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Original Article |
Departments of Obstetrics and Gynecology (S.S., M.I., K.S., H.H., M.K.S., Y.N.), Anatomy (Y.A.), and Biochemistry (S.N.), Kyorin University School of Medicine, Mitaka, Tokyo 181-8611, Japan; Department of Obstetrics and Gynecology (Y.Y.), Keio University School of Medicine, Shinanomachi, Tokyo, 160-8582, Japan; and Mitsubishi Pharma Corporation (M.U.), Aoba-Ku, Yokohama 227-0033, Japan
Address all correspondence and requests for reprints to: Shigetatsu Shiokawa, M.D., Ph.D., Department of Obstetrics and Gynecology, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka City, Tokyo 181-8611, Japan. E-mail: shiochan{at}kyorin-u.ac.jp.
Abstract
The small guanosine triphosphatase Rho controls cell adhesion and motility through reorganization of the actin cyto-skeleton and regulation of actomyosin contractility. Among the putative target molecules of Rho, a Rho-associated coiled coil-forming protein kinase (ROCK) is thought to participate in Rho-mediated cell adhesion and motility. In the present study, we explored the expression and function of RhoA and ROCK in human trophoblast cells. The colocalization of RhoA, cytokeratin 8/18, and cytokeratin 7 in some cells located in the decidual stromal region indicated that extravillous trophoblast cells expressed RhoA. In double staining for RhoA and ROCK in human chorionic villi, RhoA staining was strongly positive in the cytoplasm of cytotrophoblasts, whereas ROCK stained in the cytoplasm of cytotrophoblasts and syncytiotrophoblasts. Both RhoA and ROCK were stained in cytoplasma of cultured human cytotrophoblast. Cultured human trophoblast cells contained actin stress fibers that were lost after treatment with C3, an exoenzyme produced by Clostridium botulinum. Y-27632, a selective ROCK inhibitor, suppressed RhoA-induced formation of actin stress fibers and formation of focal contact in trophoblast cells. The trophoblast reacquired actin stress fibers and focal contact after withdrawal of Y-27632. Cultured human cytotrophoblast cells from 7–9 wk of gestation migrated into a fibronectin-coated membrane. Both C3 exoenzyme and Y-27632 inhibited cytotrophoblast migration in a dose-dependent manner. In conclusion, cyto-trophoblasts express RhoA and ROCK in their cytoplasm, and RhoA-ROCK is involved in their assembly of actin stress fibers. Suppression of RhoA-ROCK reduces trophoblast migration. These findings suggest that RhoA-ROCK signaling is a key regulator of trophoblast cell migration.
CELL-CELL AND CELL-EXTRACELLULAR matrix interactions are fundamental processes involved in cell migration and tissue remodeling. The adhesion of cells to substrata is a process mediated by cell surface integrins ligated with extracellular matrix (ECM) proteins (1). Ligated integrins are clustered and bound to cytoskeletal proteins and signaling molecules to form a complex called focal adhesions, to which thick actin bundles named stress fibers are bound. The small guanosine triphosphatase Rho works as a molecular switch in the intracellular pathway leading to integrin activation and formation of focal adhesions and stress fibers (2). Several putative target molecules of Rho have been isolated (3). Among these, a family of Rho-associated serine/threonine kinase isozymes, named Rho-associated coiled-coil forming protein kinase (ROCK), has been identified as a new class of Rho effectors that induce focal adhesions and stress fibers in cultured fibroblasts and epithelial cells (4, 5, 6, 7). ROCK works downstream of Rho and is involved in integrin-mediated cell adhesion (8).
