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Institute of Pathology (J.B., M.O., C.P., T.L., A.-M.B.), University Hospital, Hamburg-Eppendorf, 20246 Hamburg, Germany; Endokrinologikum (H.M.S.), 20246 Hamburg, Germany; and Department of Obstetrics and Gynecology (A.M.), University of Iraklion, 71202 Iraklion, Greece
Address all correspondence and requests for reprints to: Juliane Briese, Institute of Pathology, University Hospital Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany. E-mail: j.briese{at}uke.uni-hamburg.de.
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Abstract |
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Objective: The present study was designed to investigate the expression pattern of OPN in the human placental components and to correlate it with CEACAM1 expression and function in placental cell invasiveness.
Design: Immunohistochemistry with an OPN-specific antibody and immunofluorescence were performed on normal placental samples to investigate the expression pattern of OPN and CEACAM1 in the human placenta. Extravillous trophoblast (EVT) hybridoma cells transfected with CEACAM1 and stimulated with OPN were studied using the Matrigel invasion assay.
Results: All placentae presented very strong expression of OPN in the EVT at the invasion front, where it colocalized with CEACAM1. In addition, OPN was also present in the villous trophoblast, with strongest expression in the cytotrophoblast of the first trimester. Transfection with CEACAM1 followed by stimulation with OPN resulted in increased invasiveness of EVT hybridoma cells.
Conclusion: The present study shows the first systematic analysis of OPN expression pattern in the human placenta showing strong expression in the EVT at the invasion front. Colocalization of OPN with CEACAM1 in the EVT indicates that they might act together to regulate invasiveness at the maternal-fetal interface. Using an in vitro model, we also demonstrated increased cellular invasiveness after OPN treatment. We speculate that OPN and CEACAM1 may act as a functional complex involved in the regulation of placental invasiveness.
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Introduction |
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Cell adhesion molecules are important mediators of cellular contacts and cellular polarity that also modulate proliferation, differentiation, and invasion. Osteopontin (OPN) is a 70-kDa secreted extracellular matrix glycoprotein with an arginine-glycine aspartate-binding motif capable of interaction with 
v, ß1, ß3, and ß5 integrin subunits (5, 6, 7). OPN has been demonstrated to be expressed in a variety of human tissues, including the kidneys, thyroid, gastrointestinal tract, breast, and endometrium and has been implicated in mediation of cell-cell and cell-extracellular matrix communication that encompass both normal and tumorigenic developmental processes, cell adhesion, spreading, metastasis, and invasion (8, 9, 10). CEACAM1 is a member of the carcinoembryonic antigen and the immunoglobulin superfamily (11). Glycoproteins belonging to this family are expressed in epithelial tissues, such as colonic mucosa, as well as in cells of the myeloid lineage (11). CEACAM1 (CD66a, C-CAM, BGP) (12) is the human homolog of the adhesion molecule Cell-CAM (C-CAM) of the rat (13, 14), and it has been suggested to function as a ligand for E-selectin (15). In contrast to most of the genes of the carcinoembryonic antigen family, the CEACAM1 gene predicts a cytoplasmic domain containing sequence motifs involved in signal transduction (16) that has been shown to associate with pp60 c-src (17). Both OPN and CEACAM1 have been shown to interact with integrin ß3 (18, 19). Integrin studies have suggested that integrin ß3 could play an important role in the implantation and invasion process (18, 19). The present study was designed to investigate the expression pattern of OPN in the human placental components and, thus, its potential implication in placentation. To investigate the expression of OPN and to correlate it with the expression of CEACAM1, immunohistochemistry and immunofluorescence on paraffin-embedded specimens and Western blot on isolated trophoblast populations were performed using monoclonal antibodies against OPN (Akm2A1) and CEACAM1 (4D1/C2). Particular attention was given to the complex cellular structures of the placenta, and expression was assessed in the villous CT and ST and in EVT, i.e. the part that actually invades the maternal tissue. We have recently shown that CEACAM1 can act to enhance invasiveness in both melanoma (20) and trophoblast cells (21). To investigate a potential functional link between OPN and CEACAM1 in regulating trophoblast invasion, EVT hybridoma cells (22, 23) transfected with CEACAM1 have been treated with OPN and shown increased invasiveness, indicating that CEACAM1 and OPN potentially act together to enhance invasiveness of trophoblast cells.
