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EP1 Receptor-Mediated Migration of the First Trimester Human Extravillous Trophoblast: The Role of Intracellular Calcium and Calpain

Departments of Anatomy and Cell Biology (C.N., A.V.T., P.K.L.), Pathology (C.C.), and Physiology and Pharmacology (S.J.D.), University of Western Ontario, London, Canada N6A 5C1

Address all correspondence and requests for reprints to: Chandan Chakraborty, Department of Pathology, University of Western Ontario, London, Canada N6A 5C1. E-mail: cchakrab{at}uwo.ca.

	Abstract

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Context: The root cause of preeclampsia in the human lies in the placenta, where a subpopulation of cytotrophoblast cells called extravillous trophoblasts (EVT), known to be involved in the invasion of the uterine endometrium and utero-placental arteries, become less invasive, resulting in poor perfusion of maternal blood into placenta.

Objectives: Because EVT migrate into the prostaglandin (PG) E₂-rich decidua, we tested the roles of PGE₂ and PGE₂-mediated signaling in EVT migration, using our well-characterized EVT line HTR-8/Svneo as well as first trimester villus explants in culture.

Design: mRNA expression of different PGE₂ receptors (EPs) in HTR-8/Svneo cells was studied using RT-PCR. To characterize the functional significance of EP receptors in EVT, different EP receptor agonists and antagonists were used in our migration assay systems and in the measurements of intracellular concentration of Ca²⁺ ([Ca²⁺]_i) and calpain activity.

Results: Exogenous PGE₂ stimulated EVT migration both in vitro and in the villus explant cultures. Although EVT expressed mRNA for all EP receptors (EP 1–4), a functional predominance of EP1 and EP4 was demonstrated in migration assays using specific EP agonists and antagonists. EP1-receptor-mediated signaling events such as activation of phospholipase C and elevation of cytosolic free [Ca²⁺]_i were confirmed by the following findings: 1) exogenous PGE₂ or an EP1 agonist, but not an EP4 agonist, increased [Ca²⁺]_i, which could be blocked with an EP1 antagonist as well as BAPTA and thapsigargin; 2) phospholipase C inhibitor U73122, BAPTA, and thapsigargin inhibited PGE₂-mediated migratory response of EVT; and 3) PGE₂-mediated EVT migration was shown to be dependent on a class of Ca²⁺-dependent proteases called calpains, known to be involved in cell detachment from substratum during migratory responses. The presence of PGE₂ stimulated calpain activity, whereas two calpain inhibitors, calpastatin and N-Ac-Leu-Leu-methioninal (ALLM), blocked EVT migration.

Conclusion: PGE₂ stimulates EVT migration by signaling through EP1 receptors, increasing [Ca²⁺]_i, and activating calpain.

	Introduction

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A HIGHLY MIGRATORY and invasive subpopulation of cytotrophoblast cells, called extravillous trophoblast (EVT), breaks out of the chorionic villi and invades the uterine decidua and its vasculature, leading to a remodeling of the utero-placental arteries. This remodeling involves disintegration of the tunica media and a replacement of the endothelium by the trophoblast, transforming the arteries into flaccid tubes to ensure a steady flow of the maternal blood into the placental sinusoids to sustain fetal growth. Although mechanisms of EVT cell invasiveness are similar to those of tumor cells (1), in contrast to tumor cell invasion, EVT cell invasion of the uterus is stringently controlled in situ to maintain a healthy utero-placental homeostasis (2). A breakdown in this homeostasis can manifest as trophoblast hypoinvasiveness underlying preeclampsia and certain types of intrauterine growth retardation (3) or trophoblast hyperinvasiveness, a characteristic of gestational trophoblastic neoplasias including choriocarcinomas (4).

A large number of studies have investigated the cellular and molecular mechanisms regulating the proliferative, migratory, and invasive functions of EVT cells in situ. These studies revealed that a variety of molecules including growth factors, growth factor-binding proteins, and proteoglycans produced at the feto-maternal interface can positively or negatively regulate these functions in an orchestrated manner (reviewed in Refs.3 and 5). There is now a strong body of evidence to indicate that a functional deregulation of some of these molecules may contribute to the etiology of preeclampsia, a multifactorial disease (3).

Prostanoids are small lipid molecules synthesized from arachidonic acid (AA), liberated from membrane-bound phospholipids by the action of phospholipases A2 in response to various physiological and pathological stimuli. AA is subsequently converted by cyclooxygenases (COXs) and then various isomerases and reductases to different short-lived molecules collectively named prostanoids, including prostaglandins (PGs) and thromboxanes. Of these, the most well-studied is PGE₂, which was shown to play a major role in the events leading to the success of pregnancy and child birth, e.g. decidual response and implantation (6, 7), immunoprotection of the semiallogenic conceptus (8), and parturition (9). Derangements of some of these prostanoids have been reported both in the peripheral vasculature and in the placenta during the development of preeclampsia (10, 11, 12). Of these, a decline in the level of PGE₂ is believed to contribute to the increased peripheral vascular resistance in response to angiotensin II in pregnant women who are susceptible to developing preeclampsia (13). However, the possible functional relationship of low PGE₂ level in situ to trophoblast hypoinvasiveness remains unknown. PGE₂ production in the pregnant human uterus results from a number of sources, notably the fetal membranes (14) and the decidua (8). Although the role of decidua-derived PGE₂ in averting the alloreactivity of maternally derived lymphocytes against the conceptus has been well recognized (8, 15), its possible functional role in regulating EVT cell migration/invasiveness remains to be examined. This role is suggested by certain striking similarities between the EVT cells and tumor cells in the molecular mechanisms responsible for invasive and migratory functions of both cell types (1), and the findings that PGE₂ promotes tumor progression by stimulating tumor cell migration, invasiveness, and angiogenesis (16, 17).

