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Am J Physiol Regul Integr Comp Physiol 290: R1357-R1365, 2006. First published December 1, 2005; doi:10.1152/ajpregu.00630.2005
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DEVELOPMENTAL PHYSIOLOGY AND PREGNANCY

ABC drug transporter expression and functional activity in trophoblast-like cell lines and differentiating primary trophoblast

Denis A. Evseenko,2 James W. Paxton,1 and Jeffrey A. Keelan1,2

Faculty of Medical and Health Science, 1Department of Pharmacology and Clinical Pharmacology, 2Liggins Institute, University of Auckland, Auckland, New Zealand

Submitted 30 August 2005 ; accepted in final form 29 November 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
ATP-binding cassette (ABC) efflux transporters are expressed in the human placenta where they are thought to help protect the fetus from xenobiotics. To evaluate models for analysis of ABC transporter function and regulation in the placenta, we have characterized the expression and activity of multidrug resistance (MDR) 1/P glycoprotein (Pgp), MDR3/Pgp, breast cancer resistance protein (BCRP), and multidrug resistance proteins 1 and 2 (MRPs 1, 2) in differentiating primary trophoblast cells and BeWo and Jar cell lines. Real-time PCR and immunoblotting were used for analysis of mRNA and protein expression, respectively. Functional activity was measured using selective inhibitors of efflux of fluorescent substrates, calcein-AM (Pgp and MRPs) and Hoechst 33342 (BCRP). The levels of MDR1 mRNA and protein expression were much higher in trophoblast than in Jar and especially BeWo cells. Expression of MDR3 protein was also lower in BeWo cells. Levels of MDR3 expression were markedly higher than MDR1 levels in all tested cell types. Levels of both MDR1 and MDR3 expression decreased during trophoblast differentiation/syncytialization. BCRP was highly expressed in all cell types and increased with trophoblast differentiation. MRP1 expression was much lower in trophoblasts compared with both cell lines. In contrast to its abundant mRNA expression, MRP2 protein was practically undetectable in BeWo and Jar cells and was present only at very low levels in trophoblast. Functional studies confirmed the presence of active Pgp and BCRP in all studied cell types, whereas MRP functional activity was detected only in BeWo and Jar cells. Both cell lines may be useful models for studying various aspects of placental ABC transporter expression and function, but also have significant limitations. With respect to their ABC protein expression profile, Jar cells are more similar to nondifferentiated cytotrophoblast, whereas BeWo appear to more closely reflect differentiated syncytiotrophoblast.

adenosine 5'-triphosphate-binding cassette; placenta; BeWo; multidrug resistance 3; multidrug resistance 1

AS AN ORGAN OF EXCHANGE, the human placenta expresses a large number of transport proteins responsible for active translocation of endogenous substrates and a wide spectrum of pharmacological agents. The ATP-binding cassette (ABC) superfamily of transporters appears to be particularly important in this context, being specifically dedicated to removal of xenobiotics and toxic endogenous metabolites from the fetal organism (11, 22). Although the ABC pumps were initially described as drug efflux pumps, conferring resistance to cancer cells from drug-induced apoptosis (13), they have more recently been shown to be implicated in cellular protection from various proapoptotic injuries, such as exposure to endogenous toxins or hypoxic damage (10, 19). Human placenta expresses several members of the ABC superfamily; of these, the most effective for drug transfer are P-glycoprotein (Pgp), encoded by the multidrug resistance 1 (MDR1) gene, multidrug resistance proteins (MRPs) 1 and 2, and the breast cancer resistance protein (BCRP; see Refs. 3, 9, 14, 22). MDR1/Pgp, BCRP, and MRP2 are located at the apical membrane of the syncytiotrophoblast, whereas MRP1 is mainly localized on the basolateral membrane of the sycytium and the luminal membrane of fetal capillaries (22, 28). Expression of the Pgp homolog MDR3 has been reported in the placenta recently (18). However, there is nothing known of its protein expression, its function or activity, and its cellular localization within the placenta.

