, wherein lie glycerophospholipid substrates, and activation by phosphorylation. Translocation is mediated by an increase in intracellular Ca2+ concentration, whereas activation results from phosphor-ylation of Ser505 by mitogen-activated protein kinases. Depending on the cell type and effectors, either extracellular-regulated protein kinases (ERK) or p38 (also known as stress-activated protein kinase-2a) phosphorylates cPLA2 (13, 14). Along with stimulating PGE2 synthesis in Chinese hamster ovary (CHO) cells stably transfected with the rat OTR (CHO-OTR cells), OT rapidly increases the intracellular Ca2+ concentration and stimulates both ERK and p38 phosphorylation (15). OT-stimulated PGE2 synthesis in CHO-OTR cells is dependent primarily on a Gq/11/protein kinase C/ERK phosphorylation sequence of events (15). Deletion of 51 amino acid residues from the carboxyl-terminus of the rat OTR resulted in the inability of OT to stimulate PGE2 synthesis in CHO cells (16). Analysis of signaling pathways in the mutant cell line indicated that the modified receptor was not coupled to Gq/11, but remained coupled to pertussis toxin (PTX)-sensitive G protein (16). It can be argued that signal pathways mediating OT effects in cells transfected with the OT receptor are not necessarily representative of those that occur in natural target cells. To determine the mechanisms of enhanced PGE2 production, we have examined here the role of OT in the translocation and phosphorylation of cPLA2, mitogen-activated protein (MAP) kinases that are involved in activating cPLA2, and the heterotrimeric G protein type(s) that mediates OT action in a physiological target, i.e. rabbit amnion cells. We found that, like signaling in CHO-OTR cells, coupling of the OTR to a Gq/11/protein kinase C (PKC)/ERK pathway is required to stimulate cPLA2 and increase PGE2 synthesis in amnion cells. In summary, OT-stimulated PGE2 synthesis by OT in rabbit amnion cells at the end of gestation is increased at the level of both increased expression of PG-synthesizing enzymes and activation of cPLA2.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Reagents were obtained from the following sources: OT and OT antagonist, [d(CH2)5,Tyr(Me)2,Thr4,Tyr-NH29]ornithine vasotocin, Peninsula Laboratories, Inc. (Belmont, CA); PTX, GF 109203X, GP antagonist-2A, and phorbol 12-myristate 13-acetate, BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA); PD-908059, New England Biolabs, Inc. (Beverly, MA); and DMEM, Life Technologies, Inc. (Grand Island, NY). Inhibitors of arachidonic acid synthesis, including AACOCF3, aristolochic acid (8-methoxy-6-nitrophenanthro-[3,4-d]-dioxole-5-carboxylic acid), cytidine 5'-diphosphocholine, haloenol lactone suicide substrate (E-6-[bromo-methylene]-tetrahydro-3-[1-naththalenyl]-2H-pyran-2-one), oleyloxyethyl phosphorylcholine, and RHC-80267 (1,6-bis-[cyclohenyloximinocarbonyl-amino]hexane) were also purchased from BIOMOL. [3H]Arachidonic acid (200 Ci/mmol) was purchased from NEN Life Science Products (Boston, MA). All other chemicals were obtained from Sigma (St. Louis, MO).

Tissue and cell preparation
Timed pregnant New Zealand rabbits (Myrtle’s Rabbitry, Thompson Station, TN) were received on day 16 of pregnancy and killed on prescribed days of pregnancy. The rabbits were treated in accordance with the NIH Guide for the Care and Use of Laboratory Animals. The research protocol was approved by the institutional committee on animal care and use, University of Texas Medical Branch. Amnion cells were dispersed from tissue taken on day 30 of pregnancy unless otherwise noted and cultured as described previously (17). About 8 million cells were plated onto 10-cm tissue culture plates, and the cells were maintained for up to 2 weeks in DMEM containing 5% FBS, penicillin (100 U/ml), and streptomycin (100 µg/ml) at 37 C (95% humidity) in the presence of 5% CO2. The cells were serum-starved overnight (about 16 h) before OT treatment.

mRNA levels
Cytosolic PLA2 mRNA levels were measured by Northern blot analysis. PGHS-1 and PGHS-2 mRNA levels were determined by using ribonuclease protection assays by methods described previously (4). The rabbit cPLA2 probe was prepared from a clone obtained from Dr. Zhongmin Ma, Washington University (St. Louis, MO). Rabbit PGHS-1 and PGHS-2 complementary DNA clones for synthesis of riboprobes were gifts from Dr. Matthew D. Breyer, Vanderbilt University (Nashville, TN).

