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Endocrinology Vol. 144, No. 4 1620-1630
Copyright © 2003 by The Endocrine Society

ARTICLE

{alpha}vß3 Integrin Signaling Pathway Is Involved in Insulin-Like Growth Factor I-Stimulated Human Extravillous Trophoblast Cell Migration

Maryam Kabir-Salmani, Shigetatsu Shiokawa, Yoshihiro Akimoto, Keiji Sakai, Shinya Nagamatsu, Ken Sakai, Yukio Nakamura, Abbas Lotfi, Hayato Kawakami and Mitsutoshi Iwashita

Departments of Obstetrics and Gynecology (M.K.-S., S.S., K.J.S., K.S., Y.N., M.I.), Anatomy (Y.A., H.K.), Biochemistry (S.N.), Kyorin University School of Medicine, Mitaka, Tokyo 181-8611, Japan; and Department of Biochemistry (A.L.), Tarbiat Modarres University, Tehran 14115-111, Iran

Address all correspondence 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}netjoy.ne.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IGF-I and -II provide paracrine and autocrine stimuli, respectively, for extravillous trophoblast (EVT) cell migration. This study examined the role of {alpha}vß3 integrin and its signaling pathway in IGF-I-stimulated migration. Migration assays were conducted using cultured EVT cells treated with or without IGF-I in the presence or absence of {alpha}IR3, Arg-Gly-Asp (RGD) hexapeptide, and antibody against {alpha}vß3 integrin. Morphological changes were studied using scanning electron microscopy. Colocalization of {alpha}5ß1 {alpha}vß3 integrins, vinculin, focal adhesion kinase, and paxillin were determined by immuno-cytochemistry and immunoblotting. The results showed that IGF-I could stimulate EVT cell migration in a time- and dose-dependent manner and addition of {alpha}IR3, Arg-Gly-Asp hexapeptide, and antibody against {alpha}vß3 integrin attenuated the IGF-I migratory effect. Scanning electron microscopy images revealed that IGF-I promoted lamellipodia formation. Immunostaining and immunoblotting exhibited the colocalization of {alpha}vß3 integrin with phosphorylated focal adhesion kinase, paxillin, and vinculin at focal adhesions after IGF-I treatment. Immunoblotting demonstrated an increase in focal adhesion kinase and paxillin tyrosine phosphorylation followed by tyrosine phosphorylation of IGF-I receptor in a time- and dose-dependent manner. These findings indicated {alpha}vß3 integrin localization in the core of focal adhesions of EVT cells and that {alpha}vß3 integrin signaling pathways are activated in IGF-I-mediated migration of these cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MIGRATION OF SUBPOPULATION of the placental trophoblast known as extravillous trophoblast (EVT) cells into the maternal endometrium is one of the fundamental components of human implantation and placentation (1). Using first-trimester EVT cells, it has been confirmed that the migration of human EVT cells are stringently regulated by growth factors, their binding proteins, extracellular matrix (ECM), and some adhesion molecules in an autocrine and paracrine manner (2).

It has been indicated that the differentiation and net invasiveness of EVT cells during early placental development is determined, at least in part, by regulation of integrin-mediated adhesion mechanisms that accelerate or restrain invasion (3). During the differentiation of cytotrophoblast stem cells into the EVT cell pathway, they acquire a selective integrin profile; the cells lose {alpha}6ß4 and gain {alpha}5ß1 and {alpha}vß3 integrins that bind to fibronectin (FN) in vitro and can be functionally blocked by Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) hexapeptide and echistatin (4, 5). Echistatin contains an Arg-Gly-Asp (RGD) sequence that mimics the FN-binding site and blocks binding of {alpha}5ß1 and {alpha}vß3 integrins to FN (6). Integrins provide an intersection where mechanical forces, cytoskeletal organization, biochemical signals, and adhesions can meet (7). Over 50 proteins have been found to interact with integrins in cell-ECM adhesions (8). Two general classic categories of these adhesion sites have been described as: 1) classical focal adhesions (FAs) that are characterized by high level of tyrosine phosphorylation of FA kinase (FAK) and paxillin and contain vinculin as an anchoring protein, and 2) fibrillar adhesions that contain relatively low level of tyrosine phosphorylation and function in generating extracellular fibrils of FN (9, 10). Cell migration requires the continuous, coordinated formation and disassembly of these adhesions. Furthermore, traction forces, which are essential for cell movements, are transmitted to the underlying substrate through FAs (11). Understanding adhesion turnover and the mechanisms underlying this process is now a critical area of emerging interest and believed to be affected by several extrinsic and intrinsic factors including growth factors.