Choriocarcinoma Jar and BeWo cells express several ABC transporters and have been used as models for studying active drug transport in placenta (1, 25, 26). However, their suitability for this purpose remains questionable, and their similarities and differences relative to primary trophoblast cell types have not been evaluated. Expression of Pgp/MDR1 has been reported in BeWo cells by several authors, and the cell line has been used for analysis of transplacental transfer of Pgp substrates (25, 26). On the other hand, other investigators have failed to detected expression of the transporter both at mRNA and protein levels in these cells (1). No quantitative studies on protein and functional expression of Pgp, BCRP, and MRPs in primary trophoblast compared with the carcinoma cell lines have been published. In contrast to the commonly used cell lines, cytotrophoblasts do not proliferate in vitro but undergo cellular differentiation and fusion, changing their expression profile during this process. Recently, marked elevation of MRP2 mRNA and protein expression was demonstrated in cultured trophoblast with differentiation (15). No studies have yet investigated the changes in Pgp, BCRP, and MRP1 levels during trophoblast differentiation and fusion in vitro.

The aim of the present study was to investigate and characterize the expression and functional activity of BCRP, Pgp/MDR1, Pgp/MDR3, and MRP-1 and -2 in trophoblast-like BeWo and Jar cells, together with primary cytotrophoblast cultures at different stages of differentiation, to compare and validate their suitability for analysis of the ABC system function and regulation in the placenta.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reagents. Jar and BeWo cells were obtained from American Type Culture Collection. DMEM, Ham's F-12 and medium 199 (M199), FCS, normal horse serum (NHS), insulin, transferrin, human recombinant epidermal growth factor (EGF), sodium selenite, penicillin/streptomycin, Trizol, DNase I (molecular grade), and Superscript III cDNA Synthesis Kit were from Invitrogen NZ limited (Auckland, NZ). Difco trypsin 250 was obtained from Becton & Dickinson. The RNAqueous Kit for total RNA extraction was acquired from Ambion. Primers for MDR1, MDR3, MRP1, MRP2, and BCRP were designed by SuperArray. 18S rRNA primers and Sybr Green PCR Master Mix were purchased from Applied Biosystems (Warrington, UK). Complete protease inhibitor was from Roche Diagnostics (Mannheim, Germany). Nitrocellulose Hybond membrane and fluorescent Cy3-labeled goat anti-mouse antibody were purchased from Amersham Pharmacia Biotech (Buckinghamshire, UK). Monoclonal anti-MDR1 (clone 3C3.2), MDR3 (clone P3 II-26), MRP1 (clone QCRL 3), MRP2 (clone M2 I-4), and BCRP (clone BXP 21) antibodies were purchased from Chemicon. The anti-Pgp (anti-MDR1/MDR3) monoclonal antibody (clone C219) was from ID Labs (Taree, Australia). Anti-cytokeratin 7 (C7) antibodies were from DacoCytomation (Glostrup, Denmark). SuperSignal Substrate was from Pierce Biotechnology. Anti-beta-actin, peroxidase-conjugated goat anti-mouse antibody, bicinchoninic acid (BCA) reagent, and fluorescent substrates calcein-AM and Hoechst 33442 were purchased from Sigma-Aldrich. Transporter blockers Pgp-4008, MK-571, and fumitremorgin were all purchased from Alexis Biochemicals (Lausen, Switzerland). General chemicals (analytical grade) were obtained from Serva (Heidelberg, Germany), Scharlay Chemie (Barcelona, Spain), or AppliChem (Darmstadt, Germany).