Immunoblotting
Cytosolic PLA2, PGHS-1, and PGHS-2; dually phosphorylated ERK2/1; dually phosphorylated p38; p38; and PKC{alpha} protein levels were measured by Western blotting using specific antibodies: monoclonal antibody to cPLA2 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), monoclonal PGHS-1 and polyclonal PGHS-2 antibodies (Cayman Chemical Co., Ann Arbor, MI), monoclonal antibodies specific for dually phosphorylated ERK2/1 (Santa Cruz Biotechnology, Inc.), dually phosphorylated p38 and p38 antibodies (New England Biolabs, Inc., Beverly, MA), and monoclonal antibody to PKC{alpha} (BD Transduction Laboratories, Lexington, KY). The appropriate species-specific second antibodies coupled to horseradish peroxidase were obtained from Amersham Pharmacia Biotech (Arlington Heights, IL). The crude membrane preparations for cPLA2 and PKC translocation assays were prepared as described previously for PKC translocation (16). Detection of phosphorylated cPLA2 by electrophoretic mobility retardation was carried out by SDS-PAGE using 10% polyacrylamide gels (~15 cm long) and running the samples until a 78-kDa prestained marker protein migrated at about 10 cm.

Arachidonic acid release
Arachidonic acid release was measured by preincubating cells with [3H]arachidonic acid (1 µCi/250 µl·well) for 16 h. After rinsing the cells in PBS, the cells were incubated with inhibitors for 30 min. OT (100 nM) was then added, and the amount of radioactive arachidonic acid released into the medium was measured after 15 min, as described previously (18).

PGE2 assay
PGE2 concentrations were measured using an enzyme immunoassay system (Amersham Pharmacia Biotech, Aylesbury, UK), as described previously (15).

Statistical analysis
Experiments were repeated in triplicate using cells obtained from at least three rabbits. One-factor ANOVA was used to test the overall hypothesis of no group differences, followed by two-sample t tests for pairwise comparisons where the overall hypothesis was rejected. All tests were made at the 0.05 level of significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
cPLA2 mediates OT-stimulated PGE2 synthesis in cultured amnion cells
Addition of OT to amnion cells resulted in the activation of phospholipase activity, as measured by increased [3H]arachidonic acid release (Fig. 1Go). OT caused an increase in activity from 46% to 132% above nontreated control values after 15 min of stimulation. The type of phospholipase involved was determined using specific inhibitors. These included drugs inhibiting cPLA2, Ca2+-independent PLA2, secretory PLA2, ionophore-stimulated PLA2, brain PLA2, and diacylglyerol lipase (Fig. 1Go). Only the cPLA2 inhibitor was effective in blocking the effects of OT on arachidonic acid release. Accordingly, cPLA2 was the only phospholipase examined further.



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  		Figure 1. Identification of cPLA2 as the mediator of OT-stimulated arachidonic acid release by rabbit amnion cells. Amnion cells were loaded with [3H]arachidonic acid for 16 h. After rinsing of the cells, the inhibitors were added for 30 min, and the cells were then treated with OT (100 nM) for 15 min. The amount of radioactivity released into the medium was then determined. The inhibitors and concentrations were as follows: AACOCF3 (AAC; 25 µM), haloenol lactone suicide substrate (HELSS; 25 µM), oleyloxyethyl phosphorylcholine (OP; 25 µM), aristolochic acid (ARIS; 25 µM), CDP-choline (CDPC; 25 µM), RHC-80267 (RHC; 40 µM). *, P < 0.05, comparing OT-treated vs. nontreated cells.

 
Changes in cPLA2, PGHS-1, and PGHS-2 mRNA and protein levels in amnion during gestation
Cytosolic PLA2, PGHS-1, and PGHS-2 mRNA levels in rabbit amnion tissue increased during pregnancy and were maximal at the end of gestation (Fig. 2AGo). The increases in all three mRNAs were about 10-fold (Fig. 2BGo). The SE for the cPLA2 value on day 31 is larger because of the single relatively low value shown in Fig. 2AGo. Western blot analyses of cPLA2, PGHS-1, and PGHS-2 showed that the protein concentrations generally followed mRNA levels in amnion tissue during pregnancy (Fig. 2CGo).



