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Changes in Expression and Function of Syncytin and its Receptor, Amino Acid Transport System B0 (ASCT2), in Human Placental Choriocarcinoma BeWo Cells During Syncytialization
Placenta (2002), 23, 536–541 doi:10.1053/plac.2002.0839, available online at http://www.idealibrary.com on
Changes in Expression and Function of Syncytin and its Receptor, Amino Acid Transport System B0 (ASCT2), in Human Placental Choriocarcinoma BeWo Cells During Syncytialization Y. Kudo and C. A. R. Boyd Department of Human Anatomy and Genetics, University of Oxford, Oxford, UK Paper accepted 25 April 2002
Relative abundance of mRNAs encoding syncytin and its receptor, amino acid transport system B0, and activity of amino acid transport thought to be through this system have been studied in parallel in a cell model of syncytialization (BeWo cell following forskolin treatment). Relative mRNA abundance (determined by reverse transcription-polymerase chain reaction) for syncytin showed stimulation by forskolin. In contrast, the level of amino acid transporter B0 mRNA expression was lower in forskolin treated cells. Na + -dependent -(methylamino)isobutyric acid insensitive -alanine transport was similarly decreased significantly in cells treated with forskolin suggesting that there is modulation of cell surface expression of the syncytin receptor associated with syncytialization. 2002 Published by Elsevier Science Ltd. Placenta (2002), 23, 536–541
INTRODUCTION The trophoblast surface of the human placenta, in direct contact with maternal blood, is formed from the syncytiotrophoblast which is derived from the underlying cell layer, the cytotrophoblast. The syncytiotrophoblast layer plays an important role throughout pregnancy since it is the site of various placental functions, including nutrient exchange and the synthesis of steroid and peptide hormones which are required for normal foetal growth and development. The unique process of syncytialization of this tissue is very poorly understood. Mi et al. (2000) have shown that syncytin, a protein encoded by an envelope gene of the recently identified human endogenous retrovirus-W (ERV-W) (Blond et al., 1999), is specifically expressed in human placenta and may be involved in syncytialization of trophoblast. Blond et al. (2000) also showed that the ERV-W envelope gene encodes a highly fusogenic membrane glycoprotein that can induce syncytium formation upon interaction with the type D mammalian retrovirus receptor. Immunohistochemical analysis showed ERV-W envelope protein expression restricted to placenta, particularly in the cytotrophoblast and with stronger expression in the syncytiotrophoblast cell layer (Blond et al., 2000). Recently type D mammalian retrovirus receptor has been identified as amino acid transporter B0 (ASCT2) (Tailor et al., 1999). The gene encoding this protein was originally cloned from a human placental choriocarcinoma cell line (Kekuda et al., 1996). System B0 is a Na + -dependent transporter with broad substrate specificity for neutral amino acids including -alanine, -glutamine, -leucine and glycine (Yudilevich and 0143–4004/02/$-see front matter
Boyd, 1987). Anionic amino acids, cationic amino acids and N-methylated amino acids are excluded by this system. The presence of mRNA transcripts for system B0 in the human placenta has been reported (Kekuda et al., 1996). The functional expression of amino acid transporter B0 has been shown in the basal plasma membrane of human placental syncytiotrophoblast (Na + -dependent ‘system 3’, Kudo and Boyd, 1990). Immunohistochemical analysis of syncytin in normal placenta has demonstrated that this protein localizes predominantly to the basal plasma membrane of syncytiotrophoblast (Lee et al., 2001). This co-localization of syncytin and its receptor may facilitate the incorporation of the proliferating cytotrophoblast into the syncytiotrophoblast. BeWo choriocarcinoma cells can be induced to differentiate into a polarized syncytiotrophoblast in vitro by forskolin (Wice et al., 1990). We have studied both expression of mRNA encoding syncytin and its receptor, amino acid transporter B0, genes and functional changes in amino acid transport thought to be through this system using BeWo cells as a cell model of syncytialization.
MATERIALS AND METHODS Material BeWo cells (passage number approximately 40) were kindly gifted by Dr S. L. Greenwood (Academic Unit of Child Health, St Mary’s Hospital, University of Manchester). -[2,3-3H]Alanine (59.0 Ci mmol 1 or 2.18 TBq mmol 1) 2002 Published by Elsevier Science Ltd.
Kudo and Boyd: Trophoblast Syncytialization and Amino Acid Transport
was purchased from Amersham Life Science (Amersham, Buckinghamshire, UK). Forskolin and -(methylamino)isobutyric acid were obtained from Sigma-Aldrich Chemical (Poole, Dorset, UK), tissue culture supplements were from Gibco BRL (Paisley, UK) and the human chorionic gonadotropin (hCG) chemiluminescent immunoassay kit was from Euro/DPC (Llanberis, Gwynedd, UK). QuickPrep Total RNA Extraction Kit were purchased from Amersham Pharmacia Biotech (Rainham, Essex, UK), moloney murine leukemia virus (M-MLV) reverse transcriptase, oligo(dT)12–18 primer, deoxynucleotide 5 -triphosphate (dNTP) and Taq DNA polymerase were from Gibco BRL, deoxyribonuclease I (DNase I) was from Promega (Southampton, Hampshire, UK). All chemicals were of the highest purity commercially available.