Cytotrophoblast culture. Placentas were obtained with informed consent from women after delivery by caesarean section at term. Cytotrophoblasts were isolated as described by Kliman et al. (8) with some modifications. Cytotrophoblasts derived from term placenta (n = 4) were liberated by trypsin digestion (0.25% wt/vol, 8 digestions in total), and supernatants were collected in 50-ml Falcon tubes and centrifuged at 300 g for 7 min. Erythrocytes were removed by incubation of a cell pellet in a lysis buffer (50 mM NH4Cl, 10 mM NaHCO3, and 0.1 mM EDTA), and cytotrophoblasts were purified by centrifugation at 1,200 g for 20 min on a discontinuous Percoll gradient (20–60%). Cells between the 40 and 50% Percoll bands were collected and plated either in T75 Falcon flasks (107 cells), 6-well plates (2 x 106 cells/well), or 24-well plates (5 x 105 cells/well). The cells were grown in M199, supplemented with 10% FCS, EGF (10 ng/ml), insulin (5 µg/ml), transferrin (10 µg/ml), sodium selenite (0.2 nM), and penicillin/streptomycin (100 U/ml) in a 5% CO2 humidified atmosphere at 37°C. After 24 h in culture, cells were washed with PBS, and media containing 10% FCS was renewed. Cells were incubated for 5–6 days to allow complete syncytialization. Purity of the isolated cells was assessed using immunofluorescent staining with anti-C7 antibodies detected with Cy3-labeled secondary antibody. Nuclei were stained with Hoechst 33342 (5 µg/ml) for 10 min. C7-positive cells in at least 10 fields at x200 magnification were counted using an Olympus IX 71 inverted microscope (Tokyo, Japan), and the number of positive/total cells was calculated. A proportion of ≥90% cytotrophoblasts in the total cell population was considered as a sufficient purity. For analysis of transporter expression during trophoblast differentiation, samples were taken after 24, 72, and 120 h of cell incubation. For comparative analysis of transporter expression and function in trophoblast vs. cell lines, 72-h trophoblast cultures were used. Jar and BeWo cells were cultured in 1:1 DMEM-F-12 with 10% FCS until reaching 70–80% confluence before experimentation.

Quantitative real-time PCR. Total RNA from cultured cytotrophoblast, BeWo, and Jar cells was extracted using an RNAqueous kit according to the manufacturer's instructions; total RNA from placental tissue was isolated using Trizol and treated with DNase I to remove genomic DNA. First-strand cDNA synthesis with oligo(dT) primers was carried out using a Superscript III Synthesis kit for all target transporter genes and for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). For analysis of 18S RNA expression, the same samples were converted to cDNA using priming with random hexamers from the same kit according to the manufacturer's instructions. Real-time PCR for MDR1, MDR3, MRP1 and -2, BCRP, GAPDH, and 18S ribosomal RNA (18S RNA) was performed in triplicate on 0.02–1.00 µg cDNA using SybrGreen PCR Master Mix in a reaction volume of 25 µl. PCR amplification and detection was performed using an ABI Prism 7900 HT (Applied Biosystems). Exponential amplification of all PCR reactions ranged from 1.95 to 2.00 across seven serial log dilutions of template. Amplification of single product for each primer set was confirmed by dissociation curve analysis, and all products were visualized after electrophoresis on 2% agarose gel. The comparative threshold cycle (Ct) method for relative quantification (2–{Delta}{Delta}Ct) was used to quantitate gene expression according to Applied Biosystem recommendations (7900 HT Real-Time fast and SDS enterprise and database user guide). Expression of target genes was normalized to the level of 18S rRNA and expressed relative to a calibrator (sample in each set with lowest expression). Normalization to GAPDH was also performed to confirm results obtained using 18S RNA (data not shown).

Immunoblotting. Crude membrane preparations from cytotrophoblasts (72 h culture), BeWo, and Jar cells were prepared from cells cultured in T75 Falcon flasks and homogenized in 3 ml of B1 homogenization buffer (10 mM Tris·HCl, pH 7.4 with complete protease inhibitor). The nuclei and cell debris were removed from the homogenate by centrifugation at 900 g for 10 min at 4°C. The resulting supernatant was centrifuged at 100,000 g for 90 min at 4°C in Beckman ultracentrifuge. After extraction, membranes were solubilized by sonication in B2 buffer (10 mM Tris·HCl, pH 7.4, 1% Triton X-100, 1% SDS, 0.5% deoxycholic acid, and complete protease inhibitor) and maintained for a minimum of 1 h at 4°C. Insoluble material was removed by centrifugation at 14,000 g for 10 min at 4°C and stored at –80°C. Total cell lysates were also prepared by homogenization in the same buffer (B2) using primary trophoblasts after 24, 72, and 120 h of incubation. Protein concentration was measured by BCA assay calibrated to BSA. Protein (20–30 µg) was separated under reducing conditions on a 4–12% BisTris precast polyacrylamide gradient gel and transferred to a nitrocellulose membrane in an XCELL transfer module (Invitrogen). Membranes were blocked in 2% nonfat milk powder at least for 3 h and incubated overnight with the following monoclonal antibodies: anti-MDR1 (1:800), MDR3 (1:500), MRP1 (1:500), MRP2 (1:50), BCRP (1:500), and MDR1/3 (clone C219, 1:200). Membranes were then washed and incubated with horseradish peroxidase-conjugated goat anti-mouse antibody and visualized by enhanced chemiluminescence recorded on CP-BU New X-ray film (Agfa, Westerlo-Heultje, Belgium). Band intensity was quantitated by densitometry using Quantity One software from Bio-Rad. Equivalence of protein loading was confirmed by secondary immunoblotting with anti-beta-actin antibodies.