PGE₂ exerts its biological functions on cells through four membrane-bound G protein-coupled receptors (EP1–4). EP1 receptors are coupled with G_q proteins, activating phospholipase C (PLC) and elevating the intracellular concentration of Ca²⁺ ([Ca²⁺]_i); EP2 and EP4 are G_s-coupled receptors, signaling by increasing the intracellular cAMP level; EP3 consists of multiple isoforms and, in general, inhibits the activity of adenylate cyclase via G_i protein, decreasing the intracellular concentration of cAMP (18, 19, 20). Although PGE₂ stimulation of migration of breast cancer cells was shown to be mediated predominantly by EP4 receptors (17), the functional role of individual EP receptors in EVT cell migration has never been examined.

Cellular migration involves multiple processes, including adhesion, forward projection of lamellipods and filopods, cytoskeletal reorganization, cytoskeletal contraction, and detachment at the rear end (21). The calcium-dependent cysteine protease calpain, which localizes to focal adhesions, was shown to play a major role in cell locomotion by helping detachment of the rear end of the cell (22, 23). Because of its calcium-dependence, it is logical to suggest that calpain may play an intermediary role in EP1-mediated migration stimulation of cells by PGE₂.

In view of high levels of PGE₂ produced by the maternal decidua during the first trimester of pregnancy (8) and a possible functional relationship between the low PGE₂ level at the fetomaternal interface and trophoblast hypomigratory phenotype in preeclampsia, in the present study we used a well-characterized first trimester human EVT cell line as well as EVT cell sprouts in first trimester chorionic villus explants in culture to explore the role of PGE₂ and various EP receptors in EVT cell migration. Because we observed the functional predominance of EP1 and EP4, we further examined certain EP1-mediated signaling events, e.g. activation of PLC, increase in cytosolic Ca²⁺, and activation of calpain in the stimulation of EVT cell migration by PGE₂.

	Materials and Methods

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Reagents and materials

RPMI 1640 and fetal bovine serum (FBS) were purchased from GIBCO (Burlington, Ontario, Canada). PGE₂, 17-phenyl trinor PGE₂, PGE₁ alcohol, and SC-19220 (8-chloro-dibenz[b,f](24)oxazepine-10(11H)-carboxy-(2-acetyl)hydrazide) were obtained from Cayman Chemicals (Ann Arbor, MI); SC-51322 and AH-6809 were obtained from Biomol Research Laboratories Inc. (Plymouth Meeting, PA); N-Ac-Leu-Leu-methioninal (ALLM), calpastatin peptide, and its negative control were obtained from Calbiochem (San Diego, CA); and AH-23848B [[1(z), 2ß5]-7(±)-[5-[[(1,1'-biphenyl)-4-yl]methoxy]-2-(4-morpholinyl)-3-oxo-cyclopentyl]-4-hetonoic acid calcium salt] was obtained from Glaxo/Wellcome (Stevenage, UK). BSA, U73122, U73343, and indomethacin were purchased from Sigma (Oakville, Ontario, Canada). Indo-1-penta-(acetoxymethyl) ester (indo-1, AM), BAPTA-1 AM, and thapsigargin (Tg) were obtained from Molecular Probes (Eugene, OR). Mouse monoclonal antihuman Cytokeratin 7 (clone OV-TL 12/30) and 5ß1 (MAB 1999) antibodies were purchased from DakoCytomation (Glostrup, Denmark) and Chemicon International (Temecula, CA), respectively. Fluorescein isothiocyanate-conjugated goat antimouse (catalog no. CLCC30001) secondary antibodies were purchased from Cedarlane (Hornby, Ontario, Canada).

Immunofluorescence staining

Cells were allowed to grow in complete media on 12-mm glass coverslips and then were washed with ice-cold PBS. The cells were fixed with 100% methanol and then washed again with PBS. Nonspecific binding sites were blocked using normal goat serum. Primary mouse antihuman antibodies were added (1/100 dilution, 4 h at 4 C) and then secondary fluorescein isothiocyanate-labeled goat antimouse (1/100 dilution, 1 h in the dark, at room temperature). The coverslips were mounted using fluorescent mounting medium (DakoCytomation).

EVT cell line and culture

We used an EVT cell line HTR-8/Svneo (25), produced in our laboratory by SV40 Tag immortalization of a short-lived first trimester EVT cell line called HTR8. HTR-8/Svneo cells have retained all the phenotypic and functional characteristics of the parental cells HTR8, expressing all the markers of EVT in situ: cytokeratin 7, 8, and 18, placental type alkaline phosphatase, high affinity urokinase-type plasminogen activator receptor, IGF-II mRNA and protein, HLA framework antigen w6/32, and integrins 1, 3, 5, ß1, and vß3/ß5 (26). They express HLA-G when grown on laminin or Matrigel (27) and mimic the phenotypic behavior of freshly isolated cytotrophoblast cells during Matrigel invasion (28). Their responses to the migration-stimulating ligands such as IGF-II (29), IGF binding protein 1 (IGFBP-1) (30), and urokinase-type plasminogen activator (uPA) (31) and migration-inhibitory ligands TGF-ß (32) and decorin (33) are identical to those of the primary EVT cell line HTR-8. They were used at 88–106 passages during the present study and cultured in RPMI 1640 supplemented with 10% FBS, 50 U/ml penicillin, and 50 µg/ml streptomycin, unless specified otherwise.