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Materials and Methods |
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The tissue material was selected after histological review from the files of the Department of Gynecopathology (University Hospital Eppendorf, Hamburg, Germany). Only normal placentas were included. For immunohistochemistry, the specimens that had been fixed in 4% buffered formalin and embedded in paraffin were used. A total of 50 samples were analyzed, including 30 first trimester, 10 second trimester, and 10 third trimester placentae.
Immunohistochemistry
Serial sections (4–6 µm) of the 50 placentae were cut from the paraffin blocks and mounted on 3-aminopropyl-triethoxysilan-coated slides, deparaffinized in xylene, and rehydrated in graded alcohol to Tris-buffered saline [50 mmol/liter Tris, 150 mmol/liter NaCl (pH 7.4)]. For the detection of CEACAM1, all slides were microwaved for 5 x 2 min in 10 mmol/liter citrate (pH 6.0). After cooling down for 20 min, the slides were performed in the same way as the slides for OPN staining. After washing with PBS, slides were blocked for 10 min at room temperature with avidin-biotin blocking system (Dako, Glostrup, Denmark) and incubated with anti-OPN mouse monoclonal antibody (Akm2A1) (Santa Cruz Biotechnology, Santa Cruz, CA) at a dilution of 1:200 or anti-CD66a monoclonal antibody (4D1/C2) at a dilution of 1:100 for the detection of CEACAM1 for 30 min. Slides were then reacted with biotinylated secondary antibody (Dako) for 10 min. Staining was performed using peroxidase-labeled streptavidin (Dako) and 3'3''diaminobenzidine containing 0.03% H2O2. Sections were counterstained with hematoxylin (Hemalaun Meyer, Merck, Darmstadt, Germany), dehydrated, and mounted (Eukitt; Labo-Med, Leipzig, Germany). For immunohistochemical characterization of intermediate trophoblast cells, slides were additionally incubated with anticytokeratin mAb (CK7) (Dako). The staining intensity was then evaluated independently by two observers with a score given as follows: –, negative; +, positive; 1+, weak staining; 2+, moderate staining; and 3+, strong staining.
Immunofluorescence
For double staining, serial sections of 15 placentae (including five of each trimester) were pretreated as described above. Then, tissue sections were incubated with the primary antibody OPN (Akm2A1) (Santa Cruz Biotechnology) at a dilution of 1:200 for 30 min followed by secondary tetramethylrhodamine B isothiocyanate (TRITC)-conjugated rabbit antimouse IgG Ab (Dako) at a dilution of 1:1000. 4D1/C2 (CEACAM1) was incubated at a dilution of 1:100 for the detection of CEACAM1 followed by fluorescein isothiocyanate (FITC)-conjugated rabbit antimouse IgG (Dako) at a dilution of 1:1000 for 30 min. Slides were then incubated with antimouse IgG F(ab')2 [goat] (Rockland, Gilbertsville, PA) at a dilution of 1:10 for 30 min to block nonspecific binding. For nuclear staining, we used To-PRO-3 iodide (Dako) at a dilution of 1:1000 for 5 min. Staining was done by a Dako Autostainer (Serial no. 3400-9010-03). Slides were overlaid with a cover glass and a Prolong antifade mounting reagent (Leiden). Photomicrographs of representative fields of immunofluorescence staining were evaluated using a Zeiss Axioplan2 microscope (Zeiss, Jena, Germany).