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  		Figure 2. Changes in cPLA2, PGHS-1, and PGHS-2 mRNA (A and B) and protein (C) levels in rabbit amnion in the latter part of gestation. Cytosolic PLA2 was determined by Northern blot analysis, whereas PGHS-1 and PGHS-2 mRNA levels were measured by ribonuclease protection. ß-Actin and 18S RNA were used as reference standards for the Northern and ribonuclease protection assays, respectively. Protein levels were detected by immunoblotting. The values shown in B are the mean ± SE of at least three replicates. *, P < 0.05, compared with value on day 23 of pregnancy.

 
cPLA2, PGHS-1, and PGHS-2 mRNA and protein levels in amnion cells
Amnion cells were prepared on day 27 of pregnancy when cPLA2, PGHS-1, and PGHS-2 mRNA and protein levels are relatively low (Fig. 2Go, A and C). To determine whether the enzymes are up-regulated by the same mechanisms that increase OTR concentrations (4), the cells were treated with forskolin (25 µM) and cortisol (100 nM). These agents did not affect cPLA2, PGHS-1, or PGHS-2 protein (Fig. 3AGo) or mRNA levels (data not shown). However, subsequent treatment of the cells with OT (100 nM) caused a time-dependent increase in both PGHS-2 protein (Fig. 3BGo) and mRNA concentrations (data not shown). OT had no effect on cPLA2 or PGHS-1 mRNA or protein levels (Fig. 3BGo). Half-maximal stimulation occurred between 1 and 5 nM OT (data not shown), consistent with the apparent Kd value for [3H]OT binding in other tissues (19).



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  		Figure 3. A, Lack of effect of cortisol (100 nM) and forskolin (25 µM) on cPLA2, PGHS-1, and PGHS-2 levels in day 27 amnion cells. The lanes indicate increasing times after addition of the two agents. Protein concentrations were determined using immunoblotting. B, Effect of OT (100 nM) on PGHS-2 protein levels in day 27 amnion cells that were treated overnight with cortisol and forskolin. OT had no effect on either cPLA2 or PGHS-1 protein levels.

 
An examination of the time course of OT-stimulated PGE2 release showed that the effects of OT were virtually immediate, with concentrations of PGE2 in the medium reaching near-maximal values by 15 min (Fig. 4AGo). There was no further increase in PGE2 synthesis at 2 h, when OT induced PGHS-2 expression (Fig. 4AGo). Basal PGE2 levels increased sharply at about 2 h and remained high thereafter (Fig. 4AGo) despite the fact that in the absence of OT there was little induction of PGHS-2. The abrupt rise in basal PGE2 synthesis was very striking and reproducible and may be due to the release of autocrine/paracrine effectors of PGE2 synthesis into the medium. Our preliminary experiments showed that this basal PGE2 secretion was inhibited by peroxicam, a slightly selective PGHS-1 inhibitor (data not shown). The lack of involvement of PGHS-2 in OT-stimulated PGE2 production was further indicated by the absence of increased PGE2 formation from arachidonic acid in cells that were preincubated with OT for 8 h to up-regulate PGHS-2 (Fig. 4BGo). Thus, unlike other cell types in which OT stimulation of PGHS-2 synthesis is associated with increased PG synthesis (20, 21, 22), the significance of PGHS-2 induction by OT in amnion cells is not presently known.



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  		Figure 4. A, Time course of OT (100 nM)-stimulated PGE2 release. B, Lack of effect of OT-directed up-regulation of PGHS-2 on PGE2 synthesized from exogenous arachidonic acid. Day 30 rabbit amnion cells were pretreated with OT for 8 h to induce PGHS-2 synthesis. The control group was not pretreated. Arachidonic acid (1, 10, or 100 µM) was then added, and the amount of PGE2 released into the medium after 15 min was measured.

 
OT stimulation of increases in intracellular Ca2+ and translocation of cPLA2
Ca2+-mediated translocation of cPLA2 from the cytosol to the endoplasmic reticulum and nuclear envelope has been shown to be important for bringing cPLA2 into contact with glycerophospholipid substrates (23). OT stimulated an increase in intracellular Ca2+ concentration in amnion cells taken on day 30 of gestation (either 1 day before or on the day of parturition; Fig. 5AGo). This activity was not affected by overnight pretreatment of the cells with PTX (0.5 µg/ml), an inhibitor of Gi/o activation (Fig. 5AGo), or by 5-min preincubation of the cells with GF 109203X (5 µM), a protein kinase C inhibitor (Fig. 5BGo). In contrast, a 5-min preincubation with U73122 (4 µM), a phospholipase C inhibitor, blocked the OT-stimulated rise in intracellular Ca2+ (Fig. 5BGo). Injection of the cells with a selective Gq inhibitory peptide (GP antagonist-2A, 25 µM) greatly reduced the response of the cells to 100 nM OT (Fig. 5CGo). In contrast, injection of the cells with an equivalent volume of Texas Red had no effect (Fig. 5CGo).



