Hum. Reprod. Advance Access originally published online on May 24, 2007
Human Reproduction 2007 22(7):1801-1809; doi:10.1093/humrep/dem125
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Induction of prostaglandin E2 production by leukemia inhibitory factor promotes migration of first trimester extravillous trophoblast cell line, HTR-8/SVneo
1 Department of Immunology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu 807-8555, Japan 2 Department of Obstetrics and Gynecology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu 807-8555, Japan
3 Correspondence address. Tel: +81-93-691-7241; Fax: +81-93-692-2479; E-mail: yama-uki{at}med.uoeh-u.ac.jp
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Abstract |
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BACKGROUND: The invasion of first trimester extravillous trophoblast (EVT) to decidua is an important event in placentation. Leukemia inhibitory factor (LIF) is an essential factor for mouse implantation, and it is reported that LIF may be involved in human first trimester EVT invasion. Prostaglandin E2 (PGE2) is also known as a critical factor for first trimester EVT invasion. In this study, we investigated the role of LIF in PGE2 production and EVT invasion using a human first trimester EVT cell line, HTR-8/SVneo.
METHODS and RESULTS: Co-stimulation with LIF and IL-1
induced higher amounts of PGE2 production and further migration of HTR-8/SVneo cells compared with that by stimulation with LIF or IL-1 alone. Enhanced PGE2 production was most probably due to the enhanced expression of cyclooxygenase-2 (COX-2) and microsomal PGE synthase-1 (mPGES-1). PGE2 produced by HTR-8/SVneo cells promoted the migration of HTR-8/SVneo cells. A COX-2 inhibitor suppressed PGE2 production and the migration of HTR-8/SVneo cells. Agonists to PGE2 receptors, EP1, EP2 and EP4, promoted the migration of HTR-8/SVneo cells. Moreover, stimulation with LIF up-regulated EP1, EP2 and EP4 expression in HTR-8/SVneo cells.
CONCLUSIONS: It is suggested that LIF participates in placentation through EVT invasion by up-regulating PGE2 production and PGE2 receptor expression in first trimester EVT.
Key words:
extravillous trophoblast/leukemia inhibitory factor/interleukin-1/migration/prostaglandin E2
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Introduction |
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The cytokine network in the feto-maternal interface is important for mammalian implantation and for the maintenance of pregnancy. It is reported that leukemia inhibitory factor (LIF) is one of the most important factors in mammalian implantation (Bhatt et al., 1991
; Shen and Leder, 1992; Stewart et al., 1992; Vogiagis and Salamonsen, 1999; Suan, 2005). LIF is a pleiotrophic IL-6 family cytokine that induces the differentiation of mouse myeloid leukemic M1 cells to macrophages (Vogiagis and Salamonsen, 1999; Kondera-Anaz et al., 2004; Suan, 2005). The low affinity complex of LIF and LIF specific receptor (LIFR) binds to the gp130 chain, which is a common receptor for IL-6 family cytokines, forms a high affinity receptor complex (Vogiagis and Salamonsen, 1999; Cheng et al., 2001; Kondera-Anaz et al., 2004) and expresses several biological activities. In 1992, Stewart et al. reported that implantation was completely abrogated in LIF-deficient female mice. Furthermore, they indicated that embryos from LIF-deficient mice could be implanted in the uteri of wild-type female mice, but embryos from wild-type mice could not be implanted in the uteri of LIF-deficient female mice (Stewart et al., 1992). The concentration of LIF is up-regulated in mouse uterine glands at day 4 of pregnancy and in the human uterine endometrium at the mid-secretory phase of the menstrual cycle (Bhatt et al., 1991; Shen and Leder, 1992; Senturk and Arich, 1998; Chen et al., 1999; Sharkey et al., 1999; Vogiagis and Salamonsen, 1999; Cai et al., 2000; Cheng et al., 2001; Lass et al., 2001; Kondera-Anaz et al., 