The presence of IGF-I and -II at the maternal-fetal interface has been documented in both humans and a variety species of animals (12). IGF-I is a key growth factor in many reproductive events, including follicular development (13), oocyte maturation (14), sperm motility and fertilization (15), uterine endometrial proliferation (16), placental function (17), and preimplantation embryo and fetal developments (18, 19). Furthermore, IGF-I is a promoter of cell motility in vitro (20). Recently, it has been reported that placental fibroblasts at first trimester produce IGF-I in vitro that promote the migration of cytotrophoblasts from the periphery of nascent columns (21). This is in consistent with significant levels of IGF-I mRNA in this location in vivo, and further, it has been reported that a subpopulation of placental villous mesenchymal cells produce IGF-I and affect the migration of EVT cells (21, 22). Although IGF-I appears not to be produced by trophoblasts, EVT cells secrete IGF-II and expresses IGF-I receptor (IGF-IR). Because biological effects of IGF-I and -II are mediated largely by IGF-IR (23), migration of EVT cells are likely to be stimulated by their own IGF-II in an autocrine fashion as well as by neighbor cell secretion of IGF-I in an paracrine fashion (21). There are some data regarding IGF-II collaboration in EVT cell migration (2), whereas little information is available in respect of IGF-I effect on EVT cell migration and the existence of IGF-integrin interaction in human EVT cell migration.

Present studies were designed to examine the activation of {alpha}vß3 integrin signaling pathways during IGF-I-induced EVT cell migration and to determine morphological changes stimulated by IGF-I in these cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Recombinant human IGF-I was a gift from Fujisawa Pharmaceutical (Osaka, Japan). Monoclonal antibody against {alpha}-subunit of IGF-IR ({alpha}IR3) was purchased from Calbiochem Oncogene Research Products (San Diego, CA). {alpha}vß3 integrin antimouse monoclonal IgG and {alpha}5ß1 integrin antigoat polyclonal IgG were from Chemicon International (Temecula, CA). Affinity purified phosphotyrosine antimouse monoclonal IgG2b, FAK antirabbit polyclonal IgG, vinculin antigoat polyclonal IgG, paxillin antirabbit polyclonal IgG, {alpha}5 integrin antigoat polyclonal IgG, and horseradish peroxidase-conjugated goat antimouse IgG, goat antirabbit IgG, and donkey antigoat IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Phosphorylated FAK (pFAK) antirabbit polyclonal IgG was from Upstate Biotechnology, Inc. (Lake Placid, NY), which was immunodepleted from non-pFAK antibodies. Vinculin monoclonal antimouse IgG1, echistatin, and fluorescein isothiocyanate (FITC)-labeled phalloidin IgG were from Sigma (St. Louis, MO). FITC-labeled donkey antirabbit IgG and aminomethylcoumarin acetate-conjugated donkey antigoat IgG were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Alexa 568-labeled goat antimouse IgG was purchased from Molecular Probes, Inc. (Eugene, OR). Fibronectin, fibronectin inhibitor, GRGDSP hexapeptide, and its control peptide, Gly-Arg-Gly-Glu-Ser-Pro (GRGESP) hexapeptide, were synthesized by Iwaki Glass Co. (Chiba, Japan). Cell cultures reagents including medium 199, trypsin/EDTA, fetal bovine serum (FBS), and antibiotics were from Life Technologies, Inc. (Grand Island, NY), and Lab-Tek chamber slides were purchased from Nalge Nunc International (Naperville, IL). TAAB Epon 812, glutaraldehyde, and osmium tetraoxide were from TAAB Laboratories Equipment Limited (Berkshire, UK).

Primary trophoblast cell culture
Placental tissues between 6 and 10 wk of gestation were obtained at legal, 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). EVT cells were obtained by the method described previously (24), with few modifications. Briefly, the tissues were rinsed in cold medium 199 containing streptomycin (20 mg/ml) and penicillin (500 U/ml). Selected tissues were cut into small pieces, carefully removing any obvious blood vessels or clots, membranes, and decidual tissues. The fragments of villi were washed three times with medium 199 supplemented with streptomycin and penicillin and 10% FBS. These fragments were cultured in the same medium in tissue culture flasks, precoated with 20 µg/ml of FN in PBS (pH 7.4). Tissues were allowed to attach to the bottom of the flasks for 30–60 min before adding the medium. After 3–5 d, unattached cells were removed and cultured cells incubated for additional 1–2 wk in fresh medium 199 at 37 C in a humidified atmosphere containing 5% CO2. The medium was changed every 48 h until confluent. EVT cells from second to forth passage were used in this study. The medium was replaced for serum-free medium 199 18–24 h before using the cells for experiments. The identity of these cells as EVT cells was established by immunohistochemical staining using anticytokeratin 7 and 8/18, anti-{alpha}5ß1 and {alpha}vß3 integrins, antivimentin, CD9, and factor-VIII. More than 90% of the cells expressed all the markers of the EVT cells. As described previously, different morphology in the population of the primary cultured cells were observed (24).