BeWo cell culture BeWo cells were cultured at 37C as monolayers in Ham’s F-12 medium supplemented with 10 per cent foetal bovine serum (FBS), 2 m -glutamine, 100 U ml 1 penicillin and 100 U ml 1 streptomycin in a humidified atmosphere of 5 per cent CO2 and 95 per cent air. Cells were subcultured by treating with 0.05 per cent trypsin in Ca2+ - and Mg2+ -free phosphate buffer saline (PBS) containing 0.02 per cent ethylenediaminetetraacetate, seeded in 35 mm plastic culture dishes and grown for 2–3 days to the stage of 50 per cent confluence. At 50 per cent confluency, the medium was then changed to the one containing 100 forskolin or vehicle (dimethyl sulfoxide), followed by further incubation for the indicated times at 37C. At the end of the incubation period, cells were more than 95 per cent viable as assessed by trypan blue dye exclusion. Cultures were conducted in triplicate for each set of experiments to assess reproducibility. The conditioned medium was collected and centrifuged at 3000 g at 4C for 10 min to remove cellular debris and stored at 70C until use.
hCG secretion hCG secretion was determined by measuring its concentrations in the conditioned medium by an immunoassay kit which specifically detects -chain of hCG.
RNA extraction and reverse transcription– polymerase chain reaction (RT-PCR) analysis Relative mRNA abundance of syncytin and amino acid transporter B0 was analysed by semi-quantitative RT-PCR using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal standard. Total RNA was extracted from cultured cells with QuickPrep Total RNA Extraction Kit according to the manufacture’s protocol. RNA samples were
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Figure 1. Amplification of syncytin, amino acid transporter B0 and glyceraldehyde phosphate 3-dehydrogenase mRNA from BeWo cells by RT-PCR. The amplification products were separated by agarose gel electrophoresis and visualised by ethidium bromide staining. The intensity of each target gene band was quantitated using a gel documentation and analysis system. , syncytin; , amino acid transporter B0 (ASCT2); , glyceraldehyde phosphate 3-dehydrogenase (GAPDH).
treated with DNase I before RT-PCR to remove any contaminating DNA. The primers used in the subsequent RT-PCR were as follows: syncytin (Mi et al., 2000), forward, 5 -AGGAGCTTCGAAACACTGGA-3 and backward, 5 GTGAGCTAAGTTGCAAGCCC-3 ; amino acid transporter B0 (Kekuda et al., 1996), forward, 5 -GGCTTGG TAGTGTTTGCCAT-3 and backward, 5 -GGGCAAAGA GTAAACCCACA-3 ; GAPDH (Tso et al., 1985), forward, 5 -CGGGAAGCTTGTGATCAATGG-3 and backward, 5 -GGCAGTGATGGCATGGACTG-3 . The expected sizes of the PCR products are 494 bp for syncytin, 205 bp for amino acid transport system B0 and 358 bp for GAPDH. One microgram RNA was reverse transcribed into cDNA using oligo(dT)12–18 primer. The reverse transcription reaction, containing 500 dNTP, 25 g ml 1 oligo(dT)12–18 primer, 10 unit l 1 M-MLV reverse transcriptase, 3 m MgCl2, 75 m KCl, 10 m dithiothreitol and 50 m Tris-HCl (pH 8.3), was sequentially incubated at 25C for 10 min, at 42C for 50 min and at 70C for 15 min and cooled on ice. As a negative control for the absence of exogenous DNA contamination, reactions run without RNA or with RNA in the absence of the reverse transcriptase revealed no amplified product (data not shown). The synthesized cDNA (0.05 g equivalent to RNA) was used for PCR amplification in a reaction mixture containing 200 dNTP, 1 forward and backward primers, 0.05 unit l 1 Taq DNA polymerase, 1.5 m MgCl2, 50 m KCl and 20 m Tris-HCl (pH 8.4). The PCR conditions were: 94C for 3 min, 60C for 1 min and 72C for 2 min; then 22 cycles (for syncytin and amino acid transporter B0) and 20 cycles (for GAPDH) of 94C for 1 min, 60C for 1 min and 72C for 2 min; followed by a 10 min final extension at 72C. The amount of template cDNA and the number of cycles were determined experimentally so that quantitative comparison could be made during the exponential
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phase of the amplification process for both target and reference gene (Figure 1). PCR products were separated on a 2 per cent agarose gel. Gels were stained with ethidium bromide. A single band for each gene was observed at the expected size. The intensity of either the target or GAPDH band for each sample was quantitated using a gel documentation and analysis system (GDS8000, Ultra-Violet Products, Cambridge, UK) and the ratio of the two was used as a normalized value for expression of each target gene. All assays were conducted in triplicate.
Amino acid influx studies After aspirating culture medium, each dish was washed twice with pre-warmed (37C) PBS and cells were depleted of intracellular amino acids by incubating in PBS at 37C for 30 min to minimize any trans effects. The influx of amino acid was initiated by replacing this with pre-warmed PBS containing 2 -[3H]alanine, followed by further incubation at 37C. Other additions are described in the figure legends. Following aspiration of isotope solution, cells were quickly washed with ice-cold PBS with 10 m unlabelled -alanine. Then 0.1 NaOH and 0.1 per cent sodium dodecyl sulphate solution were added for solubilization and aliquots were taken for liquid scintillation counting and protein determination.