2004; Dimitriadis et al., 2005b; Suan, 2005). These time points correspond to the implantation window. In humans, a low concentration of LIF in uterine flushing fluid and mutations of the LIF gene correlate with infertility (Vogiagis and Salamonsen, 1999; Steck et al., 2004; Dimitriadis et al., 2005b; Kralickova et al., 2005; Suan, 2005). These reports indicate that maternal LIF is an important cytokine for implantation. Moreover, several reports have indicated the importance of LIF in the maintenance of early pregnancy. The LIF gene is expressed in human decidua during all stages of normal pregnancy, and a lower expression of LIF mRNA in decidua correlates with abortion (Sawai et al., 1995; Ren et al., 1997; Wang et al., 2000; Lass et al., 2001; Xia et al., 2001; Li et al., 2002; Steck et al., 2004; Dimitriadis et al., 2005b; Poehlmann et al., 2005). LIF mRNA is expressed at a higher level in the first trimester decidua than in the second trimester decidua (Ren et al., 1997; Sharkey et al., 1999; Lass et al., 2001; Kondera-Anaz et al., 2004; Suan, 2005). LIF is produced mainly by increased CD16–CD56bright NK cells and type II helper T (Th2) cells at the implantation site decidua (Sharkey et al., 1999; Kondera-Anaz et al., 2004; Dimitriadis et al., 2005b). Since human EVT express LIFR and gp130 mRNA, it is possible that LIF participates in placentation by the stimulation of first trimester EVT (Sharkey et al., 1999; Aghajanova, 2004; Poehlmann et al., 2005; Suan, 2005). However, there is a contradictory report that LIF suppresses first trimester EVT invasion (Bishop et al., 1995; Sharkey et al., 1999).
Prostaglandin E2 (PGE2) is also an important factor in the implantation process such as blastocyst attachment to the endometrium and decidualization (Jacobs and Carson, 1993; Song et al., 2000; Wang et al., 2004; Dimitriadis et al., 2005a). Recently, it has been suggested that decidua-derived PGE2 induces human first trimester EVT migration through PGE2 receptor (EP1 and EP4) in a paracrine manner (Nicola et al., 2005); however, there is a contradictory report that PGE2 inhibits the proliferation and migration of human first trimester EVT (Biondi et al., 2006). PGE2 is a member of the prostanoid family, which is synthesized from arachidonic acid (AA). First, AA is liberated from membrane-bound phospholipids by the action of cytosolic phospholipase A2 (cPLA2) or secretory PLA2 (sPLA2) (Jakobsson et al., 1999; Stichtenoth et al., 2001; Kuroda and Yamashita, 2003). Liberated AA is immediately converted to PGH2 by two cyclooxygenase (COX) isoforms, COX-1 and COX-2 (Jakobsson et al., 1999; Stichtenoth et al., 2001; Kuroda and Yamashita, 2003). PGH2 is converted to PGE2 by PGE2 synthase (PGES), which is a terminal prostanoid synthase. There are two isoforms of PGES, cytosolic PGES (cPGES) and microsomal PGES (mPGES) (Jakobsson et al., 1999; Stichtenoth et al., 2001; Kuroda and Yamashita, 2003). There are two mPGES subtypes, mPGES-1 and mPGES-2 (Tanikawa et al., 2002; Meadows et al., 2004). It has been reported that COX-2 and mPGES-1 are expressed in human first trimester EVT cells as detected by immunostaining (Meadows et al., 2004).
IL-1 is a PGE2 promoting factor at the feto-maternal interface (Ishihara et al., 1995; Meadows et al., 2004). Moreover, it is reported that IL-1 is localized in first trimester implantation site (Yagel et al., 1989; Steele et al., 1992; Kelly et al., 1995; Bennett et al., 1999; Nilkaeo and Bhuvanath, 2006). Since COX-2 expression is reduced in endometrial stromal cells in LIF-deficient mice (Song et al., 2000; Suan, 2005), we hypothesized that LIF, in addition to IL-1, plays an important role in PGE2 production in the feto-maternal interface.
In this study, we investigated the role of LIF in PGE2 production and in migration of human first trimester EVT cell line, HTR-8/SVneo, and have found that LIF participates in human first trimester EVT migration by up-regulating PGE2 production and PGE2 receptor expression.
