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Endocrinology Vol. 146, No. 11 4682-4689
Copyright © 2005 by The Endocrine Society

Up-Regulation of c-met Protooncogene Product Expression through Hypoxia-Inducible Factor-1{alpha} Is Involved in Trophoblast Invasion under Low-Oxygen Tension

Masami Hayashi, Masahiro Sakata, Takashi Takeda, Masahiro Tahara, Toshiya Yamamoto, Yoko Okamoto, Ryoko Minekawa, Aki Isobe, Masahide Ohmichi, Keiichi Tasaka and Yuji Murata

Department of Obstetrics and Gynecology (M.H., M.S., M.T., Y.O., R.M., A.I., M.O., K.T., Y.M.), Osaka University Faculty of Medicine, Suita, Osaka 565-0871, Japan; Osaka Medical Center for Cancer and Cardiovascular Diseases (T.T.), Higashinari-ku, Osaka 537-8511, Japan; and Sakai Municipal Hospital (T.Y.), Sakai, Osaka 590-0064, Japan

Address all correspondence and requests for reprints to: Masami Hayashi, M.D., Ph.D., Department of Obstetrics and Gynecology, Osaka University Faculty of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. E-mail: masami{at}gyne.med.osaka-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
During early pregnancy, the invasion of trophoblast cells into the decidua of the uterus is one of the essential steps in appropriate placentation. In this period, trophoblast cells are exposed to a relatively low-oxygen environment. The c-met protooncogene product (Met), which is a high-affinity receptor for hepatocyte growth factor, plays an important role in controlling the invasion of many types of cells. The present study was designed to investigate the effect of low-oxygen tension on Met expression and the invasiveness of trophoblast cells isolated from early-stage human placenta and trophoblast-derived BeWo cells and JEG-3 cells. RT-PCR and immunoblot analyses demonstrated that low-oxygen tension (1% O2) stimulated the expression of Met mRNA and protein, respectively. Hepatocyte growth factor production in the cells was not affected by oxygen tension. Transient transfection of BeWo cells with a hypoxia-inducible factor (HIF)-1{alpha} expression vector to induce exogenous expression of HIF-1{alpha} significantly increased the level of Met mRNA and protein, compared with transfection of a control vector. To examine whether this up-regulation of Met was directly induced by HIF-1{alpha}, we performed the chromatin immunoprecipitation assay, which revealed that HIF-1{alpha} binds to the promoter region of the Met gene under low-oxygen tension. JEG-3 cells cultured under 1% O2 showed a more invasive character than those cultured under 20% O2, whereas inhibition of Met expression by small interfering RNAs prevented the low-oxygen, tension-induced invasiveness. These results suggest that the induction of Met expression by low-oxygen tension may play an important role in the physiology of early pregnancy by promoting the invasion of trophoblast cells into the decidua of the uterus.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TROPHOBLAST CELLS ADHERE to and invade into the decidua of the uterus, thereby initiating the placentation process during early pregnancy. Before a mixture of fetal and maternal cellular elements has been established in the placental bed, trophoblast cells exist in a relatively low-oxygen environment (1). Studies in which the oxygen tension within the intervillous space was measured in vivo revealed a marked rise from less than 20 mm Hg (<3% O2) at 8 wk of gestation to 55 mm Hg at 12 wk (1, 2). To characterize the developmental system during early pregnancy, it is crucial to investigate the mechanisms regulating the invasion of trophoblast cells in this environment.

Hepatocyte growth factor (HGF) is a mesenchymal cytokine, and its receptor, c-met protooncogene product (Met), is a heterodimeric transmembrane glycoprotein with tyrosine kinase activity (3, 4). Upon stimulation of cells by HGF, Met is tyrosine phosphorylated and initiates a cascade of signals that lead to activation of cellular behaviors such as cell invasion, proliferation, and morphogenesis in several types of cells (5, 6, 7). Previous reports demonstrated that the HGF/Met pathway plays an important role in organ formation during embryogenesis (8, 9). The human placenta is one of the sources from which HGF and Met have been purified (10, 11). Furthermore, HGF has been shown to stimulate the invasiveness and motility of trophoblast cells (12), and reduced production of HGF is suggested to be one of the contributors to insufficient invasiveness of trophoblast cells in preeclampsia (12). However, the physiological significance of the HGF/Met pathway in the invasion of trophoblast cells, especially during early placentation, has not been fully elucidated.

