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BOR - Papers in Press, published online ahead of print January 25, 2006.
Biol Reprod 2006, 10.1095/biolreprod.105.050419
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BIOLOGY OF REPRODUCTION 74, 959–968 (2006)
DOI: 10.1095/biolreprod.105.050419
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

Integrin-Linked Kinase (ILK) Is Highly Expressed in First Trimester Human Chorionic Villi and Regulates Migration of a Human Cytotrophoblast-Derived Cell Line1

P.A. Elustondo 3, G.E. Hannigan 4, I. Caniggia 5, and D.J. MacPhee 2 3

Division of Basic Medical Sciences,3 Health Sciences Centre, Memorial University of Newfoundland, St. John's, Newfoundland and Labrador, Canada A1B 3V6 Department of Laboratory Medicine and Pathobiology,4 University of Toronto and Cancer Research Program, Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X5 Program in Development and Fetal Health,5 Samuel Lunenfeld Research Institute, Mt. Sinai Hospital, Toronto, Ontario, Canada M5G 1X5

ABSTRACT

The placenta represents a critically important fetal-maternal interaction. Trophoblast migration and invasion into the uterine wall is a precisely controlled process and aberrations in these processes are implicated in diseases such as preeclampsia. Integrin-linked kinase (ILK) is a multifunctional, cytoplasmic, serine/threonine kinase that has been implicated in regulating processes such as cell proliferation, survival, migration, and invasion; yet the temporal and spatial pattern of expression of ILK in human chorionic villi and its role in early human placental development are completely unknown. We hypothesized that ILK would be expressed in trophoblast subtypes of human chorionic villi during early placental development and that it would regulate trophoblast migration. Immunoblot analysis revealed that ILK protein was highly detectable in placental tissue samples throughout gestation. In floating branches of chorionic villi, from 6 to 15 wk of gestation immunofluorescence analysis of ILK expression in placental tissue sections demonstrated that ILK was highly detectable in the cytoplasm and membranes of villous cytotrophoblast cells and in stromal mesenchyme, whereas it was barely detectable in the syncytiotrophoblast layer. In anchoring branches of villi, ILK was highly localized to plasma membranes of extravillous trophoblast cells. Transient expression of dominant negative E359K-ILK in the villous explant-derived trophoblast cell line HTR8-SVneo dramatically reduced migration into wounds compared to cells expressing wild-type ILK or empty vector. Therefore, our work has demonstrated that ILK is highly expressed in trophoblast subtypes of human chorionic villi during the first trimester of pregnancy and is a likely mediator of trophoblast migration during this period of development.

developmental biology, kinases, placenta, pregnancy, trophoblast

INTRODUCTION

During pregnancy, the physiological relationship between the mother and fetus is mediated by the placenta. The placenta possesses an array of specialized metabolic, hormonal and immunological functions that ultimately control the growth and viability of the fetus and, as a result, the health of the mother (reviewed in [1–4]). The importance of the placenta to the health and well-being of the baby and mother is exemplified in conditions or diseases during pregnancy that are thought to be the result of placental abnormalities, such as preeclampsia, gestational diabetes, and intrauterine growth restriction [5]. Furthermore, proper embryonic and placental development might be closely linked to the future good health and well-being of the individual. A "developmental origins hypothesis" has been proposed [6], suggesting that adult conditions such as cardiovascular disease and type 2 diabetes might occur because of undernutrition in utero. Because the placenta is crucial for fetal growth, irregular placental development may underlie these diseases. These potential interrelationships highlight the importance of research into the various physiological, biochemical, and molecular aspects of human placental development.