Immunofluorescence staining. Placentas were washed in PBS with (0.1%) Tween 20, and small pieces were quickly frozen on dry ice in optimum cutting temperature reagent and stored at –80°C. Sections (10 µm) were then sliced with a microtome cryostat Leica CM 3050S (Leica Microsystems) at –20°C, transferred to Superfrost glass slides, and fixed in acetone at –20°C for 10 min. Sections were blocked in PBS with 5% NHS for 30 min and incubated with selective primary antibody against MDR3 (clone P3 II-26) or NHS only (for negative control) overnight at 4°C. After incubation, the sections were washed three times in PBS and incubated with secondary Cy3-labeled fluorescent antibody for 1 h at room temperature. Sections were then washed three times in PBS, counterstained with DAPI (Vector Laboratories), and mounted with Vectashield for examination with a Zeiss LSM 150 MetaLaser confocal microscope (Carl Zeiss).

Functional activity. Drug efflux activity of cells in monolayer culture was measured by selective inhibition of intracellular accumulation of fluorescent Pgp/MRP and BCRP substrates calcein-AM and Hoechst 33442, respectively. The selective Pgp, MRP, and BCRP inhibitors used were Pgp-4008 (5 µM), MK-571 (10 µM), and fumitremorgin C (5 µM), respectively (12, 21, 29). For functional studies, trophoblast cells were plated in triplicate in 24-well plates at 5 x 105 cells/well and incubated for 3 days before experimentation. BeWo and Jar cells were plated in 24-well plates (2 x 105 cells/well in triplicate) and incubated 1 day before experimentation. All cells were washed in HEPES-buffered Tyrode solution, and selective blockers were added to the same buffer. After 30 min incubation at 37°C, fluorescent substrates were added to final concentrations of 0.4 µM (calcein-AM) and 5 µg/ml (Hoechst 33442). After various incubation times (15–90 min), cells were washed three times with cold Tyrode solution and lysed in 10 mM Tris·HCl-1% Triton X-100 (pH 7.4). Accumulation of fluorescent substrates was measured on a Wallac Victor2 fluorescence reader (Wallac, Finland) at 485/535 nm for calcein-AM and 355/460 nm for Hoechst 33442.

Statistics. All studies were repeated four times, and descriptive statistics were performed for each data set. Graphs were plotted, and data were transformed using Graph Pad Prism 3.02 (GraphPad Software). Statistical analysis was performed using Sigmastat software from Systat Software. For expression studies, one-way ANOVA was applied followed by the Student-Newman-Keul's test. For functional time-course studies, statistical significance was assessed by two-way ANOVA with repeated measures. P value <0.05 was considered to be significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Comparative levels of transporter gene expression in human term placenta, cultured trophoblast, and Jar and BeWo cells. Expression of MDR1, MDR3, MRP1, MRP2, and BCRP was detected by real-time PCR in all cell/tissue types (Table 1). MDR1 expression showed the greatest dynamic range between cell types. Relative expression was highest in placenta and primary trophoblast, almost 200- and 1,000-fold greater than in Jar and BeWo cells, respectively (Fig. 1A). Levels of MDR3 gene expression were higher than MDR1 in all cell types (Table 1), with significantly less variation between primary tissues and cell lines (Fig. 1B). BCRP expression was also abundant in all samples (Table 1), being greatest in BeWo cells and lowest in the placenta (Fig. 1C). MRP1 expression was also highest in BeWo cells, almost twofold greater than in Jar cells, primary trophoblast, and total placenta (Fig. 1D). MRP2 mRNA was highly expressed in all samples (Table 1); however, in contrast to MRP1, expression of MRP2 was lowest in BeWo cells (Fig. 1E).


