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Department of Gynecology and Obstetrics (Y.S., H.F., T.H., S.Y., K.T., S.F.), Faculty of Medicine, and Institute for Frontier Medical Science (M.M.), Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan
Address all correspondence and requests for reprints to: Hiroshi Fujiwara, M.D., Department of Gynecology and Obstetrics, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan. E-mail: . fuji{at}kuhp.kyoto-u.ac.jp
Abstract
Previously, we reported that dipeptidyl peptidase IV (DPPIV), a membrane-bound peptidase, was expressed on human placental cytotrophoblasts. In the present study, we focused on DPPIV expression on extravillous trophoblasts (EVTs). In the first trimester, DPPIV was expressed in the proximal part of the cell column and some EVTs located in the deep portion of the decidua and myometrium. EVTs migrating in the decidua from the cell column were negative for DPPIV. In the second and third trimesters, almost all EVTs were positive for DPPIV. Because negative DPPIV expression was associated with migration or the invasive phenotype of EVTs, using JEG-3 cells (choriocarcinoma cell line) that endogenously produce DPPIV, the influence of DPPIV on the invasive activity was examined. When a competitive inhibitor of DPPIV, diprotin A, was added in Matrigel invasion assay system, JEG-3 cells exhibited a significant enhancement of invasion. Because hypoxia is reported to reduce trophoblastic invasion, the effect of hypoxia was examined on JEG-3 cells. JEG-3 cells became less invasive with increased expression of DPPIV when cultured under hypoxic conditions (1% O2). These results suggest that DPPIV is important for the noninvasive EVT phenotype and the down-regulation of this enzyme was strongly associated with migration or invasive EVT phenotype.
IN HUMAN PLACENTA, cytotrophoblasts show two distinct patterns of differentiation. Cytotrophoblasts differentiate into syncytiotrophoblast and form the syncytial layer in floating villi in which exchange of nutrient, waste, and gas takes place. The syncytiotrophoblast is also an important source of various kinds of hormones such as human chorionic gonadotropin and steroid hormones.
At villus-anchoring sites, cytotrophoblasts form the stratified structure that is called the cell column and differentiate into extravillous trophoblasts (EVTs). EVTs are reported to acquire invasive phenotype in the distal end of the cell column in the first trimester of pregnancy (1) and begin to invade the decidual tissue and maternal arteries. EVTs are reported to replace the endothelial and muscular linings of the spiral arteries. This replacement contributes to the enlargement of the vessel diameter to increase the placental blood flow (2). EVTs located in the decidual tissue are called interstitial trophoblasts, and those in maternal arteries are called endovascular trophoblasts. Interstitial trophoblasts have been reported to lose their invasive activity in the deep portion of the decidua or myometrium (3). In the second trimester, most EVTs begin to reside in the placental bed and show noninvasive phenotype (4); however, the endovascular trophoblasts still exhibit invasive phenotype at this stage (5).
Therefore, EVTs can be divided into two subgroups, i.e. invasive and noninvasive phenotype. Invasive EVTs include interstitial trophoblasts invading the decidua in the first trimester of pregnancy and endovascular trophoblasts in the first and second trimesters. Noninvasive EVTs include those in the proximal part of the cell column and those in the deep portion of the decidua and the myometrium in the first trimester of pregnancy. EVTs residing in the placental bed in the second and third trimesters also belong to the noninvasive phenotype. In the fetal membrane, a distinctive cell layer called the chorion laeve is present between the amnion and decidual tissue. The trophoblasts in the chorion laeve have been reported to contain noninvasive EVTs (4).
To establish a pregnancy, the development of both villous trophoblasts and EVTs is essential. The spatiotemporal development and differentiation of EVTs, particularly from noninvasive to invasive phenotype or vice versa, are important for a successful pregnancy.
For the invasion of trophoblast, cell-cell and/or cell-extracellular matrix interactions mediated by adhesion molecules, such as cadherins and/or integrins, has been considered to be important (2). For example, when trophoblasts acquire the invasive phenotype in the cell column, down-regulation of integrin 
6ß4 and up-regulation of integrin 5ß1 and integrin 1ß1 occur (1). In addition, these integrins have been reported to modulate trophoblast motility (6). However, these molecules cannot fully explain the spatiotemporal development and differentiation of EVTs, particularly from noninvasive to invasive phenotype or vice versa. For example, integrin 5 is equally expressed on interstitial trophoblasts regardless of their depth of invasion or the stage of pregnancy.