Chorionic villus explant culture to examine EVT cell outgrowth

The outgrowth of EVT cells in situ was measured using a modification of earlier-reported protocols (34, 35, 36). Small fragments of villus tips (10–15 mg wet weight) from placentae (n = 9) at 7- to 9-wk elective pregnancy terminations (collected in accordance with the local ethics review and approval guidelines set by the Department of Obstetrics and Gynecology, University of Western Ontario) were dissected to remove fetal membranes and endometrial tissue and then placed on Millicell-CM culture dish inserts (Millipore Corp., Bedford, MA). The inserts were precoated with 200 µl of 1:6 dilution of Growth Factor Reduced Matrigel (Collaborative Biomedical Products, Bedford, MA) in serum-free DMEM/F12 medium (Life Technologies, Inc., Grand Island, NY) supplemented with 50 U/ml penicillin, 50 µg/ml streptomycin, and 0.25 mg/ml ascorbic acid (Sigma) at pH 7.4. Before addition of the chorionic villi, the inserts were placed at 37 C for a half hour to allow the Matrigel to polymerize, and then were placed in 24-well plates (Becton Dickinson, Franklin Lakes, NJ) containing 800 µl of the same serum-free DMEM/F12 medium with or without different treatments. Culture media was changed every 48 h and human chorionic gonadotropin (hCG) and progesterone concentrations were measured using RIA (Coat-A-Count, DPC, Los Angeles, CA) to assess the viability of the chorionic villi. At least three placentae were used for each experiment, and triplicate explants were set up for each treatment under control and experimental conditions. Identity of EVT cells in the outgrowths was confirmed with immunostaining for cytokeratin-7. Pictures of the villus explants were taken every 24 h, up to 120 h using an inverted light microscope (x40 objective). The area of EVT cell outgrowth in live cultures was measured using Scion Image for Windows software (version ß 4.0.2; Scion Corp., Frederick, MD).

Measurement of PGE₂ production

The level of PGE₂ accumulating in 24-h serum-free culture supernatants of HTR-8/Svneo cells plated at a concentration of 1.5 x 10⁵ cells/ml was measured using Prostaglandin E₂ EIA Kit-Monoclonal from Cayman Chemicals, as reported earlier (17).

RNA isolation and RT-PCR analysis of EP receptors

RT-PCR and oligoprimers for PCR analysis of EP receptors were as reported earlier (17). Each PCR consisted of initial denaturation at 94 C for 2 min and then 40 cycles (34 cycles for EP1 only) at 94 C for 30 sec, 58 C (51 C for EP1 only) for 30 sec, 72 C for 45 sec, and an additional 5-min extension at 72 C after the last cycle, and was conducted using an Eppendorf Mastercycler Gradient (Eppendorf Scientific, Westbury, NY). GAPDH expression was used as an internal positive control for cDNAs and the sizes of the PCR products were estimated using GeneRuler (MBI Fermentas, Burlington, Ontario, Canada).

Migration assay

Migration of HTR-8/Svneo cells (chemokinesis) was measured using modification of a protocol as reported earlier (29, 30), using 24-well Falcon notched plates and Falcon cell culture inserts with microporous polycarbonate membranes having 8.0 µm pore size (Becton Dickinson). In brief, aliquots of 1 x 10⁵ cells suspended in 200 µl serum-free RPMI 1640 medium supplemented with 0.1% BSA were added in the upper chamber, while the lower chamber was filled with 800 µl serum-free medium with or without additional treatment. After incubation for 48 h at 37 C, 5% CO₂, the upper surface of the insert membranes was wiped with cotton swabs to remove nonmigratory cells, and then the membranes were stained using Harleco Hematocolor staining kit (EM Science, Gibbstown, NJ). The absolute number of migratory cells on each membrane was scored visually using light microscope (400 x magnification). Each treatment was performed in triplicates and repeated three times. For data presentation, migration indices under experimental conditions were plotted as a percentage of the controls.

Cell proliferation/survival assay

The effect of various treatments on the proliferation/survival of HTR-8/SVneo cells was assessed as reported elsewhere (33) using Cell Proliferation Kit I MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) from Roche Diagnostics (Laval, Quebec, Canada).

[Ca²⁺]_i measurement

Cytosolic [Ca²⁺] was measured in HTR-8/Svneo cells as reported by us earlier (31, 37) using a fluorometric method, after loading the cells with the Ca²⁺-sensitive fluorescent dye indo-1-AM. The treatment protocols are specified in Results. The ratio of the fluorescence intensity at 405 nm divided by the intensity at 485 nm was obtained using the system software. [Ca²⁺]_i was determined from the relationship [Ca²⁺]_i = K_d[(R – Rmin)/(Rmax – R)]ß, where K_d (the dissociation constant for the indo-1-Ca²⁺ complex) was 250 nmol/liter, Rmin and Rmax were the values of R at low and saturating concentrations of Ca²⁺, respectively, and ß was the ratio of the fluorescence at 485 nm measured at low and saturating concentrations of Ca²⁺ (38).

Calpain activity assay

Calpain activity was measured using a Calpain activity assay kit (catalog no. QIA120 from Calbiochem) as described elsewhere (39). In brief, the ability of calpain to digest the synthetic substrate Suc-LLVY AMC was measured fluorometrically at excitation 380 nm and emission 460 nm in the presence of saturating or nonsaturating levels of Ca²⁺. The calpain activity was displayed in relative fluorescence units per milligram of protein per minute.