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  		Table 1. Relative expression of ABC transporters

 

Figure 1
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  		Fig. 1. Relative expression (mean ± SD, n = 4) of multidrug resistance (MDR) 1 (A), MDR3 (B), breast cancer resistance protein (BCRP; C), multidrug resistance protein (MRP) 1 (D), and MRP2 (E) mRNA in BeWo, Jar, primary trophoblasts (Troph), and whole placenta (Pl). For quantitative analysis of gene expression, the comparative threshold cycle (Ct) method for relative quantification (2–{Delta}{Delta}Ct) was applied. Expression of the target genes was normalized to the level of 18S rRNA and relative to a calibrator (sample with lowest expression). For statistical analysis, 1-way ANOVA was used, followed by the Student-Newman-Keul's test. *P < 0.05, **P < 0.01, and ***P < 0.001.

 
ABC transporter protein expression in trophoblast, Jar, and BeWo cells. Protein expression in purified membranes extracted from BeWo, Jar, and primary trophoblasts after 72 h of incubation was analyzed by immunoblotting using specific anti-ABC transporter antibodies. Expression of Pgp/MDR1 protein was readily detectible in trophoblast cells, more so than in Jar cells, whereas in BeWo cells it was barely detectible (Fig. 2A). Using an MDR3 specific antibody, we demonstrated MDR3 protein expression in both choriocarcinoma cell lines and cultured trophoblasts (Fig. 2B). BCRP protein was highly expressed in all cell types (Fig. 2C) and was most abundant in BeWo cell extracts, consistent with its mRNA expression. MRP1 protein was readily detectible in both cell lines, but was present in much lower amounts in trophoblasts (Fig. 2D). In contrast to its abundant expression at the level of mRNA, MRP2 protein was practically undetectable in BeWo and Jar cells and was present only at very low levels in cultured trophoblasts (Fig. 2E).


Figure 2
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  		Fig. 2. Expression (mean ± SD, n = 4) of ATP-binding cassette (ABC) proteins in BeWo, Jar, and primary trophoblasts as determined by immunoblotting using specific antibodies. CaCo-2 cells (+) were used as a positive control for detection of MRP2. For statistical analysis, 1-way ANOVA was applied, followed by the Student-Newman-Keul's test. *P < 0.05, **P < 0.01, and ***P < 0.001.

 
Immunohistological localization of MDR3 in term placenta. The localization of MDR3 protein was examined by laser confocal microscopy. As shown on Fig. 3, A and B, \. immunofluorescence staining was detected predominantly at the basolateral side of the trophoblast layer using the anti-MDR3 specific primary antibody (P3 II-26). In some villi, we observed specific staining in cytoplasm of a single trophoblast nuclei or groups of nuclei (Fig. 3C). Mesenchymal cells of the trophoblast villi, presumably endothelial cells, also showed clear immunostaining compared with the negative control, which was essentially unstained (Fig. 3, D and E).


Figure 3
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  		Fig. 3. Immunofluorescent localization of MDR3 in human placenta. Cryostat sections were incubated with (A-C), or without (D and E) anti-MDR3 selective primary antibody, followed by Cy3-labeled (red) secondary antibody or Cy3-labeled antibody. Red signal represents MDR3 immunofluorescence staining. Sections were mounted with Vectashield and counterstained with DAPI (blue staining of nuclei). Arrows in A and B represent MDR3 located predominantly at the basolateral side of syncytiotrophoblasts and endothelial cells of placental vessels. Arrow in C shows cytoplasmic staining in local zones of syncytiotrophoblasts. X, times magnification.

 
Functional studies. In BeWo cells, the selective Pgp inhibitor Pgp-4008 significantly increased accumulation of calcein-AM over the duration of the experiment (Fig. 4). Functional activity of Pgp was also clearly present in Jar cells and trophoblast, with about a 40% increase in calcein-AM accumulation after 60 min incubation in the presence of Pgp-4008 (P < 0.05). Enhanced accumulation of calcein-AM (also a substrate for MRP1) was clearly detectible in BeWo and Jar cells in the presence of the MRP inhibitor MK-571 (P < 0.05). In good agreement with the immunoblotting data, MK-571 had practically no effect on calcein-AM accumulation in primary trophoblast cells. Accumulation of the BCRP substrate Hoechst 33342 was significantly higher in BeWo, Jar, and trophoblast cells in the presence of BCRP selective blocker fumitremorgin C (P < 0.05; Fig. 4).


