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Departments of Obstetrics and Gynecology (M.K.-S., S.S., Kei.S., Ken.S., M.I.) and Anatomy (Y.A.), Kyorin University School of Medicine, Mitaka, Tokyo 181-8611, Japan; and Cell Research Center (M.K.-S.), Shaheed Beheshti Medical University, Tehran 19835-177, Iran
Address correspondence to: M. Kabir-Salmani, Ph.D., Department of Obstetrics and Gynecology, Kyorin University School of Medicine, Shinkawa 6-20-2, Mitaka, Tokyo 181-8611, Japan. E-mail: kabirs_m{at}yahoo.com. Address requests for reprints to: S. Shiokawa, Department of Obstetrics and Gynecology, Kyorin University School of Medicine, Shinkawa 6-20-2, Mitaka, Tokyo 181-8611, Japan. E-mail: shiochan{at}kyorin-u.ac.jp.
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Abstract |
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Methods: The primary cultures of human placentas from 15 legal elective abortions at the first trimester of normal pregnancies and endometrial biopsies of 12 female patients in the midluteal phase as well as normal trophoblastic cell lines (CRL) were employed to address these issues using several approaches, such as scanning and transmission electron microscopies, immunostaining for light and electron microscopies, western blotting, and function assays using GRGDSP hexapeptide and an antibody against tTG.
Results: The results demonstrated tTG expression on uterine pinopodes and lamellipodia of extravillous trophoblasts. The colocalization of tTG with ß1 and ß3 integrins and its interaction with 
vß3 integrin and integrin-associated proteins at focal adhesions of the extravillous trophoblasts were illustrated in the results of immunofluorescence, immunoblot, and coimmunoprecipitation studies. Furthermore, function assays revealed that tTG mediated the adhesion and spread of the placental cells on intact FN-coated and 42- and 110-kDa FN fragment-coated wells.
Conclusion: In conclusion, our findings demonstrated for the first time that tTG actively participates in adhesion events at the embryo-maternal interface through its interaction with FN, at least in part, by activating integrin-signaling pathways.
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Introduction |
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5ß1 and vß3 integrins at this interface have central roles in regulating the above-mentioned process (1, 2). Oncofetal fibronectin (onf FN), which is produced by extravillous trophoblasts (EVTs) (3) and cytotrophoblasts of villous origin (4), serves as a substrate for wild-type integrins, called trophoblastic glue (5). On the other hand, both of the ß1 and ß3 integrin subunits as well as FN form stable complexes with tissue transglutaminase (tTG) (6). tTG appeared to play a pivotal role in the adhesion and migration of several cell types (7, 8). Furthermore, essential roles have been considered for tTG in the transendothelial migration of blood cells (9), which resembles the initial steps of embryo implantation in humans (10).
FN, a predominant constituent of the extracellular matrix (ECM) has a characteristic of being adhesive and regulates diverse biological functions (11). Onf FN is a glycosylated cellular form of FN, which is synthesized and expressed at a high level in tumors, fetal tissues, and the placenta (12). In human implantation sites, onf FN is expressed abundantly at highly specific regions of uterine-placental and fetal-decidual interfaces (5, 13). FN binds to tTG with a high affinity (Kd, 
8 nM) via its 42-kDa gelatin-binding domain (14), whereas a purified 110-kDa classical RGD-containing motif of FN interacts directly with integrins (6). The association of high-affinity tTG with integrins forming a complex strengthens their adhesion to FN at the focal adhesions (FAs) of EVTs, which could be a fundamental process for their attachment to and invasion of the maternal tissues.
tTG is a unique member of the transglutaminase family, because it is both a transamidating enzyme and a GTPase (15). Moreover, tTG is localized mostly in the cytosol; however, it is also found in the nucleus and ECM and is associated with the plasma membrane (8, 16). The complex of tTG and integrins is formed in the cytoplasm and accumulates on the cell surface and FAs (6). The biosynthesis of tTG markedly increases after an inflammatory process and an induction of cell differentiation because of its adhesion to a substrate and the exposure of cells to various cytokines, growth factors, serum retinoids, and lipopolysaccharides (17, 18).
