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Original Studies |
Departments of Obstetrics and Gynecology (S.S., K.S., N.S., H.H., M.I., Y.N.), Anatomy (Y.A., H.H.), and Biochemistry (S.N.), Kyorin University School of Medicine, Mitaka, Tokyo 181-8611; and Department of Obstetrics and Gynecology, Keio University School of Medicine (Y.Y.), Shinanomachi, Tokyo 160-8582, Japan
Address all correspondence and requests for reprints to: S. Shiokawa, M.D., Department of Obstetrics and Gynecology, Kyorin University School of Medicine, 6-20-2, Shinkawa, Mitaka, Tokyo 181-8611, Japan. E-mail: shiochan{at}kyorin-u.ac.jp
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The Rho proteins are members of a large family of small GTP-binding proteins that share approximately 30% sequence homology with Ras (5). These proteins have been described in yeast, Aplysia, and human cells, where three different Rho genes (A, B, and C) have been found by screening a peripheral T lymphocyte complementary DNA library with fragments from Aplysia Rho complementary DNA (6). Among the three isoforms of Rho, RhoA is the most ubiquitous and abundant and has been the most extensively studied. RhoA plays a prominent role in regulating organization of the cytoskeleton by promoting the assembly of focal adhesions (7) and actin stress fibers (8) and by activating focal adhesion kinase (FAK) (9). Aggregation of integrins induces the recruitment of RhoA to sites of integrin clustering (10), and activation of RhoA, in turn, regulates signaling downstream from integrins (11, 12, 13). Recent evidence indicates that RhoA modulates initial steps in integrin signaling by regulating integrin clustering (7), most likely through changes in cell contractility (14).
In human endometrium, recent studies have demonstrated that expression of ß1 integrins increases at the time of implantation (15, 16, 17, 18). We have reported that outgrowth of embryos on decidual cells, but not attachment, is inhibited by administration of antibodies recognizing components of the ß1 integrin family; this suggests that ß1 integrins on decidual cells may be important in embryonic development and differentiation after attachment (19, 20). FAK and ß1 integrin in human decidual cells are located at regions known as focal adhesions (21). Linkage of ß1 integrin to the cytoskeleton at these focal contacts on human decidual cells may be important in mediating implantation. Although many reports indicate that integrins are particularly important in both fertilization and embryogenesis, including the process of implantation (16, 17, 18, 19, 20, 21), the actions of RhoA in human endometrial cells and decidual cells during implantation remain to be clarified.
In the present study we investigated the expression and functional role of RhoA in human decidual cells. Addition of C3 exoenzyme, which ADP-ribosylates Rho proteins at amino acid Asn41, causes disappearance of actin stress fibers and contraction rounding up of the cell body in fibroblast cultures (22). Recently, nanomolar concentrations of the lysophosphatidic acid (LPA) have been shown to stimulate the formation of actin stress fibers in Swiss-3T3 cells by a pathway that requires the involvement of Rho (5, 8). We therefore used C3 exoenzyme and LPA to analyze the function of RhoA in human decidual cells. Furthermore, the effects of C3 exoenzyme and LPA on embryo attachment to and spreading on decidual cells were examined to determine the role of RhoA in implantation of embryos.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Endometrium was obtained from 28 women of reproductive age (range, 32–40 yr) at the time of hysterectomy at the Kyorin University Hospital. All hysterectomies were performed for abnormalities of nonendometrial origin, such as uterine leiomyoma and adenomyosis. The tissues were obtained from the early proliferative (days 5–7) and midsecretory phases (days 19–22). The mean cycle length in these patients was 28 days (range, 26–31 days), and no patient had received hormonal medication during the 3 months before surgery. Cyclic changes in the endometrium were determined by the endometrial dating system of Jones et al. (23). Specimens of decidua were obtained from 27 women undergoing therapeutic abortion between 7 and 11 weeks gestation. The median age of these women was 29 yr (range, 22–35). 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.