Statistical analysis

Migration scores were analyzed using one-way ANOVA, and then the values were normalized as a percent of the control (migration indices). Differences among treatment means were assessed by student t test. Differences of P < 0.05 were considered significant.

	Results

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HTR-8/Svneo cells have maintained EVT cell phenotype, produce very small amount of PGE₂, and express all EP receptors

As part of our routine phenotyping of HTR-8/Svneo cells at frequent intervals for a variety of EVT cell markers, we demonstrate here that they express cytokeratin-7 and 5ß1 integrin (Fig. 1A).

View larger version (25K): [in this window] [in a new window]   FIG. 1. A, Immunostaining of HTR-8/Svneo cells for 5ß1 (upper row) and cytokeratin 7 (lower row). B, RT-PCR showing the mRNA expression of all EP receptors (EP1-EP4) in HTR-8/Svneo cells grown in 10% FBS-supplemented RPMI medium. The transcript sizes were, respectively, 324, 655, 398, 366, and 452 bp.

PGE₂ stimulates EVT migration by using multiple EP receptors

We tested the effect of exogenous PGE₂ at concentrations of PGE₂ ranging from 10 nmol/liter to 1 µmol/liter on HTR-8/Svneo cell migration. In our chemokinesis assay conducted in serum-free media, after 48 h, PGE₂ induced a concentration (up to 1 µmol/liter)-dependent increase of migration of these cells (Fig. 2A). At each concentration level, the values were significantly higher (P < 0.05) than that of the preceding concentration. Similar stimulatory effects of exogenous PGE₂ on EVT cell outgrowth were also noted in first-trimester human chorionic villus explant cultures on growth factor-reduced Matrigel under serum-free conditions. Although control explants showed no EVT cell outgrowth (data not shown), outgrowth of EVT cells was noticed at 48 h (Fig. 2B) in the presence of 1 µmol/liter PGE₂, and the area covered by the migrant EVT cells increased further at 72 (data not shown) and 96 h (Fig. 2, B and C).

View larger version (49K): [in this window] [in a new window]   FIG. 2. A, PGE2 elicited a dose-dependent stimulation of HTR-8/Svneo cells migration in vitro at 48 h (n = 9 ± SEM). Migration indices are plotted as the ratio of experimental (with PGE2) to control (without PGE2) number of migrant cells. Significant stimulation (P < 0.05) was noted at all concentrations used. B, Chorionic villus explants cultured in the presence of 1 µmol/liter PGE2 exhibited outgrowth of EVT cells like a carpet (arrows) in the surrounding Matrigel (x40 objective). The rate of outgrowth of EVT cells in such explants was followed in real-time as illustrated here (0, 48, 96 h). C, The relative area of such outgrowths was quantified with Scion software (n = 3 ± SEM). The increase in outgrowth at 96 h was nearly 2-fold (P < 0.005) as compared to 0 h. No outgrowth was observed in control explants at any time point under serum-free conditions.

View larger version (15K): [in this window] [in a new window]   FIG. 3. A, Migration assay (48 h) showing a dose-dependent inhibition of migration of HTR-8/Svneo cells by all three EP antagonists (SC19220, EP1 antagonist; AH23848B, EP4 antagonist; AH6809, EP1/EP2/DP antagonist) in the presence of PGE2, 100 nmol/liter (at antagonist concentration 0.1 µmol/liter, P < 0.004 for SC19220, P < 0.003 for AH23848B, and P < 0.003 for AH6809). B, Migration assay (48 h) showing migration inhibition in the presence of higher concentrations (1, 10 µmol/liter) of EP antagonists alone (without PGE2) (P < 0.002 for all three antagonists). C, Migration assay (48 h) indicating a dose-dependent stimulation of HTR-8/Svneo cells migration by different EP agonists, EP1 agonist 17-phenyl trinor PGE2 and EP4 agonist PGE1 alcohol. Significant stimulation is noted at all concentrations of the agonists as well as PGE2 (P < 0.01 for PGE2, P < 0.006 for 17-phenyl trinor PGE2, and P < 0.01 for PGE1 alcohol). Data in all the graphs represent percentage of control (n = 3 ± SEM).

Using MTT assay we investigated the effect of PGE₂ (1 nmol/liter to 1 µmol/liter) on the cellularity of HTR-8/Svneo cells, because PGE₂ had been shown to have promoting effects on proliferation/survival of certain cell types (40, 41). We found that under serum-free conditions as used for migration assays, PGE₂ at these concentrations, had no effect on proliferation/survival of HTR-8/Svneo cells (data not shown). Furthermore, neither any of the EP antagonists, SC-19220, SC-51322, AH-6809, or AH-23848B, nor the agonists 17-phenyl trinor PGE₂ or PGE₁ alcohol at the concentrations used in the migration assays affected cellularity of these cells in MTT assays. In contrast, inclusion of FBS (5 and 10%) in the media significantly increased the proliferation of EVT cells (data not shown). These data, taken together, revealed that the migration-modulating effects of these agents on EVT cells were not a consequence of effects on cell proliferation/survival.

