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Endocrinology, doi:10.1210/en.2007-1136
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Endocrinology Vol. 149, No. 3 1243-1251
Copyright © 2008 by The Endocrine Society

Roles of Rho Guanosine 5'-Triphosphatase A, Rho Kinases, and Extracellular Signal Regulated Kinase (1/2) in Prostaglandin E2-Mediated Migration of First-Trimester Human Extravillous Trophoblast

Catalin Nicola, Andrei Chirpac, Peeyush K. Lala and Chandan Chakraborty

Departments of Anatomy and Cell Biology (C.N., A.C., P.K.L.) and Pathology (C.C.), University of Western Ontario, London, Ontario, Canada N6A 5C1

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Prostaglandin (PG) E2 may regulate invasiveness of human placenta because we previously reported stimulation of migration of placental trophoblasts by PGE2 acting through PGE receptor (EP)-1 and activating calpain. RhoA GTPase and its important effector Rho kinase (ROCK) have also been previously shown to regulate trophoblast migration. Using immortalized HTR-8/SVneo trophoblast cells and first-trimester human chorionic villus explant cultures on matrigel, we further examined the role of RhoA/ROCK and MAPK (ERK1/2) pathways on PGE2-mediated stimulation of trophoblast migration. Migration of cytotrophoblasts was shown to be inhibited by treatment of the trophoblast cell line and chorionic villus explants with either cell-permeable C3 transferase or selective RhoA small interfering RNA. These inhibitions were significantly mitigated by the addition of PGE2, an EP1/EP3 agonist or an EP3/EP4 agonist, suggesting that RhoA plays an important role in trophoblast migration but may not be obligatory for PGE2 action. Treatment of HTR-8/SVneo cells with nonselective ROCK inhibitor Y27632 or ROCK small interfering RNAs inhibited migration of these cells, which could not be rescued with PGE2 or the other two EP agonists, suggesting the obligatory role of ROCK in PGE2-induced migratory response. Furthermore, U0126, an inhibitor of MAPK kinases MEK1 and MEK2, abrogated PGE2-induced migration of trophoblasts, and PGE2 or the other two EP agonists stimulated ERK1/2 activation in trophoblasts, which was not abrogated by pretreatment with C3 transferase, indicating that ERK signaling pathway is an efficient alternate pathway for RhoA in PGE2-mediated migration of trophoblasts. These results suggest that ROCK and ERK1/2 play more important roles than RhoA in PGE2-mediated migration stimulation of first-trimester trophoblasts.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE HUMAN PLACENTA, primarily a fetally derived structure, is unique in its ability to invade the maternal uterus. This function is performed by trophoblast cell class derived from the chorionic villi, called the extravillous trophoblast (EVT). Interstitial invasion of the endometrial decidua and a part of the myometrium and endovascular invasion of the uterine spiral arteries by the EVT are critical for successful pregnancy. A remodeling of the spiral arteries by the EVT changes the blood flow pattern through these arteries from high resistance to low resistance. Poor placental perfusion resulting from a failure of occurrence of this change in blood flow pattern is postulated to initiate a cascade of pathological changes in the mother leading to preeclampsia and/or the result in poor fetal nourishment, manifested as intrauterine growth retardation. It is now recognized that reduced trophoblast migration/invasiveness, the earliest predictor of precclampsia, may result from aberrant functional activity of one or more of migration/invasion regulatory factors (1).

Cellular invasion involves cell-cell adhesion, cell-matrix adhesion, degradation of basement membrane proteins, and migration (2). Thus, the positive regulators of EVT cell migration will increase the invasive capacity of these cells, provided the other three functions are not inhibited. One of the positive regulators of EVT cell migration has been identified as decidua-derived prostaglandin (PG)-E2 (3). PGE2 is a vasodilator prostanoid, the concentration of which has been shown to be reduced in not only peripheral circulation (4) but also uteroplacental environment (5) in women who develop preeclampsia. These observations along with the protective ability of linoleic acid, a precursor of PGE2 against the development of preeclampsia (4), suggest that elucidation of molecular mechanisms in PGE2-mediated EVT cell migration is of physiological and clinical importance. Cell migration takes place through a complex sequence of events that starts with the adhesion of the cell to a substratum, followed by forward projection of lamellipods and filopods, cytoskeletal contraction, and detachment at the rear end. Using both a well-established first-trimester EVT cell line called HTR-8/SVneo (6) and human first-trimester chorionic villus explant cultures, we have previously shown that PGE2 stimulates migration of first-trimester human EVT cells, by increasing intracellular Ca2+ and activating calpain, a calcium-dependent cysteine protease involved in the detachment at the rear end of the cell (3). Therefore, it is also possible that PGE2 requires activation of other intracellular signaling molecules, especially those that are important for cytoskeletal reorganizations, to induce its migration stimulatory effects on EVT.