Despite reports of in vivo and in vitro analyses of human placental development and the existence of a variety of research tools such as human-derived trophoblast cell lines and null mice exhibiting placental defects, the process of trophoblast differentiation is still poorly understood [7–10]. In the fetal placenta, chorionic villi become spatially segregated into two types: floating and anchoring villi. Floating villi are bathed in maternal blood, facilitating processes such as the exchange of gases, nutrients, wastes, and hormone transport, whereas anchoring villi are involved in establishing and maintaining the fetal-maternal interface. During early placentation, two fundamental pathways of trophoblast differentiation take place [4, 11]. In the first or fusion pathway, polarized stem cytotrophoblast cells in floating branches of chorionic villi proliferate, differentiate, and fuse to form a multinucleated syncytiotrophoblast layer [12]. In the second or invasion pathway, polarized stem cytotrophoblast cells in anchoring branches of chorionic villi tips migrate off the villous basement membrane and penetrate through the syncytiotrophoblast to form columns of nonpolarized extravillous trophoblast cells that will differentiate into highly invasive trophoblast cells, thereby aiding attachment of the embryo to the uterine wall and remodeling of maternal spiral arteries [5, 13, 14]. The formation of anchoring villi is accompanied by changes in the synthesis, degradation, and spatial distribution of extracellular matrix proteins, and by the specific spatial and temporal expression of adhesion molecules in extravillous trophoblast [15–18].

The ability of trophoblast cells to adhere to and sense the surrounding extracellular environment and to produce intracellular signals is crucial to their proper differentiation [15, 16, 19–21]. Cells can sense their extracellular environment at focal contacts or adhesions, which are clusters of integrin proteins, signaling enzymes, and adapter proteins [22]. One such focal adhesion-associated, signaling enzyme is integrin-linked kinase (ILK). This serine/threonine protein kinase was originally identified and cloned from a human placental cDNA library based on its interaction with the ß1-integrin cytoplasmic domain [23]. Several laboratories have demonstrated that ILK has direct targets for its kinase activity, phosphorylating protein kinase B (AKT1), glycogen synthase kinase-3ß (GSK3B), myosin light chain, and ß-parvin [24–26]. In addition to its function as a kinase, ILK appears to be a key mediator of protein-protein interactions, and thus ILK in total can be involved in a range of signaling pathways [27]. The importance of ILK to embryonic development is exemplified by the fact that Ilk-null mutations in Drosophila, C. elegans, and mice all result in lethal embryonic phenotypes as a result of aberrant integrin-mediated muscle attachment or F-actin accumulation [28–30].

Despite the fact that ILK is recognized as a regulator of processes such as cell proliferation, survival, migration, and invasion in a variety of cell types [27, 31], the temporal and spatial pattern of expression of ILK in human chorionic villi and its role in early human placental development are completely unknown. Thus, based on such reported roles for ILK, we hypothesized that ILK would be expressed in trophoblast subtypes of human chorionic villi during early placental development and regulate trophoblast migration.

MATERIALS AND METHODS

Tissue Collection

Ethics approval for the study (Protocol #03.44) was obtained from the Human Investigation Committee of Memorial University of Newfoundland and the Health Care Corporation of St. John's Research Proposals Approval Committee. All study participants with confirmed ultrasound-dated pregnancies who were undergoing elective terminations provided written informed consent. Placental tissue was obtained from elective terminations by dilatation and curettage at wk 6–8 (n = 16), 9–12 (n = 27), and 13–15 (n = 4) of gestation, and from deliveries at 37–40 wk (i.e., term; n = 25). The tissues from elective terminations were collected in sterile PBS and transported to the laboratory within 10 min of the procedure. All tissue samples were extensively washed, then dissected with cold PBS and either fixed in 4% paraformaldehyde in PBS or frozen and stored in liquid nitrogen.

Cell Culture

The HTR8-SVneo trophoblast cell line was a gracious gift from Dr. Charles Graham (Queens University, Kingston, Canada). The cell line was originally derived from primary human villous explants [32], has been extensively characterized, and is an accepted cell line model of extravillous trophoblast [14]. It is invasive and nontumorigenic and demonstrates an extravillous trophoblast immunological and biological phenotype [7, 32–36]. Cells were maintained in RPMI 1640 (cat. no. 31800–022; Invitrogen Ltd.) supplemented with 10% fetal bovine serum (cat. no. CS-C08–500-U; Cansera International, Inc.) and 100 U penicillin/100 µg streptomycin (cat. no. 15140–122; Invitrogen) as has been described elsewhere [32, 35]. Cells were cultured at 37°C in a humidified tissue culture incubator containing 5% CO2 in air. For those experiments requiring serum starvation, cells were cultured in RPMI 1640 with 0.5% FBS according to Graham et al. [32].