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Reagents
Recombinant human LIF was purchased from Sigma (St Louis, MN, USA). Recombinant human IL-1
was purchased from Peproteche (London, England). Indomethacin, NS398, 17-phenyl trinor PGE2, CAY10399, PGE1 alcohol, anti-COX-2 antibody, anti-mPGES-1 antibody, anti-EP1 receptor antibody, anti-EP2 receptor antibody and anti-EP4 receptor antibody were purchased from Cayman Chemicals (Ann Arbor, MI, USA). All antibodies are rabbit-derived polyclonal antibodies. As a control, normal rabbit IgG were purchased from Bethyl Laboratories (Montogomery, TX, USA). Indomethacin and NS398 were dissolved in 100% ethanol at the concentration of 1 mM and were stored at –20°C. We diluted these stock solutions with PBS to the final concentration of 1 µM when used. We confirmed that the diluted ethanol (0.1%) had no adverse effect. HRP-conjugated goat anti-rabbit Ig was purchased from Amersham Biosciences (Uppsala, Sweden).
EVT cell line and culture
An immortalized first trimester EVT cell line, HTR-8/SVneo, was kindly donated by Professor Charles H. Graham (Department of Anatomy and Cell Biology, Queen's University, Kingston, Ontario, Canada). HTR-8/SVneo retained all phenotypic features and functional characteristics of the non-transformed parental trophoblast cells (Graham et al., 1993; Nicola et al., 2005). This cell line expresses all markers of EVT in situ: cytokeratin 7, 8 and 18, placental type alkaline phosphatase, high affinity urokinase-type plasminogen activator receptor, IGF-II, HLA framework antigen w6/32 and integrins 
1, 3, 5, 1 and v3/5 (Irving et al., 1995; Nicola et al., 2005). Therefore, we think that this cell line has a similar character to EVT. In addition, this cell line has also been used by other studies as a model of human first trimester EVT migration (Nicola et al., 2005; Biondi et al., 2006). Cells were cultured in RPMI 1640 medium (Nissui Pharmaceutical, Tokyo, Japan) supplemented with 10% FCS (BioWhittaker, Walkersville, MD, USA), 2 mM glutamine, 50 U/ml penicillin and 50 µg/ml streptomycin (all from Life Technologies, Rockville, MD, USA) at 37ºC, 5% CO2.
In vitro stimulation of cells
For PGE2 assay, HTR-8/SVneo cells (1.5 x 105/ml/well) were seeded into 24-well plates (Falcon 3047, BD Biosciences, Franklin Lakes, NJ, USA) and were stimulated with LIF (10 ng/ml), IL-1(10 ng/ml) or a combination of LIF and IL-1 for 48 h. As a preliminary experiment, we checked dose–response of LIF and IL-1, and found that 10 ng/ml of LIF and IL-1 showed maximum response. Therefore, we presented only the result of 10 ng/ml LIF and IL-1 in figures. In some experiments, Indomethacin (1 µM), a COX inhibitor, or NS398 (1 µM), a COX-2 selective inhibitor, was added alone or to the group of co-stimulation with LIF and IL-1. Cell-free culture supernatants were collected and used for PGE2 assay as described below. For the RT–PCR assay, HTR-8/SVneo cells (5 x 105/2 ml/well) were seeded into six-well plates (Falcon 3046), and were stimulated with LIF (10 ng/ml), IL-1(10 ng/ml) or a combination of LIF and IL-1 for 4 h. For Western blot analysis, HTR-8/SVneo cells (5 x 105/2 ml/well) were seeded into six-well plates, and were stimulated for 18 h.