Hypoxia-inducible factor (HIF)-1 is a transcriptional activator that regulates the expression of several genes in response to oxygen tension (13). It is a dimer consisting of HIF-1{alpha} and HIF-1ß subunits (14). HIF-1ß is constitutively expressed, whereas HIF-1{alpha} expression is regulated by O2 tension (15). A drop in O2 level stabilizes HIF-1{alpha} and up-regulates HIF-1{alpha} expression, leading to increased nuclear translocation and binding of HIF-1{alpha} to HIF-1ß (16, 17). Caniggia et al. (18) reported that HIF-1{alpha} expression is elevated during the early stage of the first trimester of gestation, and it decreases markedly around 10–12 wk.

Recently, one report demonstrated that the Met gene promoter contains several HIF-1 binding sites and that the Met expression is up-regulated by hypoxia in cancer cell lines (19). Thus, the present study focused on the effects of low-oxygen tension on the regulation of Met expression through HIF-1{alpha} induction in placental cells and the resultant effects on the invasiveness of the cells via the HGF/Met pathway under these conditions. In this study, we used trophoblast cells isolated from first-trimester placentas. We also used trophoblast-derived BeWo cells and JEG-3 cells as models for investigating the molecular regulatory mechanisms of placental gene and protein expression, using techniques such as transfection of DNAs. BeWo cells and JEG-3 cells have been previously used as trophoblast models for studying several aspects of placental gene expression and trophoblast invasion (20, 21).

Here we show that low-oxygen tension stimulates the expression of Met in trophoblast cells during early pregnancy and in trophoblast-derived cells, and provide additional data suggesting that the up-regulation of Met expression mediates the low-oxygen-induced cell invasion during early pregnancy.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Fetal bovine serum (FBS) was purchased from JRH Biosciences (Lenexa, KS). Vector (pcDNA3.1) was obtained from Invitrogen Corp. (Carlsbad, CA). The expression vector encoding HIF-1{alpha} was a kind gift from Dr. L. Eric Huang (22).

Tissue collection
Human placental tissues from first trimester were collected from 10 normal pregnancies that were voluntarily terminated by dilation and curettage between 6 and 9 wk of gestation. Informed consent was obtained from each patient before obtaining the placental explants. The protocol was approved by the local ethics committee of the Department of Obstetrics and Gynecology, Faculty of Medicine, Osaka University. Placental tissues were collected in ice-cold PBS, transported to the laboratory on ice, and processed within 2 h as described previously (18)

Isolation and purification of trophoblast cells
Primary trophoblast cells were prepared by the method described by Shiokawa et al. (23) with minor modifications as follows. Placental tissue was rinsed thoroughly in cold PBS, dissected from the membranes, and cut into small pieces (1–2 mm). The tissue fragments were washed three times with Medium 199 (Invitrogen) supplemented with 10% FBS. These fragments were then cultured in Medium 199 containing 10% FBS for 2–5 d until nonadherent cells could be removed and discarded. The medium was changed every 24 h. To ensure that the cultured cells possessed the invasive phenotype of extravillous trophoblast cells, adherent cells were characterized by immunofluorescence analysis (90–97% positive for cytokeratin 7 and 65–72% positive for human leukocyte antigen-G).