PGE₂ increases [Ca²⁺]_i in HTR-8/Svneo cells, and this rise in [Ca²⁺]_i is necessary for EP1-mediated cell migration

EP1 receptors are G_q-coupled receptors (19, 20) known to elevate the concentration of cytosolic free Ca²⁺. Also, [Ca²⁺]_i is an important player in cellular migration (42, 43). Hence, we tested the effect of PGE₂ on [Ca²⁺]_i in HTR-8/Svneo cells and whether this effect is correlated with the stimulation of migration of these cells. PGE₂ elicited a dose-dependent stimulation of [Ca²⁺]_i up to 1 µmol/liter (Fig. 4A). The PGE₂ effect could only be mimicked by the EP1 agonist 17-phenyl trinor PGE₂ (Fig. 4B) and not by EP4 agonist (data not shown), reinforcing the knowledge that EP4 does not signal by stimulating [Ca²⁺]_i. We further tested whether an EP1 antagonist could block the PGE₂-mediated [Ca²⁺]_i increase. Indeed, HTR-8/Svneo cells preincubated with the selective EP1 inhibitor SC-51322 (10 µmol/liter and 20 µmol/liter) showed a decrease in the [Ca²⁺]_i response elicited by PGE₂ 1 µmol/liter (Fig. 4C). To determine the source of calcium, we tested two Ca²⁺ chelators, EGTA (extracellular Ca²⁺ chelator) and BAPTA (intracellular Ca²⁺ chelator). Whereas EGTA treatment (5 mmol/liter) did not significantly influence the Ca²⁺ response to PGE₂ treatment, preincubation of the EVT cells with BAPTA (5 µmol/liter) for 30 min, although did not significantly alter the baseline [Ca²⁺]_i, eliminated the early peak and the late plateau in [Ca²⁺]_i after PGE₂ stimulation (Fig. 5A). Treatment of HTR-8/Svneo cells with Tg (1 µmol/liter), an endoplasmic reticulum (ER) Ca²⁺-ATPase inhibitor (24), evoked a large increase in [Ca²⁺]_i (Fig. 5B), demonstrating the availability of Ca²⁺ from intracellular stores. On the other hand, pretreatment of HTR-8/Svneo cells with Tg (1 µmol/liter) for 30 min blocked the effect of PGE₂ (Fig. 5B), suggesting that the transient rise of [Ca²⁺]_i mediated by PGE₂ is due to the release from ER. We also tested the effect of U-73122 (PLC inhibitor) on the Ca²⁺ release. PLC has been shown in other cell types to be activated upon EP1 stimulation by PGE₂, leading to phosphatidylinositol 4,5-bisphosphate hydrolysis, resulting in accumulation of inositol trisphosphate and diacylglycerol to elicit intracellular Ca²⁺ mobilization (18). U-73122, but not its inactive analog U-73343 (44), inhibited the PGE₂-mediated Ca²⁺ release in HTR-8/Svneo cells (Fig. 5C).

View larger version (20K): [in this window] [in a new window]   FIG. 4. A, PGE2 induced a concentration-dependent increase in [Ca2+]i in HTR-8/SVneo cells. At the time indicated by arrows, different concentrations of PGE2 (250, 500, 1000 nmol/liter) were added to the cuvette. B, EP1 agonist 17-phenyl trinor PGE2 induced a concentration-dependent increase in [Ca2+]i in HTR-8/SVneo cells. Arrows indicate addition of different concentrations of EP1 agonist (500, 1000 nmol/liter). C, Inhibition of PGE2-induced [Ca2+]i in HTR-8/SVneo cells in the presence of different concentrations of SC-51322 (EP1 antagonist). Arrows indicate addition of 1 µmol/liter PGE2. Left, No inhibitor; middle, cells preincubated for 30 min with 10 µmol/liter SC-51322; and right, cells preincubated with SC-51322, 20 µmol/liter.

View larger version (20K): [in this window] [in a new window]   FIG. 5. A, Effects of BAPTA (intracellular Ca2+ chelator) and EGTA (extracellular Ca2+ chelator) on the [Ca2+]i response to PGE2 in HTR-8/Svneo cells. Left, HTR-8/Svneo cells treated with 1 µmol/liter PGE2; middle, cells incubated with 5 µmol/liter BAPTA for 30 min concomitantly with indo-1-AM then treated with PGE2, 1 µmol/liter. BAPTA loading inhibited almost completely the PGE2-stimulated increase in [Ca2+]i. Right, Cells suspended in buffer (Na+ + 1 mM Ca2+) plus 5 mmol/liter EGTA were treated with PGE2, 1 µmol/liter. Ca2+ release was decreased but not totally inhibited. B, Effects of Tg on HTR-8/Svneo cells. Left, 1 µmol/liter PGE2; middle, Tg pretreatment (1 µmol/liter for 30 min) inhibited almost completely the PGE2-stimulated Ca2+ release; right, Tg treatment alone (1 µmol/liter) evoked a large response of [Ca2+]i. C, Effects of PLC inhibitor on [Ca2+]i after treatment with PGE2 in HTR-8/Svneo cells. Left, Cells incubated for 5 min with 1 µmol/liter of U-73122 (PLC inhibitor) were challenged with 1 µmol/liter PGE2. Note that the release of intracellular Ca2+ was almost completely abolished. Right, An inactive analog of U-73122 (U-73343, 1 µmol/liter) was used as a control.

We showed that both PGE₂ and EP1 agonist 17-phenyl trinor PGE₂ increases [Ca²⁺]_i in HTR-8/Svneo cells presumably upon activation of EP1 receptors, through a mechanism depending on PLC activation. Therefore, we tested the effect of PLC inhibitor on HTR-8/Svneo cell migration. U73122 (1 µmol/liter), and not its inactive analog U73343, inhibited migration of EVT cells (Fig. 6A) in the presence or absence of EP1 agonist 17-phenyl trinor PGE₂, supporting the hypothesis that PGE₂, acting through EP1 receptors, may activate PLC that plays an important role in the migration of HTR-8/Svneo cells. Interestingly, PGE₂ treatment partially rescued U73122 inhibition, suggesting a possible involvement of other EP receptors in EVT migration (Fig. 6A). Inhibition of basal cellular migration by PLC inhibitor (Fig. 6A) indicates that PLC is required for basal migration of HTR-8/Svneo cells.