Placental implantation in human begins with invasion of the uterine epithelium and underlying stroma by extraembryonic trophoblast cells. Villous cytotrophoblast cells at the tips of anchoring villi proliferate outwards from the underlying basement membrane to form columns, from which individual cells migrate into the decidual tissue. These interstitial trophoblast cells migrate as far as the superficial layer of the myometrium (9, 10, 11). What controls trophoblast migration is not known, although trophoblast migration appears to be tightly regulated. There is also the possibility that physical anchorage of the trophoblast to permissive ECM proteins may trigger progression into the extravillous lineage. Signals from ECM proteins, transduced by integrins, have been shown to mediate alterations in cell shape, adhesion, and migration, suggesting a role in the regulation of proliferation and differentiation of other cell types (12). Fibronectin (FN) is abundant at sites of anchoring villous formation in vivo. Because cytotrophoblast cells produce an oncofetal isoform (13) that is distinguishable by virtue of a unique glycopeptide epitope (14), it is clear that both maternal and trophoblast-derived FN are present in the ECM encountered by the trophoblast in the extravillous pathway (15). The cytotrophoblast undergoes a change in cell surface phenotype during its differentiation into the extravillous lineage (10, 16, 17, 18, 19), with loss of integrin 
6ß4 and rapid up-regulation of other integrins including the FN receptor 5ß1. The integrins themselves have no enzymatic activity and therefore must rely upon interactions with accessory proteins for generation of cytoplasmic signals. A small GTP-binding protein, Rho, has been implicated in integrin-initiated signaling events. Because Rho converts the resting integrin to an active form able to participate in cell adhesion, Rho functions as a switch to regulate processes evoked by external stimuli. Among the three isoforms of Rho, RhoA is the most ubiquitous and abundant; Rho and its downstream effector, ROCK, are considered to be critically involved in controlling cell migration.
A toxin derived from C. botulinum, C3 exoenzyme was recently to found inhibit Rho selectivity by ADP ribosylation (20). Y-27632, a pyridine derivative, selectively inhibits ROCK, and this inhibition was reversed by ATP in the competitive manner (21). These inhibitor poorly inhibit enzymes other than RhoA and ROCK (20, 21). In the present study, we investigated the expression of the RhoA-ROCK pathway in human trophoblast cells. By using above inhibitors, the role of RhoA-ROCK pathway in regulation of trophoblast cell migration was studied.
Materials and Methods
Reagents
Antihuman RhoA mouse monoclonal IgG1 antibody (200 µg IgG1 in 1.0 ml PBS) and antihuman RhoA rabbit polyclonal IgG antibody (200 µg IgG in 1.0 ml PBS) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). These antibodies react with RhoA of human origin and are not cross-reactive with RhoB, RhoC, RhoG, or other GDP/GTP binding proteins. Antihuman ROCK goat polyclonal IgG antibody (200 µg IgG in 1.0 ml PBS) and antihuman integrin 5 goat polyclonal IgG antibody (200 µg IgG in 1.0 ml PBS) were purchased from Santa Cruz Biotechnology, Inc. Antihuman vinculin mouse IgG1 antibody (8.6 mg IgG in 1.0 ml PBS) was purchased from Sigma (St. Louis, MO). Antiphospho-focal adhesion kinase (FAK) rabbit IgG antibody was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Antihuman cytokeratin 8/18 mouse monoclonal IgG1 antibody (420 µg IgG1 in 1.0 ml PBS) was purchased from Novocastra Laboratories Ltd. (Newcastle, UK). Antihuman cytokeratin 7 mouse monoclonal IgG1 antibody (1 mg IgG1 in 1.0 ml PBS) was purchased from Chemicon International (Temecula, CA). Anti-HPL (human placental lactogen) rabbit polyclonal IgG antibody (200 µg IgG in 1.0 ml PBS) was purchased from Chemicon International Inc. Fluorescein isothiocyanate (FITC)-labeled phalloidin was obtained from Sigma. The secondary antibody, FITC conjugated F(ab')2 goat antimouse IgG from DAKO Corp. A/S (Glostrup, Denmark), was affinity purified. Carbocyanine (Cy2)-labeled donkey antigoat IgG was purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Alexa 568-labeled donkey antirabbit IgG was purchased from Molecular Probes, Inc. (Eugene OR). Cy2-labeled donkey antimouse IgG, Cy2-labeled donkey antirabbit IgG, indocarbocyanine (Cy3)-labeled donkey antirabbit IgG, and Cy3-labeled donkey antimouse IgG were purchased from Jackson ImmunoResearch Laboratories, Inc. 4,6-diamidine-2-phenylindole hydrochloride (DAPI) was purchased from Roche Molecular Biochemicals (Mannheim, Germany). FN was purchased from Iwaki Glass (Chiba, Japan). C3 exoenzyme was purchased from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA). Y-27632 was a kind gift from Mitsubishi Pharma Corporation (Yokohama, Japan). Purified mouse monoclonal IgG1 antibody directed toward Aspergillus niger glucose oxidase, an enzyme that is neither present nor inducible in mammalian tissues (mouse Ig concentration; 100 mg/liter) (DAKO Corp. A/S) and normal goat serum (goat Ig concentration; 10 mg/ml) (DAKO Corp. A/S) were used as controls.