mRNA analysis
mRNA expression was assessed by RT–PCR as described previously (Kuroda and Yamashita, 2003). Since the system of real-time RT–PCR is not available for us, we compared mRNA expressions semi-quantitatively by changing PCR cycles. Briefly, total cellular RNA was extracted by TRIzol (Life Technologies) according to the manufacturer's protocol. First strand cDNA was synthesized from 5 µg of total RNA by Superscript II RNase H-reverse transcriptase (Life Technologies) according to the manufacturer's instruction using 0.25 µg of random primer (Life Technologies). One-tenth of the synthesized cDNA was amplified by PCR using 50 pmol sense and antisense primers with 1 U of Taq polymerase (Boehringer Mannheim, Mannheim, Germany) in a total volume of 50 µl. The sequences of the sense and antisense primers, product size and the number of PCR cycles were as follows: -actin, 5'-GGGTCACCCACACTG TGCCCATCTA-3' and 5'-AGCATTTGCGGTGGACGATGGAG GG-3', 838 bp, 18 and 22 cycles; LIFR, 5'-CAAAAGAGTGTCTG TGAG-3' and 5'-CCATGTATTTACATTGGC-3', 459 bp, 26 and 30 cycles; gp130, 5'-TAAAGGCATACCTTAAACAAGC-3' and 5'-GTGAATTCTGGACCATCCTTCC-3', 292 bp, 25 and 29 cycles; COX-2, 5'-TTCAAATGAGATTGTGGGAAAATTGCT-3' and 5'-AGATCATCTCTGCCTGAGTATCTT-3', 255 bp, 31 and 35 cycles; mPGES-1, 5'-CTTTTCCTGGGCTTCGTCTA-3' and 5'-ACAATCTGGAAGGAACATCAAG-3', 259 bp, 29 and 33 cycles. PCR conditions for -actin, gp130, COX-2 and mPGES-1 were 94°C for 1 min for denaturation, 55°C for 1 min for annealing and 72°C for 2 min for extension; and for LIFR, 94°C for 1 min for denaturation, 50°C for 1 min for annealing and 72°C for 2 min for extension. A portion (12 µl) of the PCR products was electrophoresed using a 1.5% agarose gel. After ethidium bromide staining (Sigma), PCR products were visualized by UV illumination. Each experiment was repeated five times.
Western blot analysis
After in vitro stimulation, the cells were lysed on ice with 100 µl of Laemmli sample buffer solution (Kuroda and Yamashita, 2003). The obtained samples were boiled for 5 min. The same amounts of proteins (40 µg) from each experimental group were loaded on e-PAGEL (ATTO, Tokyo), electrophoresed, transferred to a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA, USA) and blotted with normal rabbit IgG (1/500), anti-COX-2 Ab, anti-mPGES Ab (1/1000), anti-EP1 receptor Ab, anti-EP2 receptor Ab or anti-EP4 receptor Ab (1/500) and HRP-conjugated secondary Ab (1/1000). The reactive bands were visualized with Lumi-LightPLUS Western blotting substrate (Roche Diagnostics, Indianapolis, IN, USA) as a substrate and Fluorochem (Alpha Innotech, San Leandro, CA, USA) as a detector. Each experiment was repeated three times. Since normal rabbit IgG did not detect any bands at the position of the corresponding protein, the results with normal IgG was not shown in the Western blotting.
PGE2 assay
PGE2 was measured using a STAT-PGE2 enzyme immunoassay kit (Cayman Chemicals) according to the manufacturer's protocol. Each treatment was performed in triplicates and repeated five times.
Cell growth assay
HTR-8/SVneo cells (3 x 105/2 ml/well) were seeded into six-well plate in RPMI1640 medium supplemented with 10% FCS. Three hours later, cells were stimulated with LIF (10 ng/ml), IL-1 (10 ng/ml), Indomethacin (1 µM), NS398 (1 µM) or a combination of LIF and IL-1 in the presence or absence of Indomethacin or NS398. In some experiments, PGE2 (10 
1000 nM) was added to the cell cultures. After 48 h, cells were collected, and the cell numbers were counted using hemocytometer. Each treatment was performed in triplicates and repeated three times. Cell proliferation rates were calculated as follows: Growth rate = number of agent-treated cells/ number of non-treated cells.
Alamar blue assay
HTR-8/SV neo cells (5 x 103/200 µl/well) were seeded into 96-well plates (Falcon 3072) in RPMI1640 medium supplemented with 10% FCS. Cells were stimulated with LIF (10 ng/ml), IL-1 (10 ng/ml) or a combination of LIF and IL-1 in the presence or absence of Indomethacin (1 µM) or NS398 (1 µM). In some experiments, PGE2 (10 1000 nM) were added to the cell cultures. After 48 h, 20 µl Alamar Blue (Alamar Biosciences, Wako, Osaka) was added to each wells, and the absorbance was measured at 570 nm. Each treatment was performed in triplicate and repeated three times.