View larger version (14K): [in this window] [in a new window]   FIG. 6. A, PLC inhibitor U73122 (1 µmol/liter) significantly decreased the HTR-8/Svneo cells migration, both in the presence and absence of 17-phenyl trinor PGE2 (1 µmol/liter) (n = 3 ± SEM, P < 0.04). B, BAPTA (100 nmol/liter) and Tg (100 nmol/liter) significantly decreased the HTR-8/Svneo cells migration, both in the presence and absence of 17-phenyl trinor PGE2 (1 µmol/liter) (n = 3 ± SEM; P < 0.01).

To determine whether the rise of [Ca²⁺]_i by PGE₂ actively contributes to the migration stimulation, we also tested the effect of BAPTA (100 nmol/liter) and Tg (100 nmol/liter) on the migration of HTR-8/Svneo cells. Migration was measured in EVT cells preincubated for 45 min with BAPTA (100 nmol/liter) or with Tg (100 nmol/liter), followed by addition of PGE₂ or 17-phenyl trinor PGE₂. Both compounds inhibited cellular migration at 48 h in the presence or absence of exogenous PGE₂ or 17-phenyl trinor PGE₂ (Fig. 6B), suggesting that the intracellular Ca²⁺ stored in the ER is required for EP1-mediated stimulation of migration. Migration-inhibitory effects of both compounds in the absence of any receptor ligand can be explained by the requirements of [Ca²⁺]_i for migration in response to endogenous PGE₂ as well as other mediators. A strong inhibition by Tg alone, along with the observation that PGE₂ did not significantly rescue BAPTA or Tg effects (Fig. 6B), attest to the essential role of [Ca²⁺]_i requirement for migratory responses resulting from most or all endogenous mediators. In parallel experiments, these concentrations of BAPTA and Tg tested for effects on cell proliferation/survival were found to have no effects.

PGE₂ stimulates activation of calpain in HTR-8/Svneo cells as a migration-stimulatory event

A calcium-dependent neutral protease, calpain has recently been implicated in cell migration through its requirement for both detachment of cells at their trailing edge and cell spreading (22, 23). Also, this enzyme has been found to have an important role in epidermal growth factor (EGF)-receptor-mediated cell deadhesion and motility (45). Therefore, we first determined whether inhibition of calpain would reduce basal and also EP1-mediated EVT cell migration. We tested the effect of calpain inhibitors ALLM (10 µmol/liter) and calpastatin (1 µmol/liter) on HTR-8/Svneo cell migration. Both inhibitors, in the presence of 17-phenyl trinor PGE₂ (1 µmol/liter), decreased migration of EVT cells at 48 h to the levels of controls (Fig. 7A), suggesting that activation of calpain plays an important role in EP1-mediated migration of HTR-8/Svneo cells. Significant migration inhibition with these calpain inhibitors in the absence of exogenous ligand can be explained by the requirements of calpain activity for migration in response to endogenous mediators, including PGE₂. Minor differences in migration indexes between calpain inhibitor(s)-treated cells and PGE₂ plus inhibitor(s)-treated cells might indicate stimulation of other protease(s) along with calpain by PGE₂ in these cells or due to confounding effects of multiple EP receptors in these cells.

View larger version (19K): [in this window] [in a new window]   FIG. 7. A, 17-Phenyl trinor PGE2 activates calpain to stimulate EVT cell migration. Both calpastatin (1 µmol/liter) and ALLM (10 µmol/liter) significantly decreased the migration of HTR-8/Svneo cells at 48 h, in the presence or absence of 17-phenyl trinor PGE2 (1 µmol/liter) (n = 3 ± SEM; P < 0.04). B, Calpain activity in HTR-8/Svneo cells with or without saturating exogenous Ca2+. In both cases, exogenous PGE2 (1 µmol/liter) and EP1 agonist 17-phenyl trinor PGE2 (1 µmol/liter) increased significantly (n = 3 ± SEM, P < 0.05) calpain activity at 10 min.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Migratory and invasive functions of the human EVT are tightly regulated by a variety of locally derived molecules (3, 5). In the present paper, using a well-characterized first trimester human EVT cell line as well as first trimester chorionic villus explant cultures, it is shown for the first time that PGE₂, a small molecule produced at the fetal-maternal interface, promotes EVT cell migration in a predominantly paracrine and, to a minor extent, autocrine manner. We have further shown that EVT cells express all the EP (EP1–4) receptors, and multiple EP receptors, in particular EP1 and EP4, mediate the migration stimulatory function of PGE₂. We also have characterized some of the signals downstream of EP1 to show that activation of PLC, increase in [Ca²⁺]_i, and activation of calpain are key events in the signaling pathway responsible for migration stimulation. Signaling events resulting from activation of other EP receptors on EVT cells remain to be investigated. Results of the present study might well explain the findings of a randomized control study where daily administration of low doses of linoleic acid (which metabolizes to PG precursor AA) with calcium during pregnancy corrected the levels of PGE₂ and reduced the incidence of preeclampsia (12).