Specimens and cell culture
Specimens of first trimester placental tissues or deciduas were obtained from 27 women undergoing therapeutic abortion between 7 and 9 wk of gestation. The median age of these women was 27 yr (range, 22–33). Gestational ages were estimated from the reported date of last menstruation, uterine size, and fetal crown-rump length. All women gave informed consent for collection and investigational use of the tissues, and the study was approved by the Ethics Committee of Kyorin University School of Medicine. Isolation of primary trophoblasts was performed by methods described by Yagel et al. (22) with minor modifications. The tissues were rinsed thoroughly in cold PBS solution. Areas rich in chorionic villi were selected and minced into pieces of 
1 mm. Nonvillous materials inclusive of decidual tissue, blood clots, and membranes were carefully discarded. The fragments of villi were washed three times with Medium 199 (Invitrogen Corp., Carlsbad, CA) supplemented with streptomycin (20 mg/ml), penicillin (500 U/ml), and Amphotericin B (25 mg/ml). These fragments were then cultured in Medium 199 containing 10% fetal calf serum (Sigma;) and antibiotics in closed tissue culture flasks (Becton Dickinson and Co., Franklin Lakes, NJ) for 3–5 d until nonadherent cells could be removed and discarded. Cultures of adherent cells were expanded for 1–2 wk in these flasks in fresh medium at 37 C in humidified atmosphere containing 5% CO2 before their passage. For passages, cells were removed by trypsin-EDTA (Invitrogen Corp.) treated, washed, and replanted. The medium was changed every 48 h until confluent and cultures were continued until six passages to study of extravillous trophoblast cells. Their identity as extravillous trophoblast cells was established by immunohistochemical staining (positive for cytokeratin and HPL) (13).
Immunofluorescence analysis of tissues
Tissue was embedded in Optimum Cutting Temperature (Serologicals Corp., Kankakee, IL) and frozen in liquid nitrogen. Serial cryosections, 4–8 µm thick, were placed onto poly-L lysine-coated slides, and fixed in acetone at -20 C for 10 min. For doublestaining of decidual cells, after blocking with 5% BSA for 60 min at 37 C, cryosections were exposed to antihuman RhoA rabbit polyclonal antibody (concentration, 2 µg/ml) and antihuman cytokeratin 8/18 mouse monoclonal antibody (concentration, 2 µg/ml) at room temperature for 30 min. After rinsing in PBS, they were stained with Cy2-labeled, affinity-purified donkey antimouse IgG, and Cy3-labeled, affinity-purified donkey antirabbit IgG at 37 C for 60 min. The coverslips were washed in PBS. For double staining of decidua, the coverslips were incubated at 37 C for 60 min with antihuman RhoA rabbit monoclonal antibody (2 µg/ml) and antihuman cytokeratin 7 mouse monoclonal antibody (2 µg/ml), rinsed extensively in PBS, and then stained with Cy2-labeled, affinity-purified donkey antirabbit IgG and Cy3-labeled, affinity-purified donkey antimouse IgG for 60 min at 37 C. For double staining of villi, the coverslips were incubated at 37 C for 60 min with antihuman RhoA mouse monoclonal antibody (2 µg/ml) and antihuman ROCK goat polyclonal antibody (2 µg/ml), rinsed extensively in PBS, and then stained with Cy2-labeled, affinity-purified donkey antimouse IgG and alexa 568 labeled, affinity-purified donkey antigoat IgG for 60 min at 37 C. For controls, the coverslips were incubated at 37 C for 60 min with 2 µg/ml mouse monoclonal IgG1 antibody and normal goat serum substituted for the primary antibody. The coverslips were washed in PBS. Finally, all cells were mounted with glycerol, and then observed with an AX-80 fluorescence microscope (Olympus Corp. Optical, Tokyo, Japan). Fluorescence micrographs were taken using TMAX 400 film (Eastman Kodak Co., Rochester, NY).