Dipeptidyl peptidase IV (DPPIV, EC.3.4.14.5), which is known as T-cell activation antigen CD26 (7), is one of the membrane-bound aminopeptidases. DPPIV removes an Xaa (one unspecified amino acid)-Pro or Xaa-Ala dipeptide from the N termini of polypeptides or proteins (8). The reported physiological substrates for DPPIV are substance P, ß-casomorphin, endomorphin, NPY, peptide YY, glucagonlike peptide, gastric inhibitory peptide, GH releasing factor, vasostatin, fibrin -chain, and some chemokines (9). DPPIV can metabolize these substrates at extracellular sites and regulate their local concentration before they reach their specific receptors on the cell surface (10, 11).
We previously reported that DPPIV is expressed in the villous cytotrophoblasts as well as the chorion laeve (12). Because the trophoblasts in chorion laeve contain EVTs of noninvasive phenotype, we examined the expression of DPPIV in EVTs during various gestations to know whether DPPIV is associated with the spatiotemporal development and differentiation of EVTs. In addition, we recently found that among the cell lines of choriocarcinoma, DPPIV is expressed in JEG-3 cells but not in BeWo cells. Therefore, the effect of DPPIV on these cell lines was investigated using the Matrigel invasion assay using a competitive inhibitor of DPPIV, diprotin A. In addition, because hypoxia is reported to act negatively for trophoblastic invasion (13, 14, 15), the effect of hypoxia on the invasive activity and DPPIV expression of JEG-3 cells was examined. The effect of hypoxia was also examined on EVT-like cells that had grown out from chorionic villous explants in culture. Through these experiments, the importance of DPPIV for invasive or noninvasive EVT phenotype will be examined in this article.
Materials and Methods
Tissue samples
Placental tissues were obtained from six therapeutic hysterectomies for cervical neoplasia during normal pregnancies (9 wk gestation, n = 3; 12 wk gestation, n = 2; 16 wk gestation, n = 1), from six legal abortions (6–8 wk gestation, n = 3; 20 wk gestation, n = 3), and from four normal deliveries (37–40 wk gestation). Ectopic pregnancy samples were obtained from three unruptured tubal pregnancies (6–8 wk gestation) and one ruptured rudimentary horn pregnancy (14 wk gestation). In these ectopic pregnancies, fetal heart movements were detected by ultrasonography at the time of operation. The gestational age was calculated from the date of the last menstrual period and, if necessary, was adjusted according to ultrasonic measurements of the gestational sac and the fetal crown-rump length. Informed consent for the use of the tissue was obtained from all donors. The use of the materials was also approved by the Ethics Committee of Kyoto University Hospital.
Reagents and antibodies
Diprotin A (Ile-Pro-Ile), a competitive inhibitor of DPPIV enzymatic activity, was purchased from Peptide Institute, Inc. (Osaka, Japan) (16). Two mouse antihuman DPPIV monoclonal antibodies (mAbs), Ta1 and MA261 (both IgG1 class) were obtained from Coulter Immunology (Hialeah, FL) and Medsystems Diagnostics (Vienna, Austria), respectively (17, 18). Mouse antihuman cytokeratin 18 mAb (Ks18.04, IgG1), which reacts with all populations of trophoblasts as well as glandular epithelial cells, was purchased from Progen Biotechnik (Heidelberg, Germany) (19). In immunochemistry and flow cytometry, an anti-trinitrophenyl (TNP) mouse mAb (unrelated mAb, IgG1) (20) was used as a negative control. Fluorescein isothiocyanate (FITC)-conjugated rabbit antimouse immunoglobulin polyclonal antibody (pAb) (DAKO Corp. A/S, Glostrup, Denmark) was used as the secondary antibody.
Immunohistochemistry
Cryosections of the tissues were prepared as previously described (21). Briefly, fresh tissues were embedded in OCT compound (Tissue-Tek, Miles Inc., Elkhart, IN), snap frozen in liquid, stored at -80 C. The frozen tissues were cut into 7-µm-thick sections using a cryostat microtome (Histostat, Reichert-Jung, Heidelberg, Germany). The sections were immediately and thoroughly air dried on Neoprene (Nisshin EM Co. Ltd., Tokyo, Japan)-coated glass slides and then fixed with acetone at -20 C. The serial cryosections were incubated with the anti-DPPIV mAb (Ta1, 5 µg/ml, or MA261, 5 µg/ml), anti-cytokeratin 18 mAb (5 µg/ml), or anti-TNP mAb (5 µg/ml) as a negative control. The antibodies were diluted in RPMI (Life Technologies, Inc., Grand Island, NY) containing 10% fetal calf serum (FCS) (Dainippon Pharmaceutical Co. Ltd., Osaka, Japan) and 0.1% NaN3. After a 1-h incubation, the slides were washed with PBS three times. The sections were then incubated with FITC-conjugated rabbit antimouse immunoglobulin pAb (diluted 1:40) for 30 min at room temperature. The slides were washed extensively with PBS, mounted with an antifade agent (Perma Fluor mounting medium, Immunon, Pittsburgh, PA), and examined under a fluorescence microscope. Some serial sections were also stained with hematoxylin and eosin or used for histochemical staining.