Human endometrial tTG exhibits a regulated development and a 10-fold higher activity in the luteal phase than in the follicular phase (19). These features imply that progesterone regulates the expression of tTG in vivo (20) and suggest its potential role in endometrial receptivity. However, there is no information regarding its expression pattern and subcellular location in the human endometrium during implantation. On the other hand, human trophoblastic transglutaminase has been identified as a tTG, which was proposed to regulate the different functions of EVTs (21). The importance of tTG expression in the early events of mouse oocyte maturation (22) and during decidualization in human endometrial stromal cells (23) has been already confirmed. Moreover, it has been reported that tTG plays a specific role in masking the antigenicity of the developing embryos in the rabbit uterus (24). However, no information is available concerning the expression pattern of tTG as a binding partner of both integrins and FN in the human trophoblasts as well as its interaction in conjunction with the integrin-FN axis during their adhesion. Thus, the present study was designed to examine the subcellular localization of tTG at the components of the human embryo-maternal interface as well as to identify the interactions of tTG with integrin and FN that lead to the stimulation of their adhesive functions and signaling pathways.
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Materials and Methods |
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Antibodies and other reagents are summarized in Table 1
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Primary cell cultures of EVT were established by the method described previously (25), using placental tissues obtained from 15 legal elective abortions at the first trimester of normal pregnancies that were between 8 and 10 wk. Briefly, the placental tissues were rinsed several times in cold PBS containing streptomycin (100 µg/ml) and penicillin (100 U/ml). Selected placental tissues were then cut into small pieces and cultured in medium 199 supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% antibiotics. Tissue culture flasks were precoated with 20 µg/ml FN in PBS (pH 7.4) incubated at 37 C for 1 h before the transfer of placental fragments. After removing unattached cells, the medium was changed with a fresh one every 48 h until cells became confluent. Then EVTs were detached using EDTA and were directly used for immunofluorescent and immunogold transmission electron microscopy (TEM). We consumed cells of the first passage to prevent the contamination of EVTs with other cell types. Immunostaining revealed that more than 95% of these cells expressed the markers for EVTs (data not shown).
For functional and biochemical analysis, the normal human placental cell line (CRL) was obtained from the American Type Culture Collection (Rockville, MD). Cells were grown in Eagles’s MEM supplemented with 10% heat-inactivated FBS, streptomycin (100 µg/ml), and penicillin (100 U/ml). Cells were maintained in monolayer cultures at 37 C in a humidified atmosphere with 5% CO2. EDTA was used to detach the cultured cells because trypsin rapidly degrades cell surface tTG. Furthermore, all experiments were conducted under serum-free conditions because serum contains FN.
Endometrial biopsies were obtained from the anterior wall of the uterine cavity of 12 female patients in the midluteal phase, that is, 20–24 d of a regular menstrual cycle. All these selected patients were referred to our center at the Kyorin Medical University Hospital for some medical indication. They were fertile with regular cyclic menses and had delivered at least one live child. The mean age was 36 yr (range, 28–42 yr), and none of them had used steroidal contraceptives or intrauterine devices for at least 3 months before the sampling. Biopsies from each patient (two specimens from each case) were divided into three pieces. One was fixed in 10% neutrally buffered formaldehyde for light microscopy. The second portion was fixed in 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (pH 7.4) for immunogold staining of TEM, and the third portion was fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for scanning electron microscopy (SEM). For endometrial dating, according to the histopathological criteria described by Noyes (26), paraffin-embedded biopsies were stained with hematoxylin and eosin and analyzed by an experienced observer who was blind to the objectives of the study. All patients gave their informed consent for the collection and investigational use of endometrial tissues. This study was approved by the Ethics Committee of Kyorin University, School of Medicine, Tokyo, Japan.
SEM
SEM was performed to evaluate and confirm the presence of pinopodes in the endometrial samples. For SEM preparation, the endometrial tissues were fixed in 2.5% glutaraldehyde and postfixed in 1% OsO4 for 2 h. Then the samples were dehydrated in a graded series of ethanol (50, 70, 90, 99.5, and 100%), critical-point-dried, mounted, and coated with gold using a sputter coater (JFC-1300 Auto Fine Coater; JEOL, Tokyo, Japan). Finally, the samples were observed under a scanning electron microscope (JSM-5600 LV SEM; JEOL). One specimen of each sample was observed using this technique.