Placenta (2002), Vol. 23
time-dependent increase in amino acid transport and associated mRNA expression (Smith et al., 1973; Kudo and Boyd, 2000).] Figure 3A compares 2 -alanine influx into BeWo cells before and after forskolin treatment under initial rate condition in the presence of Na + . Data show carrier-mediated influx rate defined by subtracting the diffusional component from the total influx rate. The diffusional component was determined by measuring the influx of 2 -[3H]alanine in the presence of 20 m unlabelled -alanine. Total carrier-mediated influx was not changed following 48 h culture in either the presence or absence of forskolin. In order to separate the transport pathways contributing to total flux in the presence of Na + , the unlabelled synthetic amino acid -(methylamino)isobutyric acid (Christensen, 1979), a system A specific analogue, or unlabelled -alanine was added at 2 m. The extent of the inhibition by -(methylamino)isobutyric acid (i.e. system A mediated influx) was enhanced following forskolin treatment. In contrast the rate of Na + -dependent -(methylamino)isobutyric acid insensitive -alanine influx, defined as the difference between the influx in the presence of unlabelled -alanine and the influx in the presence of -(methylamino)isobutyric acid, was significantly decreased in cells treated with forskolin (Figure 3B).
Protein estimation
DISCUSSION
Protein concentration of the cell extract was determined by the method of Lowry et al. (1951) using bovine serum albumin as a standard.
The syncytialization of trophoblast cells in placenta is central to both structure and functional properties of this tissue. Mi et al. (2000) suggested that syncytin may mediate placental cytotrophoblast fusion in vivo, and thus may be important in human placental morphogenesis. In primary culture of isolated cytotrophoblast cells the transcript levels of syncytin increase with the differentiation and fusion of cytotrophoblasts into syncytiotrophoblasts (Frendo et al., 2001). Amino acid transport system B0 is known to be a receptor for this protein (Tailor et al., 1999). Although the mechanism involved in syncytialization is currently poorly understood, we have now asked questions concerning the expression of these two molecules during this process. Our experiments show that relative mRNA abundance for syncytin and for its receptor, amino acid transport system B0, is changed reciprocally during syncytialization. Since there appears to be concomitant decrease in transport through system B0, our results suggest that the changes in amino acid transporter functional expression appear to be closely related to changes in cell morphology. Our reasons for supposing that the Na + -dependent -(methylamino)isobutyric acid insensitive -alanine influx is through system B0 is two-fold. Firstly earlier work (Kudo and Boyd, 1990) on basal syncytiotrophoblast basal membrane vesicles identified clearly a component of transport (Na + dependent system ‘3’) that matches precisely the properties of expressed system hATB0 (Kekuda et al., 1996); however in this membrane there is no transport system which matches the properties of system ASC (this observation conflicts with those
Statistical analysis Differences between groups were analysed using an ANOVA and results were considered statistically significant at P<0.05.
RESULTS Following forskolin treatment morphological fusion of BeWo cells, appearance of multinucleated cells, was readily detectable after 24 h and is dramatic after 48 h; cell fusion was hardly detectable at 48 h in the absence of forskolin. Concomitantly an associated marked increase in hCG secretion by BeWo cells was observed but only in the presence of forskolin (4.10.2-fold increase above basal level at 24 h). The relative expression of mRNA encoding syncytin and its receptor, amino acid transporter B0, following forskolin treatment was studied by RT-PCR (Figure 2). Syncytin mRNA expression level showed stimulation by forskolin (1.90.2fold) which was maximal at 48 h. Expression level of amino acid transporter B0 mRNA at this time point was lower (0.50.1-fold) in forskolin treated compared to control cells. GAPDH mRNA was constant for each sample. [As has been found by other authors culture of placental cells causes a
Kudo and Boyd: Trophoblast Syncytialization and Amino Acid Transport
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Figure 2. Effect of forskolin treatment on the relative abundance of syncytin mRNA and amino acid transporter B0 mRNA in BeWo cells. Total RNA was extracted either from BeWo cells cultured with 100 forskolin or vehicle for the time indicated. A: RT-PCR. The relative abundance of syncytin mRNA(a), amino acid transporter B0 (ASCT2) mRNA (b) and glyceraldehyde phosphate 3-dehydrogenase (GAPDH) mRNA (c) were analysed by RT-PCR as described in Methods. The results presented are from a single representative experiment. B: Relative quantitation of syncytin mRNA (a) and ASCT2 mRNA (b). The intensity of either the target gene or the GAPDH band was quantitated by using a gel documentation and analysis system of PCR products and the ratio of the two was used as a normalised relative abundance value of each transporter gene. Data represent the means.d. of three separate experiments, expressed as percentage of control (i.e. values without culture). *Significantly different from values cultured with vehicle alone (P<0.05). †Significantly different from values at 0 h (P<0.05).
by Hoeltzli and Smith (1989) suggesting there is small contribution from system ASC activity in this membrane). Since the basal membrane of syncytiotrophoblast is known to be the site
of normal syncytin interaction with its receptor, the presence of B0 transporter in this membrane is consistent with our interpretation. Secondly the finding that mRNA for ASCT2
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Placenta (2002), Vol. 23
Figure 3. Effect of forskolin treatment on -alanine influx in BeWo cells. -Alanine influx over a 3 min period before treatment (control) and after 48 h treatment with 100 forskolin or vehicle was measured in a medium containing 2 -[3H]alanine with or without 2 m unlabelled -(methylamino)isobutyric acid (MeAIB) or -alanine in the presence of Na + . A: carrier-mediated influx rate defined by subtracting the diffusional component (determined by measuring the influx of -[3H]alanine in the presence of 20 m unlabelled -alanine) from the total influx. B: system B0 mediated influx rate defined as the difference between the influx in the presence of -alanine and the influx in the presence of MeAIB. Data represent the means.d. of three separate experiments of triplicate assay. *Significantly different from values cultured with vehicle alone (P<0.05).