Migration assay
The migration of HTR-8/SVneo cells was measured according to the report of Nicola et al. (2005) with a slight modification using 24-well Falcon notched plates (Falcon 3047) and Falcon cell culture inserts with microporous polycarbonate membranes of 8.0 µm pore size (Falcon 353097). In brief, the lower chamber was filled with 800 µl RPMI 1640 medium supplemented with 10% FCS with or without stimulators, whereas aliquots of 5 x 104 cells suspended in 200 µl RPMI 1640 medium supplemented with 10% FCS were added to the upper chamber. Cells were stimulated by LIF (10 ng/ml), IL-1 (10 ng/ml) or a combination of LIF and IL-1 in the presence or absence of Indomethacin (1 µM) or NS398 (1 µM). In some experiments, PGE2 (10 1000 nM), 17-phenyl trinor PGE2 (1 µM, an EP1 agonist), CAY10399 (1 µM, an EP2 agonist) or PGE1 alcohol (1 µM, an EP4 agonist) were added to the culture. After 48 h, the upper surface of the membranes was wiped with cotton swabs to remove non-migratory cells, and then migrated cells in the membranes were fixed by 100% methanol and stained using Giemsa's solution (MERCK, Rahway, NJ, USA). The absolute number of migratory cells on each membrane was scored visually using a light microscope (x400 magnification). Each treatment was performed in triplicate and repeated three times. The migration index (MI) under experimental conditions was calculated as follows: MI = migrated cell numbers of agent-treated cells/migrated cell numbers of non-treated cells.
Statistical analysis
All experiments were repeated three to five times, and some representative results are shown in figures. Statistical analyses were performed using Student's t-test. A confidence level of <0.05 was considered significant.
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LIFR and gp130 mRNA expression in HTR-8/SVneo cells
First, we investigated the expression of LIFR and gp130 mRNA in HTR-8/SVneo cells by RT–PCR. LIFR and gp130 mRNA were constitutively expressed, and their expression was not influenced by stimulation with LIF or IL-1
(Fig. 1).
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PGE2 production in HTR-8/SVneo cells
Next, we investigated PGE2 production in HTR-8/SVneo cells. PGE2 was not constitutively produced in HTR-8/SVneo cells, but was induced by stimulation with LIF or IL-1
(Fig. 2). Co-stimulation with LIF and IL-1 markedly enhanced PGE2 production compared with stimulation with LIF or IL-1 alone. The production of PGE2 induced by LIF and IL-1 in HTR-8/SVneo cells was markedly suppressed by Indomethacin or NS398. Indomethacin or NS398 alone suppressed PGE2 production in HTR-8/SVneo cells. Since PGE2 production by HTR-8/SVneo cells in the presence of Indomethacin or NS398 without stimulator is very low, the data are not shown in Fig. 2.
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COX-2 and mPGES-1 mRNA and protein expressions in HTR-8/SVneo cells
It is known that COX-2 and mPGES-1 are important enzymes for PGE2 production in several types of cells (Jakobsson et al., 1999
; Stichtenoth et al., 2001; Kuroda and Yamashita, 2003). Therefore, we investigated the expressions of COX-2 and mPGES-1 in HTR-8/SVneo cells by RT–PCR and Western blotting. IL-1 induced COX-2 mRNA expression in HTR-8/SVneo cells. LIF also induced COX-2 mRNA in HTR-8/SVneo cells. Co-stimulation with LIF and IL-1 further enhanced the expression of COX-2 mRNA compared with the stimulation with LIF or IL-1 alone (Fig. 3A). The expression of mPGES-1 mRNA was also enhanced by LIF or IL-1; however, the degree of expression was not significantly changed by co-stimulation with LIF and IL-1 (Fig. 3A). To compare mRNA expression levels more accurately, we changed the number of PCR amplification cycles. Each mRNA expression level was amplified by increase in the PCR cycles, and the increase of mRNA expression level by the stimulation with reagents was similarly observed in both PCR cycles. The expression of COX-2 and mPGES-1 protein was also enhanced by stimulation with LIF or IL-1, and co-stimulation with LIF and IL-1 further enhanced their expression compared with stimulation with LIF or IL-1 alone (Fig. 3B).
