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Original Article |
Departments of Obstetrics and Gynecology (M.K.-S., S.S., K.J.S., K.S., Y.N., M.I.), Anatomy (Y.A.), Biochemistry (S.N.), and Pharmacology (H.H.-N.), Kyorin University School of Medicine, Mitaka, Tokyo 181-8611, Japan; and Cell Research Center, Shahid Beheshti Medical University (A.S.), Tehran 19835-177, Iran
Address all correspondence and requests for reprints to: M. Iwashita, M.D., Ph.D., Department of Obstetrics and Gynecology, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo 181-8611, Japan. E-mail: iwashita{at}enjoy.ne.jp.
Abstract
IGF-I and IGF-II were appeared to play major roles in the adhesive and migratory events that are considered to be crucial in the implantation process. The purpose of this study was to determine the effects of IGF-I on trophoblast adhesion to extracellular matrix. Trophoblast cells obtained from early gestation at artificial abortion were incubated with the indicated doses of IGF-I at the indicated times. Trophoblast cells were treated with IGF-I in the presence or absence of RGD peptide and an antibody against 
-subunit of IGF-I receptor (IR3). Morphometric and morphological changes were studied using light and electron microscopy. Furthermore, vinculin, actin stress fibers, phosphorylated focal adhesion kinase (FAK), phosphotyrosine, and paxillin were immunolocalized in trophoblast cells after IGF-I treatment in the presence or absence of IR3. Immunoprecipitation and anti-phosphotyrosine immunoblotting were carried out to detect the phosphorylated FAK and phosphorylated paxillin contents of the IGF-I-treated and untreated trophoblast cells. The results showed that IGF-I promoted trophoblast adhesion to fibronectin substrate in a time- and dose-dependent manner, and addition of RGD peptide and IR3 monoclonal antibody abolished the effects of IGF-I in these cells. Morphological studies exhibited an increase in the lamellipodia formation upon IGF-I treatment, and confocal images of immunofluorescent staining revealed localization of phosphorylated FAK, paxillin, and vinculin at focal adhesions as well as redistribution of actin microfilaments and formation of actin stress fibers inside the cell. Western blotting, using antiphosphotyrosine demonstrated proteins with molecular masses of 125 kDa (FAK) and 68 kDa (paxillin) present in the IGF-I-treated cells, which were lacking in the control groups. In conclusion, these findings suggest that IGF-I can stimulate lamellipodia formation and promote adhesion of trophoblast cells to extracellular matrix by activating their adhesion molecules that must be activated within the implantation window.
IMPLANTATION IS THE most important biological process during the initiation of pregnancy whereby the conceptus establishes its survival as well as the maintenance of pregnancy. Embryo implantation is believed to be mediated by trophoblasts, a specialized population of cells derived from the trophoectoderm and enclosing the preimplantation blastocyst (1). The ability of trophoblast cells to attach to and invade the endometrium provides a developmental strategy that eventually leads to a mature placenta and a viable fetus. At the initial phase of embryo implantation, the trophoblast must have acquired competence for adhesion to the uterine epithelium (2). Three scenarios exist for trophoblast-uterine epithelium interaction during the initial stages of apposition and adhesion: 1) integrins expressed on the trophoectoderm bind to ligands on the uterine epithelium; 2) integrins expressed on the apical surface of the uterine epithelium bind to ligands on the trophoectoderm; or 3) integrins expressed on both cell types bind to extracellular components found in the intercellular space (3). The apical surface of the human luminal epithelium expresses the vß3 integrin (4), which localizes to the bulbous projects that have been referred to as pinopodes (5). Furthermore, the maximal expression of vß3 on the human uterine luminal epithelium coincides with the rise in progesterone during the window of implantation (3). It has been reported that 5ß1 and vß3 integrins, expressed by human trophoblasts, are functionally active and mediate the adhesion of trophoblast cells to fibronectin (FN) in vitro (6). Ligands for the mentioned integrins are secreted by both the trophoblast and the uterine epithelium (7). Therefore, the third explanation appears to be the most reasonable. Additionally, blockade of either the integrin or its ligand binding sequences reduces implantation in the mouse (8).