Immunogold staining for TEM
Immunogold staining was performed to determine the subcellular distribution pattern of tTG in the surface epithelial cells of the human endometrium as well as in human EVTs. For immunogold (TEM) preparations for endometrial biopsies, the specimens were fixed for at least 24 h at 4 C using 4% PFA in 0.1 M phosphate buffer (pH 7.4). After dehydration, the samples were dehydrated as described above and were embedded in Lowicryl white resin. Ultrathin sections were cut, washed with PBS, and then pretreated with 5% BSA for 10 min at room temperature. After a PBS rinse, they were incubated overnight at 4 C with an antirabbit polyclonal tTG antibody (1.5 µg/ml) or with normal rabbit serum as a negative control. After washing with PBS (five times, 5 min each), the sections were incubated overnight at 4 C with 12-nm colloidal gold-conjugated donkey antirabbit IgG (diluted 1:20 with PBS). Then the sections were washed with PBS (five times, 5 min each) and rinsed with distilled water (three times, 5 min each). The ultrathin sections were then stained with uranyl acetate and observed under a transmission electron microscope (JEM-1010; JEOL). Four different samples were observed under this microscope.
After the detachment of EVTs using EDTA for immunogold TEM preparation, they were seeded on FN-coated 10-cm culture plates and incubated with serum-free medium 199 for 3 h. Then cells were fixed using 4% PFA and blocked by 5% BSA for 1 h at 4 C. Thereafter, the cells were incubated overnight at 4 C with a primary antibody diluted in PBS (monoclonal anti-tTG IgG; 1.5 µg/ml), and were washed several times with PBS. Then cells were incubated overnight at 4 C with appropriate 12-nm colloidal gold-conjugated antimouse monoclonal IgG diluted in PBS (1:20). The cells were then fixed with 2.5% glutaraldehyde, postfixed with 1% OsO4, washed, and dehydrated as mentioned above. After dehydration, the cells were detached using propylene oxide and centrifuged twice for 10 min at 1500 x g. The pellets were incubated with propylene oxide-Epon resin (1:1) overnight at room temperature, centrifuged at 3000 x g for 45 min and were embedded in Epon resin. Ultrathin sections were cut and stained; grids were then observed under a transmission electron microscope. These experiments were repeated four times using different preparations of four different samples.
Immunostaining for light microscopy
To examine the expression of tTG and its colocalization with ß1 and ß3 integrins on the EVT surface, immunofluorescent staining were performed without permeabilizing the cell membrane. EVTs on FN-coated chamber slides were double stained with an anti-tTG monoclonal antibody (1 µg/ml) and one of the anti-ß1 and -ß3 integrin goat polyclonal antibodies (1 µg/ml). EVTs were fixed using 4% PFA and blocked with 5% normal donkey serum to minimize nonspecific staining. The cells were incubated overnight at 4 C with the above-mentioned primary antibodies without any permeabilization. Then EVTs were rinsed in PBS extensively, counterstained with proper fluorescent-labeled secondary antibodies (Alexa 568-conjugated goat antimouse IgG, 1.5 µg/ml; and Cy2-conjugated donkey antigoat IgG, 1.5 µg/ml) and incubated for 1 h at room temperature. After washing with PBS, rinsing in deionized water, and mounting, the cells were observed under AX-80 fluorescence microscope (Olympus Optical, Tokyo, Japan). Reproducibility was verified by repeating these experiments four times using four different samples.