A family of small GTP-binding proteins called Rho GTPases have been regarded as the master regulators of cytoskeletal reorganizations required for cellular migration (7). To date, 22 members have been described, including three Rho isoforms (A, B, and C), three Rac isoforms (1, 2, and 3), and Cdc42 (8). They cycle between an active GTP-bound state and an inactive GDP-bound state. GTP-bound Rho, Rac, and Cdc42 interact with their effector molecules to perform cellular functions. During cellular migratory responses, the activation of Rho, Rac, and Cdc42 induce stress-fiber formation, extension of lamellipodia, and filopodia, respectively (9). RhoA is one of the most well-characterized members of the RhoGTPase family. Furthermore, one of the most important downstream effectors of RhoA is Rho kinase (ROCK). Activation of ROCK by RhoA blocks the activity of myosin light-chain (MLC) phosphatase, leading to an increase in phosphorylated MLC and increased cell contractility (10). Phosphorylation of MLC can also be achieved by MLC kinase (MLCK), which is activated by phosphorylation of MAPK ERK1/2 (11).

Here we hypothesize that RhoA, ROCK, and ERK1/2 are important mediators of PGE2-mediated migration of EVT cells. We used two models: a well-established human EVT cell line and human first-trimester chorionic villus explants in culture. Interestingly, ROCK but not RhoA was found to be indispensable for PGE2-induced EVT migration. We also provided evidence for MAPK ERK1/2 as the alternate pathway for RhoA GTPase in PGE2-induced stress fiber formation and migration of these cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals
RPMI 1640 and fetal bovine serum (FBS) were purchased from GIBCO (Burlington, Ontario, Canada). PGE2, and 17-phenyl trinor PGE2, and PGE1 alcohol were obtained from Cayman Chemicals (Ann Arbor, MI). Cell-permeable C3 transferase and Y-27632 [(+)-R-trans-4-(aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide, 2HCl, H2O] were purchased from Cytoskeleton (Denver, CO) and Biomol International LP (Plymouth Meeting, PA), respectively. Cytokeratin 7 (clone OV-TL 12/30) and {alpha}5β1 (MAB 1999) antihuman antibodies were obtained from DakoCytomation (Glostrup, Denmark) and Chemicon International (Temecula, CA), respectively. Anti-ROKa/ROCK-II (rabbit polyclonal IgG, catalog no. 07–443) were purchased from Upstate (Lake Placid, NY). RhoA (catalog no. sc-418) mouse antihuman monoclonal antibody was purchased from Chemicon International and Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), respectively. Goat antirabbit IgG-horseradish peroxidase (HRP) (catalog no. sc-2004), goat antimouse IgG-HRP (catalog no. 31430), and fluorescein isothiocyanate-conjugated goat antimouse (catalog no. F-2761/6592-1) secondary antibodies were obtained from Santa Cruz Biotechnology, Pierce (distributed by Fisher Canada, Nepean, Ontario, Canada) and Molecular Probes (through Invitrogen Canada, Burlington, Ontario, Canada), respectively. RhoA activity assay kit was obtained from Cytoskeleton. All other chemicals were purchased from Sigma-Aldrich (Oakville, Ontario, Canada), unless otherwise indicated.

EVT cell line and culture
An immortalized first-trimester EVT cell line HTR-8/SVneo (6) derived from a short-lived primary EVT cell line HTR-8 was used in the present study. Phenotypic characterization has shown that HTR-8/SVneo cells express all the markers of EVT cells in situ, including: cytokeratin 7, 8, and 18 (antibodies from DakoCytomation); placental-type alkaline phosphatase, high-affinity urokinase-type plasminogen activator receptor; IGF-II mRNA and protein; human leukocyte antigen framework antigen w6/32; and integrins {alpha}1, {alpha}3, {alpha}5, β1, and {alpha}vβ3/β5 (3, 12). They also express human leukocyte antigen-G when grown on matrigel or laminin. HTR-8/SVneo cells were used between passages 90 and 115 for all the described experiments and cultured in RPMI 1640 (GIBCO) supplemented with 10% FBS (GIBCO) and 2% penicillin/streptomycin, unless specified otherwise. For experiments, cells were washed with serum-free medium. PGE2 or other PGE receptor (EP) receptor agonists, stock solutions at a concentration of 200 mmol/liter were made in dimethylsulfoxide, and further 2 x 105-fold dilutions were made into serum-free medium before performing experiments. Y-27632 and C3 transferase were dissolved in serum-free medium. Therefore, controls using serum-free medium served as vehicle controls of all the experiments.

Chorionic villus explant culture
EVT outgrowth in situ was measured as reported earlier (3). Small fragments of villus tips (10–15 mg wet weight) were dissected from placentae at 7- to 9-wk elective pregnancy terminations (collected according to the local ethics approval guidelines) and placed on Millicell-CM culture dish inserts (Millipore Corp., Bedford, MA) precoated with 200 µl of 1:6 dilution of growth factor reduced matrigel (Collaborative Biomedical Products, Bedford, MA) in serum-free DMEM/F12 (Life Technologies, Inc., Grand Island, NY) supplemented with 2% penicillin/streptomycin and 0.25 mg/ml ascorbic acid (Sigma, Oakville, Ontario, Canada) at pH 7.4 and then placed in 24-well plates (Becton Dickinson, Franklin Lakes, NJ). Culture media were changed every 48 h and human chorionic gonadotropin and progesterone concentrations in these media were measured using RIA (Coat-A-Count; Diagnostic Products Corp., Los Angeles, CA) to assess the viability of chorionic villi. Trypan blue dye exclusion of the outgrowing cells from the chorionic villi indicated cell viability of 90–95%. At least three placentae were used for each experiment, and triplicate explants were set up for each treatment under control and experimental conditions. Outgrowing cells were immunostained for cytokeratin-7 to confirm epithelial lineage of EVT. Pictures were taken every 24 h up to 72 h using an inverted light microscope (x40 objective). The area of EVT cell outgrowth was measured using Scion Image for Windows software (Scion Corp., version beta 4.0.2).