Immunofluorescence Analysis

Immunofluorescence analysis was conducted essentially according to MacPhee et al. [20]. Experiments were repeated at least 4 times with different samples obtained from wk 6–8, 9–12, and 13–15 of gestation and from deliveries at 37–40 wk. Fixed tissues were processed, embedded in paraffin wax, and sectioned (5 µm thick) by the Histology Unit of the Faculty of Medicine, Memorial University of Newfoundland. Sections were dewaxed in xylene, rehydrated in descending grades of ethanol and soaked in 1x PBS. Following tissue section rehydration, epitope retrieval was conducted by incubation of tissue sections with 0.1% Trypsin/PBS for 10 min at room temperature (RT) and a subsequent wash in PBS. Sections were blocked in 5% normal goat serum/1% horse serum/1% fetal bovine serum in PBS for 30 min at RT with constant agitation, then incubated for 1 h at RT in primary antisera (Table 1) or affinity-purified IgG of the appropriate species, at the same concentration as the primary antisera, to serve as a negative control. After three washes in PBS, the tissue sections were incubated with Rhodamine-Red-X- or FITC-conjugated secondary antisera (Table 1). The sections were then washed with PBS containing 0.2% Tween 20 (PBT) and then mounted in Vectashield (Vector Laboratories Inc.). If double immunofluorescence experiments were used, incubations in additional primary and secondary antisera were employed as described above, following washes in PBT and blocking of tissue sections for 30 min. All slides were observed using a Leica DM-IRE2 inverted microscope (Leica Microsystems) equipped for epifluorescence illumination and attached to a Retiga EXi CCD camera (QImaging). Openlab Image Analysis software (Version 3.5.1; Improvision, Inc.) was used for image capture and analysis.


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  		TABLE 1. Antisera utilized for immunofluorescence (IF) and immunoblot (IB) analysis.

Immunoblot Analysis

Immunoblot analysis was performed according to methods previously described in detail [37, 38]. Frozen tissue samples were pulverized under liquid nitrogen using a mortar and pestle and then homogenized in 500 µl of modified radioimmunoprecipitation assay (RIPA) lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, and 0.1% SDS) containing 100 µM sodium orthovanadate and complete Mini EDTA-free protease inhibitors (Roche Molecular Biochemicals).

HTR8-SVneo cells, initially seeded at 2 x 105 cells/well in 6-well plates, were transiently transfected and then grown for 48 h followed by the addition of 500 µl/well of modified RIPA lysis buffer containing inhibitors. Cells were harvested with a plastic cell scraper and homogenized; samples were cleared by centrifugation, and supernatants were retained for immunoblot analysis.

Sample protein concentrations were determined by the Bradford Assay [39] using Bio-Rad protein assay dye reagent (Bio-Rad Laboratories). Protein samples (40 µg/lane) were separated in 10% polyacrylamide gels under denaturing conditions and gels were electroblotted to Pierce 0.45 µm nitrocellulose membrane (MJS Biolynx, Inc.). Blots were rinsed in Tris-buffered saline-Tween-20 (TBST; 20 mM Tris base, 137 mM NaCl, and 0.1% Tween 20, pH 7.6) and blocked in 5% BSA/TBST for 30 min. Appropriate primary antisera (Table 1) were incubated with blots overnight at 4°C with constant agitation, and then blots were rinsed in TBST. Immunoblots were incubated in horseradish peroxidase (HRP)-conjugated secondary antisera (Table 1) for 1h at RT with constant agitation, then washed in TBST. Proteins were detected using the Pierce SuperSignal West Pico chemiluminescent substrate detection system (cat. no. 34080; MJS Biolynx, Inc.) and multiple exposures were generated to ensure the linearity of the film response.