Cell culture
The human choriocarcinoma cell line BeWo was obtained from the Health Science Research Resources Bank (Osaka, Japan). The cells were cultured in Ham’s F12 medium (Sigma Aldrich Corp., St. Louis, MO) containing 15% FBS. JEG-3 cells were obtained from the American Type Culture Collection (Rockville, MD). The cells were cultured in MEM with Eagle’s salts medium (Life Technologies, Inc., Rockville, MD) containing 10% FBS. Oxygen tension in the medium was measured with a blood gas analyzer. The measured partial pressure of oxygen (pO2) in 20% O2 incubator was 136–140 mm Hg, whereas pO2 in 1% O2 incubator was 20–23 mm Hg. These pO2 values are approximately similar to the previous study (24), and the low-oxygen condition in our system (1% O2 incubator: 20–23 mm Hg) is near to the physiological oxygen tension during early pregnancy (1, 2).

In vitro immunofluorescence
Human trophoblast cells were cultured on 35-mm plates. BeWo cells and JEG-3 cells were plated on eight-well chamber slides. For analysis of HIF-1{alpha} or Met expression, the cells were cultured under the indicated conditions (20% O2 or 1% O2) for 48 h. After incubation, cells were fixed with 3.7% paraformaldehyde in PBS for 20 min, permeabilized with 0.1% Triton X-100 for 1 h, and stained with an anti-HIF-1{alpha} mouse monoclonal antibody whose epitope maps within amino acids 432–528 of HIF-1{alpha} of human origin (NB100-105, Novus Biologicals, Littleton, CO) or an anti-Met rabbit polyclonal antibody that was raised against a peptide mapping at the carboxy terminus of c-Met p140 of human origin (sc-10; Santa Cruz Biotechnology, Santa Cruz, CA) at 4 C overnight. After washing, samples were incubated with Alexa Fluor 488-labeled goat antimouse IgG or antirabbit IgG (Molecular Probes, Eugene, OR), respectively. Specimens were double stained with rhodamine-labeled phalloidin (Molecular Probes) for 30 min at room temperature. Images were analyzed on a TE2000-U microscope (Nikon Corp., Tokyo, Japan) with an IEEE 1394 digital camera (Hamamatsu Photonics, Hamamatsu, Japan) and Lumina Vision software (Mitani Corp., Fukui, Japan).

Immunoblot analysis
Whole-cell proteins were extracted as described previously (25). Samples were electrophoresed and transferred to a nitrocellulose filter (Bio-Rad Laboratories, Hercules, CA) using standard procedures (26). For detection of Met protein, the filter was blocked with 5% (wt/vol) nonfat milk in 10 mM Tris-buffered saline containing 0.1% Tween 20 (TBS-T) for 2 h at room temperature followed by overnight incubation at 4 C with anti-Met rabbit polyclonal antibody (Santa Cruz Biotechnology). After washing three times in TBS-T, the filter was incubated with secondary antirabbit antibody (Santa Cruz Biotechnology) in TBS-T for 1 h at room temperature and developed for the detection of specific protein bands using the enhanced chemiluminescence system (Amersham Biosciences Corp., Piscataway, NJ). Whole-cell extract protein concentrations were measured with a DC protein assay kit (Bio-Rad) using a modification of the method of Lowry et al. (27).

RT-PCR
Total RNA was extracted from BeWo cells as described previously (28). RT of RNA into cDNA and PCR amplification were performed as described previously (29). The PCR primer sets used for Met and ß-actin cDNA amplification were as follows: Met (30) sense 5'-GGT CAA TTC AGC GAA GTC CT-3', antisense 5'-TTC GTG ATC TTC TTC CCA GTG-3'; ß-actin (BD Biosciences Clontech, Palo Alto, CA) sense 5'-ATC TGG CAC CAC ACC TTC TAC AAT GAG CTG CG-3', antisense 5'-CGT CAT ACT CCT GCT TGC TGA TCC ACA TCT GC-3'. The thermal cycle profile used for Met was 40 cycles of denaturation at 95 C for 25 sec, annealing at 56 C for 35 sec, and extension at 72 C for 35 sec (30). For ß-actin, 25 cycles of 45 sec at 94 C, 45 sec at 60 C, and 2 min at 72 C (29, 31, 32) were carried out. PCR amplification for Met and ß-actin was performed in the range of the linear relationship between the cycle number and the intensity of RT-PCR product (data not shown). PCR fragments were analyzed by electrophoresis on 1.8% agarose gels and stained with ethidium bromide.