Immunofluorescence staining
Cells were allowed to grow in complete media on 12-mm glass coverslips, serum starved overnight before treatment with the test substances. After washing, the cells were fixed with 4% paraformaldehyde and then permeabilized with 0.1% Triton X-100 for 10 min. Nonspecific binding sites were blocked using normal goat serum. The cells were treated with primary mouse antihuman RhoA antibody (Millipore) (10 µg/ml, 4 h at 4 C) and then secondary fluorescein isothiocyanate goat antimouse (1:100 dilution, 1 h in the dark, at room temperature). The coverslips were mounted using fluorescent mounting medium (DakoCytomation). The pictures were taken with a Zeiss Axioplan 2 microscope.

Migration assays
Migration (chemokinesis) of HTR-8/SVneo cells was measured using a protocol as reported earlier (3, 13, 14, 15). In brief, 1 x 105 cells suspended in 200 µl serum-free RPMI 1640 supplemented with 0.1% BSA were plated on Falcon cell culture inserts with microporous polycarbonate membranes having 8.0 µm pore size (Becton Dickinson) and then in 24-well Falcon notched plates. The lower chamber was filled with 800 µl serum-free medium with additional treatment or only vehicle. The cells were incubated at 37 C under 5% CO2 for 48 h. After removing the nonmigratory cells from the upper surfaces of the insert membranes by wiping with cotton swabs, the migrated cells were fixed and then stained with Harleco Hematocolor staining kit (EM Science, Gibbstown, NJ). The absolute number of migratory cells on each membrane was scored visually using light microscope (x400 magnification). Each treatment was performed in triplicates and reproduced in three different occasions. For presentation, data from one representative experiment have been used. Migration indices under experimental conditions were plotted as a percentage of the controls. Results of all the migration experiments were validated by examining the effect of various treatments on the proliferation/survival of HTR-8/SVneo cells using cell proliferation kit I from Roche Diagnostics (Laval, Québec, Canada). None of the test substances was found to have any effect on proliferation/survival of these cells when these assays were performed under same experimental conditions as those of the migration assays.

Small interfering (si) RNA preparation and transfection
The siRNA oligonucleotides duplexes (Dharmacon, Lafayette, CO) specific for human RhoA GTPase and ROCKs were designed according to literature and were purchased from Dharmacon in deprotected and desalted form. The siRNA sequences with dTdT overhanging at their 3' terminus used were: 5'-GACAUGCUUGCUCAUAGUCTT-3' to inhibit RhoA synthesis (16), 5'-GCCAAUGACUUACUUAGGATT-3' (Dharmacon siGenome reagent D-003536–05) to inhibit ROCK-I synthesis, 5'-GCAAAUCUGUAAAUACUCGTT-3' (Dharmacon siGenome reagent D-004610-05) to inhibit ROCK-II synthesis. Control siRNA, designated as scrambled siRNA, is a pool of several control siRNAs was purchased from Dharmacon. Transfection of siRNA was carried out using silencer siRNA transfection II kit (Ambion, Austin, TX). For transfection, 5 µl/ml of siPORT NeoFX and siRNA were diluted in OPTI-MEM I medium. Both were preincubated for 10 min, after which the two mixtures were combined and incubated at room temperature for additional 10 min for complex formation. The cells were detached and resuspended in RPMI 1640 containing 10% FBS (1 x 105 cells/ml), and 2.3 ml of the suspension were added to the mixture for 100 nmol/liter final concentration of siRNA. Transfections were performed for 48 h. Relative transfection efficiencies were 70–75%, as determined by the real-time RT-PCR after each siRNA transfection.

RhoA activation assay
RhoA activity was measured using a luminescence-based G-LISA kit from Cytoskeleton after the manufacturer’s protocol as described before (17, 18).

MAPK (ERK1/2) phosphorylation assays in intact cells
ERK1/2 phosphorylation was measured in intact cells using an ELISA kit (CASE) from SuperArray (SuperArray Bioscience Corp., Frederick, MD) as described by Kid et al. (19). Briefly, HTR-8/SVneo cells grown in 96-well plates were treated with PGE2 or other EP receptor agonists or vehicle for different time periods, fixed, subjected to microwave antigen retrieval, and then immunostained with either phospho-ERK1/2 or ERK1/2 antibody. After color development, the absorbance at 450 nm was determined using a plate reader. The absorbance readings were then normalized to the relative cell number by measuring the protein concentration of each well. The phosphorylated ERK1/2 signal was then compared with total ERK1/2 signal.

Western blotting
Whole-cell extracts from siRNA-transfected cells (20 µg/lane) were separated by 7.5% SDS-PAGE and transferred onto Immuno-BlotTM polyvinyl difluoride membrane (Bio-Rad, Hercules, CA). Nonspecific proteins on the polyvinyl difluoride membranes were blocked for 1 h using 20% nonfat dry milk in Tris-buffered saline (TBS). Then the membranes were incubated overnight at 4 C with the primary antibodies (Millipore) for RhoA, ROCK-I, or ROCK-II (1:1000) diluted in 1x TBS with 5% nonfat dry milk. After three washes in TBS with 0.1% Tween 20, the blots were incubated with HRP-conjugated goat antimouse IgG (1:20,000 in 5% milk in TBS) and goat antirabbit IgG (1:1000 in 5% milk in TBS) secondary antibodies. Detection was performed using enhanced chemiluminescence (ECL plus Western blotting detection system; Amersham, Oakville, Ontario, Canada).