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  		Figure 5. OT-stimulated increases in intracellular Ca2+ concentration in primary cultures of rabbit amnion cells taken on day 30 of gestation. A, The response to OT (10 nM) was not affected by pretreatment with PTX (0.5 µg/ml). Relative intracellular Ca2+ concentrations were determined by the 340/380 nm ratio. The values are the mean ± SE of 37 or 40 individual cells. B, Complete inhibition of the OT response by preincubation of amnion cells with U73122 (4 µM) for 5 min. The PKC inhibitor, GF 109203X (5 µM), had no significant effect on the Ca2+ response. The values are the mean ± SE for 17 individual cells in each group. C, Inhibition of OT-stimulated Ca2+ transients by injection of a selective Gq/11 antagonist. Control cells injected with Texas Red showed no diminution in response to OT. The values are the mean ± SE for 15 individual cells in each group.

 
The addition of 100 nM OT to day 30 amnion cells resulted in an increased concentration of cPLA2 associated with the crude membrane fraction, as determined by immunoblotting (Fig. 6AGo). The amount translocated was only a fraction of that possible, as elevation of intracellular Ca2+ concentration by treating cells with the calcium ionophore A23187 (10 µM) resulted in a substantially greater amount of cPLA2 associated with the membrane fraction (Fig. 6AGo). Uniformity of loading of the gels was verified by immunostaining the same blots for PGHS-1. OT-induced translocation of cPLA2 was not affected by pretreating the cells with either PTX (0.2 or 0.5 µg/ml) overnight or GF 109203X (1 µM) for 30 min (Fig. 6BGo). However, pretreatment with 10 µM GF 109203X blocked OT-stimulated cPLA2 translocation (Fig. 6BGo). Treatment of amnion cells with 1 and 10 µM GF 109203X progressively increased the amount of PKC{alpha} translocated to the cell membrane fraction (Fig. 7Go). OT stimulation of PKC translocation was inhibited by 10 µM GF 109203X, but not by 1 µM, possibly accounting for the lack of effect of 1 µM GF 109203X on cPLA2 translocation. The role of PKC activation in cPLA2 translocation is not clear at the present time.



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  		Figure 6. A, Effect of OT (100 nM) on the association of cPLA2 with the membrane fraction of amnion cells at 5 and 15 min after treatment. Cytoplasmic PLA2 translocation caused by elevation of intracellular Ca2+ concentrations with the Ca2+ ionophore, A23187 (10 µM), is also shown. To show uniformity of loading in the immunoblots, the nylon membranes were stripped and reprobed with antibody to PGHS-1. B, Neither PTX (0.2 or 0.5 µg/ml) nor GF109203X (1 µM) had an effect on OT-stimulated cPLA2 translocation. However, pretreatment of cells with a higher concentration of GF 109203X (10 µM) inhibited OT-stimulated cPLA2 translocation. The vehicle control for GF 109203X, dimethylsulfoxide (DMSO), is also shown.

 


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  		Figure 7. Inhibition of OT-stimulated PKC{alpha} translocation by GF 109203X. Basal translocation of PKC{alpha} was stimulated by both 1 and 10 µM GF109203X, but only 10 µM GF 109203X inhibited OT-stimulated PKC{alpha} translocation. The cells were pretreated with GF 109203X or dimethylsulfoxide vehicle for 30 min before stimulation with OT (100 nM) for 5 min. The membranes were stripped and reprobed with antibody to PGHS-1 to show protein uniformity in each lane. One group of cells was treated with phorbol 12-myristate 13-acetate (PMA) for 5 min as a positive control for PKC translocation.