Histochemical staining
Samples for histochemical staining were prepared in the same way as for immunohistochemistry. DPPIV enzymatic activity in the tissues was examined as described previously (22). Briefly, 4 mg glycyl-prolyl-4-methoxy-ß-naphthylamide (Sigma, St. Louis, MO) was dissolved in 500 µl N, N-dimethylformamide (Wako Pure Chemical Industries Ltd., Osaka, Japan). Fast Blue B Salt (10 mg, Sigma) was dissolved in 9.5 ml 0.1 M phosphate buffer (pH 7.2). The two solutions were mixed and filtered through a 0.22-µm membrane filter. After the sections were incubated in this mixture for 10 min at room temperature, the reaction was stopped by extensive washing with PBS. For the negative control, this mixture was used without substrate. The slides were mounted and observed under a light microscope.
Human chorionic villous explant culture
Villous explant cultures were established using placental tissues obtained from legal abortions (6–8 wk gestation, n = 3) by the method of Aplin et al. (23) with some modifications. Briefly, the placental tissues were placed in ice-cold RPMI and processed within 2 h of collection. The tissues were washed with sterile RPMI and aseptically dissected to remove decidual tissue. Small fragments of placental villi were teased apart and soaked in culture medium (RPMI supplemented with 10% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin), and 10 pieces of villous fragments were placed in each of four 35-mm collagen type I-coated dishes (Iwaki, Chiba, Japan). After overnight incubation in normoxic (37 C, 20% O2/5% CO2/75% N2) conditions to allow the explants to adhere to the dishes, 2 ml of culture medium was added. These culture dishes were then placed in either normoxic or hypoxic (37 C, 1% O2/5% CO2/94% N2) conditions. After 48 h of incubation, the cells in the culture dishes were gently washed with PBS. The cells were then immunostained with anti-DPPIV (Ta1) or anti-TNP mAb as described above and examined under a confocal laser scanning microscope (Carl Zeiss, Jena, Germany) without mounting.
Cell lines and culture conditions
BeWo and JEG-3, continuous cell lines established from human choriocarcinomas (24, 25), were obtained from the Japanese Cancer Research Resources Bank (Tokyo, Japan) and the American Type Culture Collection (Manassas, VA), respectively. The cells were maintained as monolayers in 10-cm cell culture dishes (Corning, Inc., Corning, NY) under normoxic conditions. For passage, the cells were dispersed with 0.05% trypsin (Difco Laboratories, Detroit, MI) and 0.05% EDTA solution, and replated in culture dishes.
To examine the effect of hypoxia, 1 x 106 JEG-3 cells were suspended in 10 ml culture medium and plated in 10-cm dishes. The dish was placed in normoxic or hypoxic conditions. After 48 h of incubation, the cells were trypsinized and collected for flow cytometry, Northern blot analysis, and Matrigel invasion assay.
Immunocytochemistry
BeWo or JEG-3 cells were grown under normoxic conditions to semiconfluence in eight-chamber slides (Lab Tek chamber slide; Nalge Nunc International, Naperville, IL). The cultured slides were gently washed with PBS, thoroughly dried, and fixed with acetone at -20 C. The specimens were then immunostained with the anti-DPPIV (Ta1) or anti-TNP mAb as described above, mounted, and examined under a fluorescence microscope.
Flow cytometry
BeWo or JEG-3 cells cultured under normoxic conditions were trypsinized and then washed in Hanks’ balanced salt solution (HBSS) with 0.1% BSA and 0.1% NaN3. The precipitated cells (1 x 105 cells/tube) were incubated with anti-DPPIV (Ta1; 100 µg/ml, 5 µl) or anti-TNP (100 µg/ml, 5 µl) mAb for 30 min at 4 C. After twice washing with HBSS, the cells were incubated with FITC-conjugated rabbit antimouse immunoglobulin pAb (diluted to 1:40) at 4 C for 30 min in the dark. The cells were then washed twice and resuspended in 500 µl HBSS. Cell surface labeling was analyzed by FITC fluorescence detection using a FACScan (Becton Dickinson and Co., Mountain View, CA). Flow cytometric data were obtained from the analysis of 2 x 104 cells per sample.
Next, levels of DPPIV expression on JEG-3 cells that had been cultured under normoxic or hypoxic conditions for 48 h were compared by flow cytometry using anti-DPPIV (Ta1; 100 µg/ml, 5 µl) or anti-TNP mAb (100 µg/ml, 5 µl) as primary antibodies.
These experiments were repeated three times.