Transwell migration assay
Transwell migration assays were conducted in 24-well fitted wells with membranes (8-µm pore size, Millipore Corp., Bedford, MA) as previously reported (25). Briefly, 2 x 105 serum-starved trophoblast cells were plated on the upper wells of transwell chambers, precoated with FN (20 µl/ml of PBS) containing either 400 µl serum-free medium 199 with 0.01% BSA, or plus various concentrations (0, 0.1, 1, 10, and 100 nM) of IGF-I. Lower chambers were loaded with medium 199-BSA with 10% FBS and incubated for 24 h. In another set of experiments, trophoblast cells were incubated for 6, 12, 18, and 24 h with or without 10 nM IGF-I. Cells on the upper surface of membranes were completely removed; migrated cells were fixed with 4% paraformaldehyde and stained with hematoxylin and eosin. Membranes were then cut from wells and mounted onto glass slides. Cellular migration indices were determined by counting the number of stained cells in 10 randomly selected nonoverlapping fields of the membranes under light microscope. These experiments were repeated six times for each group.

After the optimal concentrations and the temporal kinetics of IGF-I-stimulated migration were established, subsequent migration assays were done. To verify whether IGF-I-stimulated migration of the trophoblast cells were mediated through IGF-IR activation, the same procedures were performed in EVT cells that were preincubated with 10 nM {alpha}IR3 for 60 min. In other set of experiments, cells were preincubated with 100 µM GRGDSP hexapeptide, or its control peptide, GRGESP hexapeptide, as well as a monoclonal antibody to block {alpha}vß3 integrin specifically (25 µg/ml) for 60 min and then incubated with 10 nM IGF-I in the same condition mentioned above. Viability of the cells after preincubation with these antibodies and peptides were determined by trypan blue staining. GRGESP hexapeptide preincubated cells were considered as a corresponding control for GRGDSP hexapeptide preincubated cells. Corresponding control for {alpha}vß3 integrin blocking antibody contained the same concentration of monoclonal IgG in its IGF-I-added serum-free medium 199.

Scanning electron microscopy (SEM)
For SEM preparations, serum-starved trophoblast cells seeded on FN-coated coverslips were incubated in serum-free medium 199 with or without 10 nM IGF-I for 30 min. The cells were then fixed in cacodylate buffer (100 nM sodium cacodylate, pH 7.2; 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, and dehydrated by successive 10 min incubations with 30, 50, 70, 90, and 100% ethanol, followed by a 5-min incubation in hexamethyldisilazane, and completed by drying in a freeze dryer. Coverslips were then sputter-coated with gold/palladium and imaged using JSM, 5600 LV SEM (JEOL, Tokyo, Japan) operated at 25 kV. This experiment was repeated two times for two different preparations.

In another experiment, EVT cells seeded on FN-coated membranes of inserts that were used for migration assays were fixed as mentioned above and SEM photomicrographs were taken from the undersurface of IGF-I-treated and untreated cells to show passing of these cells through the pores.

Immunocytochemistry
Trophoblast cells were seeded on FN-coated Lab-Tek chamber slides and incubated for 24 h in serum-free medium 199. Following washing the cells with the same medium, different groups were defined by a 30-min incubation of the cells with serum-free medium 199 in the presence or the absence of 10 nM IGF-I with or without 10 nM {alpha}IR3 for double staining. For triple staining, all the cells were treated with 10 nM IGF-I for 30 min to stimulate the assembly of FAs to detect colocalization of {alpha}vß3 and {alpha}5ß1 integrins with different components of FAs of EVT cells. Then cells were fixed using 4% paraformaldehyde, permeabilized by 0.5% Triton X-100, and blocked by 5% BSA or normal donkey serum. The cells were incubated for 1–3 h at room temperature with the appropriate primary antibodies diluted in PBS (antivinculin IgG: 1 µg/ml, antipaxillin IgG: 1.7 µg/ml, anti-pFAK IgG: 1.7 µg/ml, anti-{alpha}vß3 integrin IgG: 1.5 µg/ml, and anti-{alpha}5 integrin IgG: 1.5 µg/ml). For control, the coverslips were incubated at room temperature for 1–3 h with the same concentration of corresponding antibodies, including monoclonal antimouse IgG (substituted for mouse antivinculin and {alpha}vß3 integrin), polyclonal antirabbit IgG (substituted for paxillin and pFAK), and polyclonal antigoat IgG (substituted for {alpha}5 integrin and goat antivinculin) primary antibodies. The cells were rinsed in PBS extensively and counterstained with proper fluorescent-labeled secondary antibodies (Alexa 568-labeled goat antimouse IgG, 1.5 µg/ml; FITC-labeled phalloidin, 1 µg/ml; FITC-labeled donkey antirabbit IgG, 1.7 µg/ml; aminomethylcoumarin acetate-conjugated donkey antigoat IgG, 2 µg/ml; and FITC-labeled donkey antigoat IgG, 2 µg/ml) appropriately and incubated for 1–3 h at room temperature. After washing with PBS, rinsing in deionized water and mounting, cells were observed using an AX-80 fluorescence microscope (Olympus Optical, Tokyo, Japan). These stainings were repeated for three times in three different preparations for each group.