PGE₂ is a key migration-promoting molecule for a variety of cell types, including endothelial cells participating in angiogenesis (46) as well as cancer cells (17, 47). In the latter case, it is an autocrine stimulus resulting from high levels of COX-2 expression by cancer cells, which is positively correlated with tumor progression and metastasis (17). Although EVT cells in the first trimester human placentae show microsomal PGE synthase and COX-2 immunostaining (48) and HTR-8/SVneo cells express detectable COX-2 mRNA, as shown by RT-PCR (data not shown), they produce roughly 1000 times less PGE₂ than first trimester decidual cells (8). These data, in combination with present functional data showing strong stimulatory effects of exogenous PGE₂ on migration of HTR-8/SVneo cells as well as outgrowth of EVT cells in villus explant cultures suggest that PGE₂ is predominantly a paracrine stimulus evidently provided by the decidua. It may represent one of the molecules in decidual extracts which has been shown to promote EVT cell outgrowth and matrix invasion in chorionic villus explant cultures (49).

EVT cell sprouting in villus explant cultures is a combined outcome of cytotrophoblast proliferation and differentiation of cells along the extravillous pathway with the acquisition of a migratory and invasive phenotype. Although we have not attempted to dissect the relative contribution of proliferation vs. migration in the PGE₂ stimulation of EVT cell outgrowth in our explant culture system, several observations are highly pertinent: 1) under the serum-free conditions of our explant cultures on growth factor-reduced Matrigel, no outgrowth was detected in the absence of PGE₂, although outgrowths are detectable on traditional Matrigel; 2) cells spread out like a carpet from preexisting mini-sprouts in a villus fragment (as seen at 0 h in Fig. 2B); and 3) the explant size appeared to shrink with time, indicative of cells migrating out of the preexisting sprouts rather than significant proliferation in these sprouts. Although these observations combined with the absence of PGE₂ effect on proliferation of HTR-8/SVneo cells would suggest that the increase in outgrowth size in explants with time (as shown in Fig. 2C) is primarily a result of increased migration, further studies are needed with explant cultures to identify the relative contribution of cell proliferation, if any, under the influence of PGE₂.

Using EP receptor antagonists and agonists we have established that multiple EP receptors participate in PGE₂-mediated migratory response of EVT cells. Although we have firmly established the roles of EP1 and EP4, the roles of EP2 and EP3 were not excluded. The role of EP receptor class participating in migratory responses in other cells depends on the cell type. For example, PGE₂ stimulation of migration of highly metastatic breast cancer cell lines was shown to be mediated predominantly by EP4 and, to some small extent, by EP2 (17). In the case of Langerhans cells, it was shown to be EP4 (50), whereas a role of EP3 was demonstrated in human arterial smooth muscle cells both by the use of EP3 agonist as well as by EP3 overexpression in these cells (51).

EP1 receptor is a G_q-coupled receptor and was shown in many cells to activate PLC, leading to an increase in the intracellular concentration of Ca²⁺ (18). We showed that [Ca²⁺]_i was increased upon PGE₂ stimulation in a concentration-dependent manner, and this stimulation was mimicked by EP1 agonist and not by EP4 agonist treatment. Moreover, using a selective EP1 antagonist, SC-51322, we could prevent the rise in [Ca²⁺]_i. Using a specific PLC inhibitor, U73122, we could inhibit the intracellular release of Ca²⁺, which demonstrates the requirement of PLC activity for the Ca²⁺ response. The intracellular Ca²⁺ chelator, BAPTA, and not the extracellular Ca²⁺ chelator, EGTA, almost completely inhibited the intracellular release of Ca²⁺, suggesting that, in HTR-8/Svneo cells, PGE₂ stimulates the release of Ca²⁺ mainly from the intracellular stores upon EP1 activation. Two other ligands, endothelin (ET)-1 and uPA-ATP were shown to elicit a transient increase in [Ca²⁺]_i, followed by a sustained phase in the same cell line (31, 37). We observed a similar response pattern in [Ca²⁺]_i after PGE₂ treatment. Using Tg, an ER Ca²⁺-ATPase inhibitor, we demonstrated that intracellular stores, especially ER, are important in regulating the Ca²⁺ response to PGE₂ in HTR-8/Svneo cells. Both BAPTA and Tg inhibited the migration of HTR-8/Svneo cells in the presence or absence of PGE₂, suggesting a direct involvement of the intracellular Ca²⁺ in the migration of these cells.

The present study is the first to report PGE₂-mediated cellular calpain activation. The calpain family of cysteine proteases comprises of 13 members of which m- and µ-calpain are ubiquitously expressed and are the most well-studied calpains (52). Both calpains have been implicated in cellular adhesion and migration. Integrin-mediated cellular migration was shown to require µ-calpain because of its involvement in cellular attachment as well as detachment from the substratum; a calpain-mediated proteolytic breakdown of the focal contacts at the rear end was needed for the locomotion (23, 53). A requirement of calpains for certain migratory responses has been demonstrated with the use of calpain inhibitors (54, 55). In the present study, we used two inhibitors (of both m- and µ-calpain), calpastatin and ALLM. Both inhibited PGE₂-mediated migration in EVT cells, substantiating the role of calpain in migration response. Although both m- and µ-calpain play a role in cell locomotion, they may be differentially activated upon different conditions. For example, m-calpain and not µ-calpain was shown to be activated upon stimulation of EGF receptor in a murine fibroblast cell line (56); a down-regulation of µ-calpain with antisense oligonucleotides did not significantly influence EGF-induced cellular calpain activity as well as motility in these cells (56). Initially, calpain activity was presumed to be solely dependent on [Ca²⁺]_i, micromolar levels being required for µ-calpain and millimolar levels for m-calpain activation (57). However, recent studies have identified additional mechanisms involved in the regulation of calpain activation such as binding to phospholipids, autoproteolytic cleavage, inhibition by endogenous calpastatin, and activation by phosphorylation (45, 56, 58).