Transient transfection
BeWo cells were transfected using Lipofectamine reagent (Life Technologies) as described previously (26) with slight modifications as follows. Cells (2 x 106/dish) grown in 100-mm dishes for 20 h in 15% FBS/Ham’s F12 were transfected using 20 µl of Plus reagent, 6 µg of control vector (pcDNA3.1) or the expression vector encoding HIF-1{alpha}, and 30 µl of Lipofectamine reagent with 1500 µl of Opti-MEM I medium (Life Technologies). After the cells were thoroughly washed with Opti-MEM I, they were incubated with medium containing the preincubated DNA-Lipofectamine complex. Four hours later, the medium was changed to complete medium and the cells were incubated for 48 h.

Chromatin immunoprecipitation
BeWo cells were used for the chromatin immunoprecipitation (ChIP) assay as previously described (33, 34). Cell extracts were sonicated on ice for 10 sec x 4 cycles, with 60-sec pauses between each cycle, using a Sonifier ultrasonic cell disruptor (Branson, Danbury, CT) at power level 3. The extract was divided into aliquots, and antibodies were added to the separate aliquots at 1:200 dilutions for immunoprecipitation. Mouse monoclonal human HIF-1{alpha} antibody (NB100-105) was obtained from Novus Biologicals. Normal mouse IgG (sc-2025; Santa Cruz Biotechnology) was used as a negative control. After immunoprecipitation, the immunocomplexes were treated as described (33). To separate immunoprecipitated protein from DNA, the pooled eluates were incubated at 65 C overnight. The DNA was purified using a MinElute reaction cleanup kit (QIAGEN, Santa Clarita, CA). The final volume was 15 µl [10 mM Tris-HCl (pH 8.5)]. The yield of target region DNA in each sample after ChIP was analyzed by PCR amplification, as described previously. The following primers were used for ChIP PCR analysis: Met sense 5'-GGA CAA TTC GTC CAT CCA CT-3', antisense 5'-AGA TAA GCG GGA CCG AGT CT-3'. The thermal cycle profile was 37 cycles of denaturation at 94 C for 30 sec, annealing at 53 C for 30 sec, and extension at 72 C for 60 sec.

Invasion assay
Chemotactic directional migration was assayed as described previously (35) with some modifications. Cells (1 x 105/ml) were placed on a Matrigel-coated filter (Nippon BD Biosciences, Tokyo, Japan) in 500 µl of MEM with 0.1% FBS. The lower chamber was filled with 700 µl of MEM with 10% FBS. After adhesion, cells were serum starved and incubated for an additional 48 h in the indicated conditions under 1% O2 or 20% O2. Nonpenetrating cells were removed from the upper surface of the filter with a cotton swab. Penetrating cells adherent to the underside of the filter were fixed and stained with a Diff-Quick stain kit (International Reagents Corp., Kobe, Japan) according to the manufacturer’s instructions. For quantification, the cells invaded into the lower surface were counted under a light microscope in four random fields at x100 magnification.

Met knockdown
JEG-3 cells (5 x 104/well) were grown in six-well plates in regular medium without antibiotics for 24 h. Cells at 40–50% confluency were transfected with Met-specific siRNA oligonucleotides (Met siRNA smart pool, catalog no. M-003156-01-05) (Dharmacon, Lafayette, CO) or with scramble RNA oligonucleotides as control (nonspecific control duplex-XIII, catalog no. D-001206-13-05) (Dharmacon) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. The final concentration of small interfering (si) RNA was 100 nM. Met down-regulation in transfected cells was confirmed by RT-PCR analysis, as described earlier.