RNA isolation and RT-PCR
Total RNA from JEG-3 cells was isolated using a commercial kit (TRIzol; Life Technologies, Inc.). Five micrograms of total RNA were reverse transcribed with random primers using a commercial kit (First Strand cDNA synthesis kit, Amersham Pharmacia Inc., Piscataway, NJ). Specific oligonucleotide primers were designed to amplify the sequences of human DPPIV (26). The resulting cDNA mixtures were subjected to 30 cycles of PCR amplification with human DPPIV primers (sense primer 5'-TACTCTGCTCTGTGGTGGTC-3', position 706–725; antisense primer 5'-AATACTTCGCCTCTTTACTG-3', position 1439–1458) or human S26 primers (27) (sense primer 5'-GGTCCGTGCCTCCAAGATGA-3', position 8–27; antisense primer 5'-TAAATCGGGGTGGGGGTGTT-3', position 308–327) at annealing temperatures of 52 C or 55 C, respectively. After PCR amplification, the PCR products were electrophoresed on 1% agarose gels, and amplified bands were detected by ethidium bromide staining. Subsequently, the amplified fragments were extracted from the gels, cloned, and verified by sequencing as previously described (28). The cloned DPPIV cDNA and S26 cDNA were then labeled with 32P using a commercial kit (Megaprime DNA labeling system, Amersham Pharmacia Inc.) and used as probes for the subsequent Northern blot analysis.
Northern blot analysis
Ten micrograms of total RNA from JEG-3 cells that had been cultured under normoxic or hypoxic conditions for 48 h were separated by electrophoresis on a 1% agarose-formaldehyde gel and transferred to a nylon membrane (Hybond-N+, Amersham, Arlington, IL). The membrane was incubated with prehybridization solution (rapid hybridization buffer, Amersham) for 30 min at 65 C and then hybridized with the 32P-labeled DPPIV cDNA probe for 2 h at 65 C in the same solution. After hybridization, the membrane was washed in 2x standard saline citrate [2 mM sodium citrate and 20 mM sodium chloride in distilled water (pH 7.0)] with 0.1% SDS at room temperature for 15 min and then in 0.2x standard saline citrate with 0.1% SDS at 65 C for 30 min. Then, the membrane was subjected to autoradiography. The membrane was washed and rehybridized with the S26 probe to correct for the amount of loaded RNA.
Colorimetric DPPIV enzyme assay
The inhibitory effect of diprotin A on DPPIV enzymatic activity in JEG-3 cells was examined by a colorimetric DPPIV enzyme assay as previously described (29). JEG-3 cells (1 x 105 in 200 µl RPMI with 1% FCS) were plated in each well of a 96-well culture plate (Corning, Inc.) and incubated under normoxic conditions. After 12 h of incubation to allow the cells to attach sufficiently, the culture medium was removed and the cells were washed twice with PBS(+) (PBS containing 0.9 mM Ca2+ and 0.33 mM Mg2+). Then, 100 µl of 400 µM Gly-Pro-p-nitroanilide (Sigma) dissolved in PBS(+) was added to the remaining JEG-3 cells. Diprotin A at a final concentration of 0, 5, or 20 µM was also added to the cells. After incubation at 37 C for 1 h, each incubated solution containing the reaction product was collected and mixed with an equal volume of cold sodium acetate-acetic acid buffer (pH 4.2) to stop the reaction. Then, the cultured cells were washed with PBS, trypsinized, and the cell number determined. The amount of reaction product, p-nitroaniline, was measured using an automated ELISA plate reader (Molecular Devices, Menlo Park, CA) at an OD of 385 nm. The concentration was extrapolated from the standard curve determined in the parallel assay. The enzymatic activity was expressed as p-nitroaniline (nmol) produced per 105 cells in 1 h.
Next, we examined the effect of hypoxia on DPPIV enzymatic activity in JEG-3 cells. JEG-3 cells (1 x 104 in 200 µl RPMI with 10% FCS) were cultured in each well of a 96-well plate for 48 h under either normoxic or hypoxic conditions. After removing the culture medium and extensively washing with PBS(+), the DPPIV enzymatic activity of the remaining JEG-3 cells was measured by the colorimetric assay as described above.
Each experiment was performed using triplicate samples. The average of the triplicate samples was defined as the DPPIV activity in each experimental condition. The same experiment was repeated four times, and the results were expressed as a percentage of the mean value of four controls (cultures without diprotin A or under normoxic conditions). Intra- and interassay coefficients of variation in this bioassay were 1.8% and 2.7%, respectively.
Matrigel invasion assay
The Matrigel invasion assay was carried out as previously described (30) with slight modifications. A cell culture insert (6.4 mm in diameter, Becton Dickinson and Co. Labware, Franklin Lakes, NJ) containing a polyethylene terephthalate membrane filter with 8-µm-diameter pores was placed in each well of a 24-well companion plate (Becton Dickinson and Co. Labware). We coated the upper surface of the filter with 10 µg Matrigel (Becton Dickinson and Co. Labware) and air dried the filter aseptically. Before use, the Matrigel was rehydrated with 100 µl warm RPMI for 2 h. Two sets of invasion assays were performed.