Immunofluorescence analysis of cultured cells
For immunofluorescent staining of cultured trophoblast cells, human trophoblast cells were cultured for 24 h in medium 199 containing 10% fetal calf serum (FCS) on Lab-Tek chamber slides (Nalge Nunc International, Naperville, IL) coated with FN. Coating with FN was performed by incubating the coverslips overnight at 4 C in PBS with FN 10 mg/ml. The cultured trophoblast cells were fixed with 4% paraformaldehyde in PBS for 10 min, and washed three times for 10 min with PBS. The cells were permeabilized with 0.5% Triton X-100 for 10 min. The coverslips were incubated with 5% BSA for 60 min at 37 C. Anti-RhoA and anti-ROCK immunostaining was carried out for double staining of villi. For controls, the coverslips were incubated at 37 C for 60 min with 2 µg/ml mouse monoclonal IgG1 antibody and normal goat serum substituted for the primary antibody. Antihuman integrin 5 antibody immunostaining was carried out for staining of villi. For actin localization, trophoblast cells treated with C3 exoenzyme or Y-27632 for 3 h were fixed in 4% paraformaldehyde for 10 min, and permeabilized with 0.5% Triton X-100 for 5 min. To detect focal contact, they were incubated with antihuman vinculin mouse IgG1 antibody (2 µg/ml) and antiphospho-FAK rabbit IgG antibody (2 µg/ml), then stained with Cy3-labeled donkey antimouse IgG and Cy2-labeled donkey antirabbit IgG. They were incubated with antihuman vinculin mouse IgG1 antibody (2 µg/ml), then stained with Cy3-labeled donkey antimouse IgG and FITC-labeled phalloidin (0.1 mg/ml) to detect actin stress fibers. Finally, all cells were mounted with glycerol, and then observed with an AX-80 fluorescence microscope. Fluorescence micrographs were taken using TMAX 400 film.
Immunoprecipitation
The cultured trophoblast cells were washed once with ice-cold PBS, and the cells were solubilized in lysis buffer [1% Nonidet P-40, 2 mM EGTA, 150 mM NaCl, 50 mM Tris-HCl (pH 7.5), 100 mM sodium fluoride, 10 mM sodium pyrophosphate, 2 mM sodium vanadate, 1 mM 4-(2-aminoethyl) benzenesulfonyl fluoride, 1 µg/ml pepstatin A, 1 µg/ml leupeptin, and 1 µg/ml aprotinin]. The insoluble material was removed by centrifugation at 15,000 x g for 10 min and the supernatant (1 mg protein) was incubated with anti-RhoA antibody (1 µg) or anti-ROCK antibody (1 µg) for 18 h at 4 C. The immune complexes were incubated with antimouse IgG agarose or antigoat IgG agarose for 3 h at 4 C. The immobilized antimouse IgG or antigoat IgG was sedimented by centrifugation at 7000 x g for 1 min, washed with lysis buffer without phosphatase inhibitors four times, and the proteins were resuspended in 40 µl of Laemmli sample buffer. The immune complexes were incubated with protein-A Sepharose for 3 h at 4 C. The proteins were separated on SDS-PAGE and transferred to a polyvinylidene difluoride membrane (0.45 µm pore size). The membrane was probed with the indicated antibody, and visualized with enhanced chemiluminescence (SuperSignal CL-H substrate system: Pierce Chemical Co., Rockford, IL) and exposed to TMAX 400 film.