Reagents
Antihuman RhoA mouse monoclonal IgG1 antibody (100 µg IgG1 in 1.0 mL PBS) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). This antibody reacts with proteins of human origin and is noncross-reactive with RhoB, RhoC, RhoG, or other GDP/GTP-binding proteins. Fluorescein isothiocyanate (FITC)-labeled phalloidin was obtained from Sigma (St. Louis, MO). The fluorescently labeled secondary antibody, FITC-conjugated F(ab')2 goat antimouse IgG (DAKO Corp., Glostrup, Denmark), was affinity purified. Fibronectin (FN) was purchased from Iwaki Glass (Chiba, Japan). C3 exoenzyme was purchased from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA). LPA (1-oleoyl) was obtained from Sigma. A 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/L) (DAKO Corp.), was used as a negative control.
Immunohistochemistry
Tissue was embedded in OCT (Miles Diagnostic Division, Elkhart, IN) and frozen in liquid nitrogen. Serial cryosections, 4–8 µm thick, were placed onto poly-L-lysine-coated slides, fixed in acetone at -20 C for 10 min, and stained using Strept ABC complex/HRP kits (DAKO Corp., Glostrup, Denmark). Cryosections were exposed to antihuman RhoA monoclonal antibody (concentration, 2 µg/mL) after blocking with 0.5% normal goat serum (DAKO Corp.) in phosphate-buffered saline (PBS), and allowed to bind at room temperature for 30 min. Another rinse in PBS was followed by application of a secondary antibody consisting of biotinylated goat antimouse antibody (dilution, 1:500) for 1 h. After another rinse in PBS, endogenous peroxidases were quenched by a 30-min incubation with 0.3% H2O2 in absolute methanol, followed by a 30-min of rehydration in PBS. Sections then were incubated with avidin-biotinylated horseradish peroxidase macromolecular complex for 30 min before incubation with diaminobenzadine for 3 min to complete the color reaction. Negative controls consisted of tissue sections in which a 2 µg/mL mouse monoclonal IgG1 antibody was substituted for the primary antibody; in these controls procedure-related background staining was consistently absent.
Decidual cell culture
Specimens were placed immediately in ice-cold medium 199 (Life Technologies, Inc., Grand Island, NY) containing 25 mmol/L HEPES (Sigma) and 1% antibiotic-antimycotic mixture (Life Technologies, Inc.), and transported to the laboratory within 1 h. After blood clots were manually removed, the decidual tissue was rinsed thoroughly in ice-cold medium 199. The tissue was trimmed and cut into pieces approximately 1 mm3 in size using a small pair of scissors. A portion of the tissue was stained with hematoxylin-eosin for histological examination. The remaining tissue was treated enzymatically to disperse the cells. Isolation of decidual cells was performed by methods described by Satyaswaroop et al. (24) and Braverman et al. (25), with minor modifications. The tissue was treated with 0.1% collagenase (type IA; Sigma) and 0.1% hyaluronidase (type IS; Sigma) in Ca2+-free PBS while stirring at 37 C for 1 h. At the end of this period, the cell suspension was filtered through nylon mesh (pore size, 105 µm) to remove undigested tissue debris. Cells were collected from the filtrate by centrifugation at 800 x g for 10 min, and the pellet was resuspended in medium 199 containing 10% FCS (Life Technologies, Inc.). The cell suspension was filtered through a 38-µm stainless steel sieve (Spectrum, Los Angeles, CA) to retain the glandular elements as previously described (24, 26). Stromal cells then were collected by centrifugation, washed, resuspended in 20% isotonic Percoll solution and layered above 20–60% and 40–55% Percoll gradients (25). Tubes containing gradients were centrifuged for 15 min at 30,000 x g in a Beckman Coulter, Inc., L3–50 ultracentrifuge at 4 C, using a type 65 fixed angle rotor (Beckman Coulter, Inc., Palo Alto, CA). An enriched fraction of PRL-producing decidual cells layered as a single band with a cell density of 1.033–1.048 g/mL. The band contained a nearly homogeneous population of large round mononucleated cells greater than 25 µm in diameter (25). The decidual cells were washed and suspended three times in medium 199 supplemented with 10% FCS and 1% antibiotic-antimycotic mixture. Aliquots of decidual cell suspensions were counted by the dye exclusion test using 0.4% (vol/vol) trypan blue in PBS. The stromal cells were plated at 5 x 105 cells/mL in a 35 x 10-mm plastic petri dish (Falcon 3001, Becton Dickinson and Co., Lincoln Park, NJ). The culture medium was changed every 48 h, and cultures were maintained in a humidified 95% air-5% CO2 at 37 C for 10 days. Homogeneity of the stromal cultures was verified by immunocytochemical localization of cell-specific markers, including vimentin (stroma), keratin (epithelium), muscle actin (vascular smooth muscle), and intermediate filaments of factor VIII antigen (endothelium). The contamination with endometrial epithelial or vascular cells was less than 0.1%.