In the first set of assays, the effect of diprotin A on the invasive activity of JEG-3 or BeWo cells was examined. In the upper well, JEG-3 or BeWo cells (1 x 105 in 200 µl RPMI with 1% FCS) that had been maintained under normoxic conditions were plated. Diprotin A at a final concentration of 0, 5, or 20 µM was also added. The lower chamber was filled with 800 µl RPMI with 1% FCS. The cells were allowed to invade for 12 h in normoxic conditions. The cells attached to the upper surface of the filter were then removed by scrubbing with a cotton swab. The cells remaining on the lower surface were fixed in methanol for 10 min at room temperature and stained with hematoxylin. For quantification, the cells that had migrated to the lower surface were counted under a light microscope in five predetermined fields at a magnification of x200. Parallel cultures of JEG-3 or BeWo cells were made under the same conditions as for this invasion assay as follows: JEG-3 or BeWo cells (1 x 105 in 200 µl RPMI with 1% FCS) were plated in each well of a 96-well plate the bottom of which was precoated with 10 µg Matrigel. Diprotin A at a final concentration of 0, 5, or 20 µM was also added. After 12-h incubation, the cells were trypsinized and the number of viable cells was counted using the Trypan Blue exclusion method.
In the second set of assays, the effect of hypoxia on the invasiveness of JEG-3 cells was examined. One hundred thousand JEG-3 cells that had been cultured for 48 h under normoxic conditions (normoxic JEG-3) or under hypoxic conditions (hypoxic JEG-3) were suspended in 200 µl RPMI with 1% FCS and added to the upper well. The lower chamber was filled with 800 µl RPMI with 1% FCS. Normoxic or hypoxic JEG-3 cells were allowed to invade either in normoxic or hypoxic conditions, respectively. After 12 h, the cells that reached the lower surface of the culture insert were counted as described above. Parallel cultures were also made under the same conditions and the number of viable cells was counted using the Trypan Blue exclusion method.
Each assay was performed using triplicate samples. The average of the triplicate samples was defined as the number of invaded cells or number of viable cells for each experimental condition. The same assay was repeated four times and the results were expressed as a percentage of the mean number of four controls (cultures without diprotin A or under normoxic conditions).
Statistical analysis
The data were expressed as means ± SEM. The differences were analyzed using two-tailed paired t test or one-way ANOVA for multiple comparisons, followed by Scheffé’s F test.
Results
DPPIV expression in EVTs of normal pregnancies
As reported previously, DPPIV expression was detected in villous cytotrophoblasts, intravillous stroma, and decidual glandular cells, but no staining was observed in the syncytiotrophoblast or decidual cells (12). No apparent difference in the intensity of DPPIV expression on cytotrophoblasts was observed among the placentas examined here. DPPIV expression in EVTs of normal pregnancies is summarized in Table 1
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). In the distal cell column, DPPIV expression was diminished, and no expression was detected on interstitial trophoblasts that were located in the shallow portion of the decidua (Fig. 1, C and F). In contrast to the specimens at 9 wk gestation, a large percentage of interstitial trophoblasts that were located in the deep portion expressed DPPIV (Fig. 1, I and L). In the second and third trimesters of pregnancy (both n = 4), the entire cell column as well as almost all the interstitial trophoblasts were DPPIV positive (representative data are shown in Fig. 2C).
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and Fig. 2F). In term placentas, DPPIV expression on endovascular trophoblasts could not be determined because we could not clearly detect trophoblasts in maternal vessels at this gestational stage.
These immunohistochemical analyses carried out with the two anti-DPPIV mAbs, clones Ta1 and MA261, showed similar staining profiles for DPPIV expression (data not shown). Histochemical staining for DPPIV enzymatic activity also displayed a staining pattern similar to that of the immunohistochemistry (representative data shown in Fig. 3, B and D). No staining was observed with negative controls using anti-TNP mAb or substrate-free staining solution (data not shown).
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DPPIV expression in EVTs of ectopic pregnancies is summarized in Table 2. In tubal pregnancies (6–8 wk gestation, n = 3), DPPIV expression was confined to the proximal part of the cell column. In ruptured rudimentary horn pregnancy at 14 wk gestation (n = 1), DPPIV-positive EVTs were also confined to the proximal cell column (Fig. 4C), and no DPPIV expression was detected on interstitial trophoblasts even in the deep portion of the myometrium (Fig. 4F).