Isolation of trophoblast cells
Cultures of first trimester trophoblast populations were established and characterized as previously reported (24). Briefly, eight to 10 placentas (5–12 weeks) obtained after legal termination of pregnancy were washed in sterile PBS (s-PBS), and areas rich in chorionic villi were selected, minced between scalpel blades, and subjected to three sequential 10-min treatments with 0.125% trypsin and 0.2 mg/ml DNAse I (Roche Diagnostics, Mannheim, Germany) in s-PBS containing 5 mmol/liter MgCl2. Cells released from each 10-min step were pooled and filtered through two layers of muslin, resuspended in 70% Percoll (Pfizer, Inc., Täby, Sweden) at a density of 2 x 105 cells/ml, and put under 20 ml 25% Percoll. s-PBS (10 ml) was put on top of the 25% Percoll, and a gradient was established by centrifuging for 20 min at 800 x g. Cells from the middle band (density, 1.048–1.062 g/ml) of the gradient were pooled, washed in s-PBS, and seeded at a density of 1 x 106 cells/ml keratinocyte growth medium (KGM) supplemented with 10% fetal calf serum (FCS). As previously described (24), cells were identified as being trophoblast by immunocytochemical staining using monoclonal antibodies to cytokeratin (Dako-cytokeratin, MNF 116, and 35 BH11, 1:100; Dako) as well as E-cadherin (Santa Cruz Biotechnology, Santa Cruz, CA).
In vitro invasion assay of isolated trophoblast cells
The invasive characteristics of the EVT cells were determined using an in vitro Matrigel invasion assay as already described (25). We used transwells with a polycarbonate filter of 2.5-cm diameter and 8-µm pore size. The upper surface of the filter was coated with Matrigel (Collaborative Research, Bedford, MA; diluted 1:20 with KGM). The bottom chamber was filled with 3 ml KGM containing 10% fetal calf serum. Trophoblast cells were trypsinized, washed, and resuspended at a density of 1.0 x 105 cells/ml KGM containing 10% fetal calf serum, and 2 ml of the labeled cell suspension was added to the upper well of the Transwell chamber. After 72 h of incubation, the invasive index was calculated as the amount of radioactivity (disintegrations/minute) from the lower wells divided by the sum of the total radioactivity from both the upper and lower wells plus the filter.
Western-blot analysis of isolated trophoblast cells and transfected hybridoma cells
Trophoblast cells were lysed in ice-cold sample buffer b1 [50 mmol/liter Tris (pH 6.8), 1% sodium dodecyl sulfate (SDS), and 10% sucrose] containing 20 µl protease inhibitor cocktail (Sigma Chemie, Deisenhofen, Germany). Protein concentration was determined following standard protocols and using bovine serum albumin standards. Extraction of proteins for Western-blot analysis was carried out in PBS in the presence of 1% NP40 and protease inhibitors as previously described (26). SDS-PAGE was performed in a 7.5% polyacrylamide gel under reducing conditions, applying 50 µg of each sample of the concentrated protein extract. For electrophoresis, samples of trophoblast and hybridoma cells were diluted with a 1:1 mixture of sample buffers b1 and b2 [containing 50 mmol/liter Tris (pH 6.8), 3% SDS, 10% sucrose, 10% ß-mercaptoethanol, and 0.01% bromophenol blue] to a final volume of 100 µl and a final protein concentration of 400 µg/ml. Equal amounts of protein (40 µg) of each sample were loaded per well, and equal loading was verified by immunoblotting with actin antibodies (not shown). After electrophoretic transfer to nitrocellulose and blocking in Tris-buffered saline containing 10% FCS for 2 h, antirabbit-specific OPN monoclonal antibody (Akm2A1) (Lab Vision, Fremont, CA) was added at a dilution of 1:1000 and incubated overnight at 4 C. Detection was carried out with goat-antimouse alkaline phosphatase coupled antibody applying the chemiluminescent substrate and enhancer kit (Pierce, Rockford, IL). Films (Hyperfilm, Amersham, Braunschweig, Germany) were exposed for 15 min. Extract of osteosarcoma cells (ATCC, Braunschweig, Germany) were used as a positive control and rabbit serum as negative control. To demonstrate that the transfections of EVT-based hybridoma cells were successful, a total cell lysate of 50 µg protein was used for Western-blot analysis.