Immunofluorescent staining of cultured decidual cells
Human decidual cells were cultured for 60 min or 24 h in medium 199 containing 10% 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 µg/mL. The cultured decidual cells were fixed with 4% paraformaldehyde in PBS for 10 min and washed three times for 10 min each time 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 for blocking nonspecific staining. The coverslips then were incubated at 37 C for 60 min with antihuman RhoA mouse monoclonal antibody (2 µg/mL), rinsed extensively in PBS, and stained with a fluorescein-labeled, affinity-purified goat antimouse IgG for 60 min at 37 C. For negative control, the coverslips were incubated at 37 C for 60 min with 2 µg/mL mouse monoclonal IgG1 antibody substituted for the primary antibody. The coverslips were washed in PBS, rinsed in deionized water, and mounted with glycerol. Coverslips were observed with an AX-80 fluorescence microscope (Olympus Corp., Tokyo, Japan). Fluorescence micrographs were taken using TMAX 400 film (Eastman Kodak Co., Rochester, NY). For actin localization, decidual cells treated with C3 exoenzyme or LPA for 24 h were fixed in 4% paraformaldehyde for 10 min, permeabilized with 0.5% Triton X-100 for 5 min, and incubated with 0.1 µg/mL FITC-labeled phalloidin for 40 min. The coverslips were rinsed extensively in PBS. Cells were viewed with an Olympus Corp. microscope and photographed using TMAX 400 film.
Immunoblotting
Endometrium, decidua, decidual cells cultured for 10 days, and HeLa cells (for a positive control), which was obtained from Japan Health Sciences Foundation (Osaka, Japan), were disrupted by sonication, boiled in SDS sample buffer with 10 mmol/L dithiothreitol, and transferred to nitrocellulose filters. Protein concentrations were determined using a protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA). In each lane 50 µg total cell protein were studied. The filters were incubated with antihuman RhoA monoclonal antibody (2 µg/mL), followed by appropriate horseradish peroxidase-conjugated secondary antibody. For a negative control, the filters were incubated with 2 µg/mL mouse monoclonal IgG1 antibody substituted for the primary antibody. Bands were quantitated using a chemiluminescence detection system (NEN Life Science Products, Boston, MA).
Assays for embryo attachment and spreading on human decidua
Embryo attachment and spreading assays were performed using cultured human decidua. Superovulation was induced in female ICR mice (8 weeks old; Clea Japan, Tokyo, Japan) by an injection of 5 IU PMSG (Teikoku-zoki, Tokyo, Japan). After 48 h an injection of 2.5 IU hCG (Teikoku-zoki) was given, after which the mice were caged with ICR males. Embryos at the late morula or early blastocyst stage were obtained by flushing of the uterine horns 96 h after the hCG injection. Collected embryos were rinsed in medium 199 supplemented with 0.4% BSA.