Figure 4
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  		Fig. 4. Functional expression of Pgp, MRPs, and BCRP in BeWo, Jar, and primary trophoblasts (mean ± SD, n = 4). Statistical significance was assessed by 2-way ANOVA with repeated measures. P < 0.05 was considered to be significant. Drug efflux activity was measured by selective inhibition of intracellular accumulation of fluorescent Pgp/MRP and BCRP substrates calcein-AM and Hoechst 33442, respectively. The selective Pgp, MRP, and BCRP inhibitors used were Pgp-4008 (5 µM), MK-571 (10 µM), and fumitremorgin C (FTC, 5 µM), respectively.

 
Influence of trophoblast differentiation on ABC transporter expression. Term cytotrophoblast cells differentiated between days 1 and 3 of culture and formed multinucleated syncytium by day 5. The effect of trophoblast differentiation on expression of ABC transporters was assessed by determining their expression levels (mRNA and protein in total cell lysates) at 24, 72, and 120 h of incubation. Trophoblast MDR1 and MDR3 mRNA levels at 120 h of incubation decreased by >60% (P < 0.05), relative to those at 24 h (Fig. 5, A and B). In contrast, levels of BCRP mRNA increased during in vitro differentiation by fourfold (P < 0.001; Fig. 5C). The changes in protein expression with differentiation were even more significant. The MDR3 selective antibody indicated that MDR3 protein was present in abundance at 24 h but was reduced by ~50% by day 3 and by 80% on day 5 (Fig. 6A). Immunoblotting with C219 antibody, which detects Pgp/MDR1 with high affinity/sensitivity and also cross-reacts with the MDR3 isoform of Pgp, revealed a >40% reduction of expression with differentiation (72 h) to almost undetectable levels after syncytialization at the 120-h time point (Fig. 6B). In contrast, immunodetectable BCRP increased by approximately threefold in abundance with differentiation (Fig. 6C). Immunoblotting studies indicated very low expression of both MRP transporter proteins in all trophoblast samples during differentiating, below the reliable limit of quantitation (data not shown).


Figure 5
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  		Fig. 5. Changes in MDR1 (A), MDR3 (B), and BCRP (C) mRNA expression (mean ± SD, n = 4) with trophoblast differentiation calibrated to the level at 120 h (MDR1 and -3) and 24 h (BCRP). For statistical analysis, 1-way ANOVA was applied followed by the Student-Newman-Keul's test. *P < 0.05, **P < 0.01, and ***P < 0.001.

 

Figure 6
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  		Fig. 6. Changes in Pgp MDR3 (A), MDR1/MDR3 (B), and BCRP protein expression (C) (mean ± SD, n = 4) with trophoblast differentiation. Expression of Pgp was detected either by anti-MDR3 specific antibody (P3 II-26) or C219 antibody, which preferentially recognizes the MDR1 isoforms of Pgp. *P < 0.05, **P < 0.01, and ***P < 0.001. For statistical analysis, 1-way ANOVA was applied followed by the Student-Newman-Keul's test.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, we have characterized and compared ABC transporter gene and protein expression, together with drug efflux activity, in two commonly used choriocarcinoma cell lines and primary trophoblast cells in culture. BeWo and Jar cells are frequently employed as models of trophoblast biology and have been used by many investigators to study trophoblast transport in vitro (1, 17, 25). Our findings indicate that, although these cell lines share some similarity with trophoblast cells with respect to their drug efflux capacity, their differences are notable and significant; care must be taken, therefore, to use these cell models appropriately in light of their limitations. We have also shown that ABC expression varies significantly as trophoblasts differentiate in culture, likely reflecting the phenotypic differences between cytotrophoblasts and syncytialized trophoblast, in addition to other putative functions related to regulation/maintenance of cellular viability and differentiation status. Functional studies, employing specific inhibitors to achieve selectivity, confirmed the presence of efflux capacity attributable to specific ABC transporters (Pgp, BCRP, and MRP) in the cell types studied. However, caution must be exercised in attempting to compare efflux activity between cell types using these data. Significant variations in apparent efflux rates could occur as a result of different factors relating to culture conditions and metabolic status (5) that could render cell-to-cell comparisons invalid.