(encoding system B0) decreases proportionally to amino acid transport is compatible with this proposal. However, further study to confirm this interpretation will require with experiments with -leucine as an inhibitor. Additionally possible mechanisms underlying this will require further studies aimed at determining protein abundance, subcellular localization and stability. Importantly our assay technique measures function only of the syncytin receptor protein present in the plasma membrane of BeWo cells, that is protein localized to the same position as is necessary for syncytin binding. Previous work (Furesz et al., 1993) has shown that system A-, ASC- and -like transporters are responsible for -alanine transport in primary cultures of trophoblast cells isolated from full-term human placenta and that during in vitro differentiation, formation of microvillous membrane and syncytium, system ASC activity is decreased. These authors also showed that system ASC activity is decreased in forskolin treated BeWo cells. Furesz et al. thus concluded that membrane specialization accompanying fusion and differentiation of the cytotrophoblast to form syncytiotrophoblast results in a polarization of neutral amino acid transport systems. Trophoblast invasion of the maternal decidua and myometrium is a carefully orchestrated process, with insufficient infiltration of the uterine wall implicated in placental disorders such as pre-eclampsia, and uncontrolled trophoblast invasion observed in choriocarcinoma and other gestational tropho-
blastic diseases. Amino acid transport system B0 as well as syncytin might limit invasiveness by inducing trophoblast fusion and terminal differentiation. It has also been proposed that trophoblast syncytialization is the first step of a sequence leading to syncytial apoptosis which in turn is necessary for syncytial renewal (Huppertz et al., 1998). Lee et al. (2001) have shown that in pre-eclampsia syncytin gene expression in trophoblast is reduced and that syncytin is localized improperly to the apical syncytiotrophoblast microvillous membrane as opposed to its normal location on the basal syncytiotrophoblast cytoplasmic membrane. They suggest that altered expression of the syncytin gene and altered cellular location of its protein product may contribute to the aetiology of preeclampsia. Very recently it also has been reported that placental syncytin mRNA expression level is reduced in pregnancies complicated with haemolysis, elevated liver enzymes and low platelets (HELLP) syndrome (Knerr et al., 2002) or trisomy 21 (Frendo et al., 2001) in which there is a defect in syncytiotrophoblast formation. It therefore will be of interest to analyse the expression of amino acid transport system B0 immunohistochemically and functionally in placentae from these pregnancies. Interestingly a reduction of placental amino acid transport activity is well demonstrated in intrauterine growth-restricted pregnancies (Glazier et al., 1997; Norberg et al., 1998) and this disorder is commonly accompanied by pre-eclampsia (Lin et al., 1991).
ACKNOWLEDGEMENT Yoshiki Kudo was supported by Oxford Kobe Scholarship.
REFERENCES Blond JL, Beseme F, Duret L, Bouton O, Bedin F, Perron H, Mandrand B & Mallet F (1999) Molecular characterization and placental expression of HERV-W, a new human endogenous retrovirus family. J Virol, 73, 1175–1185.
Blond JL, Lavillette D, Cheynet V, Bouton O, Oriol G, Chapel Fernandes S, Mandrand B, Mallet F & Cosset FL (2000) An envelope glycoprotein of the human endogenous retrovirus HERV-W is expressed in the human placenta and fuses cells expressing the type D mammalian retrovirus receptor. J Virol, 74, 3321–3329.
Kudo and Boyd: Trophoblast Syncytialization and Amino Acid Transport Christensen HN (1979) Exploiting amino acid structure to learn about membrane transport. Adv Enzymol Relat Areas Mol Biol, 49, 41–101. Frendo JL, Therond P, Bird T, Massin N, Muller F, Guibourdenche J, Luton D, Vidaud M, Anderson WB & Evain Brion D (2001) Overexpression of copper zinc superoxide dismutase impairs human trophoblast cell fusion and differentiation. Endocrinology, 142, 3638–3648. Furesz TC, Smith CH & Moe AJ (1993) ASC system activity is altered by development of cell polarity in trophoblast from human placenta. Am J Physiol, 265, C212–C217. Glazier JD, Cetin I, Perugino G, Ronzoni S, Grey AM, Mahendran D, Marconi AM, Pardi G & Sibley CP (1997) Association between the activity of the system A amino acid transporter in the microvillous plasma membrane of the human placenta and severity of fetal compromise in intrauterine growth restriction. Pediatr Res, 42, 514–519. Hoeltzli SD & Smith CH (1989) Alanine transport systems in isolated basal plasma membrane of human placenta. Am J Physiol, 256, C630–C637. Huppertz B, Frank HG, Kingdom JC, Reister F & Kaufmann P (1998) Villous cytotrophoblast regulation of the syncytial apoptotic cascade in the human placenta. Histochem Cell Biol, 110, 495–508. Kekuda R, Prasad PD, Fei YJ, Torres Zamorano V, Sinha S, Yang Feng TL, Leibach FH & Ganapathy V (1996) Cloning of the sodiumdependent, broad-scope, neutral amino acid transporter Bo from a human placental choriocarcinoma cell line. J Biol Chem, 271, 18657–18661. Knerr I, Beinder E & Rascher W (2002) Syncytin, a novel human endogenous retroviral gene in human placenta: evidence for its dysregulation in preeclampsia and HELLP syndrome. Am J Obstet Gynecol, 186, 210–213. Kudo Y & Boyd CAR (1990) Characterization of amino acid transport systems in human placental basal membrane vesicles. Biochim Biophys Acta, 1021, 169–174. Kudo Y & Boyd CAR (2000) Heterodimeric amino acid transporters: expression of heavy but not light chains of CD98 correlates with induction of amino acid transport systems in human placental trophoblast. J Physiol, 523, 13–18.