Statistical analysis
We used SPSS software (version 12.0; SPSS, Chicago, IL) for statistical analysis. Migration assay results were converted to indices by normalizing each value as a percent of the control. The results were expressed as means ± SEM. Fold increases of migration indices in PGE2- or other EP agonist-treated cells, compared with controls without inhibitor treatments, were log transformed and compared with those with inhibitor treatments, and the significant differences between these two groups were identified using two-way ANOVA. Extents of phosphorylation of ERK1/2 after EP agonist treatments were compared between controls and C3 transferase-treated cells using two-way ANOVA. Statistical significance of the extent of ERK1/2 phosphorylation between the different time points was evaluated with one-way ANOVA followed by Newman-Keuls test for multiple groups. Differences of P < 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of cell permeable C3 transferase on PGE2-mediated migration of HTR-8/SVneo cells
We first investigated the role of RhoA in PGE2-mediated stimulation of migration of first-trimester human extravillous trophoblast by examining the migration of HTR-8/SVneo cells after treatment with cell-permeable C3 transferase (Cytoskeleton) that was shown to robustly inhibit the activities of RhoA, RhoB, and RhoC by irreversibly ADP-ribosylating these enzymes at asparagine 41 in the effector binding domain and thereby selectively inactivating them. The treatment with C3 transferase for 4 h significantly inhibited the migration of HTR-8/SVneo cells (Fig. 1AGo) without affecting their proliferation, suggesting that Rho proteins play an important role in the migration of these cells. Because cell migration requires polymerization of actin and reorganization of cytoskeleton containing F-actin, we also examined the effect of cell permeable C3 transferase on the actin cytoskeleton. C3 transferase treatment for 4 h (Fig. 1CGo) led to complete disorganization of F-actin that appeared bundled-up in the cytosol. Interestingly, migration assays performed by preincubating the cells for 4 h with C3 transferase followed by treatment with PGE2, 17-phenyl trinor PGE2 (EP1 and EP3 agonist), or PGE1 alcohol (EP4 and EP3 agonist) (Fig. 1AGo) showed that some of the migratory responses were partially rescued, suggesting that other endogenous molecule(s) partially substitute for activities of Rho proteins in these cells. However, this restoration was far below control levels or the stimulated levels in the presence of the individual ligands alone (Fig. 1AGo), indicating again the important role of Rho protein(s) in EVT cell migration. Also, in support of the migration data, phalloidin staining of HTR-8/SVneo cells preincubated with C3 transferase and then challenged with PGE2 (Fig. 1DGo), 17-phenyl trinor PGE2 (Fig. 1EGo), and PGE1 alcohol (Fig. 1FGo) showed that some of the morphological characteristics of the untreated cells (Fig. 1BGo), such as spreading of the cells, reappearance of stress fibers, and cortical actin, in concordance with the migration data.


Figure 1
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  		FIG. 1. Effects of cell permeable C3 transferase on PGE2-induced migration of HTR-8/SVneo cells. A, Migration assay showing that PGE2, 17-phenyl trinor PGE2 (an EP1 and EP3 agonist), and PGE1 alcohol (an EP3 and EP4 agonist) strongly stimulated migration (P < 0.01), whereas 3.5 mg/ml C3 transferase significantly inhibited (n = 3 ± SEM, P < 0.001) the migration of first-trimester human EVT at 48 h. The migratory responses were partially but significantly (P < 0.001) rescued by 1 µmol/liter PGE2, 17-phenyl trinor PGE2, or PGE1 alcohol treatment. *, Significantly different from the control. *a, Significantly different from only C3 transferase (C3) treatment. B–F, Phalloidin staining showing that incubation with 3.5 mg/ml C3 transferase for 4 h of HTR-8/SVneo cells disrupted the actin cytoskeleton (C), compared with controls (B). The cells have a round shape with almost no stress fibers and a weak cortical actin. The preincubation for 4 h with 3.5 mg/ml C3 transferase followed by treatment for 10 min with 1 µmol/liter PGE2 (D), 17-phenyl trinor PGE2 (E), or PGE1 alcohol (F) induced the reappearance of stress fibers and cortical actin, the cells regaining a polygonal shape. Results shown are representative of three independent experiments.