 
OT-stimulated MAP kinase phosphorylation
Cytosolic PLA2 is activated by a wide variety of agents that stimulate its phosphorylation on Ser505 by MAP kinases (13, 24, 25). To determine the identity of the MAP kinase potentially involved in OT-stimulated PLA2 phosphorylation, immunoblotting of amnion cell lysates was performed using antibodies to dually phosphorylated ERK2/1 and p38 MAP kinases. Treatment of rabbit amnion cells with OT (100 nM for 5 min) resulted in the phosphorylation of ERK2 and ERK1 (pp42 and pp44, respectively; Fig. 8AGo) and p38 (Fig. 8BGo). Phosphorylation of ERK2/1 was completely inhibited by the MEK1 inhibitor PD98059 (10 µM) and was partially blocked by the p38-selective inhibitor SB202190 (1 µM; Fig. 8AGo). In contrast, OT-stimulated p38 phosphorylation was completely blocked by SB202190 and was partially blocked by PD98059 (Fig. 8BGo). Preincubation of cells with PTX (0.5 µg/ml, overnight) reduced OT-stimulated phosphorylation of ERK2/1 (Fig. 8CGo). The selective PKC inhibitor, GF 109203X (10 µM), also partially reduced OT-stimulated ERK2/1 phosphorylation (Fig. 8CGo). There was no apparent effect of PTX on OT-stimulated p38 phosphorylation. However, basal levels of phosphorylated p38 were increased, suggesting that Gi/o normally suppress basal levels of p38 phosphorylation (Fig. 8DGo). GF 109203X had no effect on OT-stimulated p38 phosphorylation (Fig. 8DGo). The tyrosine protein kinase inhibitor genestein (10 µM) had no effect on OT stimulation of either ERK2/1 or p38 phosphorylation (data not shown), indicating that the process is specific for Gq/11 {alpha}-subunits and not Gq/11 ß{gamma}-subunits (26).



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  		Figure 8. OT-stimulated phosphorylation of ERK2/1 (A) and p38 (B) MAP kinases as shown by immunoblot analyses using antibodies to the dually phosphorylated MAP kinases. The effects of OT on ERK1/2 and p38 phosphorylation were completely blocked by PD98059 (10 µM) and SB202190 (1 µM), respectively (A and B). The effects of pretreatment with PTX (0.5 µg/ml) and GF 109203X (1 and 10 µM) on OT-stimulated ERK2/1 (C) and p38 (D) phosphorylation were also studied. Immunostaining for total p38 was used as a loading/transfer control.

 
Pathways mediating OT-stimulated PLA2 phosphorylation
Phosphorylated cPLA2 has a retarded electrophoretic mobility compared with the nonphosphorylated form, allowing the two to be distinguished from each other (24, 27). Incubation of day 30 amnion cells with OT stimulated cPLA2 phosphorylation within 1 min of treatment (Fig. 9AGo). This effect was maintained for at least 5 min. Pretreatment of cells with PD98059 resulted in complete inhibition of OT-stimulated PLA2 phosphorylation; SB202190 was without effect at a dose that completely blocked p38 phosphorylation (Fig. 9BGo). These findings indicate the importance of ERK2/1 and not p38 in cPLA2 phosphorylation. Inhibition of PKC activity by GF 109203X (10 µM) also blocked OT-stimulated cPLA2 phosphorylation (Fig. 9CGo), consistent with the preceding data showing the role of ERK2/1 in cPLA2 phosphorylation. Although PTX treatment partially inhibited OT-stimulated ERK2/1 phosphorylation (Fig. 8CGo), it had no effect on OT-stimulated cPLA2 phosphorylation (Fig. 9BGo).



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  		Figure 9. Pathways mediating OT-stimulated phosphorylation of cPLA2. A, Phosphorylation, which was measured by retardation of the electrophoretic mobility of cPLA2 after immunoblot analysis, was apparent within 1 min after OT treatment. B, Pretreatment of amnion cells with PD98059 (10 µM) blocked OT-stimulated phosphorylation of cPLA2, whereas SB202190 (1 µM) had no effect. Phosphorylation was measured once at 5 min. C, Pretreatment of amnion cells with PTX (0.2 or 0.5 µg/ml) had no effect on OT-stimulated cPLA2 phosphorylation, whereas 10 µM GF 109203X was completely inhibitory.

 
Inhibition of OT-stimulated PGE2 synthesis by the same inhibitors that block cPLA2 phosphorylation
Both PD98059 (10 µM) and GF 109203X (10 µM) were potent inhibitors of OT-stimulated PGE2 synthesis in rabbit amnion cells (Fig. 10Go). These results further indicate a causal relationship between the phosphorylation of cPLA2 and PGE2 synthesis.