541 Lee X, Keith JC Jr, Stumm N, Moutsatsos I, McCoy JM, Crum CP, Genest D, Chin D, Ehrenfels C, Pijnenborg R, van Assche FA & Mi S (2001) Downregulation of placental syncytin expression and abnormal protein localization in pre-eclampsia. Placenta, 22, 808–812. Lin CC, Su SJ & River LP (1991) Comparison of associated high-risk factors and perinatal outcome between symmetric and asymmetric fetal intrauterine growth retardation. Am J Obstet Gynecol, 164, 1535–1541. Lowry OH, Rosebrough NJ, Farr AL & Randall RJ (1951) Protein measurements with the Folin phenol reagent. J Biol Chem, 193, 265–275. Mi S, Lee X, Li X, Veldman GM, Finnerty H, Racie L, LaVallie E, Tang XY, Edouard P, Howes S, Keith JC Jr & McCoy JM (2000) Syncytin is a captive retroviral envelope protein involved in human placental morphogenesis. Nature, 403, 785–789. Norberg S, Powell TL & Jansson T (1998) Intrauterine growth restriction is associated with a reduced activity of placental taurine transporters. Pediatr Res, 44, 233–238. Smith CH, Adcock EWR, Teasdale F, Meschia G & Battaglia FC (1973) Placental amino acid uptake: tissue preparation, kinetics, and preincubation effect. Am J Physiol, 224, 558–564. Tailor CS, Nouri A, Zhao Y, Takeuchi Y & Kabat D (1999) A sodium-dependent neutral-amino-acid transporter mediates infections of feline and baboon endogenous retroviruses and simian type D retroviruses. J Virol, 73, 4470–4474. Tso JY, Sun XH, Kao TH, Reece KS & Wu R (1985) Isolation and characterization of rat and human glyceraldehyde-3-phosphate dehydrogenase cDNAs: genomic complexity and molecular evolution of the gene. Nucleic Acids Res, 13, 2485–2502. Wice B, Menton D, Geuze H & Schwartz AL (1990) Modulators of cyclic AMP metabolism induce syncytiotrophoblast formation in vitro. Exp Cell Res, 186, 306–316. Yudilevich DL & Boyd CAR (1987) Amino Acid Transport in Animal Cells. Manchester University Press: Manchester.
Changes in Expression and Function of Syncytin and its Receptor, Amino Acid Transport System B0 (ASCT2), in Human Placental Choriocarcinoma BeWo Cells During Syncytialization Y. Kudo and C. A. R. Boyd Department of Human Anatomy and Genetics, University of Oxford, Oxford, UK Paper accepted 25 April 2002
Relative abundance of mRNAs encoding syncytin and its receptor, amino acid transport system B0, and activity of amino acid transport thought to be through this system have been studied in parallel in a cell model of syncytialization (BeWo cell following forskolin treatment). Relative mRNA abundance (determined by reverse transcription-polymerase chain reaction) for syncytin showed stimulation by forskolin. In contrast, the level of amino acid transporter B0 mRNA expression was lower in forskolin treated cells. Na + -dependent -(methylamino)isobutyric acid insensitive -alanine transport was similarly decreased significantly in cells treated with forskolin suggesting that there is modulation of cell surface expression of the syncytin receptor associated with syncytialization. 2002 Published by Elsevier Science Ltd. Placenta (2002), 23, 536–541
INTRODUCTION The trophoblast surface of the human placenta, in direct contact with maternal blood, is formed from the syncytiotrophoblast which is derived from the underlying cell layer, the cytotrophoblast. The syncytiotrophoblast layer plays an important role throughout pregnancy since it is the site of various placental functions, including nutrient exchange and the synthesis of steroid and peptide hormones which are required for normal foetal growth and development. The unique process of syncytialization of this tissue is very poorly understood. Mi et al. (2000) have shown that syncytin, a protein encoded by an envelope gene of the recently identified human endogenous retrovirus-W (ERV-W) (Blond et al., 1999), is specifically expressed in human placenta and may be involved in syncytialization of trophoblast. Blond et al. (2000) also showed that the ERV-W envelope gene encodes a highly fusogenic membrane glycoprotein that can induce syncytium formation upon interaction with the type D mammalian retrovirus receptor. Immunohistochemical analysis showed ERV-W envelope protein expression restricted to placenta, particularly in the cytotrophoblast and with stronger expression in the syncytiotrophoblast cell layer (Blond et al., 2000). Recently type D mammalian retrovirus receptor has been identified as amino acid transporter B0 (ASCT2) (Tailor et al., 1999). The gene encoding this protein was originally cloned from a human placental choriocarcinoma cell line (Kekuda et al., 1996). System B0 is a Na + -dependent transporter with broad substrate specificity for neutral amino acids including -alanine, -glutamine, -leucine and glycine (Yudilevich and 0143–4004/02/$-see front matter
Boyd, 1987). Anionic amino acids, cationic amino acids and N-methylated amino acids are excluded by this system. The presence of mRNA transcripts for system B0 in the human placenta has been reported (Kekuda et al., 1996). The functional expression of amino acid transporter B0 has been shown in the basal plasma membrane of human placental syncytiotrophoblast (Na + -dependent ‘system 3’, Kudo and Boyd, 1990). Immunohistochemical analysis of syncytin in normal placenta has demonstrated that this protein localizes predominantly to the basal plasma membrane of syncytiotrophoblast (Lee et al., 2001). This co-localization of syncytin and its receptor may facilitate the incorporation of the proliferating cytotrophoblast into the syncytiotrophoblast. BeWo choriocarcinoma cells can be induced to differentiate into a polarized syncytiotrophoblast in vitro by forskolin (Wice et al., 1990). We have studied both expression of mRNA encoding syncytin and its receptor, amino acid transporter B0, genes and functional changes in amino acid transport thought to be through this system using BeWo cells as a cell model of syncytialization.