ELISA
The concentration of HGF in supernatants of placental cells was estimated using an ELISA kit (R&D Systems, Minneapolis, MN), which recognizes both natural and recombinant human HGF, according to the manufacturer’s protocol. The minimum detectable dose of HGF was 40 pg/ml. Values were expressed as HGF concentration adjusted for cell protein concentration. To validate the assay, samples that were either serially diluted or to which known amounts of the HGF standard were added were compared with the standard curve to demonstrate appropriate parallelism. The intra- and interassay coefficients of variation were 7.1 and 7.2%, respectively.

Statistical analysis
Data were expressed as the mean ± SEM. Statistical analysis was performed using the Student’s t test or one-way ANOVA for multiple comparisons followed by Fisher’s post hoc test. Differences were considered statistically significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Low-oxygen tension increases Met protein levels
To investigate the effects of low-oxygen tension on the expression of Met protein in trophoblast cells, we performed immunofluorescence analysis. For this purpose, primary cultured trophoblast cells and trophoblast-derived cells (BeWo cells and JEG-3 cells), used as trophoblast models, were incubated under 20% O2 or 1% O2. As shown in Fig. 1Go, 1% O2 treatment increased the levels of not only HIF-1{alpha} protein but also Met protein in the cells. To examine the kinetics of Met induction under these conditions, we performed time-course analysis of immunoblotting. As shown in Fig. 2AGo, 1% O2 caused a significant increase in Met protein expression, and the induction began to appear after 12 h of exposure of trophoblast cells to 1% O2. The level of Met protein in BeWo cells was also increased under 1% O2, compared with that under 20% O2 (Fig. 2BGo).



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  		FIG. 1. Immunofluorescent detection of HIF-1{alpha} and Met. A, Trophoblast cells and trophoblast-derived BeWo cells were incubated under 20% O2 or 1% O2, and double stained with rhodamine-labeled phalloidin (red) and anti-HIF-1{alpha} antibody followed by Alexa 488-labeled antimouse IgG (green). B, Trophoblast cells and trophoblast-derived cells (BeWo cells and JEG-3 cells) were incubated under the indicated conditions, and double stained with rhodamine-labeled phalloidin (red) and anti-Met antibody followed by Alexa 488-labeled antirabbit IgG (green). Photographs were taken at a 100-fold magnification under a fluorescence microscope. Scale bar, 100 µm.

 


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  		FIG. 2. Time-course analysis of Met protein expression in first-trimester human trophoblast cells and BeWo cells. Cells were incubated under 20% O2 (lanes 1, 3, and 5) or 1% O2 (lanes 2, 4, and 6) for the indicated times. Whole-cell extract proteins (50 µg) were subjected to immunoblot analysis using anti-Met and anti-ß-actin antibodies. A, Trophoblast cells isolated from human first-trimester placental tissue were cultured under 20% O2 or 1% O2. The lower panel shows densitometric analysis assessing relative protein expression levels. The density of the control bands (20% O2 for 12 h) was set at 100. Values shown represent the mean ± SEM from three separate experiments. *, P < 0.05; **, P < 0.01 (vs. 20% O2). B, Parallel experiments and quantitative analyses were performed using BeWo cells. Values shown represent the mean ± SEM from three separate experiments. Significant differences from the control band density are indicated by asterisks. *, P < 0.05; **, P < 0.01 (vs. 20% O2).

 
Low-oxygen tension increases Met mRNA levels
The effect of low-oxygen tension on the expression of Met mRNA was analyzed by RT-PCR and quantified by densitometric analysis. As shown in Fig. 3Go, BeWo cells expressed higher levels of Met mRNA under 1% O2 (lanes 2, 4, and 6) than under 20% O2 (lanes 1, 3, and 5).