To identify the involvement of tTG in the assembly of FAs in the absence of a direct interaction of integrin with FN, EVTs were seeded on 42-kDa gelatin-binding FN fragment-coated chamber slides for 3 h and were fixed with 4% PFA, permeabilized with 0.5% Triton X-100, and blocked using 5% normal donkey serum. Then the EVTs were incubated overnight at 4 C with the appropriate primary antibodies: polyclonal antipaxillin IgG, 1.5 µg/ml; polyclonal anti-phosphorylated focal adhesion kinase (pFAK) IgG, 1.5 µg/ml; monoclonal anti-vß3 integrin IgG, 1.5 µg/ml; and both polyclonal and monoclonal anti-vinculin IgG, 1.5 µg/ml. Then cells were rinsed in PBS thoroughly and counterstained with appropriate fluorescence-labeled secondary antibodies: Alexa 568-conjugated goat antimouse IgG, 1.5 µg/ml; and fluorescein isothiocyanate-conjugated donkey antirabbit IgG, 1.5 µg/ml. The subsequent steps were the same as those mentioned above. For negative controls, the cells were incubated overnight at 4 C with nonimmune IgGs using the same concentrations as those of their corresponding antibodies, including antimouse IgG (substituted for vß3 integrin and vinculin), antigoat IgG (substituted for vinculin), and antirabbit IgG (substituted for paxillin and pFAK) primary antibodies. These experiments were repeated four times using samples from four different preparations.
Immunoprecipitation and immunoblotting
After the detachment of the CRL cells using EDTA, 2 x 105 cells were plated on 10-cm tissue culture plates precoated with FN or a 42-kDa gelatin-binding FN fragment. For a time-course study, serum-starved CRL cells were incubated for 1, 2, and 3 h with serum-free medium 199. To determine the significance of tTG in the tyrosine phosphorylation of FAK, cells were preincubated for 30 min with a monoclonal antibody against tTG, GRGDSP, or GRGESP hexapeptides and nonimmune IgG in another set of experiments. After the incubation of the cells for 3 h at 37 C with serum-free medium 199 containing 0.05% BSA, adherent or suspended cells were solubilized using 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, 1 mM 4-(2-aminoethyl)benzene sulfonyl fluoride, 0.5 µg/ml pepstatin, 2 mM sodium orthovanadate, and 100 mM sodium fluoride). The insoluble materials were removed by centrifugation at 15,000 x g for 10 min, and supernatants were incubated overnight at 4 C with 1 µg/ml anti-FAK or anti-paxillin polyclonal antibodies. The immunocomplexes were incubated for 2 h at 4 C with protein A-Sepharose. Then the immobilized protein A-Sepharose was sedimented and washed with the same lysis buffer. After the resuspension of the pellets in 20 µl of Laemmli buffer, the same amount of immunoprecipitated proteins were subjected to 7.5% SDS-PAGE under reducing conditions and electrophoretically transferred to polyvinylidene difluoride membranes. Then the membranes were blocked with Tris buffer saline (10 mM Tris and 140 mM NaCl, pH 7.4) containing 3% BSA and 0.05% Tween 20 for 2 h at room temperature and then probed with an antiphosphotyrosine monoclonal antibody diluted with blocking buffer (1:1000) overnight at 4 C. After several washes with washing buffer (Tris buffer saline containing 0.05% Tween 20), immunoreactive proteins were identified by 1 h of incubation at room temperature with horseradish peroxidase (HRP)-conjugated goat antimouse monoclonal IgG diluted with blocking buffer (1:5000). After several washes, the membranes were visualized using enhanced chemiluminescence ECL reagents and exposed to Kodak X-AR film for 1–15 min. For the control of each group, the antiphosphotyrosine antibody was stripped using Western blot stripping buffer and reblotted with anti-FAK and -paxillin antibodies. These experiments were carried out three times to determine reproducibility.
To analyze the association of cell surface tTG with v, 5, ß1, and ß3 integrin subunits in CRL cells, the coimmunoprecipitation experiments were conducted as follows. The cells were lysed using the lysis buffer B [0.2% Triton X-100, 1 mM EGTA, 150 mM NaCl, 50 mM HEPES (pH 7.5), 1 mM MgCl2, and protease inhibitors], and the above-described procedures were performed for protein extraction. Then supernatants were incubated with 1 µg/ml of one of the polyclonal anti-v, -5, -ß3, or -ß1 integrins or anti-tTG antibodies overnight at 4 C. The immune complexes were immobilized with protein G-Sepharose after a 2-h incubation at 4 C and sedimentation. The membranes were blotted with an anti-tTG monoclonal antibody diluted at 1:1000, and the immunoreactive complexes were identified using the HRP-conjugated donkey antimouse IgG diluted at 1:5000. After several washes, the membranes were visualized using enhanced chemiluminescence ECL reagents as described above.