Focal adhesions or focal contacts are the contact points between cultured cells and their underlying substratum and sites of intense tyrosine phosphorylation that provide a convenient model for studying the mechanisms of cell adhesion process (9). By immunofluorescence, focal adhesions are readily seen as vinculin-containing plaques at the ends of actin filament stress fibers (10). A diverse inventory of proteins, which includes integrins, cytoskeletal proteins, protein kinases, and phosphatases, and signaling molecules colocalize with vinculin in the adhesion plaque (11). The most relevant trophoblast-extracellular matrix (ECM) adhesion molecule is integrin in which its ligation by ECM molecules results in downstream tyrosine phosphorylation of focal adhesion proteins (12) and accumulation of cytoskeletal molecules at the sites of cell adhesion to ECM (13). Binding of trophoblast cells to purified plasma FN is mediated by 5ß1 and vß3 integrins (14). Adhesion to FN in vitro can be inhibited by monoclonal antibodies to the mentioned integrins as well as by synthetic peptides that mimic the FN-binding sites (e.g. RGD) (15).
IGF-I is a low molecular weight, single chain polypeptide whose extensive local production is consistent with its functioning in an autocrine or paracrine mode in addition to a more classical endocrine fashion. IGF-I elicits its action on cells by binding to the IGF-I receptor (IGF-IR), which consists of an - and ß-subunit heterotetramer with ligand-dependent tyrosine kinase activity in the ß-subunit (16). Furthermore, IGF-II is secreted by extravillous trophoblast cells and exerts its biological effects through binding to IGF-IR (11). It has been reported that in the preimplantation period, maternally produced IGF-I is present in the human uterine fluid at concentration of 10.9 nM, at which concentration the IGF-IRs could be activated (17). By the time of implantation in the rat, IGF-I is strongly expressed in the basal lamina, the site of trophoblast invasion into the maternal stroma, and also in the apical epithelium, the site of initial trophoblast attachment (18). IGF-I is involved in the growth and differentiation of trophoblast cells both in vitro (19, 20) and in vivo (21). However, little information is available on the role of IGF-I and its interaction with integrins expressed in human trophoblast cells at the time of implantation. The current study was undertaken to investigate the effects of IGF-I on human trophoblast cell adhesion to extracellular matrix, concerning focal adhesion assembly and integrin involvement during the initial stage of attachment. The results of this study might help to clarify some clues in understanding the human implantation paradox as a target for both contraception and in vitro fertilization-embryo transfer.
Materials and Methods
Reagents
Recombinant human IGF-I was a gift from Fuji Photo Film Co., Ltd. (Osaka, Japan). Monoclonal antibody against the -subunit of IGF-I receptor (IR3) was purchased from Calbiochem Oncogene Research Products (San Diego, CA). Affinity-purified phosphotyrosine antimouse monoclonal IgG2b, focal adhesion kinase (FAK) antirabbit polyclonal IgG, paxillin antirabbit polyclonal IgG, and horseradish peroxidase-conjugated goat antimouse IgG were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Phospho-FAK antirabbit polyclonal IgG was obtained from Upstate Biotechnology, Inc. (Lake Placid, NY), and was immunodepleted from nonphospho-FAK antibodies. Vinculin monoclonal antimouse IgG1 and fluorescein isothiocyanate (FITC)-labeled phalloidin IgG were obtained from Sigma (St. Louis, MO). FITC-labeled donkey antirabbit IgG was purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA), and Alexa 568-labeled goat antimouse IgG was purchased from Molecular Probes, Inc. (Eugene, OR). Fibronectin, Gly-Arg-Gly-Asp-Ser-Pro, and Gly-Arg-Gly-Glu-Ser-Pro peptide were synthesized by Iwaki Glass Co. (Chiba, Japan). Cell culture reagents including medium 199, trypsin/EDTA, fetal bovine serum, and antibiotics were obtained from Life Technologies, Inc. (Grand Island, NY), and Lab-Tek chamber slides were purchased from Nalgen Nunc International (Naperville, IL). TAAB Epon 812, glutaraldehyde, and osmium tetraoxide were purchased from TAAB Laboratories Equipment Ltd. (Berkshire, UK).