Immunoprecipitation and immunoblotting
Trophoblast cells were grown to 80% confluent on 10-cm tissue culture plates, rinsed three times with serum-free medium 199, and incubated for 24 h with same medium. The cells were washed three times with serum-free medium 199 and incubated with the same medium in the presence or the absence of 10 nM IGF-I for 0, 5, 15, 30, and 60 min to determine the optimum time for IGF-I stimulation. In another set of experiments, the cells were incubated for 30 min with 0, 0.1 1, 10, and 100 nM IGF-I to determine the dose response. To detect the importance of IGF-IR and RGD-related integrins, in another groups the cells were preincubated with {alpha}IR3 (10 nM) and echistatin (10 nM) for 1 h and then 10 nM IGF-I was added to their medium. Cells were washed using ice-cold PBS for three times, solubilized with 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 M 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, 0.5 mg/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 supernatant was incubated overnight at 4 C with 6 µg/ml anti-FAK, anti-IGF-IRß, and antipaxillin polyclonal antibodies separately. The immunocomplexes were incubated with protein A-Sepharose (Sigma, St. Louis, MO) at 4 C for 2 h and immobilized protein A-Sepharose was sedimented by centifugation at 10,000 x g for 1 min, washed four times with the same lysis buffer without phosphatase and protease inhibitors, and were resuspended in 20 µl of 4x reducing-sodium dodecyl sulfate sample buffer. The same amount of immunoprecipitated proteins were subjected to 7.5% SDS-PAGE under the reducing condition and electrophoretically transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Inc., Hercules, CA). The membranes were blocked with Tris buffered saline (10 mM Tris; 140 mM NaCl, pH 7.4) containing 1% BSA for 4 h at room temperature, then incubated overnight at 4 C with antiphosphotyrosine monoclonal antibody diluted with blocking buffer (1:1000). After several washes with washing buffer [Tris buffer saline containing 0.5% (vol/vol) Tween 20], immunoreactive proteins were identified by 2 h incubation with horseradish peroxidase-conjugated goat antimouse monoclonal IgG diluted with blocking buffer (1:5000) at room temperature. Following several washes, the membranes were visualized using enhanced chemiluminescence reagents (Amersham Biosciences KK, Tokyo, Japan) and exposed to Kodak X-AR film (Eastman Kodak Co., Rochester, NY) for 1–15 min. For control of each group, we stripped the phosphotyrosine antibody using Western blot stripping buffer (Pierce Chemical Co., Rockford, IL) and reblotted the membranes with the same antibodies that were used for immunoprecipitation (FAK, paxillin, or IGF-IR). We obtained the same intensity of each band in different group, suggesting that equal amount of protein were loaded.

For coimmunoprecipitation of {alpha}5ß1 and {alpha}vß3 integrins with different components of focal adhesions including FAK, paxillin, and vinculin, the same procedures were performed using modified lysis buffer (1% Triton X-100, 1 mM EGTA, 150 mM NaCl, 50 mM HEPES, and 1 mM MgCl2). The supernatants were incubated with 6 µg/ml of anti-{alpha}5ß1 and {alpha}vß3 integrins overnight at 4 C and precipitated with protein G-Sepharose by 2 h incubation at 4 C followed by sedimentation. The membranes were blotted with appropriate primary antibody diluted at 1:1000 and the proteins were identified using horseradish peroxidase-conjugated goat antimouse IgG, goat antirabbit IgG, and donkey antigoat IgG diluted at 1:5000.

Statistics
In time-course and dose-response migration assays, migrated cells were expressed as mean ± SEM of six assays. Statistical significance were evaluated using ANOVA with Scheffé’s test and were considered statistically significant if P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Migration assay
Time-course migration assay demonstrated that the optimum time for maximum transwell migration of the trophoblast cells was 24 h (Fig. 1CGo). Therefore, dose-response experiments were performed to incubate with indicated doses of IGF-I for 24 h (Fig. 1Go, A and D). The results showed that IGF-I significantly stimulated trophoblast cells migration in a dose-dependent manner. Maximal response was obtained at 100 nM IGF-I that was about 2-fold of control with ED50 value of 1.8 nM. Inhibition of IGF-IR by {alpha}IR3 demonstrated that the numbers of migrated cells were significantly decreased comparing to its corresponding control (Fig. 1Go, B and E). To investigate the association of integrins in IGF-I-stimulated migration of trophoblast cells, the cells were preincubated with GRGDSP and GRGESP hexapeptides and {alpha}vß3 integrin antibody before addition of 10 nM IGF-I to their medium. As shown in Fig. 1Go, B and E, numbers of migrated cells in GRGDSP hexapeptide preincubated cells were significantly decreased comparing to GRGESP hexapeptide preincubated cells as its corresponding control. Number of migrated cells in the cells preincubated with {alpha}vß3 integrin functional blocking antibody was significantly lesser than its corresponding control cells, which were preincubated with same concentration of mouse IgG.