Migration assay
To quantify migration, cytotrophoblasts were plated on cell culture inserts (6.3 mm; Becton Dickinson and Co. containing polycarbonate filters with 8-µm pores. The ability of cells to penetrate the FN and/or migrate through to the underside of the filter has been positively correlated with invasive potential in several studies (23). In this study, the upper surface of the filters was coated with FN (13 µg/cm2). Coated filters were incubated at 37 C for 30 min. Cytotrophoblast cells (2x105) were plated on the filter in 400 µl of Medium 199. Medium was added to both the top and bottom of the cell culture insert, and the cells were allowed to migrate in the presence or absence of the appropriate materials. After cell culture for 24 h, the cell culture inserts were washed twice with PBS, and the cells were fixed with 2.5% hematoxylin and eosin for 30 min. The upper surface of the filter was then wiped with a cotton-tip applicator to remove noninvasive cells. The filters were cut off with a scalpel blade and mounted with glycerol. The numbers of stained cells were counted under the microscope. The cells on the underside of the filter were stained with antibody against cytokeratin and HPL to identify first trimester trophoblast cells. Migration assay was repeated at least three times and the result were expressed as the mean ± SEM of quadruplicate assays of each concentration of C3 exoenzyme or Y-27632.
Statistical analysis
The number of migrated cytotrophoblast cells is expressed as the mean ± SEM. Statistical analysis was performed by ANOVA with Scheffé’s test. Differences were considered statistically significant at P < 0.05.
Results
To investigate the location of RhoA in extravillous trophoblast cells, a double immunofluorescent study was performed in decidua. In decidua, the expression of RhoA was more pronounced in stromal cells and also in the glandular epithelium. RhoA, cytokeratin 8/18, and cytokeratin 7 colocalized in some cells located in the stromal region of the decidua indicating that extravillous trophoblast cells expressed RhoA (Fig. 1
). The double immunofluorescent study was performed in trophoblasts. The expression of RhoA was more prominent in cytotrophoblast cells, whereas ROCK was present in both cytotrophoblast and syncytiotrophoblast cells (Fig. 2). Nuclei were stained by DAPI. There was no staining by antibodies against RhoA or ROCK except nuclei stained by DAPI in controls. In cultured human cytotrophoblast cells, expression of RhoA in trophoblast cells showed a fibrillar pattern throughout the cytoplasm, especially in the perinuclear region. The expression of ROCK was observed intensely in the nucleus and perinuclear regions and was distributed diffusely in the cytoplasm (Fig. 3a). RhoA and ROCK protein levels in cultured trophoblast cells were evaluated by immunoblot analysis of total cellular protein. Trophoblast cells expressed appreciable amounts of RhoA protein and ROCK protein. A band of RhoA protein with a molecular mass of 21 kDa (Fig. 3b, lane 1) and a band of ROCK protein with a molecular mass of 160 kDa (Fig. 3b, lane 2) were detected in cell lysates. The expression of 5 integrin in the cultured cytotrophoblast cells showed a punctuate staining pattern in the cytoplasm (Fig. 4). There was no staining for 5 integrin in controls.
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). Treatment with C3 exoenzyme for 3 h alter the structure of actin stress fibers and reduced the enlargement and spreading of cytotrophoblast cells on FN (Fig. 5B). Phospho-FAK and vinculin clearly colocalized in focal adhesion of the cultured cytotrophoblast cells [Fig. 6a (A and B)]. Focal adhesion and actin stress fibers were observed in trophoblast cells stained with anti-vinculin antibody or FITC-labeled phalloidin, respectively [Fig. 6b (A and D)]. Focal adhesion and actin stress fiber formation was abolished by treatment with Y-27632 for 3 h [Fig. 6b (B and E)]. The structural changes of actin stress fibers were observed in none of 101 cells in control experiment, 98 of 120 cells in C3 treatment (P < 0.001 vs. control), and 93 of 98 cells in Y-27632 treatment (P < 0.001 vs. control), respectively. To negate the possibility of toxicity, Y-27632 was withdrawn from the culture medium, and cytotrophoblast cells were recultured in medium without Y-27632. Three hours after withdrawal of Y-27632, trophoblast cells reacquired their activated phenotype, including stress fiber and focal adhesion formation [Fig. 6b (C and F)].