MDR1/Pgp is believed to be the most pharmacologically active xenobiotic efflux pump and has been localized to the apical surface of the syncytial membrane (16). Our findings, at both the mRNA and protein level, indicate that, although it is present in the placenta, the expression of this transporter is extremely variable across the cell types studied. MDR1 expression in BeWo cells in particular was extremely low, consistent with the work of Atkinson et al. (1). These data highlight the inappropriateness of the use of BeWo cells as a model to study placental Pgp activity. In contrast, other authors have reported MDR1/Pgp expression and apparent activity in BeWo cells using classical Pgp substrates (25, 26). Our finding that BeWo cells (and the other cells studied) express MDR3 raises the possibility that the Pgp activity previously reported could reflect some contribution from the MDR3 Pgp homolog. Although it was readily detectible, we found levels of MDR3 protein expression in BeWo cells to be significantly lower than in Jar cells, which paradoxically had the lowest level of MDR3 mRNA expression. Levels of Pgp activity in BeWo, Jar, and trophoblast cells were not markedly dissimilar. These discrepancies suggest that translational regulation plays an important role in determining functional MDR3 expression and are a salient reminder of the importance of confirming mRNA expression data with appropriate protein/activity studies. MDR3 has been reported to have a more restricted drug transport profile than MDR1/Pgp, being mainly involved in the efflux of phosphatidylcholine from cells (20). However, several Pgp substrates, such as digoxin and vinblastine, have been shown to be substrates for this transporter (20), and it may prove to be more active in this regard than originally perceived. To the best of our knowledge, calcein-AM has not been tested as a MDR3/Pgp substrate, and no direct studies have examined Pgp-4008 inhibition of MDR3 activity, although archetypal MDR1 blockers cyclosporine A, PSC 833, and verapamil do inhibit MDR3 activity (20). It is, therefore, not possible to conclude with absolute confidence that the Pgp-4008-mediated effects on calcein-AM accumulation that we observed represent evidence of MDR3 activity in BeWo cells, although it remains a likely and plausible explanation for our findings.

Although MDR3 is generally considered a liver-specific transporter, its expression in human term and preterm placenta has been described recently, with levels increasing toward term (18). Interestingly, this expression profile is directly opposite to MDR1/Pgp, the abundance of which decreases pregnancy progression (14). Unexpectedly, immunoreactive MDR3 was identified predominantly in the basolateral membrane of the syncytium of the term placenta, in contrast to the apical orientation of MDR1. Therefore, MDR3 presumably mediates the vectorial transport of substrates toward the fetus, rather than out of the fetus/placenta as brought about by MDR1/Pgp. This novel finding has important clinical implications with respect to placental drug efflux.

BCRP expression was found at very high levels in all three placental cell types studied, consistent with the placenta being identified as a major site of expression of this transporter. Levels of activity appeared to be relatively similar in cell lines and primary trophoblasts, although BeWo cells expressed considerably higher levels of BCRP mRNA and protein. The reasons for this are not known. The roles and natural substrates for this transporter in the human placenta have not yet been identified. Studies in animal models have suggested that BCRP may contribute to the placenta's ability to reduce fetal-to-maternal drug passage (7). Recently, BCRP has been reported to confer resistance to hypoxia via removal of toxic products of heme metabolism from cytoplasm (10), a function that may be important for trophoblast survival in some pregnancy complication.

Very low levels of expression and minimal functional activity of MRP1 were detected in trophoblasts, whereas BeWo and Jar cells both had considerable expression and activity. The functional significance of MRP1 in human placenta remains unclear, although several authors have found this transporter in the basal syncytiotrophoblast membrane and the luminal membrane of fetal endothelium, suggesting maternal-to-fetal vectorial transport (16). Because MRP1 is expressed not only in placental trophoblast but also in placental endothelium (16, 22), the use of trophoblast-like lines to study MRP function in the placenta must be undertaken with caution, particularly in light of the much greater levels of expression and activity identified in Jar and BeWo cells.