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Under normoxic (20% O2) conditions, outgrowth of trophoblastic cells occurred from the explanted villous tip, resulting in the formation of a cell sheet. From the distal end of the cell sheet, the migration of elongated spindle-shaped cells was observed (Fig. 5A). DPPIV was expressed only in the proximal region of the cell sheet, and it disappeared in the distal part (Fig. 5B). Under hypoxic (1% O2) conditions, the number of the elongated spindle-shaped cells was decreased (Fig. 5C), and the DPPIV-positive population was markedly increased (Fig. 5D).
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Flow cytometric analysis revealed that more than half the JEG-3 cells were DPPIV positive (Fig. 6A), but BeWo cells were completely DPPIV negative (Fig. 6B). These findings were also confirmed in immunocytochemistry (data not shown).
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The ability of diprotin A to inhibit DPPIV enzymatic activity in JEG-3 cells was confirmed using a colorimetric enzyme assay. In the presence of 5 or 20 µM diprotin A, DPPIV enzymatic activity in JEG-3 cells was reduced to 73.8% or 41.5% (P < 0.05) of the control value, respectively (Fig. 7A). When 5 or 20 µM diprotin A were added to the invasion assay, the number of JEG-3 cells that had invaded Matrigel was increased significantly in a dose-dependent manner (Fig. 7B). Diprotin A had no significant effect on the proliferation of JEG-3 cells incubated under the same conditions as for this invasion assay (Fig. 7C). As for DPPIV-negative BeWo cells, diprotin A had no effect on their invasiveness or proliferation (data not shown).
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Northern blot analysis showed that JEG-3 cells cultured under hypoxic (1% O2) conditions expressed a higher level of DPPIV mRNA than those cultured under normoxic (20% O2) conditions (Fig. 8A). The enhancement of DPPIV expression was also observed at the protein level using flow cytometric analysis (mean fluorescence intensity 30.6 ± 3.4 vs. 58.2 ± 7.2, P < 0.05, Fig. 8B). DPPIV enzymatic activity in JEG-3 cells was also significantly increased under the hypoxic conditions, as assessed by the colorimetric enzyme assay (P < 0.01, Fig. 8C). Matrigel invasion assay revealed that JEG-3 cells that had been cultured under hypoxic conditions were significantly less invasive than those cultured under normoxic conditions (P < 0.05, Fig. 8D). In parallel cultures, no significant differences were observed in the numbers of viable JEG-3 cells (Fig. 8E).
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This study demonstrated an intriguing expression profile of DPPIV in EVTs. In the first trimester of pregnancy, DPPIV was most highly expressed in the proximal part of the cell column, expressed at a lower level in the distal part, and not detectably expressed on migrating interstitial trophoblasts. DPPIV expression, however, appeared again on interstitial trophoblasts as they migrated more deeply or as the length of gestation increased. In contrast to interstitial trophoblasts, endovascular trophoblasts lacked DPPIV expression at least up to 20 wk gestation.
The EVTs located in the proximal part of the cell column and deep portion of maternal tissue in the first trimester pregnancy have been reported to be of the noninvasive phenotype (1, 3). EVTs residing in the placental bed in the second and third trimester of pregnancies are also considered to be of the noninvasive phenotype (4). In contrast, the EVTs migrating in the decidua in the first trimester and endovascular trophoblasts until 20 wk gestation are considered to be of the invasive phenotype (5). Therefore, the EVTs stained positively for DPPIV in this study belong to the noninvasive phenotype. On the other hand, the EVTs of invasive phenotype are negative for DPPIV staining. Therefore, positive or negative DPPIV staining seems to coincide with the spatiotemporal differentiation of EVTs, particularly from noninvasive to invasive phenotype or vice versa. We also examined DPPIV expression in villous explant culture experiments, which reproduce trophoblast differentiation occurring in the cell column, i.e. from the noninvasive to invasive phenotype (31, 32, 33). The same kind of tendency for DPPIV staining that the noninvasive phenotype is positive but the invasive phenotype is negative for DPPIV was observed in this in vitro model. DPPIV expression was detected in the proximal region of the cell sheet, diminished in the distal region, and undetectable on the migrating cells. These results suggest that the DPPIV expression level is down-regulated as trophoblasts may acquire migratory activity in vitro.