Cell adhesion and spreading assays
Adhesion assays were performed as described previously (25). Briefly, 2 x 105 cells were plated on 24-well plastic wells, which were either precoated with FN, a 110-kDa FN fragment, or a 42-kDa gelatin-binding FN fragment (10 µg/ml in PBS, 1 h at 37 C) or noncoated, designed as a negative control. Before seeding the cells on the FN-coated wells, CRL cells were preincubated for 30 min at 37 C with serum-free medium 199 containing 0.01% BSA and one of the following: 1) a monoclonal blocking antibody against tTG (10 µg/ml), 2) nonimmune IgG as a control (10 µg/ml), 3) 100 µM GRGDSP hexapeptide, and 4) 100 µM GRGESP hexapeptide as a control for the GRGDSP hexapeptide group. CRL cells in the negative control group were kept in a suspension of serum-free medium 199 containing 0.01% BSA for 30 min at 37 C. At the end of the preincubation period, more than 95% of the cells were viable as assessed by trypan blue dye exclusion. After 1 h of plating on precoated plastic wells, cells were fixed and stained using a Diff Quick kit. Adherent cells were counted under a phase contrast microscope. To assess reproducibility, the experiments included six replicates in each experimental group.
For spreading assays, detached cells were cultured for 3 h on 10-cm tissue culture plates and the above-mentioned treatments. After fixation and staining, the cell areas of randomly chosen nonadjacent cells were analyzed using Image-Pro Plus microscopy software (Media Cybernetics, Baltimore, MD). The software was calibrated with an Applied Micro Stage micrometer (Applied Image, Inc., Rochester, NY). Using a measurement slide containing squares of known dimensions, the accuracy of the area measurements was confirmed. To assess reproducibility, the experiments included six replicates in each experiment.
Statistical analysis
In the adhesion assay, statistical analysis was performed by taking the mean of 60 fields per test substance for six replicates. In the spreading assay, statistical analysis was performed by taking the mean cell area of randomly chosen 200 nonadjacent cells from each group. The number of adhered cells and area of cells were expressed as mean ± SEM. Statistical significance was evaluated using ANOVA with Scheffé’s test, and differences were considered statistically significant if P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, all the specimens were examined under a scanning electron microscope because although pinopodes on the uterine epithelial surface were visible by light microscopy, other structures could be mistaken for pinopodes. Thus, SEM was performed to select the samples that contained these ultrastructures for use in immunogold TEM. SEM images demonstrated that the endometrial epithelium in the midluteal phase exhibited two types of cell, ciliated and nonciliated cells, and the latter type of cell covered the major area of the uterine luminal surface, many of which showed apical projections called pinopodes (Fig. 1A).
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). No reactivity was detected on the endometrial surface using the nonimmune control antibody in these specimens (Fig. 1C).
Furthermore, ultrathin immunogold TEM images exhibited the distribution of tTG in the lamellipodia of the human EVTs as a coreceptor at the embryo-maternal interface (Fig. 1D). Immunoreactive nanogold particles were not observed in the control images, in which nonimmune IgG was used instead of the primary antibody (results not shown).
Immunocytochemistry
Immunocytochemical staining revealed that tTG was abundant on the EVT surface, which colocalized with ß1 or ß3 integrin subunits on the cell surface (Fig. 2). In the negative controls, no staining was observed.
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vß3 integrin, which assembled at the periphery of these cells corresponding to FAs (Fig. 3). These structures were not stained using nonimmune antibodies in the negative control group.
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The results of phosphotyrosine immunoblotting demonstrated that the adhesion of cells to the isolated 42-kDa FN fragment could trigger the tyrosine phosphorylation of FAK and paxillin in a time-dependent manner (Fig. 4). Of course, the cells that were cultured on wild-type FN-coated plates exhibited larger amounts of tyrosine phosphorylation of both FAK and paxillin. In a complimentary set of experiments, it was demonstrated that the pretreatment of the cells with either GRGDSP hexapeptide or a function-blocking antibody against cell surface tTG decreased the tyrosine phosphorylation of FAK (Fig. 5A). In Fig. 5B, for which total content of FAK in the cells was measured by Western blotting, it is illustrated that after treatment of the cells with an antibody against tTG, GRGDSP and GRGESP hexapeptides, and nonimmune IgG, the contents of FAK were almost the same as that of the control group, which received no treatment. The same intensity of each band in the different groups suggested that equal amounts of protein were loaded.