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  		FIG. 3. Time-course analysis of Met mRNA expression in BeWo cells. Cells were incubated under 20% O2 (lanes 1, 3, and 5) or 1% O2 (lanes 2, 4, and 6) for the indicated times. RT-PCR amplification was performed using primers for Met or ß-actin. The lower panel shows densitometric analysis. The density of the control bands (20% O2 for 8 h) was set arbitrarily at 100. Values shown represent the mean ± SEM from separate experiments. **, P < 0.01 (vs. 20% O2).

 
Exogenous HIF-1{alpha} increases the levels of Met protein and mRNA
To examine whether the induction of HIF-1{alpha} mediates an increase of Met expression in placental cells, we analyzed the expression of Met protein and mRNA after transfection of BeWo cells with an expression vector encoding HIF-1{alpha} or a control vector lacking HIF-1{alpha} sequence. As shown in Fig. 4AGo, enhanced Met protein expression was changed under 20% O2 in cells transfected with the HIF-1{alpha} expression vector (lane 2), compared with that in cells transfected with the control vector (lane 1) (P < 0.05). We also investigated the effect of HIF-1{alpha} on the expression of Met at the transcriptional level. As shown in Fig. 4BGo, cells expressing HIF-1{alpha} (lane 2) showed an increased level of Met mRNA, compared with control vector transfectants (lane 1) under 20% O2 (P < 0.05). These data demonstrate that HIF-1{alpha} is directly involved in the induction of the Met gene and its protein products.



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  		FIG. 4. Increased levels of Met mRNA and protein in BeWo cells transfected with an expression vector encoding HIF-1{alpha}. Cells were transfected with 1 µg of expression vector for HIF-1{alpha} (lane 2) or control vector (lane 1). A, Whole-cell extract proteins (50 µg) from the cells were subjected to immunoblot analysis using anti-Met and anti-ß-actin antibodies. The lower panel shows densitometric analysis of relative protein expression levels. The density of the control transfectants (open bars) was set at 100. Values shown represent the mean ± SEM from three separate experiments. *, P < 0.05 (vs. control values). B, RT-PCR amplification was performed using primers for Met or ß-actin. The lower panel shows quantitative analysis of the data using densitometric analysis. The density of the control transfectants (open bars) was set at 100. Values shown represent the mean ± SEM from three separate experiments. *, P < 0.05 (vs. control values).

 
HIF-1{alpha} is recruited to the promoter region of the Met gene under low-oxygen tension
To investigate the precise mechanism through which HIF-1{alpha} regulates Met gene expression under low-oxygen tension, we performed ChIP assays. A pair of primers spanning a 250-bp region of the Met gene promoter encompassing the hypoxia response element (19) was used for PCR (Fig. 5AGo). BeWo cells were incubated under 20% O2 or 1% O2 for 16 h and then subjected to formaldehyde cross-linking. Met DNA cross-linked with HIF-1{alpha} was efficiently detected after1% O2 incubation but not when a nonimmune mouse IgG control serum was used for ChIP, indicating that HIF-1{alpha} binds to the promoter region of the Met gene under low-oxygen tension.



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  		FIG. 5. Low-oxygen, tension-induced recruitment of HIF-1{alpha} to the Met promoter in BeWo cells. A, Schematic representation of the human Met promoter and the locations of the primers used to amplify the promoter region. Nucleotide +1 corresponds to the start site. B, BeWo cells cultured under 20% O2 or 1% O2 for 16 h were subjected to formaldehyde cross-linking and then to sonication and immunoprecipitation with normal mouse IgG or anti-HIF-1{alpha} antibody. The ChIP products were amplified by PCR using primers flanking the promoter region of the Met gene. HRE, Hypoxia response element.

 
Low-oxygen tension enhances motility
Because Met plays an important role in cell motility, we investigated whether up-regulation of Met under low-oxygen tension affected the motility of trophoblast-derived JEG-3 cells (Fig. 6Go). As shown in Fig. 6AGo, the invasiveness of the cells was stimulated under 1% O2, compared with that under 20% O2. To further explore the role of Met in the increase of invasiveness under low-oxygen tension, we selectively down-regulated Met in JEG-3 cells using siRNAs of Met (Fig. 6Go, B and C). In the Met siRNA-transfected cells, the elevated invasiveness under low-oxygen tension was inhibited, compared with that in cells transfected with scramble RNA oligonucleotides.