Cell Migration Assay

HTR8-SVneo cells were initially seeded on 22 x 22 cm glass coverslips (1 x 105 cells/coverslip) seated in 35-mm tissue culture dishes and cultivated in RPMI 1640, as described above, with the omission of antibiotics during transfection. When cells reached approximately 85% confluence, they were transiently transfected with empty enhanced green fluorescent protein-C3 (pEGFP-C3) vector (BD Clontech, cat. no. 6082–1), pEGFP-C3 containing human wild-type (WT) ILK or pEGFP-C3 containing dominant-negative human ILK (E395K) constructs using Lipofectamine 2000 (cat. no. 11668–027; Invitrogen Ltd.) according to the manufacturer's detailed instructions. Twenty-four hours after transfection, a wound was created in the confluent cell monolayers with a sterile pipette tip and cells were cultivated under serum starving conditions to study migration. The wounds were photographed (migration time = 0 h) using a 10x objective under phase contrast and epifluorescence illumination with a DM-IRE2 inverted microscope (Leica Microsystems) equipped with a Retiga EXi CCD camera (QImaging). A second set of pictures was taken 24 h later (total of 48 h posttransfection) to assess cell migration into the wounds. The ratio of the number of transfected cells (i.e., EGFP-ILK fusion protein-containing cells) to the total number of cells in the wound after 24 h was subsequently determined from the captured images using Openlab Image Analysis software (Improvision). Migration assays were completed in triplicate and the experiment was repeated a total of four times.

MTT Assay

HTR8-SVneo cells were seeded (2 x 104 cells/well) in a 96-well plate in RPMI 1640 media under serum-starving conditions and the assay conducted following the manufacturer's detailed instructions (Cell Proliferation Kit I [MTT], cat. no. 1 465 007; Roche Diagnostics, Laval, Quebec, Canada). The absorbance measurements (550 nm) were completed with a POLARstar Optima Plate Reader (BMG Laboratories, Offenburg, Germany). Assays were done in triplicate and the experiments repeated twice.

Statistical Analysis

Statistical significance in cell migration and MTT assays was determined with a one-way ANOVA and a Tukey-Kramer multiple comparisons test. Values were considered significantly different if P < 0.05.

RESULTS

Immunoblot and Immunofluorescence Analysis of ILK Expression in Human Chorionic Villi

Immunoblot analysis was used to determine whether or not there were any changes in the temporal pattern of ILK expression in placental tissue during the first trimester, early second trimester, and at term. This analysis demonstrated that ILK was readily and comparably detected, at the expected ~55 kDa, at all gestational timepoints examined (Fig. 1). We also used immunofluorescence analysis to examine the temporal and spatial pattern of ILK localization in human chorionic villi during gestation. From 6 to 15 wk of human gestation, ILK was highly detectable in floating branches of chorionic villi. Specifically, ILK was localized to cells of the villous stroma and cytoplasm and plasma membranes of villous cytotrophoblast cells (Fig. 2A), where ILK expression colocalized with the cytotrophoblast marker cytokeratin-7 (data not shown). As a whole, ILK was barely detectable, compared to villous cytotrophoblast, in overlying syncytiotrophoblast. ILK was also inconsistently detected, at a low level, on the apical surface of overlying syncytiotrophoblast cells that also expressed the syncytiotrophoblast marker human placental lactogen (CSH1) (Fig. 2B). This result may be artifactual because a number of experiments using different antisera in our laboratory have also displayed this inconsistency. Because of the presence of microvilli in this layer, it is possible that some residual nonspecifically bound primary antisera remained trapped despite washes during the experiments, although we cannot definitively rule out the presence of ILK in this region. In anchoring branches of chorionic villi, ILK was primarily localized to the plasma membranes of extravillous trophoblast cell columns (Fig. 3A). Specifically, in proximal extravillous trophoblast cells of the villous tip, ILK expression colocalized with {alpha}v-integrin (ITGAV) expression (data not shown), whereas ILK colocalized with {alpha}5-integrin (ITGA5) in more distal regions of the cell columns (Fig. 3B).