MATERIALS AND METHODS Material BeWo cells (passage number approximately 40) were kindly gifted by Dr S. L. Greenwood (Academic Unit of Child Health, St Mary’s Hospital, University of Manchester). -[2,3-3H]Alanine (59.0 Ci mmol 1 or 2.18 TBq mmol 1) 2002 Published by Elsevier Science Ltd.
Kudo and Boyd: Trophoblast Syncytialization and Amino Acid Transport
was purchased from Amersham Life Science (Amersham, Buckinghamshire, UK). Forskolin and -(methylamino)isobutyric acid were obtained from Sigma-Aldrich Chemical (Poole, Dorset, UK), tissue culture supplements were from Gibco BRL (Paisley, UK) and the human chorionic gonadotropin (hCG) chemiluminescent immunoassay kit was from Euro/DPC (Llanberis, Gwynedd, UK). QuickPrep Total RNA Extraction Kit were purchased from Amersham Pharmacia Biotech (Rainham, Essex, UK), moloney murine leukemia virus (M-MLV) reverse transcriptase, oligo(dT)12–18 primer, deoxynucleotide 5 -triphosphate (dNTP) and Taq DNA polymerase were from Gibco BRL, deoxyribonuclease I (DNase I) was from Promega (Southampton, Hampshire, UK). All chemicals were of the highest purity commercially available.
BeWo cell culture BeWo cells were cultured at 37C as monolayers in Ham’s F-12 medium supplemented with 10 per cent foetal bovine serum (FBS), 2 m -glutamine, 100 U ml 1 penicillin and 100 U ml 1 streptomycin in a humidified atmosphere of 5 per cent CO2 and 95 per cent air. Cells were subcultured by treating with 0.05 per cent trypsin in Ca2+ - and Mg2+ -free phosphate buffer saline (PBS) containing 0.02 per cent ethylenediaminetetraacetate, seeded in 35 mm plastic culture dishes and grown for 2–3 days to the stage of 50 per cent confluence. At 50 per cent confluency, the medium was then changed to the one containing 100 forskolin or vehicle (dimethyl sulfoxide), followed by further incubation for the indicated times at 37C. At the end of the incubation period, cells were more than 95 per cent viable as assessed by trypan blue dye exclusion. Cultures were conducted in triplicate for each set of experiments to assess reproducibility. The conditioned medium was collected and centrifuged at 3000 g at 4C for 10 min to remove cellular debris and stored at 70C until use.
hCG secretion hCG secretion was determined by measuring its concentrations in the conditioned medium by an immunoassay kit which specifically detects -chain of hCG.
RNA extraction and reverse transcription– polymerase chain reaction (RT-PCR) analysis Relative mRNA abundance of syncytin and amino acid transporter B0 was analysed by semi-quantitative RT-PCR using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal standard. Total RNA was extracted from cultured cells with QuickPrep Total RNA Extraction Kit according to the manufacture’s protocol. RNA samples were
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Figure 1. Amplification of syncytin, amino acid transporter B0 and glyceraldehyde phosphate 3-dehydrogenase mRNA from BeWo cells by RT-PCR. The amplification products were separated by agarose gel electrophoresis and visualised by ethidium bromide staining. The intensity of each target gene band was quantitated using a gel documentation and analysis system. , syncytin; , amino acid transporter B0 (ASCT2); , glyceraldehyde phosphate 3-dehydrogenase (GAPDH).