Decidual cells had been cultured in medium 199 supplemented with 10% FCS for 10 days. In experimental cultures, subconfluent decidual cell monolayers were incubated with either C3 exoenzyme at a concentration of 1–100 ng/mL or LPA at a concentration of 1–100 µmol/L for 12 h before addition of embryos. Subconfluent decidual cell monolayers that had not been treated with either C3 exoenzyme or LPA were used as controls. Five to eight embryos were placed in prepared dishes with a subconfluent monolayer culture of decidual cells and cocultured for 96 h. Embryo attachment was identified by gentle flushing of each embryo with a small amount of medium, using a glass pipette pulled to a very fine bore. Embryos that showed no movement upon flusing during observations under an inverted phase contrast microscope (Olympus Corp.) were considered attached. Embryos were classified as spreading if migration of individual cells or monolayers of trophoblasts from the ectoplacental cone rudiment was observed. The extent of spreading was determined by photographing the embryos at a magnification of x200 and printing each negative at the same size. The area of outgrowth was measured using a color image-analyzing system (SP500, Olympus Corp.) as described by Imamura et al. (27). The same observer (S.S.) produced each tracing. The final value for each embryo was calculated as the mean of three tracings. The result for each treatment represents the mean final area values for at least 45 embryos. Occurrences of embryo attachment and spreading were assessed at 24 and 48 h of incubation, respectively. The area of embryo outgrowth was determined between 48 and 96 h of incubation.
Statistical analysis
The percentages of embryo hatching, attachment, and spreading and the area of embryo spreading are expressed as the mean ± SEM. To obtain a normal distribution, the percentages of embryo showing attachment and spreading were transformed using an arcsine transformation. Statistical analysis was performed by ANOVA with Scheffe’s test. Differences were considered statistically significant at P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. In the proliferative and
midsecretory phases, RhoA was present mainly in glandular epithelium
(Fig. 1, A and B). In decidua, RhoA was expressed more prominently in
the stromal cells and also was present in the glandular epithelium
(Fig. 1C). There was no staining in negative control (Fig. 1, D–F).
RhoA protein levels in the proliferative and midsecretory phases as
well as in decidua were evaluated by performing immunoblot analysis of
total cellular protein. HeLa cell (as a positive control; Fig. 2, lane 1), proliferative endometrium
(Fig. 2, lane 2), midsecretory endometrium (Fig. 2, lane 3), and
decidua (Fig. 2, lane 4) expressed appreciable amounts of RhoA protein.
A single band of RhoA protein with a molecular mass of 21 kDa was
detected in all cell lysates, although any positive bands were not
observed in midsecretory endometrium with mouse monoclonal IgG1
antibody (Fig. 2, lane 5).
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). RhoA in human
decidual cells showed a floccular staining pattern throughout the
cytoplasm, with a small portion located at the plasma membrane (Fig. 3B), whereas there was no staining in negative control (Fig. 3, C and
D). Immunoblots showed an appreciable amount of RhoA protein in
cultured decidual cells (Fig. 4, lane 1).
HeLa cells, the positive control, also expressed an appreciable amount
of RhoA protein (Fig. 4, lane 2), in contrast to no band detected with
mouse monoclonal IgG1 antibody (Fig. 4, lane 3).
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).
Treatment with C3 exoenzyme for 24 h made cultured decidual cells
round up, associated with loss of actin stress fiber formation (Fig. 5B). In contrast, new stress fibers appeared within 24 h after the
addition of LPA (Fig. 5C).