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Effects of LIF and PGE2 on HTR-8/SVneo cell proliferation
To study the autocrine effect of increased PGE2 production by co-stimulation with LIF and IL-1
on the growth of HTR-8/SVneo cells, we investigated the proliferation of HTR-8/SVneo cells. In a cell growth assay, LIF slightly stimulated the proliferation of HTR-8/SVneo cells, but IL-1 did not (Fig. 4). The co-stimulation with LIF and IL-1 stimulated the proliferation compared with the non-treated group; however, it was not significantly different from those treated with LIF or IL-1 alone. Moreover, the co-stimulation with LIF and IL-1 + Indomethacin stimulated the proliferation compared with the non-treated group; however, this was also not significant from the groups treated with LIF, IL-1 or LIF + IL-1. Treatment with PGE2 did not affect the growth of HTR-8/SVneo cells. Treatment with Indomethacin or NS398 alone did not affect the growth of HTR-8/SVneo cells (data not shown). In the Alamar Blue assay, all reagents did not have any cell growth effects on HTR-8/SVneo cells (data not shown).
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Effects of LIF and PGE2 on HTR-8/SVneo cell migration
Next, we investigated the effect of LIF and PGE2 on the migration of HTR-8/SVneo cells. PGE2 enhanced the migration of HTR-8/SVneo cells in a dose-dependent manner (Fig. 5). LIF or IL-1
also stimulated HTR-8/SVneo cell migration, and co-stimulation with LIF and IL-1 markedly enhanced it. The enhanced migration was reduced by Indomethacin or NS398. Indomethacin or NS398 alone did not influence the migration of HTR-8/SVneo cells (data not shown).
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Role of PGE2 receptors in HTR-8/SVneo cell migration
It is known that PGE2 expresses its function through four types of PGE2 receptors, EP1, EP2, EP3 and EP4 (Kuroda and Yamashita, 2003
; Nicola et al., 2005; Biondi et al., 2006). It is suggested that EP1, EP2 and EP4 are concerned with first trimester EVT migration (Nicola et al., 2005; Biondi et al., 2006); therefore, we investigated the role of PGE2 receptors in HTR-8/SVneo cell migration. 17-Phenyl trinor PGE2 (an EP1 agonist), CAY10399 (an EP2 agonist) and PGE1 alcohol (an EP4 agonist) stimulated HTR-8/SVneo cell migration (Fig. 6). However, these reagents did not have any growth effects on HTR-8/SVneo cells (data not shown).
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PGE2 receptor protein expression in HTR-8/SVneo cells
Finally, we investigated the role of LIF in PGE2 receptor protein expressions in HTR-8/SVneo cells. It is reported that all types of EP receptor mRNAs are expressed in HTR-8/SVneo cells (Nicola et al., 2005
). In our study, EP1, EP2 and EP4 receptors were expressed in HTR-8/SVneo cells and were enhanced by LIF stimulation (Fig. 7).
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Discussion |
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It is reported that LIF plays an important role in implantation. In this communication, we studied the role of LIF and its relationship with PGE2 in the invasion of EVT cells using a human first trimester EVT cell line, HTR-8/SVneo. We suggest that LIF participates in the migration of HTR-8/SVneo cells by inducing PGE2 production through expression of COX-2 and mPGES-1. We also found that LIF up-regulates the expression of PGE2 receptors, EP1, EP2 and EP4.
LIFR and gp130 mRNAs were constitutively expressed in HTR-8/SVneo cells. So, we considered that this cell line may respond to stimulation with LIF. There are some reports concerning the action of LIF on trophoblasts in implantation and placentation. LIF promotes differentiation from cytotrophoblasts to anchoring trophoblasts (Senturk and Arich, 1998; Lass et al., 2001). There is a report that LIF has no effect on trophoblast proliferation (Sharkey et al., 1999), whereas another report indicates that LIF promotes the proliferation of gestational trophoblastic neoplasia, a choriocarcinoma cell line, Jeg 3 cells (Fitzgerald et al., 2005a,b). Fitzgerald et al. reported that LIF promotes first trimester EVT invasion (Fitzgerald et al., 2005a,b; Poehlmann et al., 2005); however, Sharkey et al. reported that LIF suppressed EVT invasion. In this way, evidence for the role of LIF in first trimester EVT invasion is contradictory.