 
PGE2 stimulates the activity of RhoA and induces cytoskeletal changes in HTR-8/SVneo cells
To investigate whether PGE2 or other EP agonists activate RhoA, we measured the activity of RhoA after 1 µmol/liter PGE2, and 17-phenyl trinor PGE2, or PGE1 alcohol treatment. PGE2 as well as the other two EP agonists modestly but significantly stimulated the activity of RhoA starting at 5 min (Fig. 2AGo). We have previously shown that PGE2 can increase the migration of HTR-8/SVneo cells, acting through EP1 and EP4 (3). Rho GTPases play an essential role in cytoskeletal reorganization of F-actin during cell migration. Therefore, we studied the cytoskeletal changes in HTR-8/SVneo after treatments with PGE2 or two other EP agonists. Untreated cells (Fig. 2BGo) presented with a disorganized network of F-actin, thin cortical actin, and a polygonal shape. The treatment for 10 min with 1 µmol/liter PGE2 (Fig. 2CGo), 17-phenyl trinor PGE2 (Fig. 2DGo), or PGE1 alcohol (Fig. 2EGo) increased the organization of stress fibers that became orientated along the long axis of the cell. Also, the cortical actin was better represented under these treatments. This pattern of F-actin reorganization after EP agonist treatment also provides evidence for potential activation of RhoA GTPase because majority of inactive Rho GTPase is believed to be coupled with Rho GDI in the cytosol, whereas the majority of the active GTPase is membrane bound.


Figure 2
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  		FIG. 2. Effect of PGE2 on RhoA activation in HTR-8/SVneo cells. A, Treatment with 1 µmol/liter PGE2, 17-phenyl trinor PGE2, or PGE1 alcohol increased the activity of RhoA at 5, 10, and 15 min in HTR-8/SVneo cells, compared with their respective controls (n = 3 ± SEM, P < 0.05). B–E, HTR-8/SVneo cells stained with phalloidin showed increased stress fibers and cortical actin after 10 min of treatment with 1 µmol/liter PGE2 (C), 17-phenyl trinor PGE2 (D), or PGE1 alcohol (E), compared with control (B), which also give indication of Rho activation. Results shown are representative of three independent experiments.

 
Effect of siRNA-mediated silencing of RhoA on PGE2-mediated migration of HTR-8/SVneo cells
Because C3 transferase inhibits RhoA, RhoB, and RhoC and any pharmacological inhibitor may always have potential nonspecific effects, we further characterized the specific involvement of RhoA in mediating the PGE2 stimulatory effects on EVT cell migration by selectively knocking down RhoA using siRNA transfections into HTR-8/SVneo cells. Forty-eight hours after siRNA transfection, more than 70% down-regulation of RhoA mRNA level, compared with control, was observed by real-time PCR (data not shown), and 72 h after siRNA transfections, significant down-regulation of RhoA protein level was observed by Western blot analysis (Fig. 3BGo). Specific siRNA treatment did not alter the protein or mRNA levels of other Rho GTPases like Rac1 or Cdc42 (data not shown). RhoA knockdown resulted in a significant decrease in the migration of HTR-8/SVneo cells (Fig. 3AGo), suggesting again that RhoA plays an important role in the migration of these cells. The addition of exogenous PGE2, 17-phenyl trinor PGE2, or PGE1 alcohol partially restored some of the inhibition in the migration of HTR-8/SVneo cells (Fig. 3AGo), suggesting that RhoA is an important but not the only player in the migration of these cells when stimulated with these ligands.


Figure 3
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  		FIG. 3. Effect of RhoA siRNA treatment on migration of first-trimester human EVT. A, Migration assay showing that inhibition of RhoA silencing caused a significant reduction in the migration of HTR-8/SVneo cells at 48 h. Migration of these cells was only partially restored by 1 µmol/liter PGE2, 17-phenyl trinor PGE2, or PGE1 alcohol treatment (n = 3 ± SEM, P < 0.05). B, Western blot showing that down-regulation of RhoA protein after RhoA-specific siRNA transfection of HTR-8/SVneo cells at 72 h. Scrambled siRNA means a pool of four different control siRNA (obtained from Dharmacon). Results shown are representative of three independent experiments.

 
Effect of ROCK inhibition on PGE2-mediated migration of HTR-8/SVneo cells
It has been shown that RhoA induces actin polymerization in response to a variety of stimuli like lysophosphatidic acid, platelet-derived growth factor, insulin, bradykinin, and IL-1 signaling (20, 21, 22, 23) through ROCK and mDia (Drosophila gene Diaphanous). We investigated the effect of inhibition of ROCK on HTR-8/SVneo cell migration with a specific pharmacological inhibitor Y-27632 and also by siRNA-mediated gene silencing. siRNA-mediated down-regulations of ROCK-I and ROCK-II proteins are shown in Fig. 4Go, D and E, respectively. As shown in Fig. 4Go, A–C, the migration of these cells was significantly inhibited by both types of treatment. However, there was no restoration of migration with the addition of PGE2, or 17-phenyl trinor PGE2, or PGE1 alcohol. These data suggested that both ROCK-I and ROCK-II are essential for the PGE2-mediated migration of first-trimester human EVT. We also investigated the effect of Y-27632 on the F-actin in HTR-8/SVneo cells, and as shown in Fig. 4GGo, the cells appeared to have long processes and lose the F-actin stress fibers, compared with controls (Fig. 4FGo). Additional treatment with PGE2, 17-phenyl trinor PGE2, or PGE1 alcohol induced some cell spreading, but no stress fibers were observed (Fig. 4Go, H–J). These morphological data therefore are in support of the functional data on migration.