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  		Figure 1. Morphometric evaluation of EVT cell migration stimulated with different doses (0.1–100 nM) of IGF-I (A and D), incubated for different times (6, 12, 18, 24 h) at 37 C (C). Preincubation of EVT cells with 10nM {alpha}IR3, GRGDSP hexapeptide, and antibody against {alpha}vß3 integrin significantly decreased the number of migrated cells (B and E). Migrated cells were expressed as mean ± SEM of six assays. a, Not significant difference vs. corresponding control; b, significant difference vs. corresponding control; c, not significant vs. control; d, significant difference vs. 10 nM IGF-I. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Scale bars, 10 µm.

 
Morphological changes of trophoblast cells
Upon IGF-I treatment, trophoblast cells underwent morphological changes assuming a flattened contour with lamellipodia-like extensions. SEM highlighted an increase in lamellipodia formation in the IGF-I-treated trophoblast cells, which were subjected to serum starvation (Fig. 2Go, A–D). These macrographs also revealed that the bodies of these cells did not adhere to the substratum, but the cell-subtrate attachment was mediated mostly by the lamellipodia. Figure 3Go demonstrated passing of the trophoblast cell through membrane pores of inserts using for transwell migration assay in the IGF-I-treated (A) and untreated cells (B).



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  		Figure 2. SEM micrographs of serum-starved human EVT cells untreated (A and C) and treated with 10 nM IGF-I (B and D). Panels C and D are higher magnification of A and B, respectively. Notice the formation and extension of lamellipodia in the IGF-I-treated EVT cells. Bar, 10 µm.

 


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  		Figure 3. SEM photomicrographs from undersurface of millipore membranes showing passing of 10 nM IGF-I-stimulated EVT cell through the membrane pore (A), whereas control serum-starved cells showed no or little lamellipodia formation (B). Both photos were taken 24 h after seeding of the cells on upper chamber of inserts that were used for migration assays. Bar, 2 µm.

 
Immunocytochemistry
To correlate the data obtained from migration assays and SEM with molecular and cytoskeletal dynamics changes at focal adhesions, immunocytochemical analysis was performed. Immunofluorescent images from fluorescein phalloidin, which shows high affinity for the actin cytoskeleton, double stained with human antivinculin monoclonal antibody revealed a redistribution of the actin cytoskeleton toward the leading processes of IGF-I-treated cells that were anchored to the vinculin molecules in the FAs, whereas in the control and {alpha}IR3-treated cells, actin stress fibers were not formed (Fig. 4Go). Furthermore, vinculin molecules were localized at focal adhesions in IGF-I-stimulated cells whereas in untreated and {alpha}IR3-treated cells, vinculin molecules did not show such organization and were not related to actin filaments.



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  		Figure 4. Immunolocalization of pFAK, paxillin, vinculin, and actin stress fibers in serum-starved trophoblast cells incubated for 30 min with serum-free medium 199 without IGF-I (left column), plus 10 nM IGF-I (middle column), and plus 10 nM IGF-I treatment in the cells preincubated with 10 nM {alpha}IR3 for 60 min (right column). The IGF-I-treated EVT cells showed localization of immunoreactive pFAK, paxillin, and vinculin in the cell periphery, corresponding to focal adhesions. Untreated cells and the cells that were preincubated with {alpha}IR3 did not exhibit such organizations. Scale bar, 10 µm.

 
pFAK and paxillin can be detected in the FAs of the trophoblast cells treated with 10 nM IGF-I (Fig. 4Go), whereas in the cells preincubated with {alpha}IR3 and untreated trophoblast cells, these organizations were not detectable in FAs of EVT cells (Fig. 4Go).

Triple staining of {alpha}vß3 and {alpha}5ß1 integrins with pFAK, paxillin, and vinculin exhibited colocalization of {alpha}vß3 integrin with pFAK, paxillin, and vinculin in the FAs of IGF-I-stimulated EVT cells, but {alpha}5ß1 integrin did not significantly show colocalization with these proteins in FAs of the IGF-I-treated trophoblast cells (Fig. 5Go). Antibody that was used to immunostain {alpha}vß3 integrin required both {alpha}v and ß3 subunits to get stained; thus, it was specific to {alpha}vß3 integrin rather than other subfamily of its subunits.



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  		Figure 5. Immunodetection of {alpha}vß3 integrin, {alpha}5 integrin, pFAK, paxillin, and vinculin in cultured human EVT cells at 6–10 wk of gestation after treatment with 10 nM IGF-I for 30 min. Notice that {alpha}vß3 integrin but not {alpha}5 integrin colocalized with pFAK, paxillin, and vinculin in the cell periphery, corresponding to focal adhesions of the EVT cells (shown by arrowheads). Cells in the right column are computer-combined images of the EVT cells that were immunostained in the left three columns. Colocalized area can be detected in the right column as combination of red and green colors as violet. Combined colors in the cell periphery represent the focal adhesions of these cells, which are assembled following IGF-I stimulation. Scale bars, 10 µm.