Recently, it has been reported that PGE2 is involved in first trimester EVT proliferation, migration and invasion (Meadows et al., 2004; Nicola et al., 2005; Biondi et al., 2006). Meadows et al. indicated that COX-2 and mPGES-1 proteins were detected in first trimester EVT by immunostaining. They considered that PGE2 produced by COX-2 and mPGES-1 expression in first trimester EVT may cause EVT invasion in an autocrine manner. Biondi et al. (2006) reported that PGE2 production from HTR-8/SVneo cells was stimulated by IL-1. So, we used IL-1 as a PGE2 stimulation factor in HTR-8/SVneo cells. Other factors that induce PGE2 in first trimester EVT remain to be clarified. In this study, we found that stimulation with LIF enhanced PGE2 production most probably through the actions of COX-2 and mPGES-1 in HTR-8/SVneo cells, as did stimulation with IL-1. Though LIF enhanced PGE2 production significantly in HTR-8/SVneo cells, the amount of PGE2 produced by LIF stimulation was lower than that of PGE2 produced by IL-1 stimulation. Therefore, in order to investigate the action of LIF in particular in the PGE2 production, we also examined the synergistic effect of LIF and IL-1 stimulation to the PGE2 production in HTR-8/SVneo cells. We demonstrated that co-stimulation with LIF and IL-1 markedly enhanced PGE2 production compared with stimulation with LIF or IL-1 alone in HTR-8/SVneo cells. We consider two possibilities for the reason why a synergistic effect is seen when LIF and IL-1 are added together. One possibility is an interaction of several transcription factors. The combination of cytokines induces different transcription factors, which interact each other, and consequently, gene expression is increased more efficiently compared with the stimulation with one cytokine alone. Another possibility is that one cytokine up-regulates the expression of other cytokine receptors. Although the expression of total LIFR and gp130 mRNA were not influenced by co-stimulation with LIF or IL-1, the sensitivity and capability of promoting signal transduction through these receptors may be affected. Moreover, there is a possibility that LIF may enhance the expression of IL-1 receptors. Since PGE2 production from HTR-8/SVneo cells was suppressed by NS398, it is considered that COX-2 induced by co-stimulation with LIF and IL-1 play an important role in PGE2 production by HTR-8/SVneo cells. These results suggest that LIF participates in implantation or placentation through PGE2 production in first trimester EVT.
It is well known that PGE2 promotes the proliferation and migration of several cell types including cancer cells (Tsuji et al., 1998; Timoshenko et al., 2003); however, the role of PGE2 in first trimester EVT proliferation and migration remains to be clarified. There are two different reports concerning the role of PGE2 in proliferation and migration in HTR-8/SVneo cells (Nicola et al., 2005; Biondi et al., 2006). Nicola et al. (2005) demonstrated that PGE2 promoted HTR-8/SVneo cell migration through EP1 and EP4 receptors. They also indicated that PGE2 was markedly produced by first trimester decidua in human pregnancy, and suggested a functional relationship between a low concentration of PGE2 in first trimester decidua and the progression of pre-eclampsia (Nicola et al., 2005). On the other hand, Biondi et al. (2006) demonstrated that PGE2 suppressed HTR-8/SVneo cell proliferation and migration through EP2 and EP4 receptors, by increasing intracellular cAMP. They concluded that PGE2 worked as an important control factor for EVT functions by preventing excessive proliferation and migration (Biondi et al., 2006). It seems that the differences of these results are due to different experimental conditions such as the concentration of PGE2 used or the stimulation period with PGE2.
We found that LIF slightly promoted HTR-8/SVneo cell proliferation; however, PGE2 did not influence their proliferation. These results are consistent with Nicola's report which indicates that PGE2 does not influence HTR-8/SVneo cell proliferation (Nicola et al., 2005), although Biondi et al. (2006) have shown that PGE2 suppresses HTR-8/SVneo cell proliferation. Moreover, IL-1 also did not show any cell growth effect on HTR-8/SVneo cells. Therefore, it is considered that the effect of IL-1 and PGE2 on cell growth do not affect the migration assay performed afterwards.