Figure 4
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  		FIG. 4. Effect of ROCK inhibition on migration of HTR-8/SVneo cells. A, Treatment with ROCK inhibitor (Y-27632, 10 µmol/liter) showed a significant decrease in migration of EVT cells at 48 h (n = 3 ± SEM, P < 0.01). The inhibition could not be mitigated by the treatment with 1 µmol/liter PGE2, 17-phenyl trinor PGE2, or PGE1 alcohol. B and C, Knockdown of either ROCK-I or ROCK-II by siRNA transfection induced a significant reduction in the migration of HTR-8/SVneo cells at 48 h (n = 3 ± SEM, P < 0.01). This inhibition was not significantly mitigated in the presence of PGE2, 17-phenyl trinor PGE2, or PGE1 alcohol. D and E, Western blot for ROCK-I and ROCK-II proteins, showing a significant reductions in the protein levels at 72 h after specific siRNA transfections. F–J, Staining for the actin cytoskeleton with phalloidin showing that the incubation of EVT cells for 30 min with 10 µmol/liter Y-27632 (G) induced a reduction in the number of stress fibers, stellate shape appearance with a small cytoplasm, compared with controls (F). The cells preincubated with 10 µmol/liter ROCK inhibitor for 30 min and then treated with PGE2 (H), 17-phenyl trinor PGE2 (I), or PGE1 alcohol (J) for 10 min showed polygonal shapes with few stress fibers and less abundant cortical actin, which correlated with the migration assay results. Results shown are representative of three independent experiments.

 
PGE2, EP1/EP3 agonist, or EP3/EP4 agonist increases the spreading of first-trimester human EVT from villus explants
Chorionic villus explants from first-trimester elective terminations of pregnancy have been used extensively to study EVT cell proliferation, adhesion, and migration (24). Proliferation studies using 5-bromo-2'-deoxyuridine labeling indicate that the number of dividing EVT cells decreases after 2 d of explant culture (25); therefore, the cells that outgrow derive mainly from cell columns already present by reorganization of these cell aggregates into a directionally migrating cell population. The dissecting technique and the selection of the villi become extremely important, and this may explain why not all villous explants produce viable outgrowths. We have shown previously that PGE2 stimulates the outgrowth of EVT cells in these explants primarily by stimulating their migration (3). In the present study, we have further demonstrated that an EP1/EP3 agonist or an EP3/EP4 agonist can mimic the effects of PGE2 to stimulate the outgrowth of EVT cells in these explants (Fig. 5AGo). As depicted in Fig. 5AGo, villi placed on matrigel and grown in DMEM/F12 show little cell spreading in the control wells at 24 and 48 h. The addition of 1 µmol/liter of PGE2, or 17-phenyl trinor PGE2, or PGE1 alcohol increased the migration of EVT cells, as indicated by the arrows (Fig. 5AGo) and quantified in Fig. 5BGo. The migrating cells stained positively for cytokeratin 7, an epithelial cell marker for cytotrophoblasts (1, 26) (Fig. 5CGo).


Figure 5
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  		FIG. 5. Effects of PGE2, 17-phenyl trinor PGE2, or PGE1 alcohol on the spreading of cytotrophoblast cells in chorionic villus explants in culture. A, Explant cultures of first-trimester human placentae showing that 1 µmol/liter PGE2 (second row), 17-phenyl trinor PGE2 (third row), or PGE1 alcohol (fourth row) increase the carpeting of EVT cells breaking out of the villi, compared with controls (first row), visible at 24 h and more obvious at 48 h. The arrows indicate the cells spreading from the tips of the villi. B, The graph shows quantification of the outgrowth area, plotting the area of outgrowth for each treatment divided by the area of outgrowth in control experiments (n = 6 ± SEM, P < 0.05). C. Confocal microscopic image of an explant at 48 h, stained with cytokeratin 7, an epithelial cell marker of EVT cells.

 
Cell-permeable C3 transferase inhibits the outgrowth of first-trimester human EVT cells in villus explants
We have shown earlier in the present study that C3 transferase inhibits the migration of HTR-8/SVneo cells, suggesting that RhoA is important for the migration of first-trimester human EVT cells. We also investigated the effect of C3 transferase on the EVT outgrowth. As shown in Fig. 6AGo, the villi treated with 3.5 µg/ml C3 transferase exhibited no cell spreading. Interestingly, the addition of 1 µmol/liter PGE2, and 17-phenyl trinor PGE2, or PGE1 alcohol to the cells preincubated for 4 h with 3.5 µg/ml C3 transferase caused a stimulation of the outgrowth, in concordance with the in vitro migration data on the EVT cell line, suggesting that PGE2 may activate an alternate signaling pathway, overcoming the RhoA inhibition by C3 transferase. Quantification of the area covered by the migrating cells is represented in Fig. 6BGo as a migration index. Migrating EVT cells that are breaking out of the villi have been magnified and represented in Fig. 6CGo.


Figure 6
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  		FIG. 6. Effect of C3 transferase on the spreading of cytotrophoblast cells. A, Explants from first-trimester human placentae incubated with 3.5 µg/ml C3 transferase showed reduced carpeting of EVT cells (first row) at 24 and 48 h. However, when the explants were preincubated for 4 h with 3.5 mg/ml C3 transferase and then treated with 1 µmol/liter PGE2 (second row), 17-phenyl trinor PGE2 (third row), or PGE1 alcohol (fourth row) showed increased carpeting at 24 and 48 h (see arrows), reaching the levels comparable with the explants that were incubated without C3 (see Fig. 4AGo). B, The graph quantifies the outgrowth area of the cytotrophoblast cells, plotted against controls (n = 6 ± SEM, P < 0.05). C, Inset depicted in the fourth row is shown at a higher magnification to show the spreading of the EVT cells, breaking out of the villi.