In the present study, we obtained modest but significant up-regulation of calpain activity by PGE₂. These effects could be mimicked only by the EP1 agonist 17-phenyl trinor PGE₂ and not by EP4 agonist PGE₁ alcohol. Modest stimulatory effects of PGE₂ and EP1 agonist on calpain activity in whole cell homogenate are likely to be due to its localized stimulation on the cell membrane at the rear end of the cell as opposed to its activation throughout the cell. However, involvement of other Ca²⁺-dependent proteases like matrix metalloproteases in PGE₂-mediated cellular migration cannot be ruled out. These findings of EP1-mediated calpain activation are consistent with a previous report of G_q-mediated activation of calpain-induced proteolysis (59). The observed increase of calpain activity in the absence of exogenous Ca²⁺ cofactor (Fig. 7B) is most likely due to the rise of intracellular Ca²⁺ upon stimulation of cells by the agonists. Its increase in the presence of enzyme saturating concentrations of exogenous Ca²⁺ (Fig. 7B) indicate novel mechanism(s) of activation of calpain that may not be directly dependent on the rise of [Ca²⁺]_i. Calpain phosphorylation could be one such mechanism of its activation because of the presence of several putative phosphorylation sites in this protein (56). A direct phosphorylation of m-calpain by ERK activation remains a possibility in the cascade of PGE₂-mediated events, as had been shown with EGF (56). Calpain has been suggested to cleave focal adhesion proteins like talin, ezrin, and pp125^FAK (30, 53, 58). Whereas involvement of pp125^FAK was previously demonstrated to play an important role in ligand-induced EVT cell migration (30) it is not known at the moment whether PGE₂-induced calpain activation can cleave pp125^FAK in EVT cells. These data taken together suggest that PGE₂-induced activation of calpain involves an increase of intracellular concentration of Ca²⁺ via EP1 receptors. However, calpain activation by PGE₂ even in the presence of saturating concentrations of exogenous Ca²⁺ further suggests additional events such as calpain phosphorylation, in the activation process.

A balanced migration/invasiveness of EVT cells into the uterine decidua and uteroplacental arteries appears to be achieved in situ by a large number of regulating factors produced at the fetomaternal interface. Of these, the EGF family acting as autocrine as well as paracrine mediators promotes proliferation of cytotrophoblast (60) and its differentiation into the EVT cell pathway, as evidenced by acquisition of a migratory/invasive phenotype (61, 62, 63). Other factors which promote EVT cell migration/invasiveness without an influence on proliferation are IGF II, IGFBP-1, uPA, ET-1 and hepatocyte growth factor (HGF), all of which require activation of ERK (an activator of MAPK) for migration stimulation (29, 30, 31, 37, 64). IGF II is an autocrine mediator acting via IGF type II receptors and by inhibiting adenylyl cyclase and stimulation of MAPK (29). IGFBP-1 is a paracrine (decidua-derived) mediator acting via 5/ß1 integrin receptor, and activation of focal adhesion kinase (FAK) (30). uPA is an autocrine/paracrine mediator promoting invasiveness by its catalytic function, and migration by binding to the noncatalytic domain of urokinase-type plasminogen activator receptor, activation of PLC, phosphatidylinositol 3-kinase (PI3-K), and rise in [Ca²⁺]_i (31). ET-1 is an autocrine/paracrine mediator acting by activation of ET A/B receptors, increase in [Ca²⁺]_i (37). HGF is a paracrine (mesenchyme-derived) mediator that promotes EVT cell motility and invasiveness (64, 65) by activating c-met receptors, and at least in part by induction of NO (64). The present study adds PGE₂ (primarily as a paracrine mediator) to this list of migration-promoting factors. The role of [Ca²⁺]_i increase appears to be common for numerous migration-promoting ligands, e.g. heparin-binding EGF-like growth factor (62), uPA (31), ET-1 (31), and PGE₂ (present study). In summary, there is substantial commonality and possibly cross-talk among the signaling molecules activated by these migration-promoting factors. A number of inhibiting factors were shown to control EVT cell migration/invasiveness in situ: decidua-derived TGFß (66, 67), decorin (33), and TNF (68). Thus, it is evident that the migration-promoting molecules are produced both by the EVT cells and the decidua, whereas migration-inhibitory factors have primarily a decidual origin. A detailed understanding of the signaling by the migration-promoting and inhibitory ligands as well as their possible derangements in trophoblast hypomigratory/hypoinvasive disorders should be of significant value in identifying molecular targets for prevention and intervention of these diseases.

	Footnotes

 
This work was supported by Canadian Institutes of Health Research (CIHR) Grants MOP 36446 and MOP 69091 (to P.K.L.), MOP 68997 (to C.C.), and a CIHR.UWO Strategic Training Initiative in Cancer Research and Technology Transfer fellowship (to C.N.).

First Published Online May 10, 2005

Abbreviations: AA, Arachidonic acid; ALLM, N-Ac-Leu-Leu-methioninal; [Ca²⁺]_i, intracellular concentration of Ca²⁺; COX, cyclooxygenase; EGF, epidermal growth factor; EP, PGE₂ receptor; ER, endoplasmic reticulum; ET, endothelin; EVT, extracellular trophoblast; FAK, focal adhesion kinase; FBS, fetal bovine serum; HGT, hepatocyte growth factor; IGFBP-1, IGF binding protein 1; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide; PG, prostaglandin; PLC, phospholipase C; Tg, thapsigargin; uPA, urokinase-type plasminogen activator.

Received February 28, 2005.

Accepted April 28, 2005.

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