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v, and 5 integrin subunits (Fig. 6, lanes 2–5) exhibited a close association between tTG and ß1 and ß3 integrin subunits in these cells. The coimmunoprecipitation of tTG with polyclonal anti-tTG IgG (Fig. 6, lane 1) was considered as a positive control. The coimmunoprecipitation with nonimmune IgG (Fig. 6, lane 6) was performed to confirm the specificity of the used antibodies.
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In vitro assays, including adhesion and spreading assays, were performed to evaluate the functional significance of cell surface tTG in these processes in the trophoblastic cell line. Because tTG on the cell surface interacts with FN by binding to its 42-kDa gelatin-binding domain rather than to the 110-kDa RGD-containing fragment (5), we compared cell binding and spreading on intact FN-coated and 42- and 110-kDa FN fragment-coated wells. Furthermore, to identify the RGD-independent and tTG-dependent adhesion and spreading of these cells, we performed these assays by pretreating the cells with either GRGDSP hexapeptide, the function-blocking antibody against tTG or the mixture of both as well as their corresponding controls. Finally, to examine the effects of the endogenous production of FN by these cells, the cells were seeded on the uncoated wells in the negative control group. The statistical analysis of both adhesion and spreading assays demonstrated that the pretreatment of these cells with GRGDSP hexapeptide significantly (P < 0.01) reduced the number of the adherent cells as well as their spreading both on intact FN-coated and 110-kDa FN fragment-coated wells (Figs. 7 and 8). No statistically significant difference (P < 0.05) was noted in the extent of cell spreading and the number of adherent cells, which were pretreated with GRGDSP hexapeptide before their seeding on the 42-kDa FN fragment-coated wells. GRGESP hexapeptide and control IgG had essentially no effect on the spreading of these cells on the intact FN-coated and 42- and 110-kDa fragment-coated wells. The preincubation of CRL cells with the blocking anti-tTG antibody significantly (P < 0.05) decreased the rate of their spreading and adhesion to 42-kDa FN fragment-coated wells, whereas it had no significant effects on their spreading and adhesion to the 110-kDa FN fragment-coated wells. In the negative control group, in which the CRL cells were cultured on noncoated plastic wells under a serum-free condition, the number of adherent cells and their rate of spreading were not significantly different (P < 0.05) compared with those of the nonimmune IgG-treated cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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vß3 integrin and integrin-associated proteins as well as its role in the assembly of FAs in the absence of a direct integrin-FN interaction were illustrated. Considering the report that human endometrial tTG displays a 10-fold higher activity in the luteal phase than during the follicular phase (19, 20), its subcellular localization in the surface epithelium of the uterus in the midluteal-phase biopsies was detected in this study. Immunogold TEM images clearly exhibited a high-level tTG expression in the human uterine pinopodes. This is the first illustration of the molecular localization of tTG in the bulbous ultrastructure of the human endometrial epithelium called pinopodes. As pinopodes appeared to be the preferred site of embryo-endometrial interaction in vitro (27) and is considered as a biological marker of uterine receptivity in vivo (28), tTG expression on these structures may indicate its role in the cascade of the implantation process. It is tempting to hypothesize that tTG on pinopodes may provide a binding site for onf FN that is secreted by the embryonic EVTs. However, the precise mechanism underlying its function in this regard and its regulation during uterine epithelial development and pinopode formation remain to be elucidated.