Primary trophoblast culture
Placental tissues between 6 and 10 wk of gestation were obtained at elective termination of pregnancy. All patients gave informed consent for collection and investigational use of tissues. This study was approved by the ethics committee of Kyorin University School of Medicine (Tokyo, Japan). Cytotrophoblast cultures were established by growing the villous explants without enzymatic digestion as described previously (22), with few modifications. Briefly, the tissues were rinsed in cold PBS, and selected tissues were cut into small pieces, carefully removing any obvious blood vessels or clots, membranes, and decidual tissues. The fragments of villi were then washed three times with medium 199 supplemented with streptomycin (20 mg/ml) and penicillin (500 U/ml). These fragments were cultured in the same medium containing 10% fetal bovine serum in tissue culture flasks precoated with 20 µg/ml FN in PBS. Tissues were allowed to attach to the bottom of the flasks for 30–60 min before adding the medium. After 3–5 d, nonadherent cells were removed, and culture was continued for additional 1–2 wk in fresh medium at 37 C in a humidified atmosphere containing 5% CO2. The medium was changed every 48 h until confluent, and cultures were continued for three or four passages. Identity as extravillous trophoblast cells was established by immunohistochemical staining (using anticytokeratin 7 and 8/18, anti-5ß1 and -vß3 integrins, antivimentin, CD9, and factor VIII). The medium was replaced with serum-free medium 18–24 h before using the cells for experiments.
Adhesion assay
Serum-starved trophoblast cells were detached using trypsin-EDTA solution (0.05% trypsin and 0.02% EDTA) and were washed three times with serum-free medium. After trypsinization, cells were seeded in FN-coated (20 µg/ml in PBS, 1 h at 37 C), 6-well plates and incubated with 0.1, 1, 10, and 100 nM IGF-I for 2 h for the dose-response study or for 15, 30, 60, and 120 min with or without IGF-I (10 nM) for the time-course adhesion assay. Then cells were washed twice with PBS and fixed with 4% paraformaldehyde for 10 min, and adhered cells were counted under a phase contrast microscope using an eyepiece. This experiment was repeated six times for each group.
To determine whether IGF-I-stimulated adhesion of trophoblast cells was due to IGF-IR activation, the same experiment was performed in the presence of 10 nM IR3. In another set of experiments, cells were incubated with 10 nM IGF-I in the presence of 100 µM Arg-Gly-Arg (RGD), a peptide that mimics the FN-binding site and blocks the binding of 5ß1 and vß3 integrins to FN in vitro, or Arg-Gly-Glu (RGE), a peptide used as the control in the conditions mentioned above.
Scanning and transmission electron microscopy
For scanning electron microscopy preparations, serum-starved trophoblast cells were seeded in coverslips coated with FN and incubated in medium 199 with or without 10 nM IGF-I for 2 h. The cells were then fixed in cacodylate buffer (100 nM sodium cacodylate, pH 7.2, and 120 mM CaCl2) containing 2.5% glutaraldehyde overnight at 4 C. After washing with cacodylate buffer, coverslips were postfixed in 1% OsO4 for 2 h, washed, dehydrated by successive 10-min incubations with 30%, 50%, 70%, 90%, and 100% ethanol, followed by 5-min incubation in hexamethyldisilazane, and dried in a freeze dryer. Coverslips were then sputter-coated with gold/palladium and imaged using scanning electron microscopy (5600 LV, JSM, Tokyo, Japan) operated at 25 kV. Two coverslips from two different preparations were prepared and compared.
For transmission electron microscopy preparations, the same procedure was carried out on the FN-coated, 6-cm cell culture dishes. After dehydration, the cells were detached using propylene oxide and centrifuged twice for 10 min each time at 1500 x g. The pellet was then incubated in propylene oxide-Epon resin (1:1) overnight at room temperature. Cells were pelleted by centrifugation at 3000 x g for 45 min and were embedded in Epon resin. Ultrathin sections were cut using a diamond knife, and a routine double staining procedure was performed. Grids were observed using a transmission electron microscope (JEM 1010, JEOL, Tokyo, Japan) operated at 80 kV. Three blocks were prepared from three different preparations for each group.