It is reported that there are a large number of promoting factors on trophoblast migration produced at the feto-maternal interface, such as EGF family, IGF II, IGFBP-1, uPA, ET-1 and hepatocyte growth factor (HGF) (Gleeson et al., 2001; Mckinnon et al., 2001; Nicola et al., 2006). On the other hand, a number of inhibiting factors are also reported, such as decidua-derived TGF and TNF (Xu et al., 2001; Bauer et al., 2004). However, we have not found any report concerning the role of IL-1 on trophoblast migration or invasion. We found that exogenous PGE2, LIF and IL-1 each stimulated HTR-8/SVneo cell migration. Moreover, we also demonstrated that the co-stimulation with LIF and IL-1 markedly enhanced HTR-8/SVneo cell migration compared with the stimulation with LIF or IL-1 alone. Since the migration of HTR-8/SVneo cells stimulated with LIF and IL-1 was suppressed by Indomethacin or NS398, it is suggested that PGE2 produced from HTR-8/SVneo cells through the action of COX-2 promotes their migration in an autocrine manner. Our results are consistent with the report of Nicola et al. (2005), which indicated that PGE2 stimulates HTR-8/SVneo cell migration. Although Biondi et al. (2006) reported that PGE2 suppressed HTR-8/SVneo cell migration, we consider that PGE2 stimulates the migration of HTR-8/SVneo cells, because PGE2 promoted their migration in a dose-dependent manner and COX inhibitor inhibited their migration in our experiments. Cell migration is usually determined over a shorter time frame so that the migration is not influenced by cellular proliferation. We carried out migration assays at 48 h according to the report of Nicola et al. (2005). We also carried out migration assay at 6 h, but none of the reagents had stimulating effects on HTR-8/SVneo cell migration at this culture interval. As we carried out migration assays at 48 h, there is a possibility that the increased cell number on the membrane after treatment with LIF is affected by cell proliferation. However, since the cell numbers in the migration assay are much more than the increase in cell numbers in cell growth assay, we consider that most of the cells in the migration assay is caused by cell migration, not by cell proliferation.
There are four membrane-bound G protein-coupled PGE2 receptors, EP1, EP2, EP3 and EP4, to express the biological functions of PGE2. The actual role of these receptors in the proliferation and migration of first trimester EVT also remains to be clarified. Using a PGE2 receptor agonist, we suggest the possibility that PGE2 promotes HTR-8/SVneo cell migration through EP1, EP2 and EP4. We also found that the stimulation with LIF increased EP1, EP2 and EP4 protein expression in HTR-8/SVneo cells detected by Western blotting. Therefore, it is suggested that LIF promotes the migration of HTR-8/SVneo cells through the induction of EP1, EP2 and EP4 expression in HTR-8/SVneo cells.
The presence of PGE2 receptors (EP2 and EP4) in human first trimester chorionic villi (Biondi et al., 2006) has been reported. But there is no report to indicate the presence of EP1 receptor in vivo. However, all receptors (EP1, EP2, EP3 and EP4) are reportedly expressed in second and third trimester placenta and fetal membranes (Grigsby et al., 2006). The expression of PGE receptors, LIF and IL-1 in first trimester chorionic villi are almost similar to their expression in HTR-8/SVneo cells (Yagel et al., 1989; Steele et al., 1992; Kelly et al., 1995; Sawai et al., 1995; Bennett et al., 1999; Biondi et al., 2006; Nilkaeo and Bhuvanath, 2006). So, we consider that our study using a first trimester EVT cell line, HTR-8/SVneo, is relevant to the in vivo scenario.
In conclusion, we found that LIF stimulates HTR-8/SVneo cells to induce PGE2 production by up-regulating COX-2 and mPGES-1 expression. And we suggested that PGE2 produced from HTR-8/SVneo cells by LIF stimulation promoted their migration through EP1, EP2 and EP4. LIF also increased the expression of EP1, EP2 and EP4 proteins in HTR-8/SVneo cells. Therefore, it is suggested that LIF participates in placentation and the maintenance of early pregnancy by stimulation of first trimester EVT invasion through PGE2 production and PGE2 receptor expression. Our data suggest that LIF may be useful for the treatment of not only implantation failure but also diseases that retard EVT invasion such as early abortion, pre-eclampsia or certain types of intrauterine growth retardation. Hereafter, it will be necessary to study the role of LIF in these diseases.
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We thank Dr Charles H. Graham (Department of Anatomy and Cell Biology, Queen's University, Kingston, Ontario, Canada) for generously supplying HTR-8/SVneo cells. This study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (18591618) and a Grant for Advanced Research from the University of Occupational and Environmental Health, Japan.
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Submitted on November 28, 2006; resubmitted on April 10, 2007; accepted on April 17, 2007.
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