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  		Figure 10. Inhibition of OT (100 nM)-stimulated PGE2 synthesis in day 30 rabbit amnion cells by preincubation with PD98059 (10 µM) or GF 109203X (10 µM) for 30 min. PGE2 was measured in the medium 10 min after addition of OT.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previous work has shown that OTRs in rabbit amnion are up-regulated about 200-fold at the end of gestation (17). These results explain in part the extraordinary increase in OT-stimulated PGE2 production by rabbit amnion cells at term. The present studies indicate that up-regulation of cPLA2 and PGHS-1 concentrations also contribute to increased PGE2 synthesis. The up-regulation of cPLA2 and OT receptors allows enhanced activation of cPLA2 through OT-induced Gq/11 activation, which results in transient increases in intracellular Ca2+ concentrations and cPLA2 translocation. Increased PKC activity resulting from Gq/11 activation also leads to successive ERK2/1 and cPLA2 phosphorylations. The resulting activated cPLA2 catalyzes increases in arachidonic acid levels, which leads to greater PGE2 synthesis through up-regulated PGHS. Although the concentrations of both types of PGHS rose in the amnion at the end of pregnancy, it is likely that PGHS-1 is the only form that mediates OT-stimulated PGE2 synthesis, at least in cultured amnion cells.

Exposure of cultured amnion cells to cortisol and forskolin elevates OT receptor concentrations (4). This treatment also causes a disproportionately large rise in OT-stimulated PGE2 synthesis (5). As cortisol and forskolin treatment did not up-regulate PG-synthesizing enzymes, the mechanisms of enhanced PGE2 production might reside at the level of G protein-coupled/OT receptor interactions. These mechanisms remain to be characterized. Indeed, agents up-regulating cPLA2 and PGHS-1 synthesis in the rabbit amnion at the end of gestation are not presently known. Cytosolic PLA2 is a widely distributed enzyme that is expressed in all human tissues (28, 29). A variety of cytokines and mitogens have been shown to both induce activation and increase the synthesis of cPLA2 in different cell types (30). In some cell types, concomitant up-regulation of cPLA2 and PGHS-2 occurs, suggesting that both genes are induced by the same stimuli (30). The proximity of both genes on human chromosome 1q25 (31) lends support to this idea. However, only PGHS-2 was induced by OT treatment in amnion cells, suggesting that there are independent pathways for regulating cPLA2 and PGHS-2 expression in these cells.

PGHS-1 is generally considered to be constitutively expressed, but as the present studies and others have shown, it is also developmentally regulated (32). OT induction of PGHS-2 expression has been shown to coincide with stimulation of PG synthesis in several cell types. For example, OT stimulation of PGF2{alpha} synthesis by cultured bovine endometrial cells was attributed to OT induction of PGHS-2 (20). Similar conclusions were obtained from studies in which OT was administered to sheep (21). OT induction of PGHS-2 has also been shown to mediate OT-stimulated prostacyclin production by human uterine smooth muscle cells in culture (22). Generally, utilization of PGHS-1 is associated with the early phase of PG synthesis, occurring within several minutes of stimulation, whereas PGHS-2-dependent PG synthesis proceeds over several hours in parallel with the induction of PGHS-2 expression (33, 34, 35, 36, 37). However, in our studies OT induction of PGHS-2 synthesis did not enhance PGE2 synthesis over the course of a number of hours. A lack of correlation between induction of PGHS-2 expression and PG synthesis has also been observed in other cell types. In murine 3T3 cells, maximal induction of PGHS-2 caused only a relatively small increase in net prostanoid synthetic capacity (7). Similarly, there was a lack of correlation between induction of PGHS-2 expression in transformed rat liver cells and PGE2 production. On the basis of these findings, Smith and co-workers concluded that it is unlikely that PGHS-2 is induced solely to augment PG synthesis catalyzed by PGHS-1 (7). Because PGHS-1 and -2 are often coexpressed in the same cell, it has been suggested that they may act as parts of separate prostanoid biosynthetic systems, using separate arachidonic acid pools, that function independently to channel prostanoids to the extracellular milieu (PGHS-1) and the nucleus (PGHS-2) (7). In support of this concept, the two forms of PGHS are coupled to different extracellular stimuli and appear to operate independently. Alternative functions of PGHS-2 in amnion cells remain to be determined.