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Addition of C3 exoenzyme or LPA to the cultured decidual cells did not
affect their hatching ratio. Attachment of hatched blastocysts was
slightly, but not significantly, reduced when decidual cell cultures
were treated with C3 exoenzyme or LPA. The spreading of trophoblasts
from attached blastocysts was observed 48–96 h after coculture with
decidual cells. The area of trophoblast outgrowth increased with
increasing the duration of coculture with decidual cells. Exposure of
the decidual cells to C3 exoenzyme decreased the area of trophoblast
outgrowth in a dose-dependent manner at 96 h after coculture
(Figs. 6 and 7). Furthermore, exposure of the decidual
cells to LPA increased the area of trophoblast outgrowth in a
dose-dependent manner at 96 h after coculture (Figs. 6 and 7).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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4ß1 and
vß3 integrin has been
suggested to coincide with the putative implantation window
(17). We recently demonstrated that
ß1 integrins were expressed predominantly in
glandular epithelial cells in the secretory phase and in stromal cells
in the decidua (18). These data in conjunction with our
present data indicate that expression of RhoA coincides with integrin
expression in human endometrium and decidua. In Rat-2 fibroblasts, RhoA was reported to show a floccular pattern fluorescent staining throughout the cytoplasm, with a small proportion located at the plasma membrane (29). RhoB, on the other hand, showed a very different pattern of fluorescence when expressed in Rat-2 fibroblast cells; staining was predominantly associated with cytoplasmic vesicles that typically clustered around the nucleus, becoming less numerous toward the cell periphery (29). In the present study RhoA in cultured human decidual cells showed a pattern of immunofluorescent localization similar to that in Rat-2 fibroblasts. Immunoblots detected a single band of RhoA protein with a molecular mass of 21 kDa in all cell lysates, and similar amounts of the protein were observed in the early proliferative phase, midsecretory phase, and decidua. In human implantation, the embryo makes contact with the endometrial ECM in the midsecretory phase and then burrows into the decidualized stromal cells. In the present study the amount of RhoA protein did not change during the endometrial cycle, although the expression of RhoA shifted from glandular epithelium to stromal cells with decidualization. Thus, RhoA in human endometrium and decidua may act in part to regulate blastocyst outgrowth.
Rho proteins trigger the formation of contractile actin stress fibers, resulting in the regulation of cell motility (8, 30). Actin stress fibers consist of long bundles of filaments that traverse the cell and are linked to the ECM through integrins. C3 exoenzyme and LPA have been used to explore the role of Rho proteins in various types of cultured cells (8, 31). C3 exoenzyme, which specifically ADP-ribosylates members of the Rho family and inhibits their biological activity, induces morphological change when incubated with or microinjected into cultured cells. A typical change is rounding up of cell bodies associated with disassembly of actin stress fibers (22, 32). In the present study decidual cells growing in medium supplemented with FCS contained actin stress fiber. Treatment with C3 exoenzyme made these decidual cells round up, in association with loss of actin stress fibers. Changes in actin stress fibers induced in human decidual cells by C3 exoenzyme were very similar to those seen in other cell lines. In contrast, at nanomolar concentrations, LPA, a well characterized phospholipid, stimulates the assembly of focal adhesions and formation of actin stress fibers in Swiss-3T3 cells by a pathway that requires the function of Rho (5, 8). In the present study LPA potently stimulated actin stress fiber accumulation in decidual cells, a change comparable to that seen in Swiss-3T3 cells. Thus, treatment of decidual cells with C3 exoenzyme or LPA induces changes in the pattern of actin stress fibers by changing the biological activity of RhoA. However, the possibility that C3 exoenzyme and LPA induce these changes by modifying the formation of contractile stress fiber directly, but not through RhoA, remains to be clarified.