 
Immunoblotting of pFAK and paxillin
To examine whether IGF-I might activate integrin signaling pathway in trophoblast cells, Western blotting was performed in mentioned time and dose of IGF-I treatment as well as in cells preincubated with {alpha}IR3 antibody and echistatin. Western blot using antiphosphotyrosine demonstrated that 10 nM IGF-I stimulated the phosphorylation of tyrosine residues in ß-subunit of IGF-IR after 5 min and both paxillin and FAK proteins were phosphorylated 15 min following IGF-I stimulation (Fig. 6Go). The results of antiphosphotyrosine immunoblotting in IGF-IR, FAK, and paxillin immunoprecipitated groups showed a dose-dependent manner activation of these proteins (Fig. 7Go). Inhibition of integrin and IGF-IR by echistatin and {alpha}IR3 antibody respectively, exhibited inhibition of tyrosine pFAK and paxillin following IGF-IR inhibition (Fig. 8Go). Results shown are representative of three independent experiments.



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  		Figure 6. Time-course study of IGF-I-stimulated tyrosine phosphorylation of IGF-IR (A), FAK (B), and paxillin (C). Serum-starved EVT cells were treated for 5–60 min (lane 2–5) with serum-free medium containing 10 nM IGF-I. The 0 time point (lane 1) indicates EVT cells treated with serum-free medium alone. Trophoblast cells were lysed and proteins were immunoprecipitated with antibodies specific to the FAK, paxillin, or IGF-IR separately. Equal amounts of total cellular protein were loaded in each lane. Isolated proteins were separated by SDS-PAGE, transferred electrophoretically to polyvinylidene difluoride, and analyzed by antiphosphotyrosine immunoblotting. IGF-I stimulated transient tyrosine phosphorylation of FAK and paxillin and affected IGF-IR tyrosine phosphorylation time dependently. For control, the membranes were reprobed with IGF-IR, FAK, and paxillin antibodies, respectively.

 


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  		Figure 7. Dose-response of IGF-I-stimulated tyrosine phosphorylation of IGF-IR (A), FAK (B), and paxillin (C). Serum-starved trophoblast cells were treated for 30 min with serum-free medium containing 0–100 nM IGF-I (lanes 1–5). The same procedures that were mentioned for time-course study were performed in this experiment. Tyrosine phosphorylation of IGF-IR, FAK, and paxillin exhibited dose-dependent relationship to IGF-I treatment. For control, the membranes were reprobed with IGF-IR, FAK, and paxillin antibodies, respectively.

 


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  		Figure 8. Effects of the IGF-IR blocking antibody ({alpha}IR3) and disintegrin (echistatin) on IGF-I-stimulated tyrosine phosphorylation of IGF-IR (A), FAK (B), and paxillin (C). Serum-starved trophoblast cells were treated for 60 min with serum-free medium containing no addition or 10 nM {alpha}IR3 or 10 nM echistatin. Media were then adjusted to 10 nM IGF-I as indicted, and tyrosine phosphorylation was assessed after 30 min. In this experiment, IGF-IR tyrosine phosphorylation was assessed in whole-cell lysates because the addition of {alpha}IR3 precluded IGF-IR immunoprecipitation. The same procedures that were mentioned for time-course study were performed in this experiment. Inhibition of IGF-IR by {alpha}IR3 reduced the tyrosine phosphorylation of IGF-IR, FAK, and paxillin. Preincubation of EVT cells with echistatin reduced the tyrosine phosphorylation of FAK and paxillin but not IGF-IR. For control, the membranes were reprobed with FAK and paxillin antibodies, respectively.

 
Coimmunoprecipitation of FAK, vinculin, and paxillin with {alpha}vß3 integrin and {alpha}5ß1 integrin in the IGF-I-treated and untreated EVT cells showed that these proteins are more correlated with {alpha}vß3 integrin rather than {alpha}5ß1 integrin and appeared more clearly in IGF-I-treated cells (Fig. 9Go). Results shown are representative of three independent experiments.



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  		Figure 9. Coimmunoprecipitation of the FAK, paxillin, and vinculin (A, B, and C, respectively) are shown with {alpha}vß3 integrin or {alpha}5ß1 integrin in the IGF-I-treated and untreated EVT cells. Serum-starved trophoblast cells were treated for optimum period resulted from time-course study (15–30 min) with serum-free medium containing no addition or 10 nM addition of IGF-I. Trophoblast cells were then lysed and immunoprecipitated with {alpha}vß3 integrin or {alpha}5ß1 integrin. Immunoprecipitates were separated by SDS-PAGE and analyzed by immunoblotting with anti-FAK, antipaxillin, and antivinculin as mentioned for time-course study.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The human placenta is an invasive structure in which highly proliferative and invasive EVT cells invade the uterus and its vasculature. EVT cell migration similar to tumor cell migration can be regarded as a multistep cycle, including extension of a protrusion, formation of stable attachments, translocation of the cell body, release of adhesions, and retraction at the cell rear (26). It has been shown that explants of placental villi from the first trimester pregnancy represent selective outgrowth of the invasive trophoblast population, which can progress into extravillous lineage providing an appropriate in vitro model for studying migratory events during human implantation.