Immunocytochemistry
Serum-strarved trophoblast cells were seeded on FN coated Lab-Tek chamber slides and incubated for 2 h with serum-free medium in the presence or absence of 10 nM IGF-I with or without 10 nM IR3 mAb. Cells were washed with PBS, fixed using 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 for 5 min, and then incubated with 5% BSA for 60 min. After washing with PBS, the cells were incubated for 1 h at room temperature with the appropriate primary antibodies diluted in PBS (antivinculin IgG, 1:400; anti-paxillin IgG, 1:200; antiphosphotyrosine, IgG 1:200; antiphospho-FAK IgG, 1:100). For the negative control, the coverslips were incubated at room temperature for 1 h with 2 µg/ml monoclonal antimouse IgG (substituted for vinculin) and polyclonal antirabbit IgG (substituted for paxillin and phospho-FAK) primary antibodies. The cells were rinsed in PBS extensively, counterstained with fluorescent-labeled secondary antibodies (Alexa 568-labeled goat antimouse IgG, 1:200; FITC-labeled phalloidin, 1:50; FITC-labeled donkey antirabbit IgG, 1:100, respectively), and incubated for 3 h at room temperature. After washing with PBS, rinsing in deionized water, and mounting, cells were observed using an AX-80 fluorescence microscope (Olympus Corp., Tokyo, Japan). Each staining was repeated three times for three different preparations for each group.
Immunoprecipitation and immunoblotting
Trophoblast cells were grown to 80% confluence on 10-cm tissue culture dishes, rinsed three times with serum-free medium 199, and incubated overnight in the same medium. After trypsinization, the cells were washed three times with serum-free medium, and the same number of cells was seeded in 10-cm tissue culture dishes and incubated with serum-free medium in the presence or absence of 10 nM IGF-I for 2 h at 37 C. The attached cells were washed with ice-cold PBS three times and solubilized with RIPA lysis buffer [50 mM Tris-HCl (pH 7.5), 1% Nonidet P-40, 150 mM NaCl, 1 mM EGTA, 0.25% sodium deoxycholate, and 50 mM HEPES (pH 7.5)] containing various phosphatase and protease inhibitors (1 µg/ml aprotinin and leupeptin, 100 µM 4-(2-aminoethyl)-benzensulfonyl fluoride, 0.5 mg/ml pepstatin, 2 mM sodium orthovanadate tyrosine phosphatase, and 100 mM sodium fluoride serine/threonine phosphatase), and the floating cells were solubilized with the same buffer after centrifugation. The insoluble materials were removed by centrifugation at 15,000 x g for 10 min, and supernatants were immunoprecipitated by overnight incubation at 4 C with 6 µg/ml anti-FAK and anti-paxillin polyclonal antibodies separately. The immune complexes were incubated with protein A-Sepharose at 4 C for 2 h, and immobilized protein A-Sepharose was sedimented by centrifugation at 10,000 x g for 1 min, washed four times with the same lysis buffer without phosphatase and protease inhibitors, and resuspended in 20 µl 4x reducing sodium dodecyl sulfate sample buffer. The immunoprecipitated proteins were subjected to 7.5% SDS-PAGE under reducing conditions and electrophoretically transferred to polyvinylidene difluoride membranes. The membranes were blocked with Tris buffer [10 mM Tris and 140 mM NaCl (pH 7.4)] containing 1% BSA for 4 h at room temperature, then incubated overnight at 4 C with antiphosphotyrosine mouse monoclonal antibody diluted with blocking buffer (1:1,000). After several washes with washing buffer [Tris buffer containing 0.5% (vol/vol) Tween 20], immunoreactive proteins were identified by 3-h incubation with horseradish peroxidase-conjugated goat antimouse monoclonal IgG diluted with blocking buffer (1:5,000) at room temperature. After several washes, the membranes were visualized with enhanced chemiluminescence ECL reagents (Amersham Pharmacia Biotech, Tokyo, Japan) and exposed to Kodak X-AR film (Eastman Kodak Co., Rochester, NY) for 1–5 min at room temperature.
Statistics
In time-course and dose-response adhesion assays, attached cells were expressed as the mean ± SEM of six assays. Statistical significance were evaluated using ANOVA with Scheffé’s test and were considered statistically significant at P < 0.05.