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and 8).
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Extravillous trophoblast cells migrate deeply into the uterine mucosa, and thus encounter the many types of maternal cells present in the endometrium (11). Extravillous trophoblast cells are believed to be derived from the cell column of the anchoring villi, and they differentiate into interstitial trophoblasts in the placental bed. In the present study, RhoA and cytokeratin 8/18 were expressed in some cells in the stromal region of the decidua. Furthermore, RhoA and cytokeratin 7 were expressed in the same cells. Cytokeratin 7 is a marker for cytotrophoblast (24). Cytokeratin 8/18 is not specific to extravillous trophoblast cells, but cytokeratin 8/18 is specific to epithelial cells. The colocalizations of RhoA, cytokeratin 8/18 and cytokeratin 7 in some cells located in decidual stromal region indicated the expression of RhoA in extravillous trophoblast cells invading into the maternal decidua. We have demonstrated in a previous study that human decidual cells express RhoA (25). These findings indicate that RhoA is expressed in both human decidual stromal cells and the invading trophoblast at least in vivo.
In the double immunostaining of trophoblast cells, cytotrophoblast cells expressed RhoA and ROCK in vivo and in vitro. Cytotrophoblast cells also expressed 5ß1 integrin. Expression of 5ß1 integrin in various types of cells appears to be associated with cell motility. Burrows et al. (26) demonstrated that the 5ß1 integrin expressed by trophoblast cells is functionally active and mediates adhesion to FN in vitro. Irving et al. (27) demonstrated that cell surface 5ß1 integrin is essential for trophoblast cell migration. The Rho-ROCK pathway works as a molecular switch in the intracellular pathway leading to integrin activation and plays a central role in the migration of many cells. These data, in conjugation with our data, demonstrate that cytotrophoblast expresses 5ß1 integrin and Rho/ROCK pathway; this pathway is one of the important pathways that regulate the migration of cytotrophoblast, although we did not demonstrate the direct interaction between 5ß1 integrin and FN in this culture systems.
Actin stress fibers, consisting of long bundles of filaments that traverse the cell, are linked to ECM proteins through integrins. Rho proteins trigger the formation of contractile actin stress fibers, resulting in regulation of cell motility (2, 28). In the present study, trophoblast cells growing in medium supplemented with FCS contained actin stress fiber. Treatment with C3 exoenzyme, which specifically ADPribosylates members of the Rho family and inhibits their biologic activity (2, 29), made these trophoblast cells round up, in association with alteration of the structure of actin stress fibers. Changes in actin stress fibers induced in human trophoblast cells by C3 exoenzyme were very similar to those seen in other cell lines (2). Furthermore, we investigated the involvement of the downstream effector of RhoA in the intracellular translocation of lysosomes using Y-27632, a selective ROCK inhibitor. The addition of Y-27632 to the culture medium also blocked actin stress fiber bundling and focal adhesion assembly in the trophoblast. The inhibitory effects of Y-27632 were reduced after drug exclusion from the medium at the end of preincubation, implying that ROCK is required for actin stress fiber formation and assembly of focal adhesion in cytotrophoblast cells. These data suggest that the RhoA-ROCK pathway in trophoblast cells is involved in the assembly of actin stress fibers. Thus, the conformational change in actin stress fibers may affect trophoblast migration.