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  		FIG. 6. The effects of low-oxygen tension and Met down-regulation by specific siRNAs on the invasion of JEG-3 cells. A, JEG-3 cells were seeded onto porous filters coated with matrigel. After incubation under 20% O2 or 1% O2 for 48 h to permit penetration of the filters, nonpenetrating cells were removed from the upper chamber. Penetrating cells adherent to the underside of the filters were counted as described in Materials and Methods, and values were expressed as a percentage of the control (mean ± SEM from three separate experiments performed in triplicates). *, P < 0.05 (vs. 20% O2). B, JEG-3 cells were treated with Met siRNA or with control RNA oligonucleotides (scramble RNA). Cells were lysed and evaluated for Met mRNA and ß-actin mRNA expression by RT-PCR analyses. C, Invasion analysis of JEG-3 cells after Met down-regulation. The cells treated with either scramble RNA oligonucleotides or Met siRNA were seeded onto porous filters coated with Matrigel. After incubation under 20% O2 or 1% O2 for 48 h to permit penetration of the filters, penetrating cells adherent to the underside of the filters were counted. The numbers of cells that had invaded through Matrigel are expressed as a percentage of the control (mean ± SEM from three separate experiments performed in triplicate). a, P < 0.01 vs. 20% O2 from scramble RNA transfectants; b, P < 0.05 vs. scramble RNA transfectants under 20% O2; c, P < 0.01 vs. scramble RNA transfectants under 1% O2.

 
Low-oxygen tension does not affect HGF production
To examine the effect of low-oxygen tension on the production of HGF, which is an activator of trophoblast invasiveness, we examined the HGF concentration in the culture supernatants of BeWo cells (A) and JEG-3 cells (B) after 48 h of incubation under 20% O2 or 1% O2. The results of ELISA showed that there was no significant difference in the ability to produce HGF in cells under 20% O2 vs. 1% O2 (Fig. 7Go).



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  		FIG. 7. HGF concentrations as determined by ELISA. BeWo cells (A) and JEG-3 cells (B) were cultured under 20% O2 or 1% O2 for 48 h. Values are expressed as HGF concentration adjusted for cell protein concentration. Data are expressed as the mean ± SEM from three separate experiments with three parallel wells for each condition in each experiment.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Successful pregnancy depends on placental growth and development, with appropriate invasion of trophoblast cells. The major finding of this study is that low-oxygen tension stimulated the invasiveness of trophoblast cells. HGF is known to be a pleiotropic growth factor that enhances cell proliferation and migration. However, the regulation of HGF production under low-oxygen tension has not yet been clearly demonstrated in trophoblast cells. A previous study demonstrated that HGF production in vascular smooth muscle cells and endothelial cells was decreased under low-oxygen tension (36). In the present study, there was no significant difference in HGF production in trophoblast-derived cells cultured under 1% O2 vs. 20% O2. What are the molecular mechanisms of that explain the discrepancy between the increased invasiveness and decreased HGF production under low-oxygen tension in placental cells? The present study revealed that the expression of Met, the high-affinity HGF receptor, was significantly increased in trophoblast cells under 1% O2, compared with those under 20% O2 through HIF-1{alpha} induction. These observations are in agreement with a recent study reporting the relationship between low-oxygen tension and stimulated invasiveness of cancer cells (19). In addition, our ChIP assays demonstrated for the first time that low-oxygen tension induces recruitment of HIF-1{alpha} to the Met gene promoter, resulting in increased Met expression under low-oxygen tension. Given the pivotal role of Met in cell invasion and motility, the results obtained in this study suggest that low-oxygen tension promotes invasion of trophoblast cells, at least in part through increased Met expression, resulting in placentation.