In an attempt to confirm the relationship between the motility of EVTs and DPPIV, we used the villous explant culture system. The cell outgrowth from each of the explanted villous tips, however, was highly fluctuated and this prevented us from demonstrating the effect of DPPIV in this model. Fortunately, we found that DPPIV is expressed on JEG-3 cells but not on BeWo cells. Therefore, in these choriocarcinoma cell lines, the effect of diprotin A was investigated using the Matrigel invasion assay system. Diprotin A is considered to be a potent peptidase inhibitor with good selectivity for DPPIV (34, 35, 36). JEG-3 cells, which endogenously express DPPIV, showed significant enhancement of invasion if the cells were treated with diprotin A. In parallel cultures under the same conditions, diprotin A did not affect the proliferation of JEG-3 cells. Colorimetric enzyme assays confirmed the inhibitory effects of diprotin A on the DPPIV enzymatic activity of JEG-3 cells. Because it is unclear whether diprotin A has some nonspecific effect(s) other than enzyme inhibition, we also examined the effect of diprotin A on DPPIV-negative BeWo cells. The invasive activity or proliferation of BeWo cells was not affected by diprotin A. This finding strongly suggested that the down-regulation of DPPIV enzymatic activity by diprotin A results in the enhancement of the invasive activity of JEG-3 cells. In other words, it seems that the cell surface DPPIV enzymatic activity plays some invasion-restraining role.
In this study, we could not determine the actual substrate(s) for DPPIV in this invasion assay system using JEG-3 cells. It is plausible, however, that DPPIV modulates the bioactivity of some invasion-regulating substance(s) that is endogenously produced by JEG-3 cells. Our preliminary experiments revealed that the mRNA of chemokine RANTES (regulated on activation, normal T cell expressed and secreted) was expressed by JEG-3 cells (data not shown). Because it has been reported that DPPIV inactivates RANTES (37) and that receptors for RANTES are expressed on JEG-3 cells (38), RANTES could well be one of the substrates involved in the invasion assay system of JEG-3 cells.
Migration or invasion of human trophoblasts has been reported to be regulated by several factors such as decidua-derived factors, cytokines, growth factors, and oxygen environment (13, 14, 15, 39, 40). Therefore, we examined the relation between the expression of DPPIV on JEG-3 cells and these factors. Preliminary experiments involving coculturing with decidual tissues did not affect DPPIV expression in JEG-3 cells (data not shown). IL-1, IL-6, IL-10, TNF-, and TGF-ß, all of which have been reported to affect trophoblast invasiveness (39, 40), also had no effect on the expression of DPPIV in JEG-3 cells (data not shown). Because hypoxia is reported to reduce the invasiveness of trophoblasts (13, 14, 15), the effect of hypoxia on DPPIV expression in JEG-3 cells was also examined. Interestingly, DPPIV expression on JEG-3 cells was significantly up-regulated in hypoxic conditions. Similar hypoxic effect was also observed in the villous explant culture system. These results suggest that trophoblastic DPPIV expression is at least in part regulated by surrounding oxygen tension. This could explain why endovascular trophoblasts that are exposed to high oxygen tension lack DPPIV expression up to 20 wk gestation when almost all the interstitial trophoblasts in the placental bed become DPPIV positive. Oxygen tension in the intervillous space is considered to increase at around 10 wk gestation when maternal arterial transformation occurs and placental circulation is established (41). However, we could not observe any apparent difference in the intensity of DPPIV expression on cytotrophoblasts between 6 and 12 wk gestation. Furthermore, DPPIV reexpression on interstitial trophoblasts in the deep portion or in later pregnancy could not be explained by a change in the surrounding oxygen tension. Therefore, several factors, which might include environmental oxygen tension, may cooperatively regulate trophoblastic DPPIV expression.
We have demonstrated that JEG-3 cell invasiveness is reduced in hypoxic conditions. We also observed that in the villous explant culture system the number of spindle-shaped migrating trophoblasts was markedly decreased under hypoxic conditions, in agreement with previous reports (14, 15). In other words, the up-regulation of DPPIV expression induced by hypoxia is accompanied by a reduction of the invasive/migratory activity of JEG-3 cells and primary trophoblasts. It is possible that hypoxia may decrease invasion/migration and increase DPPIV expression independently. However, considering that DPPIV plays some invasion-restraining role in JEG-3 cells, DPPIV up-regulation might in part contribute to the hypoxic reduction of invasive activity.
In ectopic pregnancy, exaggerated invasive activity of EVTs is reported to be one of the causes of the eventual rupture (42). In this pathological condition, DPPIV expression on interstitial trophoblasts is scarcely observed even at 14 wk gestation. This finding also supports the preventative role of DPPIV in trophoblastic invasion.
In conclusion, this study demonstrates that DPPIV is a novel marker for EVTs of noninvasive phenotype and down-regulation of this enzyme is strongly associated with migration or invasion. Therefore, DPPIV may contribute to the spatiotemporal development and differentiation of EVTs, particularly from noninvasive to invasive phenotype or vice versa. Further study is necessary to clarify the molecular mechanisms by which DPPIV controls the invasive phenotype or noninvasive phenotype of EVTs during the establishment of successful pregnancy.
Acknowledgments
Footnotes
This work was supported in part by Grants-in-Aid for Scientific Research (12470342, 13557140, 13671709, and 13671710).