Results
Adhesion assay
The time-course adhesion assay indicated that the optimum time for maximum attachment of the trophoblast cells was 2 h (Fig. 1A
). Therefore, a dose-response adhesion assay was performed by 2-h incubation of trophoblast cells (Fig. 1B). The results showed that IGF-I significantly stimulated trophoblast cells adhesion in a dose-dependent manner. The maximal response was obtained at 100 nM IGF-I and was about 3-fold of the control value (50% efffective concentration, 1.9 nM). Inhibition of IGF-IRs byIR3 demonstrated that the number of attached cells was significantly decreased comparing to the corresponding control number (Fig. 1B). To investigate whether 5ß1 and vß3 integrins were involved in IGF-I-stimulated cell-ECM adhesion, 100 µM RGD peptide was added to 10 nM IGF-I-containing medium. As shown in Fig. 1C, the number of attached cells in the RGD group significantly decreased comparing to that in the 10 nM IGF-I group whereas adding 100 µM RGE peptide produced no significant difference compared with 10 nM IGF-I-treated cells.
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Upon IGF-I treatment, trophoblast cells underwent morphological changes, assuming a flattened contour with lamellipodia-like extensions. Scanning electron microscopy highlighted a dramatic increase in lamellipodial extension in the IGF-I-treated cells comparing to controls (Fig. 2). These macrographs also revealed that the bodies of these cells did not adhere to the substratum, but the cell-substrate attachment was mediated mostly by the lamellipodia. Transmission electron microscopic macrographs showed that the distribution of actin microfilaments in the untreated trophoblast cells was not organized, whereas in the 10 nM IGF-I-treated group, polymerization of these microfilaments resulted in the formation of actin stress fibers that migrated to the lamellipodia (Fig. 3). IGF-I-treated cells also showed a dominant Golgi apparatus and endoplasmic reticulum comparing with untreated trophoblast cells.
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To correlate the data obtained from the adhesion assay and from transmission and scanning electron microscopy with cytoskeletal dynamics and changes at focal contact sites, immunofluorescent staining were performed. Immunofluorescent images from fluorescein phalloidin, which shows high affinity for the actin cytoskeleton, double stained with human antivinculin mAb revealed a redistribution of the actin cytoskeleton toward the leading processes of IGF-I-treated cells that were anchored to vinculin molecules in the lamellipodia (Fig. 4), whereas in the control and IR3 cells, actin stress fibers were not formed (Fig. 4). Furthermore, vinculin molecules were localized at focal adhesions in IGF-I-stimulated cells, whereas in untreated and IR3-treated cells, vinculin molecules did not show such organization and were not related to actin filaments.
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), whereas in the cells preincubated with IR3 and untreated trophoblast cells, no pFAK was detectable in the cell periphery corresponding to focal adhesions (Fig. 5).
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IR3-treated cells (Fig. 5
), suggesting that paxillin was phosphorylated in the IGF-I-treated cells. Immunoblotting of phosphorylated FAK and paxillin
Western blotting using antiphosphotyrosine demonstrated that 10 nM IGF-I stimulated the phosphorylation of tyrosine residues in both paxillin and FAK proteins with apparent molecular masses of 125 and 68 kDa, respectively (Fig. 6, A and B). In the untreated trophoblast cells, these bands were lacking.
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Embryo implantation and placentation are hypothesized to be successfully achieved through a cascade of adhesive and migratory events involving interaction between the trophoblast and ECM (23). Because of easy accessibility, trophoblast cells from isolated human placental tissue have been widely used to overcome some of the restrictions placed on clinical research, whereas they can also provide answers that are difficult to obtain in the complex in vivo set-up.
The present study demonstrated that IGF-I stimulated adhesion of serum-starved trophoblast cells to FN in a time- and dose-dependent manner. This was confirmed to be regulated by IGF-IRs by preincubation of trophoblast cells with antibody against the -subunit of IGF-IR, IR-3. The 5ß1 and vß3 integrins was shown to mediate binding of trophoblast cells to FN (14). Integrins collaborated or synergized functionally with some growth factors in a variety of biological processes, and the cross-talk between IGF-I and integrin signal transduction pathways has been observed (24). The 5ß1 and vß3 integrins recognize ECM ligands that contain the three-amino acid sequence Arg-Gly-Asp (RGD), and it was reported that the RGD peptide could block ligation of 5ß1 and vß3 integrins to FN in vitro (3, 25). Thus, in the present study the inhibitory effect of RGD peptide on trophoblast adhesion induced by IGF-I suggests that 5ß1 and vß3 integrins are important for IGF-I-mediated cell adhesion to FN. These data are in agreement with the previously reported interaction between integrins and IGF-I in a breast cancer cell line and in epithelial cells of lens (26, 27). Additionally, the attachment and outgrowth of murine trophoblasts to FN and vitronectin can be blocked by RGD-containing peptides in vitro (28, 29).