Cytotrophoblast cells that migrate out of chorionic villi in situ selectively express 5ß1 integrin (16, 18). This discovery led to an increased interest in the possible functional relationship of this receptor to the migratory phenotype of extravillous trophoblast cells. FN, which is produced by extravillous trophoblast cells in situ, is used by these cells for anchorage via the 5ß1 integrin receptor before migration (27). The cytotrophoblast cells used in the present study expressed 5ß1 integrin. Cytotrophoblast cells were plated on FN-coated filters containing pores to quantify migration. The ability of cells to migrate through defined-size pores is positively correlated with migration potential (30). Hocking et al. (31) suggest that FN polymerization triggers integrin-mediated cytoskeletal reorganization and enhances cell contractility through a mechanism involving the action of RhoA. From this migration assay, it is possible to speculate that the interaction between integrins in cytotrophoblast and FN promotes migration. Damsky et al. (18, 32) have demonstrated that the expression of 5ß1 integrin on extravillous trophoblasts is maintained during the invasion process. Treatment of extravillous cells with a blocking antibody against 5ß1 integrin showed nearly complete inhibition of basal migration (30), suggesting that 5ß1 integrin is involved in trophoblast invasion. In this study, cytotrophoblast migration was inhibited by C3 exoenzyme in a dose-dependent manner, indicating that C3 exoenzyme modifies the migration of trophoblast by regulating RhoA activity. Rho is involved in molecular switching of integrin activation (33). Activated integrins bind to ECM, triggering the assembly of focal adhesion proteins and concern the actin stress fiber formation (33). Thus RhoA may implicated in integrin-mediated cytotrophoblast migration by regulation of actin stress fibers in cytotrophoblasts.
A recent study has demonstrated that Y-27632 blocks Rho-mediated activation of actomyosin and invasive activity of MM1 cells (34), indicating that ROCK plays an essential part in tumor cell invasion. By using this specific inhibitor, we found that Y-27632 reduced cytotrophoblast cell migration in a dose-dependent manner. Decreased motility of cytotrophoblast cells by Y-27632 can be ascribed to the impaired formation of intracellular structures required for cell movement, typically focal adhesions and actin stress fibers. Integrin-mediated signaling events are thought to be RhoA-ROCK pathway dependent (35). This pathway regulates cell migration. We have now demonstrated that activation of the RhoA-ROCK pathway is an essential step during migration of cytotrophoblast cells. Implantation of the human blastocyst into the endometrium has similarities to tumor invasion of host tissue (36). Implantation and metastatic processes share common biochemical intermediates, but invasion by trophoblasts is tightly controlled. Regulation of the expression of integrins and interaction with individual ECM variants play a key role in ensuring adequate, but not excessive, invasion of the maternal uterine decidua. Further work is needed to identify the downstream effects of ECM-integrin ligation on trophoblast. For example, binding of IGFBP-1 to 5ß1 integrin was shown to promote trophoblast migration by activation of FAK and stimulation of MAPK pathway (30). A detailed analysis of signals transduced from the matrix and decidual microenvironment will provide a better understanding of the factors that determine behavior of the trophoblast in vivo and may lead to treatment of disorders of pregnancy associated with inadequate or overinvasion by the trophoblast. Use of inhibitors of ROCK activity may thus provide novel therapeutic strategies.
In conclusion, the formation of focal contacts and actin bundles in cytotrophoblast cells is regulated by the RhoA-ROCK pathway. C3 exoenzyme and Y-27632 suppress migration of cytotrophoblast cells by inhibiting this pathway. These findings suggest that the RhoA-ROCK pathway in human cytotrophoblast cells may be an important mediator of implantation.
Acknowledgments
Footnotes
This work was supported in part by Grants-in-Aid (C) 11671648 (S.S.) from the Ministry of Education, Science and Culture (Tokyo, Japan).
Abbreviations: Cy2, Carbocyanine; Cy3, indocarbocyanine; DAPI, 4,6-diamidine-2-phenylindole hydrochloride; ECM, extracellular matrix; FAK, focal adhesion kinase; FCS, fetal calf serum; FITC, fluorescein isothiocyanate; FN, fibronectin; HPL, human placental lactogen; ROCK, Rho-associated coiled coil-forming protein kinase.
Received March 20, 2002.
Accepted September 4, 2002.
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