treated with DNase I before RT-PCR to remove any contaminating DNA. The primers used in the subsequent RT-PCR were as follows: syncytin (Mi et al., 2000), forward, 5 -AGGAGCTTCGAAACACTGGA-3 and backward, 5 GTGAGCTAAGTTGCAAGCCC-3 ; amino acid transporter B0 (Kekuda et al., 1996), forward, 5 -GGCTTGG TAGTGTTTGCCAT-3 and backward, 5 -GGGCAAAGA GTAAACCCACA-3 ; GAPDH (Tso et al., 1985), forward, 5 -CGGGAAGCTTGTGATCAATGG-3 and backward, 5 -GGCAGTGATGGCATGGACTG-3 . The expected sizes of the PCR products are 494 bp for syncytin, 205 bp for amino acid transport system B0 and 358 bp for GAPDH. One microgram RNA was reverse transcribed into cDNA using oligo(dT)12–18 primer. The reverse transcription reaction, containing 500 dNTP, 25 g ml 1 oligo(dT)12–18 primer, 10 unit l 1 M-MLV reverse transcriptase, 3 m MgCl2, 75 m KCl, 10 m dithiothreitol and 50 m Tris-HCl (pH 8.3), was sequentially incubated at 25C for 10 min, at 42C for 50 min and at 70C for 15 min and cooled on ice. As a negative control for the absence of exogenous DNA contamination, reactions run without RNA or with RNA in the absence of the reverse transcriptase revealed no amplified product (data not shown). The synthesized cDNA (0.05 g equivalent to RNA) was used for PCR amplification in a reaction mixture containing 200 dNTP, 1 forward and backward primers, 0.05 unit l 1 Taq DNA polymerase, 1.5 m MgCl2, 50 m KCl and 20 m Tris-HCl (pH 8.4). The PCR conditions were: 94C for 3 min, 60C for 1 min and 72C for 2 min; then 22 cycles (for syncytin and amino acid transporter B0) and 20 cycles (for GAPDH) of 94C for 1 min, 60C for 1 min and 72C for 2 min; followed by a 10 min final extension at 72C. The amount of template cDNA and the number of cycles were determined experimentally so that quantitative comparison could be made during the exponential
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phase of the amplification process for both target and reference gene (Figure 1). PCR products were separated on a 2 per cent agarose gel. Gels were stained with ethidium bromide. A single band for each gene was observed at the expected size. The intensity of either the target or GAPDH band for each sample was quantitated using a gel documentation and analysis system (GDS8000, Ultra-Violet Products, Cambridge, UK) and the ratio of the two was used as a normalized value for expression of each target gene. All assays were conducted in triplicate.
Amino acid influx studies After aspirating culture medium, each dish was washed twice with pre-warmed (37C) PBS and cells were depleted of intracellular amino acids by incubating in PBS at 37C for 30 min to minimize any trans effects. The influx of amino acid was initiated by replacing this with pre-warmed PBS containing 2 -[3H]alanine, followed by further incubation at 37C. Other additions are described in the figure legends. Following aspiration of isotope solution, cells were quickly washed with ice-cold PBS with 10 m unlabelled -alanine. Then 0.1 NaOH and 0.1 per cent sodium dodecyl sulphate solution were added for solubilization and aliquots were taken for liquid scintillation counting and protein determination.
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time-dependent increase in amino acid transport and associated mRNA expression (Smith et al., 1973; Kudo and Boyd, 2000).] Figure 3A compares 2 -alanine influx into BeWo cells before and after forskolin treatment under initial rate condition in the presence of Na + . Data show carrier-mediated influx rate defined by subtracting the diffusional component from the total influx rate. The diffusional component was determined by measuring the influx of 2 -[3H]alanine in the presence of 20 m unlabelled -alanine. Total carrier-mediated influx was not changed following 48 h culture in either the presence or absence of forskolin. In order to separate the transport pathways contributing to total flux in the presence of Na + , the unlabelled synthetic amino acid -(methylamino)isobutyric acid (Christensen, 1979), a system A specific analogue, or unlabelled -alanine was added at 2 m. The extent of the inhibition by -(methylamino)isobutyric acid (i.e. system A mediated influx) was enhanced following forskolin treatment. In contrast the rate of Na + -dependent -(methylamino)isobutyric acid insensitive -alanine influx, defined as the difference between the influx in the presence of unlabelled -alanine and the influx in the presence of -(methylamino)isobutyric acid, was significantly decreased in cells treated with forskolin (Figure 3B).
Protein estimation
DISCUSSION
Protein concentration of the cell extract was determined by the method of Lowry et al. (1951) using bovine serum albumin as a standard.
The syncytialization of trophoblast cells in placenta is central to both structure and functional properties of this tissue. Mi et al. (2000) suggested that syncytin may mediate placental cytotrophoblast fusion in vivo, and thus may be important in human placental morphogenesis. In primary culture of isolated cytotrophoblast cells the transcript levels of syncytin increase with the differentiation and fusion of cytotrophoblasts into syncytiotrophoblasts (Frendo et al., 2001). Amino acid transport system B0 is known to be a receptor for this protein (Tailor et al., 1999). Although the mechanism involved in syncytialization is currently poorly understood, we have now asked questions concerning the expression of these two molecules during this process. Our experiments show that relative mRNA abundance for syncytin and for its receptor, amino acid transport system B0, is changed reciprocally during syncytialization. Since there appears to be concomitant decrease in transport through system B0, our results suggest that the changes in amino acid transporter functional expression appear to be closely related to changes in cell morphology. Our reasons for supposing that the Na + -dependent -(methylamino)isobutyric acid insensitive -alanine influx is through system B0 is two-fold. Firstly earlier work (Kudo and Boyd, 1990) on basal syncytiotrophoblast basal membrane vesicles identified clearly a component of transport (Na + dependent system ‘3’) that matches precisely the properties of expressed system hATB0 (Kekuda et al., 1996); however in this membrane there is no transport system which matches the properties of system ASC (this observation conflicts with those
Statistical analysis Differences between groups were analysed using an ANOVA and results were considered statistically significant at P<0.05.