Figure 1
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  		FIG. 1. Immunoblot analysis of ILK protein expression. A representative immunoblot demonstrating that ILK is detectable in human placental tissue lysates from the first and early second trimester and at term. The lower panel also illustrates the detection of ß-actin (ACTB) on the same representative immunoblot, which was used as a loading control.


Figure 2
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  		FIG. 2. Immunolocalization of ILK in floating branches of chorionic villi. A) Representative images of ILK expression in floating villi of human placenta from 6, 8, 10, 14, and 41 wk (term) of gestation. During the first and early second trimester, ILK was highly expressed in the villous cytotrophoblast layer but was also detectable in cells of the villous stroma and barely detectable in the syncytiotrophoblast layer. In term placenta, ILK was localized to endothelial cells of fetal blood vessels. B) ILK was also inconsistently detected, at a low level, on the apical surface of overlying syncytiotrophoblast cells that also expressed the syncytiotrophoblast marker CSH1. Control, representative nonspecific mouse IgG control—term (week 40) placenta; CT, villous cytotrophoblast layer; ST, syncytiotrophoblast layer; S, stroma. Bar = 50 µm.


Figure 3
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  		FIG. 3. Immunolocalization of ILK in anchoring branches of chorionic villi. A) Representative images of ILK expression in anchoring villi of human placenta from 6, 8, 11, 15, and 37 wk (term) of gestation. During the first and early second trimester, ILK was highly expressed in the villous cytotrophoblast and extravillous trophoblast but was also detectable in cells of the villous stroma and barely detectable in the syncytiotrophoblast layer. In the fetal side of the term placenta (basal plate), ILK was virtually undetectable. B) Representative images from double immunofluorescence experiments (e.g., 11 wk gestation) illustrating that ILK coimmunolocalized (yellow) with ITGA5 in the extravillous trophoblast cell column. Control, representative nonspecific mouse IgG control—first trimester (week 9) placenta; CT, villous cytotrophoblast layer; EVT, extravillous trophoblast; ST, syncytiotrophoblast layer; S, stroma. Bar = 50 µm.

From 6 to 15 wk of human gestation, ILK was also readily detectable in developing blood vessels in chorionic villi, specifically endothelial cells, as confirmed by the coimmunolocalization of ILK with von Willebrand factor (VWF) (Fig. 4). Similarly, in term placental chorionic villi, ILK immunolocalized to endothelial cells of blood vessels, but was markedly decreased in trophoblast subtypes (Figs. 2A, 3A, and 4).


Figure 4
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  		FIG. 4. Immunolocalization of ILK in blood vessels of chorionic villi. Representative images of chorionic villi from 14 and 40 wk gestation illustrating the coimmunolocalization (yellow and wedge) of ILK with VWF in blood vessel endothelial cells. BV, blood vessel; CT, villous cytotrophoblast layer; ST, syncytiotrophoblast layer. Bar = 50 µm.

Immunoblot and Immunofluorescence Analysis of the ILK Substrate AKT1

AKT1 is a substrate for ILK and is phosphorylated by the enzyme on Ser-473 [24]. Thus, we determined the temporal and spatial localization of Ser-473 phosphorylated AKT1 (pAKT1) in first and early second trimester human chorionic villi to serve as a gauge of the activation state of ILK in this tissue during these timepoints. Immunoblot analysis demonstrated that pAKT1 was detectable, at the expected ~60 kDa, in human chorionic villus tissue lysates at all gestational timepoints examined (Fig. 5). A lower molecular weight protein band of unknown identity was consistently detected in all placenta tissue lysates, but this may also potentially represent an AKT1 degradation product. Similarly to ILK immunolocalization, from 6 to 15 wk of human gestation pAKT1 was also detectable in the villous stromal cells, cytoplasm and plasma membranes of villous cytotrophoblast cells, and plasma membranes of extravillous trophoblast cells, and was virtually undetectable in the syncytiotrophoblast layer of chorionic villi (Fig. 6).