 
PGE2 activates MAPK (ERK1/2) and cell-permeable C3 transferase does not affect PGE2-mediated MAPK (ERK1/2) signaling
MAPK (ERK1/2) is another signaling molecule, besides ROCK, that can activate MLCK and induce the phosphorylation of myosin, increasing the contractile capacity of the cell during migration (27, 28). The results presented earlier in this study indicated that PGE2 can induce both the migration of EVT cells and stress fiber formation, even in the presence of RhoA inhibition (Fig. 1Go, D–F). Therefore, we tested the effect of PGE2 and the other two EP agonists on the level of ERK1/2 phosphorylation. As shown in Fig. 7AGo, PGE2, 17-phenyl trinor PGE2, or PGE1 alcohol induced the phosphorylation of ERK1/2, with a peak at 10 min and then decreasing by 30–60 min. To test whether RhoA played any role in PGE2-induced ERK1/2 phosphorylation, we incubated HTR-8/SVneo cells with 3.5 µg/ml C3 transferase for 4 h, and then the cells were challenged with either PGE2 or any one of the other two EP agonists. Whereas C3 treatment alone did not affect the level of ERK phosphorylation at 10, 30, and 60 min, the addition of PGE2 or any one of the other two EP agonists to the C3 pretreated cells stimulated ERK1/2 phosphorylation (Fig. 7AGo), suggesting that MAPK ERK1/2 could serve as an alternate pathway for RhoA action during PGE2-induced migration of HTR-8/SVneo cells. As a negative control, the cells were incubated for 2 h with U0126, a highly selective MAPK kinase inhibitor, followed by the challenge with EP agonist, showing no change in the phosphorylation status (data not shown). Furthermore, we tested the effect of U0126 on the migration of HTR-8/SVneo cells. U0126 treatment decreased the migration of EVT cells at 48 h, and the inhibition could not be mitigated by the addition of PGE2, 17-phenyl trinor PGE2, or PGE1 alcohol, suggesting that MAPK (ERK1/2) is an important mediator of PGE2-induced migration of EVT cells (Fig. 7BGo).


Figure 7
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  		FIG. 7. Effects of PGE2, 17-phenyl trinor PGE2, and PGE1 alcohol on ERK phosphorylation in HTR-8/SVneo cells. A. Treatment of HTR-8/SVneo cells with 1 µmol/liter PGE2, 17-phenyl trinor PGE2, or PGE1 alcohol for 10, 30, and 60 min increased ERK phosphorylation, compared with controls. Preincubation of HTR-8/SVneo cells with 3.5 µg/ml C3 transferase did not affect the level of phosphorylated ERK. Rho inhibition with C3 transferase did not affect PGE2-, 17-phenyl trinor PGE2-, or PGE1 alcohol-mediated ERK signaling. The data represent the level of phospho-ERK relative to the total ERK, measured with an ELISA (n = 3 ± SEM, P < 0.05). B, Presence of 10 µmol/liter MAPK kinase inhibitor U0126 decreased migration of HTR-8/SVneo cells at 48 h. Further addition of 1 µmol/liter PGE2, 17-phenyl trinor PGE2, or PGE1 alcohol to the cells, which were preincubated for 2 h with U0126, was not able to abrogate the migration inhibitory effect of UO126 (n = 3 ± SEM, P < 0.001). Results shown are representative of three independent experiments.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It is well known that defective placentation due to impaired EVT invasion of the uterine spiral arteries is a key feature underlying preeclampsia (29). On the other hand, hyperinvasive EVT cells are characteristic of gestational neoplasias including choriocarcinomas (30). Hence, a tight regulation of the invasive and migratory functions of EVT cells must be maintained in situ. A large number of regulatory factors produced at the fetomaternal interface have been shown to control EVT cell migration, invasiveness, and/or survival (1, 3, 31, 32, 33). PGE2 has been shown to stimulate the migration of a variety of cells, including both normal and cancer cells (34, 35, 36), acting through four types of membrane-bound G protein-coupled receptors, EP1–4 (37). We have also previously shown that PGE2 uses at least two receptors, EP1 and EP4, to stimulate the migration of first-trimester EVT cells (3). A selective EP1 antagonist (SC-19220), a nonselective EP1/EP2/DP antagonist (AH-6809), or a selective EP4 antagonist (AH-23848B) was shown to dose-dependently inhibit PGE2-mediated migration of HTR-8/SVneo cells (3). Furthermore, 17-phenyl trinor PGE2 (nonselective EP1/EP3-agonist) and PGE1 alcohol (nonselective EP3/EP4-agonist) were shown to mimic PGE2 action on migration of these cells (3). These results along with those in the present study do not exclude, however, the role(s) of EP2 and EP3 receptors in PGE2-mediated migration of EVT cells.

Rho GTPases, major regulators of cell cycle, cell morphogenesis, and cell migration are regarded as major downstream targets of G protein-coupled receptors (38). Shiokawa et al. (39) suggested an important role for RhoA and ROCK in the migration of first-trimester EVT cells. The present study provides for the first time evidence that RhoA and ROCK mediate the PGE2-mediated stimulation of EVT cell migration and that ROCK is a more important mediator than RhoA for this function. Furthermore, effects of inhibition of Rho protein activation in EVT cells may be compensated by PGE2-mediated activation of MAPK (ERK1/2).