In contrast to MRP1, MRP2 protein was virtually undetectable in both cell lines and present at very low levels in cultured trophoblasts. This finding is somewhat at odds with the very high levels of MRP2 gene expression detected in all cell types, suggesting the presence of inhibitory processes operating at the level of translation. Expression of MRP2 in the human placenta has been reported by several authors in the apical membrane of syncytiotrophoblasts and most recently has been shown to be higher in term vs. preterm placenta (15). However, other authors have failed to find this protein in purified apical membranes obtained from term placenta and BeWo cells (17), and whether MRP2 plays a significant role in the placenta remains unclear. MRP2 polymorphisms have recently been identified in the human placenta (15), providing a potential explanation for the differences in levels of MRP2 expression and activity noted in various studies.

BeWo and Jar cells proliferate in culture and do not spontaneously fuse, whereas term trophoblast cells start to fuse from day 2 in culture and form differentiated syncytium by days 4–5. Dramatic changes in Pgp and BCRP expression with trophoblast maturation were revealed in the present work, raising the question as to whether this reflects phenotypic differences between cytotrophoblast and syncytiotrophoblast cell types, or whether the transporters actively participate in the process of trophoblast differentiation. Certainly, our findings appear to be consistent with the higher abundance of Pgp reported in first-trimester placenta (14), where the number of nondifferentiated cytotrophoblasts cells is far greater than at term.

The formation of the syncytium entails activation of caspases, loss of the asymmetry of the cell membrane, and externalization of phosphatidylserine (very similar to apoptosis; see Refs. 2, 6, 23). The structure and composition of the cell membrane is critical for cell fusion, differentiation, and apoptosis (23). Recently, sphingolipids such as ceramides and their metabolites (lipid mediators of cell differentiation/proliferation/apoptosis) have been shown to be substrates for ABC transporters, in particular for Pgp (24). Pgp and BCRP have also been shown to maintain asymmetry of lipid bilayers by intramembrane translocation of lipid molecules, including sphingomyelin and phosphotidylserine (4, 20, 27). We speculate, therefore, that ABC transporters may be actively involved in the process of trophoblast differentiation, regulating intracellular levels of phospholipids and mediating changes in lipid composition of the membrane during cell fusion. It remains unclear why expression of both isoforms of Pgp (MDR1/MDR3) should virtually cease with trophoblast fusion, since this is a trend that is directly opposite to the pattern of the other ABC transporters studied (expression increases with trophoblast maturation). Perhaps this phenomenon reflects the requirement for high levels of Pgp expression early in gestation, when the fetus is at its most vulnerable and when trophoblast differentiation is not complete.

In summary, our data highlight both similarities and differences in ABC efflux pump expression and activity in primary trophoblasts and trophoblast-like cell lines, suggesting that BeWo and Jar cells have restricted usefulness as models of trophoblast drug transport. BeWo cells demonstrate an expression pattern similar to the syncytiotrophoblast in vitro, whereas Jar cells are more similar to the nondifferentiated cytotrophoblasts. All cell types studied showed evidence of functional expression of Pgp and BCRP, whereas MRP1 and 2 protein expression was very low throughout, and MRP activity was detected only in the cell lines. Importantly, we also demonstrated that the MDR3 isoform of Pgp is more highly expressed in the placenta and cultured trophoblasts/cell lines than MDR1, suggesting that this is the dominant Pgp isoform in the term placenta. Intriguingly, its orientation suggests it effluxes in the opposite direction to MRP1/Pgp. The changes observed in Pgp and BCRP expression with cellular fusion and syncytialization, which may reflect either phenotypic changes or functional roles in cell differentiation, are consistent with gestational age-related changes reported for these transporters.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the Maurice and Phyllis Paykel Trust. D. A. Evseenko is a recipient of a Top Achiever Doctoral Scholarship from the Foundation for Research Science and Technology, New Zealand.


   ACKNOWLEDGMENTS
 
We thank the staff of the Labour and Birthing Unit, Auckland Hospital, New Zealand, for assistance in collection of placentas and Dr. Starling Emerald, Claire Osepchook, and Mohan Kumar for excellent technical advice.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. Evseenko, Liggins Institute, Univ. of Auckland, Private bag 92019 (e-mail: d.evseenko{at}auckland.ac.nz)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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