Implantation requires a molecular dialogue between trophoblast cells and decidual cells. Hatched mouse blastocysts cultured in vitro attach and exhibit trophoblast outgrowth on human decidual cells, representing a model for implantation (19, 20). In the present study, therefore, we used human decidual cells as a model for studying embryo attachment and implantation. Mouse trophoblast invasion differs in some respects from human trophoblast invasion, but ethical considerations precluded the use of large numbers of human embryos. The trophoblastic outgrowth of embryos on decidual cells was inhibited by C3 exoenzyme in a dose-dependent manner. On the other hand, the outgrowth of embryos on decidual cells was increased dose dependently by the addition of LPA. C3 exoenzyme and LPA affected the formation of actin stress fibers of decidual cells, suggesting that these substances may modify the outgrowth of trophoblast cells by regulating the RhoA activity of decidual cells. However, the possibility that C3 exoenzyme and LPA directly modify the outgrowth of trophoblast cells, not indirectly through endometrial stromal cells cannot be excluded.
Our previous studies have demonstrated that decidual cells express ß1 integrins on the cell surface (18, 19). Outgrowth, but not attachment, of embryos on decidual cells was inhibited in a dose-dependent manner by the addition of an antibody against the ß1 chain, implying that ß1 integrins are important in blastocyst development and differentiation after attachment (19, 20). The integrins themselves have no enzymatic activity and therefore must rely upon interactions with accessory proteins for generation of cytoplasmic signals. Two pathways have been proposed for integrin activation. One is outside-in signaling, in which binding of integrin with ECM components activates integrin and triggers signaling events based on the formation of focal adhesion structures. In the other pathway, inside-out signaling, stimuli such as growth factor signals are transmitted within cells (33). We previously demonstrated the localization of FAK and ß1 integrin in human decidual cells to the regions known as focal adhesions. ß1 integrin-cytoskeleal linkage in focal contacts on human decidual cells may be important in mediating implantation (21). Integrin-mediated activation of FAK is an early step in a signal transduction cascade that permits flow of information from the ECM to the cell interior. Thus, our previous study demonstrated that an outside- in signaling cascade is important in mediating implantation. In the present study RhoA proved to be highly influenced in embryonic development and differentiation after attachment. Here RhoA acted as the key protein in inside-out signaling, which concerns integrin activation. Activated integrins then bind to the ECM, and this process implicates cell motility. Thus, the inside-out signaling cascade is also important in mediating implantation. Our data indicate that both pathways of integrin activation are important in mediating implantation.
Invasion by tumor cells is one of the essential processes underlying metastasis. Imamura et al. showed a significant association of invasion of a mesothelial cell monolayer by tumor cells such as rat ascites hepatoma cells (MM1) with Rho-mediated tyrosine phosphorylation, especially phosphorylation of 110- to 130-kDa proteins (34). Furthermore, FAK is a major protein that is phosphorylated at tyrosine residues in response to LPA in MM1 cells. Rho regulates actin stress fibers, leading to transmigration of tumor cells, and also controls signal transduction pathways during LPA-induced transmigration of tumor cells. Thus, Rho plays a significant role in invasion of tumor cells. Implantation of the human blastocyst into the endometrium has similarities to tumor invasion of a host tissue (35), and implantation and metastatic processes share common biochemical intermediates. However, invasion by trophoblasts is tightly controlled by unknown mechanisms. We found that the RhoA pathway is involved in trophoblast outgrowth. Mechanisms involving this pathway may restrict the trophoblast invasion to a precisely circumscribed region of the uterine wall. Precise cause-effect relationships within this important signal transduction pathway require much further investigation.
In conclusion, our findings indicate that the expression of RhoA coincides with integrin function in the human endometrial cycle. Outgrowth of embryos among decidual cells was inhibited by C3 exoenzyme treatment, whereas addition of LPA to decidual cells increased the outgrowth of embryos. Although C3 and LPA may affect RhoA in trophoblasts, these findings suggest that RhoA in decidual cells may be an important mediator of implantation.
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Footnotes |
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Received March 8, 2000.
Revised June 13, 2000.
Revised August 24, 2000.
Accepted September 2, 2000.
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) or with C3
exoenzyme (A) at a concentration of 1 ng/mL (•), 10 ng/mL (
), or
100 ng/mL (
) or with LPA (B) at a concentration of 1 µmol/L (•),
10 µmol/L (