Cell culture and transient transfections
The cells used in this study were EVT-based hybridoma cells that we have already described elsewhere (22, 23). The cells (clone ACI.88) were kept in culture and split twice a week. Transfections of placental hybridoma cells were performed with LipofectAMINE PLUS reagent (Life Technologies, Karlsruhe, Germany). On the day before transfection, cells were plated in six-well culture plates at a density of 500.000 cells/well. After 24 h, the medium was replaced by 0.8 ml FCS-free medium per well, and the cells were transfected with 1 µg of the plasmid CEACAM1 with PLUS reagent and lipofectamine, as suggested by the manufacturer. After 3-h incubation with this mixture, aliquots of 1 ml medium containing 20% FCS were added per well. The cells were harvested after 24 h.
The construction of the expression vectors containing the full coding region of the CEACAM1 gene in the plasmid pcDNA3.1(+) was recently described (18). As negative control for transfection experiments, the mock expression vector pcDNA3.1(+) (Invitrogen, Karlsruhe, Germany) was used. To demonstrate that the transfections were successful, cells were analyzed by Western blot.
In vitro invasion assay of transfected cells
The invasiveness of cells was assayed in the membrane invasion culture system using polyethylene terephthalate membrane (8-µm pore size) in the upper compartment of a transwell coated with Matrigel (BD BioCoat Matrigel Invasion Chamber, BD Biosciences, Heidelberg, Germany). Cells were harvested by trypsin-EDTA buffer and washed with RPMI 1640. The cells were then seeded at 2.5 x 104 cells per 500 µl RPMI 1640 on the Matrigel-coated PET membrane in the upper compartment. Human OPN (2 µg) was given directly into the medium. The lower compartment was filled with RPMI 1640 medium, and the plates were incubated at 37 C for 48 h. At the end of the incubation time, the upper surface of the membrane was wiped with a cotton-tip applicator to remove nonmigratory cells. Cells that migrated to bottom side of membrane were fixed and stained by Diff-Quick (Dade Behring AG, Düdingen, Switzerland). The cells were quantified by counting seven high-powered fields (460 µm x 700 µm) in the center of each well using a Zeiss microscope. Each measurement was performed in triplicate, and experiments were repeated five times.
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Results |
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Immunohistochemistry was performed on paraffin-embedded samples with the monoclonal antibody OPN (Akm2A1), which specifically recognizes only OPN, and the monoclonal antibody 4D1/C2, which recognizes only CEACAM1 (24). The staining results for OPN are summarized in Table 1
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and Fig. 1, A and B). Note strong OPN expression in an EVT island with strongest expression in the second trimester (Fig. 1C). Strong OPN expression could be found in the cellular subpopulations of EVT (proximal trophoblast, showed 60% positive cells; interstitial trophoblast, showed 80% positive cells; endovascular trophoblast, showed 30% positive cells) (Table 1 and Fig. 1, B, D, and E). In addition, OPN was present in the villous trophoblast, with strongest expression in the CT (80% positive cells, 3+ staining intensity), where it also was strongest in immunofluorescence analysis with localization especially at the interface between CT and ST (Fig. 2D).
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The expression pattern of CEACAM1 in the human placenta was recently described by our group (24) showing that CEACAM1 is specifically expressed in the cellular populations of the EVT, whereas it is absent from villous trophoblast as we now confirmed (Fig. 2, E and F). By using immunofluorescence, we could demonstrate that in EVT at the maternal-fetal interface, CEACAM1 and OPN are being colocalized (see Fig. 2A, showing expression of OPN; Fig. 2B, showing expression of CEACAM1; and Fig. 2C, showing colocalization of OPN and CEACAM1).