Figure 5
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  		FIG. 5. A representative immunoblot illustrating the detection of Ser-473 phosphorylated AKT1 (pAKT1) and total AKT in tissue lysates prepared from human placenta of 8, 9, 10, 12, 13, and 39–40 (term) wk gestation. pAKT+, Phosphorylated AKT1 control Jurkat cell extract; pAKT–, nonphosphorylated AKT1 control Jurkat cell extract (cat. no. 9273, Cell Signaling Technology).


Figure 6
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  		FIG. 6. Immunolocalization of Ser-473 phosphorylated AKT1 (pAKT1) in chorionic villi. Representative images of chorionic villi from the first and early second trimester of gestation (9 and 14 wk gestation). pAKT1 was highly detectable in villous cytotrophoblast, extravillous trophoblast, and cells of the villous stroma of chorionic villi, cell types that highly express ILK. CT, Villous cytotrophoblast layer; EVT, extravillous trophoblast; ST, syncytiotrophoblast layer; S, stroma. Bar = 50 µm.

Cell Migration Assays

Transient expression of pEGFP-WT-ILK or pEGFP-E359K-ILK for 48 h in HTR8-SVneo cells subsequently resulted in significant detection of these fusion proteins on immunoblots at the expected ~80 kDa, compared to endogenous levels of ILK, thus confirming the viability of the expression vectors (Fig. 7). Furthermore, the immunoblot analysis demonstrated that detection levels of pEGFP-WT-ILK and pEGFP-E359K-ILK fusion proteins in the respective cell lysates were similar.


Figure 7
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  		FIG. 7. Transient overexpression of wild-type (WT)- and dominant negative (E359K)-EGFP-ILK fusion proteins in HTR8-SVneo cells (HTR8). Representative immunoblots demonstrating that the two EGFP-ILK fusion proteins were readily detectable in HTR8-SVneo cells 48 h posttransfection and absent in the empty vector-transfected cells (HTR8-EGFP). Endogenous ILK (ILK) was detectable in all three sets of transfected cells. Immunoblot analysis of ß-actin (ACTB) was used as a loading control.

Over a 24-h period, transient expression of dominant negative pEGFP-E359K-ILK in HTR8-SVneo cells dramatically reduced the migration of these EGFP-labeled cells into wounds compared to cells expressing EGFP-WT-ILK or pEGFP vector itself (one-way ANOVA, P < 0.05; Figs. 8 and 9A). MTT viability assays demonstrated that the observed reduction in cell migration of HTR8-SVneo cells expressing pEGFP-E359K-ILK was attributable to the effects of dominant negative ILK expression, because these cells did not display any impaired viability as a result of E359K-ILK expression compared to pEGFP-WT-ILK- or pEGFP- expressing cells (Fig. 9B).


Figure 8
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  		FIG. 8. Transient overexpression of E359K-ILK in HTR8-SVneo cells dramatically inhibits cell migration. Phase contrast and corresponding immunofluorescence images of EGFP from wound assays with HTR8-SVneo cells transiently overexpressing the EGFP-ILK fusion proteins are shown. HTR8-SVneo cells were transiently transfected with either pEGFP (empty vector; EGFP) or pEGFP vector containing wild-type (WT) ILK or dominant negative (E359K) ILK. Following creation of a wound with a pipette tip, the wound area was photographed (0 hrs). Wounds were then photographed 24 hrs later (24 hrs) and the number of EGFP-positive cells in the wound area counted relative to total cells in the wound. Yellow bars indicate the position of the wound in the monolayer. Bar = 100µm.


Figure 9
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  		FIG. 9. A) Quantitative analysis of wound assays with HTR8-SVneo cells transiently overexpressing EGFP-ILK fusion proteins. The number of EGFP-expressing cells were counted in each photographed wound area and expressed relative to the total number of cells present in that wound area. Transient overexpression of E359K-ILK (E359K) dramatically inhibited cell migration into the wound (ANOVA, P < 0.05; Tukey-Kramer posttest) compared to cells overexpressing wild-type (WT)-ILK or empty vector (EGFP). B) MTT assays. Transient overexpression of E359K- or WT-ILK in HTR8-SVneo cells did not significantly decrease cell viability/proliferation relative to HTR8-SVneo cells overexpressing EGFP. Columns in A and B represent averages ± SEM.