In this study, we used trophoblast-derived human BeWo and JEG-3 cells as trophoblast models for studying invasion because they are well-accepted and frequently used models for studying trophoblast behavior during early pregnancy (37, 38). Also, we observed by immunofluorescence analysis that they expressed human leukocyte antigen-G (data not shown), as reported previously (39, 40). Although these cells have several characteristics of normal trophoblast cells, they were derived from human choriocarcinomas. Further studies using extravillous trophoblasts in all experiments would provide additional support for the notions suggested by our present data.

Previous studies revealed that low-oxygen tension stimulates the invasion of first-trimester trophoblast cells by increasing the expression of plasminogen activator inhibitor (PAI)-1 (41). PAI-1 has been shown to be one of the target genes of HIF-1{alpha} (42). PAI-1 participates in cell migration by being released at the receding end of the cell, which results in detachment of the cell membrane from the substratum (43).

In a previous study (19), Met was also identified as a target gene of HIF-1{alpha}. It has also been shown that the expression of HIF-1{alpha} in human placenta is increased during the first trimester and decreases with gestational age (44). Therefore, it is likely that the increased expression of HIF-1{alpha} may play a compensatory role in the cellular response by increasing the levels of motility factors such as PAI-1 and Met aimed at escaping the low-oxygen environment and attempting to move toward places in which the oxygen supply is not limited.

The small guanosine triphosphatase Rho functions as a key factor in the regulation of the actin cytoskeleton and actomyosin contractility. Shiokawa et al. (23) reported that RhoA is one of the important factors regulating the invasion of trophoblast cells. We previously demonstrated that RhoA was up-regulated under low-oxygen tension and that RhoA in turn up-regulated the HIF-1{alpha} expression in trophoblast cells (29). These findings, together with those of our present study, support the idea that RhoA might participate in the invasion of trophoblast cells not only in a direct manner but also in an indirect one via stimulation of Met expression through HIF-1{alpha} induction.

Invasion of trophoblast cells during early placentation has similarities to tumor cell invasion (45). However, the invasion of trophoblast cells is well controlled; that is, their invasion is confined to the endometrium, the first third of the myometrium, and the associated uterine spiral arteries (46). Some unknown factor such as an antagonistic antibody against Met, which has been proposed for therapeutic use against cancer (47), might be involved in this limited physiological invasion. To understand the adequate but not excessive invasion of the trophoblast cells, further analyses will be needed.

After the second trimester of pregnancy, pathological hypoxia of trophoblast cells is closely linked with pregnancy complications such as preeclampsia (48). In this disease, major uteroplacental pathology is characterized by the coexistence of poor uterine arterial remodeling, which results in placental hypoxia (49) and poor invasion of trophoblast cells into the decidua (50). Previous studies showed that the expression of HGF was reduced in the placenta of preeclamptic pregnancy (51) and that the expression of Met was not increased, compared with that in normal pregnancy (52). These findings suggest that impairment of the HGF/Met pathway may be involved in the pathogenesis of preeclampsia. Up-regulation of the HGF/Met pathway may have implications for the development of new therapeutic strategies for preeclampsia.

In summary, expression of Met in trophoblast cells is stimulated by low-oxygen tension, resulting in increased invasiveness of the cells. Our data may provide a molecular explanation for the physiological regulation of trophoblast invasion through up-regulation of Met expression.


   Footnotes
 
First Published Online August 11, 2005

Abbreviations: ChIP, Chromatin immunoprecipitation; FBS, fetal bovine serum; HGF, hepatocyte growth factor; HIF, hypoxia inducible factor; Met, c-met protooncogene product; PAI, plasminogen activator inhibitor; pO2, partial pressure of oxygen; si, small interfering; TBS-T, Tris-buffered saline containing Tween 20.

Received April 11, 2005.

Accepted for publication August 2, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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