Using cultured EVT cells in the present study, SEM figures revealed that IGF-I stimulated the formation of lamellipodia in the serum-starved EVT cells. Furthermore, cell-ECM adhesion sites were found to be present mostly in the lamellipodia of these cells. Formation of lamellipodia has been reported previously as an early event after addition of a migratory stimulus similar to IGF-I. Moreover, existence of FAs in the lamellipodia has been reported in other migrating cells as well (27). Formation of a protrusion (lamellipodium) initiates the migration cycle in the motile cell and for migration to occur the protrusions must be stabilized by attaching to their underlying substratum. Thereafter, cytoplasmic extension in the form of pseudopodia leads to the formation of new cell-substrate adhesions known as FAs (26).

Assembly of FAs as integrin-based structures was detected in this study to determine the effect of IGF-I on formation of FAs in EVT cells. Results obtained from immunofluorescent staining demonstrated an increase in assembly of FAs upon IGF-I stimulation in these cells. This supports previous findings that reported IGF-I could stimulate formation of FAs in neuronal cells (27). Addition of the {alpha}IR3 antibody inhibited IGF-I-induced formation of FAs and confirmed that the activation of IGF-IR is necessary for IGF-I-mediated assembly of these structures. Reorganization of actin microfilaments to form actin stress fibers and their anchorage to vinculin at FAs following IGF-I treatment has been reported both in the present and previous studies (28). In this context, reorganization of actin microfilaments, increase in lamellipodia formation and extension, and assembly of FAs in IGF-I-treated EVT cells could be considered as a part of procedures involved in IGF-I-mediated migration of these cells.

To clarify colocalization of {alpha}vß3 or {alpha}5ß1 integrins in respect to different component of FAs including pFAK, paxillin, and vinculin, immunofluorescent staining and immunoblotting following coimmunoprecipitaion of mentioned proteins with {alpha}vß3 or {alpha}5ß1 integrins were performed. The results of immunofluorescent staining exhibited that the dominant integrin in FAs of EVT cells was {alpha}vß3 integrin, and {alpha}5ß1 integrin was excluded from the core of FAs of these cells. To our knowledge, these results provided the first demonstration regarding colocalization of paxillin, vinculin, and pFAK with {alpha}vß3 integrin in FAs of human EVT cells. Further, the results of immunoblotting showed that FAK, paxillin, and vinculin coimmunoprecipitated with {alpha}vß3 integrin but not {alpha}5ß1 integrin in human EVT cells. These results are in consistent with the previous report that showed coimmunoprecipitation of FAK, paxillin, and vinculin with {alpha}vß3 integrin in IGF-I-treated chondrocytes (29). Although it is in contrast with the results of others that reported colocalization of {alpha}5 integrin with pFAK in human trophoblast cells (30). This controversy might be due to the different specimens that have been used in these studies (two-dimensional in vitro-cultured cells compared with in vivo cells in a three-dimensional environment). Further, different pattern of integrin association has been reported previously in three-dimensional cell-ECM adhesions in vitro (31). The specific type of integrin present in matrix adhesions can vary, depending on the nature of the underlying ECM and type of association of integrins with ECM in different cells can display different patterns (32). In two-dimensional culture of fibroblastic cells, focal and fibrillar adhesions exhibited distinct molecular compositions: FAs characteristically contain integrin {alpha}vß3, whereas fibrillar adhesions composed of {alpha}5ß1 integrin (10); and FAs and fibrillar adhesions appeared to be colocalized in NIL-8 cells (33). Hereby, we suggest further studies to localize the {alpha}vß3 and {alpha}5ß1 integrins in respect to different components of FAs in three-dimensional culture system of human EVT cells to clarify their more distinct roles in migration cycles and implantation in vivo.

The results of migration assays in the present study revealed that IGF-I stimulated migration of EVT cells in a time- and dose-dependent manner. Because preincubation of EVT cells with {alpha}IR3 have significantly reduced increased-number of migrating cells induced by IGF-I; it was concluded that IGF-I-induced migration was attributed to activation of IGF-IR. This is in agreement with previous reports that showed importance of IGF-IR for majority of biological effects of IGF-I (34). Moreover, the results of this study indicated that binding of {alpha}vß3 integrin to their ligands is essential for the IGF-I-mediated migration to occur. This is consisted with the results of immunostaining of {alpha}vß3 integrin in the present study, showing increased assembly of FAs after IGF-I stimulation. Assembly of FAs seems to be mediated by {alpha}vß3 integrin activation. In vivo, at the terminal end of the trophoblastic column where the trophoblast cells fan into the decidua, the cells express {alpha}vß3 and {alpha}5ß1 integrins indicating the importance of both integrins in promoting migratory phenotype of the EVT cells. There are some controversies regarding the role of {alpha}5ß1 integrin in respect of EVT cell migration and invasion (3, 35, 36). However, there was no report in respect of the role of {alpha}vß3 integrin in this regard and in respect to collaboration of {alpha}vß3 integrin with IGF-I in EVT cell migration. Taking our findings together with others, it is tempting to speculate that adhesion turnover in EVT cell requires the coordinated cycling of {alpha}vß3 and {alpha}5ß1 integrins. However, it seems that the ratio of {alpha}vß3 to {alpha}5ß1 integrins might be also important in the migratory function of these cells. There are some reports that demonstrated IGF-I increased migration of smooth muscle cell by increasing {alpha}vß3 integrin affinity in these cells and showed that ligand occupancy of {alpha}vß3 integrin was necessary for the biological activities of IGF-I (37). Whether IGF-I regulates activation of {alpha}vß3 integrins of EVT cells through an increased avidity or affinity of these receptors remains to be elucidated.