On the other hand, the adhesion of endometrial epithelial cells to embryonic trophoblastic cells or to the ECM bridge, which exists between them can be a result, at least in part, of the formation of FAs. Thus, tTG expression in the lamellipodia of human EVTs as well as its involvement in the assembly of FAs in these cells was detected. Images obtained by immunogold TEM in this study demonstrated that tTG was distributed in the lamellipodia of EVTs, where it was reported that FAs assembled (29). Furthermore, our results of the immunofluorescence experiments indicated that the tyrosine-phosphorylated forms of FAK and paxillin could be detected in the periphery of the EVTs, which were cultured on 42-kDa FN fragment-coated chambers. This result was further confirmed by performing phosphotyrosine immunoblotting, which demonstrated that even in the absence of a direct integrin-FN interaction, the association of tTG on the trophoblastic cell membrane with 42-kDa FN fragment was sufficient to trigger the tyrosine phosphorylation of FAK and paxillin. Moreover, our results demonstrated that the blocking of tTG accessibility by a monoclonal antibody could inhibit the phosphorylation of FAK in these cells. This could indicate the importance of the involvement of tTG in regulating the integrin signaling pathways. This is consistent with the previous report, which demonstrated that tTG could potentiate integrin-mediated FAK phosphorylation in fibroblasts (6). Apparently, tTG lacks a transmembrane domain that activates the intracellular signaling pathways directly (30). Then, considering the fact that integrins span the cell membrane and are physically and functionally associated with tTG (6), it is tempting to hypothesize that an integrin-tTG-FN axis exists in these cells. Consistent with this view, another previous study (6) has also suggested an interaction between tTG with integrins through their extracellular domains. In these previous studies, two models were suggested for the ternary adhesion complexes concerning these interactions (as modified in Fig. 9B). In this model, tTG as a bridge between integrin and FN could strengthen these adhesion complexes first through its higher affinity for FN and by simultaneously providing the second integrin molecule with the RGD site of the same FN chain. According to this model, tTG can trigger and mediate the integrin signaling pathways. However, the precise molecular mechanism of the tTG-mediated formation of FAs on the 42-kDa FN fragment-coated plates is as yet unknown. On the basis of recent reports, it is considered that RhoA regulates the function of FN-bound tTG and is a substrate for tTG in vivo (31). On the other hand, a recent study conducted at our laboratory demonstrated that RhoA-Rho-associated coiled coil-forming protein kinase signaling has fundamental roles in the formation of FAs in human EVTs (32). Hereby, we propose the idea that the activation of RhoA by tTG could be considered as one of the most probable pathways involved in the assembly of FAs in these cells. However, the exact role of the Rho-family GTPases in the tTG-mediated adhesion remains to be elucidated.
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vß3 integrin as the core integrin in the assembly of FAs at the lamellipodia of human EVTs (29, 33), we further investigated the existence of an interaction between tTG with the ß-subunit of vß3 integrins in these cells. The colocalization of tTG with vß3 integrin was shown in the immunofluorescence images, which was confirmed by our Western blotting results as well. These results indicated a direct association of tTG with both ß1 and ß3 integrin subunits. Likewise, in a previous study, the analysis of the formation of a complex between ß integrin heterodimers and tTG in rat smooth muscle cells showed that tTG was associated with 1ß1, 3ß1, and 5ß1 as well as with vß3 integrins (6). These findings indicate an interaction between tTG and vß3 integrins in its outside-in signal transduction pathways and to some extent delineate the role of tTG in the assembly of FAs. The interaction of tTG with vß3 integrin and the localization of this integrin at the apical plasma membrane of both the maternal endometrial epithelium (1, 34) and the embryonic trophoblasts (35) suggest a significant role of tTG as a mediator of the adhesive events at the embryo-maternal interface.