Transmission and scanning electron microscopic images elucidated some morphological and structural changes in IGF-I-treated trophoblast cells. It was revealed that the bodies of trophoblast cells did not adhere strongly to the substratum, but the cell-substrate adhesion was mediated mostly by the lamellipodia. These findings are consistent with other reports that showed the importance of lamellipodia in the adhesion of neuronal cells to ECM (30). Morphological study of trophoblast cells also exhibited an increase in lamellipodia formation upon IGF-I treatment. Thus, the increase in the number of adherent serum-starved trophoblast cells after IGF-I treatment could be due to the increase in lamellipodia formation stimulated by IGF-I in these cells. The increase in lamellipodia formation in IGF-I-treated trophoblast cells is believed to be due at least in part to the formation of actin stress fibers and the redistribution of actin microfilaments toward the cell periphery. These were confirmed by transmission electron microscopy as well as immunolocalization of actin stress fibers, as shown in Figs. 3 and 4. Our findings are in accordance with previous reports that IGF-I promoted recruitment of actin microfilaments into the membrane ruffles, followed by protrusion of lamellipodial extension (31).
It has been previously reported that many cell types lose their focal adhesions after incubation with serum-free medium (32). The formation of focal adhesions in such cells could be stimulated by the addition of serum or purified growth factors. The formation of focal adhesion after trophoblast cell adhesion to FN also requires the presence of serum, and in the absence of serum it had been reported that trophoblasts did not adhere to FN (33). The results of our morphological studies showed that the lamellipodia extensions, but not the trophoblast cell body, were the points of trophoblast cell-FN adhesion sites. Consequently, it was predicted that focal adhesions appeared at the lamellipodia of trophoblast cells after IGF-I treatment. The immunofluorescent staining performed in this study demonstrated that IGF-I stimulates vinculin, phosphorylated FAK, and phosphorylated paxillin molecules assembly in the focal adhesions presented in the lamellipodia extension. However, the precise molecular mechanism(s) that is directly or indirectly involved in the activation and migration of these molecules to the focal adhesions is as yet unknown. We propose that one of the likely substances to mediate IGF-I action in focal adhesion assembly is vinculin, an important anchoring protein that contains binding sites for both actin and paxillin. Vinculin molecules were shown to exist in folded (inactive form) and unfolded (active form) forms. Vinculin-binding sites could be exposed only after extension of the tail domain induced by phosphatidylinositol-4,5-biphosphate (34). Activation of IGF-IR by IGF-I has been reported to activate phosphatidylinositol-4,5-biphosphate synthesis (35). Activated vinculin migrates to the focal adhesion sites where actin stress fibers are anchored. The double staining of vinculin and actin stress fibers found in the current study indicated migration and activation of vinculin molecules after IGF-I treatment in trophoblast cells.
Immunoblotting results showed tyrosine phosphorylation of FAK and paxillin in IGF-I-treated trophoblast cells. This was confirmed by our immunofluorescent staining results. IGF-I was shown to either phosphorylate or dephosphorylate FAK depending on the cell type and the adhesion status (36, 37). It was reported that IGF-I stimulated the phosphorylation of FAK and paxillin, as lamellipodia are extended in neuronal cells (36), whereas, in contrast, IGF-I dephosphorylated FAK in fibroblasts as cells attached and organized their cytoskeleton (37). The current study demonstrated tyrosine phosphorylation of FAK in trophoblasts as the lamellipodia advanced over the substrate upon IGF-I stimulation. The tyrosine phosphorylation of FAK is critical for its function and is thought to stimulate cytoskeletal remodeling and recruitment of other proteins, including paxillin and vinculin (38). Phosphorylated FAK is colocalized with integrin in focal adhesions, and its phosphotyrosine content and enzymatic activity were reported to be elevated upon integrin-dependent cell adhesion as well as IGF-I stimulation (31, 39). These findings in conjunction with other reports (40) showed that tyrosine phosphorylation of FAK and paxillin accompanies cell adhesion to ECM. Furthermore, It was reported that a human embryonal RD cell line, BT474 human breast ductal carcinoma cells, and a C8161 human melanoma cell line significantly lost their adhesion to ECM after attenuation of FAK expression using different antisense oligonucleotides to FAK (41).