Western-blot analysis of isolated trophoblast cells
Western-blot analysis was performed on cultivated villous trophoblasts and on EVT cells, which had been characterized with an invasive phenotype, using the monoclonal antibody (Akm2A1). Extract of osteosarcoma cells was used as positive control. A representative Western blot is shown in Fig. 3. Western-blot analysis confirms strong expression of OPN in EVT cells, as had been previously shown for CEACAM1(24). In addition, expression of OPN was seen in CT and a very weak expression in ST cells (Fig. 3). The molecular weight of the protein OPN was 60 kDa.
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We used the established cell line ACI.88 of extravillous hybridoma cells that displayed an expression of OPN confirmed by Western-blot analysis (Fig. 4A) but was completely negative for CEACAM1 (Fig. 4B) and was of low invasive potential. Thus, these cells represent a good model for testing the effects of OPN and CEACAM1 upon cellular invasiveness and to investigate the connection between OPN and CEACAM1. In the present study, a potential functional link between OPN and CEACAM1 in regulating trophoblast invasion was investigated using EVT hybridoma cells (22, 23) transfected with CEACAM1. In these cells, we have recently shown that transfection of a CEACAM1 expression vector results in enhanced invasiveness on a Matrigel invasion assay (21). Transfection efficiency of hybridoma cells was confirmed by Western blot (Fig. 4, A and B). Invasion was studied using the Matrigel in vitro invasion assay. Figure 5A demonstrates the results of testing different OPN concentrations and different stimulation times. The optimal concentration was 2 µg OPN for 48 h. Figure 5B shows the results of a representative in vitro invasion assay experiment. Transfection with CEACAM1 followed by stimulation with OPN resulted in higher invasion level of the hybridoma cells compared with cells that were only transfected with CEACAM1.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, we investigated for the very first time the systematic expression pattern of the glycoprotein OPN in the human placenta and its correlation with the expression of CEACAM1. As shown by immunohistochemistry, all placentas presented a strong expression of OPN in the EVT cells correlating with the expression of CEACAM1 (24). Strong expression was observed in interstitial and proximal trophoblast and in EVT islands (Fig. 1, A–C). The villous trophoblast showed strongest expression of OPN in the CT cells (Fig. 1C), which was confirmed by immunofluorescence analysis with strongest localization at the interface between CT and ST (Fig. 2, D and F). CEACAM1 was absent from CT and ST in all placentas as we have recently found (24). We demonstrated colocalization of OPN and CEACAM1 in the EVT cells using immunofluorescence (see Fig. 2, A and B, showing OPN and CEACAM1 as single staining; and Fig. 2C, presenting an immunofluorescence double staining of both antibodies). OPN expression in EVT and villous CT cells was confirmed by Western-blot analysis on isolated trophoblast populations (Fig. 3). To investigate a potential functional link between OPN and CEACAM1 in regulating trophoblast invasion, EVT-based hybridoma cells (22, 23) transfected with CEACAM1 have been treated with OPN and shown increased invasiveness (Fig. 5B), indicating that CEACAM1 and OPN potentially act together to enhance invasiveness of trophoblast cells.
Very recently, we have also investigated the expression pattern of OPN in correlation with the adhesion molecule CEACAM1 in gestational trophoblastic diseases (GTDs) (27). GTDs (including hydatidiform moles, choriocarcinomas, placental site trophoblastic tumor, and placental site nodule) are the result of pathological placental development and are associated with abnormal proliferation and/or invasion of trophoblast (5, 28, 29, 30, 31). We have found that in hydatidiform moles, OPN is highly expressed in the villous CT and in the trophoblast proliferations on the villous surface that is considered to be a mixture of CT, ST, and intermediate trophoblast (27). Also the implantation site showed a positive nuclear staining as did the invasion site in three cases of an invasive mole. CEACAM1 was absent from villous trophoblast, and only a few cells of trophoblast proliferations were positive, as we have recently shown (32). In addition, OPN expression has also been found in the choriocarcinomas with an inhomogeneous expression pattern correlating with the expression of CEACAM1 (27).