Whereas RhoA inhibition with either cell-permeable C3 transferase or siRNA impaired basal HTR-8/SVneo cell migration, PGE2 stimulation through EP receptors partially restored some of this inhibition. This indicates that although RhoA plays an important role mediating EP receptor-induced migration of first-trimester EVT cells, PGE2 also has RhoA-independent action(s). Results of our present study suggest that MAPK (ERK1/2) can be one such pathway operative in EVT cells. Indeed, both ERK1/2 (27) and ROCK (28, 40, 41) can cause phosphorylation of MLCK that can induce actin stress fiber formation and contraction of these fibers to generate contractile force required for cellular migration. PGE2- or PGE1 alcohol-mediated stimulation of EP receptors in EVT cells, in which Rho proteins were inactivated by C3 transferase, induced a sustained activation of ERK1/2 (at least up to 60 min; Fig. 7Go), whereas the control cells not treated with C3 transferase exhibited a transient activation of ERK1/2 (that peaked at 10 min and declined thereafter). This may suggest that RhoA has a restraining effect on the maintenance of ERK activation by PGE2 in EVT cells. Other MAPKs like p38-MAPK, Jun N-terminal kinase, and ERK5 or big MAPK-1 have also been reported to play important roles in the regulation of cell migration (11). For example, both Jun N-terminal kinase and p38-MAPK cascades can phosphorylates paxillin at the focal adhesion sites, and ERK5 activation can lead to disruption of actin stress fibers (11). Whether PGE2 action is also mediated through these MAPK pathways remains to be elucidated.

Interestingly, first-trimester villus explants challenged with PGE2 or nonselective EP1 or EP4 agonist after 4 h preincubation with C3 transferase showed almost complete restoration of PGE2 actions that was not evident with the EVT cell line in migration assays. The villi were placed on growth factor-reduced matrigel and grown in serum-free conditions. It is possible that the presence of matrigel was responsible for this increased response of the C3 transferase-treated cells to PGE2 and various EP agonists. Whether a specific component of the ECM is responsible for these effects remains to be determined. It is also possible that cell permeability of C3 transferase was higher in the migration assays, leading to a more efficient inhibition of RhoA activity.

GTP-bound RhoA has been shown to interact with numerous downstream effectors (42). One of these effectors, ROCK, has been extensively cited in studies involving RhoA and migration. However, because Rho-independent activation of ROCK has also been reported before (43, 44, 45, 46), our data raise the possibility that PGE2-mediated activation of ROCK in EVT cells is RhoA independent. Two ubiquitously expressed isoforms of ROCK have been described: ROCK-I or Rho kinase-β and ROCK-II or Rho kinase-{alpha}. Most studies revealed that ROCK inhibition mimics RhoA inhibition. In our experiments, ROCK inhibition with Y-27632 or siRNA for ROCK-I or ROCK-II could not be rescued with PGE2 or various agonist treatments, suggesting that ROCK is essential for PGE2-mediated stimulation of EVT cell migration. ROCK-I and ROCK-II are highly homologous but have distinct functions on actin cytoskeletal reorganization (47). ROCK-I is more important than ROCK-II in the formation of focal adhesion and stress fiber (47). Furthermore, although null mutation of either ROCK-I or ROCK-II causes perinatal problems in mice, only ROCK-II mutants show placental defect (48, 49). The fact that down-regulation of either ROCK-I or ROCK-II totally blocks PGE2-mediated migration of EVT cells also indicate that one ROCK may not be able to compensate for the loss of the other ROCK. Indeed, the two ROCKs were shown to cooperate with each other by coordinating distinct signaling pathways to regulate actin dynamics (50).

Results of the present study are likely to have clinical significance. Decreased RhoA expression has been reported in human umbilical arteries of patients with preeclampsia (51). ROCK-II overexpression, reported in placentas of patients with clinically manifest preeclampsia (52), is likely a compensatory mechanism rather than a cause of the disease. Indeed, an important role of ROCK in the formation of placental microvilli-like structures (53) and migration of cytotrophoblast cells (39) have been suggested. Our present study identifies for the first time ROCK and ERK as major players in PGE2-mediated stimulation of first-trimester human EVT migration. Further studies are needed to investigate whether derangements in PGE2-ROCK axis and PGE2-ERK axis may play mechanistic roles in preeclampsia.


   Acknowledgments
 
We thank Dr. Fraser Fellows for providing the placental samples.


   Footnotes
 
First Published Online December 13, 2007

Abbreviations: EP, PGE receptor; EVT, extravillous trophoblast; FBS, fetal bovine serum; HRP, horseradish peroxidase; MLC, myosin light chain; MLCK, MLC kinase; PG, prostaglandin; ROCK, Rho-associated coiled-coil protein kinase; si, small interfering; TBS, Tris-buffered saline.

This work was supported by Grants MOP 69091 (to P.K.L.) and MOP 68997 (to C.C.) from the Canadian Institutes of Health Research. C.N. is the recipient of a Canadian Institutes for Health Research/Strategic Training Program scholarship in cancer research at the University of Western Ontario.

Disclosure Statement: The authors have nothing to disclose.

Received August 15, 2007.

Accepted for publication November 30, 2007.


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