RESULTS Following forskolin treatment morphological fusion of BeWo cells, appearance of multinucleated cells, was readily detectable after 24 h and is dramatic after 48 h; cell fusion was hardly detectable at 48 h in the absence of forskolin. Concomitantly an associated marked increase in hCG secretion by BeWo cells was observed but only in the presence of forskolin (4.10.2-fold increase above basal level at 24 h). The relative expression of mRNA encoding syncytin and its receptor, amino acid transporter B0, following forskolin treatment was studied by RT-PCR (Figure 2). Syncytin mRNA expression level showed stimulation by forskolin (1.90.2fold) which was maximal at 48 h. Expression level of amino acid transporter B0 mRNA at this time point was lower (0.50.1-fold) in forskolin treated compared to control cells. GAPDH mRNA was constant for each sample. [As has been found by other authors culture of placental cells causes a
Kudo and Boyd: Trophoblast Syncytialization and Amino Acid Transport
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Figure 2. Effect of forskolin treatment on the relative abundance of syncytin mRNA and amino acid transporter B0 mRNA in BeWo cells. Total RNA was extracted either from BeWo cells cultured with 100 forskolin or vehicle for the time indicated. A: RT-PCR. The relative abundance of syncytin mRNA(a), amino acid transporter B0 (ASCT2) mRNA (b) and glyceraldehyde phosphate 3-dehydrogenase (GAPDH) mRNA (c) were analysed by RT-PCR as described in Methods. The results presented are from a single representative experiment. B: Relative quantitation of syncytin mRNA (a) and ASCT2 mRNA (b). The intensity of either the target gene or the GAPDH band was quantitated by using a gel documentation and analysis system of PCR products and the ratio of the two was used as a normalised relative abundance value of each transporter gene. Data represent the means.d. of three separate experiments, expressed as percentage of control (i.e. values without culture). *Significantly different from values cultured with vehicle alone (P<0.05). †Significantly different from values at 0 h (P<0.05).
by Hoeltzli and Smith (1989) suggesting there is small contribution from system ASC activity in this membrane). Since the basal membrane of syncytiotrophoblast is known to be the site
of normal syncytin interaction with its receptor, the presence of B0 transporter in this membrane is consistent with our interpretation. Secondly the finding that mRNA for ASCT2
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Figure 3. Effect of forskolin treatment on -alanine influx in BeWo cells. -Alanine influx over a 3 min period before treatment (control) and after 48 h treatment with 100 forskolin or vehicle was measured in a medium containing 2 -[3H]alanine with or without 2 m unlabelled -(methylamino)isobutyric acid (MeAIB) or -alanine in the presence of Na + . A: carrier-mediated influx rate defined by subtracting the diffusional component (determined by measuring the influx of -[3H]alanine in the presence of 20 m unlabelled -alanine) from the total influx. B: system B0 mediated influx rate defined as the difference between the influx in the presence of -alanine and the influx in the presence of MeAIB. Data represent the means.d. of three separate experiments of triplicate assay. *Significantly different from values cultured with vehicle alone (P<0.05).
(encoding system B0) decreases proportionally to amino acid transport is compatible with this proposal. However, further study to confirm this interpretation will require with experiments with -leucine as an inhibitor. Additionally possible mechanisms underlying this will require further studies aimed at determining protein abundance, subcellular localization and stability. Importantly our assay technique measures function only of the syncytin receptor protein present in the plasma membrane of BeWo cells, that is protein localized to the same position as is necessary for syncytin binding. Previous work (Furesz et al., 1993) has shown that system A-, ASC- and -like transporters are responsible for -alanine transport in primary cultures of trophoblast cells isolated from full-term human placenta and that during in vitro differentiation, formation of microvillous membrane and syncytium, system ASC activity is decreased. These authors also showed that system ASC activity is decreased in forskolin treated BeWo cells. Furesz et al. thus concluded that membrane specialization accompanying fusion and differentiation of the cytotrophoblast to form syncytiotrophoblast results in a polarization of neutral amino acid transport systems. Trophoblast invasion of the maternal decidua and myometrium is a carefully orchestrated process, with insufficient infiltration of the uterine wall implicated in placental disorders such as pre-eclampsia, and uncontrolled trophoblast invasion observed in choriocarcinoma and other gestational tropho-
blastic diseases. Amino acid transport system B0 as well as syncytin might limit invasiveness by inducing trophoblast fusion and terminal differentiation. It has also been proposed that trophoblast syncytialization is the first step of a sequence leading to syncytial apoptosis which in turn is necessary for syncytial renewal (Huppertz et al., 1998). Lee et al. (2001) have shown that in pre-eclampsia syncytin gene expression in trophoblast is reduced and that syncytin is localized improperly to the apical syncytiotrophoblast microvillous membrane as opposed to its normal location on the basal syncytiotrophoblast cytoplasmic membrane. They suggest that altered expression of the syncytin gene and altered cellular location of its protein product may contribute to the aetiology of preeclampsia. Very recently it also has been reported that placental syncytin mRNA expression level is reduced in pregnancies complicated with haemolysis, elevated liver enzymes and low platelets (HELLP) syndrome (Knerr et al., 2002) or trisomy 21 (Frendo et al., 2001) in which there is a defect in syncytiotrophoblast formation. It therefore will be of interest to analyse the expression of amino acid transport system B0 immunohistochemically and functionally in placentae from these pregnancies. Interestingly a reduction of placental amino acid transport activity is well demonstrated in intrauterine growth-restricted pregnancies (Glazier et al., 1997; Norberg et al., 1998) and this disorder is commonly accompanied by pre-eclampsia (Lin et al., 1991).
ACKNOWLEDGEMENT Yoshiki Kudo was supported by Oxford Kobe Scholarship.
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