Previous studies from this laboratory have shown that the OTR is coupled to both Gi/o and Gq/11 heterotrimeric proteins in the rat myometrium (38) and in CHO cells transfected with the rat OTR (16). Using a carboxyl-terminal truncated mutant of rat OTR, it was possible to demonstrate converging as well as distinct signal pathways emanating from Gi/o and Gq/11 activation (16). Neither OT stimulation of intracellular Ca2+ transients nor ERK2/1 phosphorylation in rabbit amnion cells were affected by PTX, indicating that Gi/o does not mediate these processes. Instead, increases in intracellular Ca2+ concentrations were completely blocked by Gq/11 and phospholipase C inhibitors. The PKC inhibitor GF 109203X blocked both OT-stimulated ERK2/1 and cPLA2 phosphorylation, indicating that these processes are also linked to Gq/11/phospholipase C activation. Thus, both the translocation and activation of cPLA2 were mediated by Gq/11. Although CHO cells stably transfected with the rat OTR are a contrived cell system to study OTR-regulated signal pathways, natural OT target cells such as rabbit amnion appear to share the same pathways with respect to stimulation of PGE2 synthesis.

In some cell systems, the phosphorylation of cPLA2 is mediated by p38 (13, 14). Although OT stimulated p38 phosphorylation in rabbit amnion cells, complete inhibition of p38 phosphorylation by SB202190 had no effect on OT-stimulated cPLA2 phosphorylation. The significance of OT-stimulated p38 phosphorylation in rabbit amnion cells remains to be established. It seems clear that the process is not mediated by PKC, but we cannot determine whether Gi/o is involved, as PTX pretreatment elevated basal levels of p38 phosphorylation. Inhibition of ERK2/1 phosphorylation by PD98059 resulted in inhibition of cPLA2 phosphorylation, indicating a causal relationship. Pretreatment of cells with either PTX or GF 109203X partially inhibited ERK2/1 phosphorylation, yet only GF 109203X blocked cPLA2 phosphorylation. These findings indicate that there might be more than one pool of ERK2/1, one phosphorylated by a Gi/o-mediated step and the other phosphorylated by a Gq/11/PKC-dependent pathway. Only the latter appears to be associated with cPLA2 phosphorylation. Because a fraction of phosphorylated ERK2/1 is translocated from the cytosol to the nuclear compartments upon stimulation (39, 40), we might surmise that the two compartments are distinct targets for PTX-sensitive and PKC-sensitive phosphorylation.

In summary (Fig. 11Go), the increased ability of rabbit amnion cells to produce PGE2 in response to OT treatment at the end of gestation involves complementary mechanisms acting at several levels. The major components, OT receptors, cPLA2, and PGHS-1, are all up-regulated near term. In conjunction with the up-regulation of OTRs, there is enhanced Ca2+ signaling and activation of PKC. These two processes, which are stimulated by OT via Gq/11 activation, result in increased translocation and phosphorylation of cPLA2 (via ERK2/1 phosphorylation), respectively. Increased levels of cPLA2 and PGHS-1 thereby allow commensurate increases in arachidonic acid and PGE2 levels, respectively. As a result, the rabbit amnion become strikingly sensitive to OT at the end of pregnancy through coordinated events that lead to a large increase in the capacity to produce PGE2.



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  		Figure 11. Model of OT action in rabbit amnion cells. OT-stimulated increases in intracellular Ca2+, translocation of cPLA2 to the membrane fractions, and phosphorylation of cPLA2 require coupling of the OTR to Gq/11. The phosphorylation of cPLA2 is mediated by PKC and ERK2/1 phosphorylation. OT also stimulates p38 phosphorylation, but the significance of p38 activation in amnion cells is not presently known. At the end of gestation, up-regulation of OTR, cPLA2, PGHS-1, and PGHS-2 (arrows) contributes to the greatly increased capacity of amnion cells to synthesize PGE2 in response to OT treatment. Up-regulation of PGHS-2 results from OT stimulation, but the function of PGHS-2 in rabbit amnion cells is currently unknown. The translocated form of cPLA2 is signified by cPLA2Ca; phosphorylated forms are indicated by the addition of a circled P. PL, Plycerophospholipid; AA, arachidonic acid.

 


   Footnotes
 
1 This work was supported in part by NIH Grant HD-26168 (to M.S.S.). Back

2 Present address: Department of Obstetrics and Gynecology, University of Illinois, Chicago, Illinois 60612-7313. Back

Received February 17, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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