Here we propose that IGF-I might activate integrin signaling to promote trophoblast cell adhesion to ECM. Previously it was reported that IGF-I increased vß3 integrin affinity for ligand by increasing the amount of integrin-associated protein in smooth muscle cells (42). Uterine fluid in the cavum is reported to contain a considerable amount of IGF-I at the time of implantation (17). Taken together, these reports might indicate that IGF-I is involved in the process of embryo attachment in the initial stage of implantation. Furthermore, there is increasing evidence indicating that unsuccessful implantation is one of the major causes of failure of in vitro fertilization-embryo transfer procedures. The results of this study together with those of others (17) suggest that addition of human IGF-I could benefit clinical in vitro fertilization culture and results.
In conclusion, our results demonstrate that binding of IGF-I to its receptors could stimulate the formation of lamellipodia and the assembly of focal adhesions as well as the tyrosine phosphorylation of paxillin and FAK. These findings might predict an interaction between IGF-I and integrin signaling pathways that lead to adhesion of trophoblast cells to ECM. As integrins play key roles in different stages of human implantation phenomena, including embryo adhesion, these results might indicate some clinical application for IGF-I to obtain a better outcome from assisted reproductive technology as well as to improve contraceptive methods.
Acknowledgments
We are particularly grateful to Mr. Fukuda (Department of Anatomy, Kyorin University School of Medicine, Tokyo, Japan) for his valuable help with this study.
Footnotes
This work was supported in part by Grant-in-Aid (C) 11671648 (to S.S.) from the Ministry of Education, Science, and Culture (Tokyo, Japan).
Abbreviations: ECM, Extracellular matrix; FAK, focal adhesion kinase; FITC, fluorescein isothiocyanate; FN, fibronectin; IGF-IR, IGF-I receptor; IR3, -subunit of IGF-I receptor; pFAK, phosphorylated focal adhesion kinase.
Received April 8, 2002.
Accepted August 15, 2002.
References
v are apically distributed in endometrial epithelium. Mol Hum Reprod 2:527–534v ß3 integrin adversely affects implantation in the mouse. Biol Reprod 62:1285–1290vß3 affinity by increasing the amount of integrin-associated protein that is associated with non-raft domains of the cellular membrane. J Biol Chem 277:1800–1805This article has been cited by other articles:
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  A. N. Sferruzzi-Perri, J. A. Owens, P. Standen, R. L. Taylor, G. K. Heinemann, J. S. Robinson, and C. T. Roberts Early treatment of the pregnant guinea pig with IGFs promotes placental transport and nutrient partitioning near term Am J Physiol Endocrinol Metab, March 1, 2007; 292(3): E668 - E676. [Abstract] [Full Text] [PDF]
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 M. Kabir-Salmani, S. Shiokawa, Y. Akimoto, K. Sakai, K. Sakai, and M. Iwashita Tissue Transglutaminase at Embryo-Maternal Interface J. Clin. Endocrinol. Metab., August 1, 2005; 90(8): 4694 - 4702. [Abstract] [Full Text] [PDF]
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 M. Kabir-Salmani, S. Shiokawa, Y. Akimoto, K. Sakai, and M. Iwashita The role of {alpha}5{beta}1-integrin in the IGF-I-induced migration of extravillous trophoblast cells during the process of implantation Mol. Hum. Reprod., February 1, 2004; 10(2): 91 - 97. [Abstract] [Full Text] [PDF]
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 M. Kabir-Salmani, S. Shiokawa, Y. Akimoto, K. Sakai, S. Nagamatsu, K. Sakai, Y. Nakamura, A. Lotfi, H. Kawakami, and M. Iwashita {alpha}v{beta}3 Integrin Signaling Pathway Is Involved in Insulin-Like Growth Factor I-Stimulated Human Extravillous Trophoblast Cell Migration Endocrinology, April 1, 2003; 144(4): 1620 - 1630. [Abstract] [Full Text] [PDF]
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