Abbreviations: DPPIV, Dipeptidyl peptidase IV; EVT, extravillous trophoblast; FITC, fluorescein isothiocyanate; mAb, monoclonal antibody; pAb, polyclonal antibody; RANTES, regulated on activation, normal T cell expressed and secreted; TNP, trinitrophenyl.
Received January 14, 2002.
Accepted June 13, 2002.
References
) functions of murine B cells. J Immunol 144:4571–4578[Abstract]
3 and integrin 5. Mol Hum Reprod 5:162–1675ß1. Biol Reprod 60:828–838This article has been cited by other articles:
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  M. Stankovic, P. Vlahovic, V. Avramovic, and M. Todorovic Distribution of Dipeptidyl Peptidase IV in Patients with Chronic Tonsillitis Clin. Vaccine Immunol., May 1, 2008; 15(5): 794 - 798. [Abstract] [Full Text] [PDF]
|
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|
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|
 I. Kryczek, S. Wei, E. Keller, R. Liu, and W. Zou Stroma-derived factor (SDF-1/CXCL12) and human tumor pathogenesis Am J Physiol Cell Physiol, March 1, 2007; 292(3): C987 - C995. [Abstract] [Full Text] [PDF]
|
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 |
|
 S. Yoshioka, H. Fujiwara, T. Nakayama, K. Kosaka, T. Mori, and S. Fujii Intrauterine administration of autologous peripheral blood mononuclear cells promotes implantation rates in patients with repeated failure of IVF-embryo transfer Hum. Reprod., December 1, 2006; 21(12): 3290 - 3294. [Abstract] [Full Text] [PDF]
|
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 |
|
 F. Shido, T. Ito, S. Nomura, E. Yamamoto, S. Sumigama, K. Ino, A. Itakura, A. Hattori, M. Tsujimoto, S. Mizutani, et al. Endoplasmic Reticulum Aminopeptidase-1 Mediates Leukemia Inhibitory Factor-Induced Cell Surface Human Leukocyte Antigen-G Expression in JEG-3 Choriocarcinoma Cells Endocrinology, April 1, 2006; 147(4): 1780 - 1788. [Abstract] [Full Text] [PDF]
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Y. Huang, X.-Y. Zhu, M.-R. Du, X. Wu, M.-Y. Wang, and D.-J. Li Chemokine CXCL16, a scavenger receptor, induces proliferation and invasion of first-trimester human trophoblast cells in an autocrine manner Hum. Reprod., April 1, 2006; 21(4): 1083 - 1091. [Abstract] [Full Text] [PDF]
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|
 E. Yamamoto, T. Ito, A. Abe, F. Sido, K. Ino, A. Itakura, S. Mizutani, S. Dovat, S. Nomura, and F. Kikkawa Ikaros is expressed in human extravillous trophoblasts and involved in their migration and invasion Mol. Hum. Reprod., November 1, 2005; 11(11): 825 - 831. [Abstract] [Full Text] [PDF]
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 Y. Sato, H. Fujiwara, B.-X. Zeng, T. Higuchi, S. Yoshioka, and S. Fujii Platelet-derived soluble factors induce human extravillous trophoblast migration and differentiation: platelets are a possible regulator of trophoblast infiltration into maternal spiral arteries Blood, July 15, 2005; 106(2): 428 - 435. [Abstract] [Full Text] [PDF]
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J. A. Horcajadas, A. Riesewijk, J. Polman, R. van Os, A. Pellicer, S. Mosselman, and C. Simon Effect of controlled ovarian hyperstimulation in IVF on endometrial gene expression profiles Mol. Hum. Reprod., March 1, 2005; 11(3): 195 - 205. [Abstract] [Full Text] [PDF]
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Y. Nishioka, T. Higuchi, Y. Sato, S. Yoshioka, K. Tatsumi, H. Fujiwara, and S. Fujii Human migrating extravillous trophoblasts express a cell surface peptidase, carboxypeptidase-M Mol. Hum. Reprod., December 1, 2003; 9(12): 799 - 806. [Abstract] [Full Text] [PDF]
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 Y. Sato, T. Higuchi, S. Yoshioka, K. Tatsumi, H. Fujiwara, and S. Fujii Trophoblasts acquire a chemokine receptor, CCR1, as they differentiate towards invasive phenotype Development, November 15, 2003; 130(22): 5519 - 5532. [Abstract] [Full Text] [PDF]
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 H. Kajiyama, F. Kikkawa, E. Khin, K. Shibata, K. Ino, and S. Mizutani Dipeptidyl Peptidase IV Overexpression Induces Up-Regulation of E-Cadherin and Tissue Inhibitors of Matrix Metalloproteinases, Resulting in Decreased Invasive Potential in Ovarian Carcinoma Cells Cancer Res., May 1, 2003; 63(9): 2278 - 2283. [Abstract] [Full Text] [PDF]
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