DISCUSSION

Trophoblast differentiation during early human placental development involves the two fundamental pathways of fusion or invasion [4, 11–14]. The ubiquitous cytoplasmic serine/threonine kinase named ILK is known to be a regulator of cell proliferation, cell-cell adhesion, migration, and invasion [27]—processes that are highly pertinent to villous and extravillous trophoblast development via these pathways. Therefore, we hypothesized that ILK would be expressed in trophoblast subtypes in human chorionic villi during early placental development and that it would regulate trophoblast migration.

Implications of ILK Expression in Human Chorionic Villi

Cytotrophoblast proliferation. Our immunofluorescence results demonstrated that in floating villi, ILK protein was highly localized to villous cytotrophoblast cells and was barely detectable in the syncytiotrophoblast layer. In extravillous trophoblast cell columns of anchoring villi, ILK was also highly detectable. These subtypes of cytotrophoblast cells are known to be highly proliferative [12, 20, 40]. The overexpression of ILK is known to promote anchorage-independent cell cycle progression through upregulation of cyclin D1, and it also suppresses anoikis [41, 42]. AKT1 is a mediator of cell survival and is also a substrate for ILK, becoming phosphorylated on Ser-473, a critical residue for AKT1 activation [24]. Notably, pAKT1 was also readily detectable in placental tissue lysates in all gestational timepoints examined and was immunolocalized to both villous and extravillous trophoblast cells. In both villous cytotrophoblast and the proximal tips of extravillous trophoblast cell columns we also found that ILK protein colocalized with ITGAV (data not shown) and Cruet-Hennequart et al. [43] has recently described that ITGAV regulates proliferation of ovarian cancer cells through ILK.

Distal to the villous tip of extravillous trophoblast cell columns ILK colocalized with ITGA5. This region of extravillous trophoblast cell columns, marked by readily detectable ITGA5B1, is known to highly express focal adhesion kinase, paxillin, and matrix metalloproteinase 2, and to possess readily detectable fibronectin (FN1), a ligand for ITGA5B1 [15, 20]. As such, this region of the extravillous trophoblast cell column has been characterized as possessing an intermediate, proliferative, migratory phenotype [19, 20]. Integrins, such as ITGA5B1, and ILK are known mediators of FN1 matrix deposition and, as a result, promote cell proliferation [44, 45]. Thus, ILK, via several mechanisms, may promote cell cycle progression and trophoblast survival in villous and extravillous cytotrophoblast.

Cytotrophoblast differentiation. During differentiation of villous cytotrophoblast and fusion into a multinucleated syncytium (reviewed in [12]), expression of the cell-cell adhesion molecule E-cadherin (CDH1) and the associated protein ß-catenin (CTNNB1) decreases significantly [46–48]. Furthermore, in extravillous trophoblast cell columns, CDH1 is highly detectable, but it is significantly downregulated in extravillous trophoblast cells that have undergone an epithelial-mesenchymal transition (EMT) and are present in the decidua [17]. Hannigan et al. [23] have demonstrated that overexpression of ILK in epithelial cells results in disrupted cell-cell adhesion. ILK can also downregulate CDH1 expression through activation of the transcriptional repressor Snail, independently of CTNNB1/T cell factor (TCF7) regulation [49]. Furthermore, ILK can induce EMT in a variety of epithelial cell types (reviewed in [27]). EMT is a critical event in extravillous trophoblast differentiation because these trophoblast cells must undergo EMT in the cell column to facilitate their change in phenotype to less cohesive, single pleomorphic trophoblast cells that will migrate and invade the decidua and maternal spiral arteries. Thus, ILK may mediate trophoblast cell differentiation by initiating downregulation of CDH1 expression in villous and extravillous cytotrophoblast to facilitate the formation of syncytiotrophoblast and induction of EMT, respectively. We also cannot rule out the possibility that ILK expression in these two subtypes of cytotrophoblast may, additionally or alternatively, affect CTNNB1-TCF7-mediated gene expression in these cells. The viability of this pathway is controlled by GSK3B, which is inhibited, upon phosphorylation, by ILK, thus preventing the proteasomal degradation of CTNNB1 [49, 50].