Because the vß3 integrin recognizes only those ligands that contain the Arg-Gly-Asp (RGD) sequence, it binds to the 110-kDa FN fragment rather than to the 42-kDa FN fragment, which binds to tTG with a high affinity. Thus, next we performed functioning in vitro quantitative assays using the 110- and 42-kDa FN fragments as well as intact FN as one the most important adhesive substrates in the process of embryo implantation. The results of these assays demonstrated that inhibiting the cell surface tTG could attenuate and abolish the attachment and spreading of trophoblasts on the FN-coated and 42-kDa FN fragment-coated wells, respectively. While following the blockade of the cell surface tTG by a monoclonal antibody against tTG, no statistically significant changes were detectable in the adhesion and spreading of these cells on the 110-kDa RGD-containing FN fragment-coated wells. In turn, the pretreatment of the trophoblasts with GRGDSP hexapeptide showed no effect on their adhesion or spreading on the 42-kDa FN fragment-coated wells but attenuated and abolished the integrin-dependent adhesion and spreading of these cells on FN and its 110-kDa fragment, respectively. Because both adhesion and spreading events are active processes that require the activation of the outside-in signaling pathways; these results indicated the association of tTG with integrin during the adhesion and spreading of the cells on FN. As illustrated in a schematic model in Fig. 9A, these findings support the notion that the tTG-binding site on the FN substrate involves sequences outside the integrin ligand-binding pocket, which contains the Arg-Gly-Asp (RGD) sequence (6, 36). Accordingly, the association of tTG with the FN substrate was not perturbed by the RGD-containing peptides, which affect the interaction of integrins with both intact FN and the 110-kDa FN fragment. Collectively, these findings demonstrated that trophoblasts could attach, spread, and form FAs on the isolated 42-kDa FN fragment possibly through a tTG-integrin-FN axis. This hypothesis strengthens the proposal that the presence of tTG on the surface of EVTs and epithelial pinopodes will double the number of binding sites on the FN substrate, to which these cells may have access. Hereby, we suggest that the interactions of cell surface adhesion molecules, such as tTG, integrins, and L-selectin, which have different affinities for their substrates, can partly regulate the strength of the adhesion requirements of the cells during the complex process of implantation.
In reproductive medicine, embryo implantation yet remains a major limiting factor for the success of assisted reproductive therapies. Retinoids and epidermal growth factor are the most powerful inducers of transglutaminase activity, which could result in a rapid accumulation of tTG-integrin complexes up to 1% of the total cellular protein mass (17, 18). Thus, the regulation of the activity and biosynthesis of tTG could be considered for modifying several physiological aspects of embryo implantation, including the adhesion and invasion processes as a target for either contraception or assisted reproduction. Furthermore, in clinical medicine, changes in tTG expression were reported to contribute to the metastatic potential of tumors and wound healing (37). It appeared that the regulation of the proteolysis of cell surface tTG by matrix metalloproteinases at the boundary of tumors could modify the invasiveness of tumors by affecting migratory events (38). Taken together, clarifying the function and the mechanisms underlying the regulation of cell surface tTG may lead to the understanding and modification of several physiological and pathological phenomena.
In conclusion, the data reported in this study elucidated a novel role of the cell surface tTG in integrin-dependent adhesive events of the human implantation process, at least in part, by a direct interaction of tTG with FN or through a tTG-integrin-FN axis by activating integrin signaling pathways and by enhancing the assembly of FAs at the embryo-maternal interface.
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Acknowledgments |
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Footnotes |
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First Published Online May 10, 2005
Abbreviations: ECM, Extracellular matrix; EVT, extravillous trophoblast; FA, focal adhesion; FBS, fetal bovine serum; FN, fibronectin; HRP, horseradish peroxidase; onf, oncofetal; PFA, paraformaldehyde; pFAK, phosphorylated focal adhesion kinase; SEM, scanning electron microscopy; TEM, transmission electron microscopy; tTg, tissue transglutaminase.
Received February 4, 2005.
Accepted May 3, 2005.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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vß3 Integrin signaling pathway is involved in insulin-like growth factor-I-stimulated human extravillous trophoblast cell migration. Endocrinology 144:1620–16305ß1 integrin in the IGF-I-induced migration of extravillous trophoblast cells during the process of implantation. Mol Hum Reprod 10:91–97This article has been cited by other articles:
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  M. Kabir-Salmani, M. N. Fukuda, M. Kanai-Azuma, N. Ahmed, S. Shiokawa, Y. Akimoto, K. Sakai, S. Nagamori, Y. Kanai, K. Sugihara, et al. The Membrane-Spanning Domain of CD98 Heavy Chain Promotes {alpha}v{beta}3 Integrin Signals in Human Extravillous Trophoblasts Mol. Endocrinol., March 1, 2008; 22(3): 707 - 715. [Abstract] [Full Text] [PDF]
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 M. Kabir-Salmani, H. Nikzad, S. Shiokawa, Y. Akimoto, and M. Iwashita Secretory role for human uterodomes (pinopods): secretion of LIF Mol. Hum. Reprod., August 1, 2005; 11(8): 553 - 559. [Abstract] [Full Text] [PDF]
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