OPN and CEACAM1 were expressed in both normal and pathological interstitial EVT, and their expression pattern can be potentially useful as an additional diagnostic marker in GTD. A clinical investigation could demonstrate that the OPN expression pattern of trophoblast cells is disrupted in pregnancies complicated by preeclampsia and preeclampsia in conjunction with fetal growth retardation (33).
Several studies have defined OPN as an important glycoprotein with multiple functions and to play a role in basic cellular processes, such as neovascularization and tissue remodeling, which are essential to placental morphogenesis and embryo implantation (34, 35). From our results, implications for the functional role of OPN at the maternal-fetal interface can be discussed. OPN could play a role in mediating cell adhesion at the maternal-fetal interface. Furthermore, several lines of evidence have implicated OPN in angiogenesis, and vascular endothelial growth factor may induce expression of OPN as well as vß3 integrin in endothelial cells (36, 37, 38). Apparao et al. (39) have demonstrated the coexpression of OPN and vß3 integrin in the human endometrium during menstrual cycle. Using the Ishikawa cell line, they demonstrated a different regulation for both. OPN was regulated through progesterone, whereas vß3 integrin was modulated through growth factor epidermal growth factor. Omigbodun et al. (40) have found that the secretion of progesterone by ST regulates the expression of OPN by CT.
There are several groups who have found that OPN could be a diagnostic marker for malignant tumors like ovarian cancer or breast cancer and have recently demonstrated the production of OPN mRNA in ovarian cancer cells (38, 41, 42). Batorfi et al. (43) presented a down-regulation of OPN in trophoblast proliferation of hydatidiform mole.
CEACAM1 is an adhesion molecule belonging to the immunoglobulin gene family that is specifically expressed in epithelial tissues such as colonic mucosa (17). Specific expression of CEACAM1 in EVT and endometrial and endothelial cells implicates a possible role in regulating the normal processes taking place at the maternal-fetal interface during implantation and placentation (24). We have recently demonstrated an increased invasiveness of CEACAM1-transfected melanoma and corticotropin-releasing hormone-stimulated trophoblast cells (20, 21). The present study shows the first systematic analysis of OPN expression pattern in the human placenta with its localization in cellular populations showing strong expression in the EVT and CT. Colocalization of OPN and CEACAM1 in the EVT indicates that they might act together to regulate invasiveness at the maternal-fetal interface. With the very recent demonstration of the correlated expression pattern of OPN and CEACAM1 in trophoblastic lesions, we have shown that OPN and CEACAM1 may act as a functional complex in these lesions (23). Using an in vitro model with CEACAM1-expressing EVT-based hybridoma cells, we have now demonstrated increased cellular invasiveness after OPN treatment, indicating a probable functional link between OPN and CEACAM1 in regulating trophoblast invasion.
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Acknowledgments |
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Footnotes |
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First Published Online June 14, 2005
Abbreviations: CT, Cytotrophoblast; EVT, extravillous trophoblast; FCS, fetal calf serum; FITC, fluorescein isothiocyanate; GTD, gestational trophoblastic disease; KGM, keratinocyte growth medium; OPN, osteopontin; SDS, sodium dodecyl sulfate; s-PBS, sterile PBS; ST, syncytiotrophoblast; TRITC, tetramethylrhodamine B isothiocyanate.
Received December 14, 2004.
Accepted June 3, 2005.
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v ß 3 in smooth muscle cell migration to osteopontin in vitro. J Clin Invest 95:713–724
vß3 integrin are coexpressed in the human endometrium during the menstrual cycle but regulated differentially. J Clin Endocrin Metabol 86:4991–5000This article has been cited by other articles:
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