To more functionally address the role of ILK in the differentiation of extravillous trophoblast exhibiting an intermediate, migratory phenotype, we conducted cell migration assays with HTR8-SVneo cells, an accepted cell line model of extravillous trophoblast [14], transiently expressing different ILK-EGFP fusion proteins. Our novel results clearly demonstrated that ILK can indeed mediate trophoblast cell motility. These results also add to the documented evidence of a role for ILK in cell migration [27].

ILK Expression in Blood Vessels of Chorionic Villi

During the first trimester of placental development, ILK protein was highly detectable in villous stromal cells and was also detectable in blood vessels of first trimester and term chorionic villi, where ILK colocalized with VWF. Vascular endothelial growth factor (VEGF) is highly expressed in villous cytotrophoblast during the first trimester, and its receptors fms-related tyrosine kinase 1 and kinase insert domain receptor are also highly detectable in vasculogenic and angiogenic precursor cells and detectable, albeit to a lesser extent, in villous stromal cells [51, 52]. The transcription factor hypoxia-inducible factor 1{alpha} (HIF1A) is also detectable at the mRNA and protein levels in villous mesenchyme and trophoblast layers during the early first trimester [53, 54], coincident with a low oxygen environment at this time, and is also reportedly detectable in second and third trimester placentas [54]. Expression of VEGF has recently been shown to be mediated by HIF1A independently of hypoxia, through a mechanism involving phosphatidylinositol 3-kinase, ILK, and AKT1 [55–57], and recent knockout of Hif1a, Epas1 or Arnt in mice resulted in defective placental vascularization in addition to aberrant trophoblast cell fates [58]. Interestingly, gene microarray analysis has shown that ILK expression can be highly upregulated by hypoxic conditions [59], adding another layer of complexity to regulation of ILK expression. Finally, endothelial cell-targeted knockout of ILK in mice results in embryonic lethality as a result of defective placental vascularization [60]. Therefore, these reports and our experimental data together strongly suggest that ILK could be involved in placental vascularization during early human gestation.

In summary, we are the first laboratory to establish that during early placental development, ILK protein is highly expressed in a trophoblast subtype (villous cytrotrophoblast) that will enter the fusion pathway and in a subtype that has entered the invasive pathway of trophoblast differentiation (extravillous trophoblast), and that ILK regulates trophoblast migration. These research findings now serve as a foundation for future research to elucidate the role of ILK in placental vasculogenesis, syncytiotrophoblast differentiation, and the differentiation of invasive extravillous trophoblast.

ACKNOWLEDGMENTS

We would like to acknowledge the invaluable assistance of Drs. A. Gill and F. Kum; Robin Hiscock, R.N.; and Maxine Kelly, R.N., for help with first trimester placental tissue acquisition. In addition, we would like to thank Chungyee Leung-Hagesteijn, Yuan Wu, Michael Organ, and Trina Kirby for their technical assistance in the laboratory, and Judy Foote and Art Taylor (Histology Unit, Faculty of Medicine, Memorial University of Newfoundland) for their assistance in tissue processing and paraffin sectioning of human placental tissue.

FOOTNOTES

1 Supported by Canadian Institutes of Health Research grant 64731, Industrial Research and Innovation Fund grant 0405-014 (Province of Newfoundland and Labrador), and Canada Foundation for Innovation New Opportunities Fund 74119. Back

2 Correspondence: Daniel MacPhee, Division of Basic Medical Sciences, Health Sciences Centre, Rm. 5340B, 300 Prince Philip Dr., St. John's, Newfoundland and Labrador A1B 3V6, Canada. FAX: 709 777 7010; dmacphee{at}mun.ca Back

Received: 21 December 2005.

First decision: 9 January 2006.

Accepted: 25 January 2006.

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