Integrins and growth factors share many common elements in their signaling pathways, suggesting the possibility of cross-talk between integrins and growth factors signaling pathways. Previous studies have documented the existence of a cross-talk between components of the integrin-mediated and the IGF-I signaling pathways (38). The binding of IGF-I to its receptor activates the intrinsic kinase activity of the receptor and results in autophosphorylation of several intracellular tyrosine residues (39). The results obtained from immunoblotting in this study were in agreement with and provide indirect proof for this hypothesis. Tyrosine phosphorylation of FAK and paxillin upon IGF-I stimulation are critical for their migratory function and has been reported to stimulate cytoskeletal remodeling and recruitment of other proteins including vinculin (40) as well. pFAK and paxillin are colocalized with {alpha}vß3 integrin in FAs of EVT cells, and preincubation of these cells with echistatin attenuated the tyrosine phosphorylation of FAK and paxillin following tyrosine phosphorylation of IGF-IR. These results are in agreement with previous reports (28, 41). FAK and paxillin proteins were highly expressed and active from 5–8 wk of gestation in developing trophoblast cells at a time when these cells are highly proliferative and migratory and in IGF-I-induced EVT cell-ECM adhesions (30, 42). Collectively, our findings suggest a straightforward sequence of events to explain how IGF-I stimulates transient FAK and paxillin tyrosine phosphorylation and EVT cell migration: 1) IGF-I promotes the extension of lamellipodia; 2) the advancing lamellipodia bind to the ECM; and 3) lamellipodial adhesion leads to FAK and paxillin tyropine phosphorylation, which in turn stimulate migratory phenotype in these cells. Because tyrosine phosphorylation of FAK and paxillin are associated with assembly of adhesion foci, they may help stabilizing the advancing lamellipodia. Such a role for FAK and paxillin in lamellipodial advance could explain why expression of these proteins correlates with cell motility. FAK is known to enhance cell migration through FA turnover, and fibroblast from FAK-deficient mice exhibited markedly reduced cell migration and large focal adhesions (43). Further, high levels of FAK expression were found in invasive human tumors, suggesting its role in metastasis (44). Thus, it is likely that adhesion turnover require the coordinated cycling of key regulators between phosphorylated and dephosphorylated states. IGF-I has been shown to cross-talk with integrins during migration of different cell types to modulate transient phosphorylation of components of integrin pathways (27, 45). Moreover, it has been reported that the {alpha}vß3 integrin regulates IGF-IR phosphorylation (46). Our findings in conjunction with others suggest the importance of tyrosine phosphorylation of FAK and paxillin as activated integrin-dependent signaling pathways in IGF-I-mediated EVT cell migration.

In conclusion, the results presented in this study indicated that IGF-I stimulated migration of EVT cells in a time- and dose-dependent manner in vitro and could be considered as an important potent paracrine regulator in vivo. Furthermore, migratory effect of IGF-I is, at least in part, mediated by IGF-IR and {alpha}vß3 integrin. Tyrosine phosphorylation of FAK and paxillin following IGF-I treatment may indicate the interaction between IGF-I and integrin signaling pathways. These findings assume an added interest in view of the increasing evidences implicating the role of integrins in mediating the migratory effects of IGF-I.


   Footnotes
 
Address requests for reprints to: S. Shiokawa, 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: shiochan{at}kyorin-u.ac.jp.

This work was supported in part by Grants-in-Aid (C) 11671648 (to S.S.) from the Ministry of Education, Science, and Culture (Tokyo, Japan).

Abbreviations: ECM, Extracellular matrix; EVT, extravillous trophoblast; FA, focal adhesion; FAK, FA kinase; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; FN, fibronectin; GRGDSP, Gly-Arg-Gly-Asp-Ser-Pro; GRGESP, Gly-Arg-Gly-Glu-Ser-Pro; IGF-IR, IGF-I receptor; {alpha}IR3, {alpha}-subunit of IGF-IR; pFAK, phosphorylated FAK; RGD, Arg-Gly-Asp; SEM, scanning electron microscopy.

Received October 25, 2002.

Accepted for publication December 11, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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