Placenta 35 (2014) 324e330
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Effect of oxygen on multidrug resistance in term human placenta M. Javam a, M.C. Audette a, M. Iqbal a, E. Bloise a, W. Gibb b, c, S.G. Matthews a, d, e, f, * a
Department of Physiology, University of Toronto, Toronto, Canada Dept Ob-Gyn, University of Ottawa, Ottawa, Canada c Dept Cellular & Molecular Medicine, University of Ottawa, Ottawa, Canada d Department of Ob-Gyn, University of Toronto, Toronto, Canada e Department of Medicine, University of Toronto, Toronto, Canada f Fraser Mustard Institute for Human Development, University of Toronto, Toronto, Canada b
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 23 February 2014
Introduction: The placenta contains efflux transporters, including P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), that limit the passage of xenobiotics, certain hormones and nutrients from the maternal to the fetal circulation. The expression of these transporters changes with gestational age, yet the mechanisms involved remain unknown. However, the changes in P-gp and BCRP transporter expression coincide with those of oxygen tension in the placenta, and oxygen tension has been shown to modulate P-gp and BCRP expression in other tissues. The objective of this study was to investigate the effects of oxygen tension on P-gp and BCRP expression in the term human placenta. Methods: Following equilibration in culture (96 h), term placental explants (n ¼ 7) were cultured in 3% or 20% oxygen for 24 and 48 h. Culture medium was collected every 24 h to measure lactate dehydrogenase (LDH; explant viability) and human chorionic gonadotropin (hCG; syncytiotrophoblast function). P-gp (encoded by ABCB1) and BCRP (encoded by ABCG2) protein and mRNA, as well as VEGFA mRNA were measured using western blot and qRT-PCR. P-gp localization was determined using immunofluorescence. Results: Oxygen tension had a significant effect on P-gp expression, with ABCB1/P-gp mRNA and protein levels increased in the hypoxic condition (3% O2) after 48 h (p < 0.05). VEGFA mRNA was elevated by hypoxia at both 24 and 48 h (p < 0.05). In contrast, placental ABCG2/BCRP mRNA and protein expression were stable with changes in oxygen tension. We identified profound differences in the glycosylation of Pgp between cultured and non-cultured placental tissue, with cultured explants expressing deglycosylated P-gp. Conclusions: These findings demonstrate that, at term, the expression of placental P-gp, is regulated by oxygen tension. This suggests that changes in oxygenation of the placenta in the third trimester may alter levels of placental P-gp, and in doing so alter fetal exposure to P-gp substrates, including xenobiotics and certain hormones. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: P-glycoprotein BCRP Third-trimester placenta Hypoxia Multidrug resistance
1. Introduction One of the many roles of the placenta throughout gestation is to act as a protective barrier. In addition to acting as a barrier that separates maternal blood from fetal blood, the placenta also contains a variety of efflux transporters that are localized to the syncytiotrophoblast and fetal capillary endothelium, which limit the passage of molecules from the maternal to the fetal circulation [1].
* Corresponding author. Department of Physiology, Faculty of Medicine, Medical Sciences Building, Rm 3302, University of Toronto, 1 King’s College Circle, Toronto, ON M5S 1A8, Canada. Tel.: þ1 416 978 2025; fax: þ1 416 978 4940. E-mail address: [email protected] (S.G. Matthews). http://dx.doi.org/10.1016/j.placenta.2014.02.010 0143-4004/Ó 2014 Elsevier Ltd. All rights reserved.
Many of these efflux transporters belong to the ATP-binding cassette (ABC) family of transporters, which decrease intracellular substrate concentrations through an active, energy-dependent mechanism [2]. Two of these important ABC efflux transporters are P-glycoprotein (P-gp encoded by ABCB1) and breast cancer resistance protein (BCRP encoded by ABCG2) [1]. Prior studies have shown these transporters play a critical role in fetal protection throughout pregnancy [3e6]. For example, all the offspring of mice deficient in placental P-gp showed the cleft palate phenotype when exposed to Avermectin, a known substrate of P-gp, whereas the offspring from wild type animals was normal [3]. In BCRP-deficient pregnant mice there was a five-fold increase in fetal concentrations of the BCRP substrate, nitrofurantoin, compared with wild type animals [5].
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We have previously shown that the expression of these transporters in the human placenta changes throughout pregnancy [7,8]. ABCB1 mRNA and P-gp protein expression decrease with advancing gestation [7,8]. This decrease in P-gp levels has a potential to leave the fetus susceptible to teratogens that may be present in the maternal circulation. The mechanism behind this decrease in P-gp expression remains unknown. However, the decrease in P-gp expression coincides with changes in oxygen tension in the placenta. In the first trimester, the fetus is highly sensitive to molecular oxygen and a low oxygen environment helps to protect against oxidative stress [9]. At this early stage of pregnancy, the partial pressure of oxygen is about 20 mmHg (w3% O2) due to the occlusion of the terminal portion of maternal spiral arteries by the invading extravillous trophoblast (EVT) [9]. In the early second trimester, the de-plugging of the spiral arteries allows maternal blood to enter the intervillous space, causing an increase in oxygen tension to about 60 mmHg (w8% O2) [9]. However, improper or inadequate conversion of spiral arteries in the first trimester can lead to chronic placental ischemia and hypoxia later in gestation, which can cause deleterious outcomes such as intrauterine growth restriction (IUGR), preeclampsia, unexplained miscarriage, and preterm labor [10e13]. Furthermore, these hypoxic placentas may have altered transporter expression patterns compared to placentas from normal pregnancies [14]. Studies in other cell types, especially cancer cells, have provided valuable insight into the relationship between oxygen tension and transporter expression. Both P-gp and BCRP are highly expressed in a number of cancer cells and expression has been shown to increase under conditions of low oxygen tension [15e18]. This increase in expression is mediated through the binding of hypoxia inducible factor (HIF-1a) to the HIF responsive element (HRE) in the ABCB1 and ABCG2 promoters [15,18]. We have recently shown that oxygen tension affects P-gp and BCRP transporter expression in first trimester human placental explants [19]. An increase in oxygen tension resulted in an increase in ABCG2 expression with no change in ABCB1 mRNA expression. At the protein level, hypoxia induced Pgp and BCRP expression in proliferating cytotrophoblasts [19]. Term placenta is subjected to higher oxygen tension than first trimester placenta and it is not known whether oxygen tension regulates P-gp and BCRP expression at term. Moreover, uteroplacental hypoxia, as a result of occlusion or inadequate trophoblast invasion of maternal spiral arteries, can lead to chronic placental ischemia and hypoxia later in gestation [20]. Altered P-gp and BCRP expression in response to hypoxia would impact fetal exposure to xenobiotics and hormones present in the maternal circulation. The purpose of the present study was to examine the effect of oxygen on the expression of these transporters in term placenta using an in vitro model of intact placental explants. We hypothesized that a decrease in oxygen tension would result in an increase in P-gp and BCRP levels. 2. Methods 2.1. Placental collection and tissue culture Term C-section placentas (n ¼ 7) were collected within 30 min of delivery from the Research Centre for Women’s and Infants’ Health (RCWIH) BioBank program at Mount Sinai Hospital. Written informed consent was obtained from all subjects and ethical approval was obtained from the Mount Sinai Hospital and the University of Toronto Research Ethics Board. Placental tissue biopsies (1 cm3) were further dissected in the laboratory into approximately 8 mm3 fragments. Villous explants were then cultured in individual Costar Netwells (Corning, New York) in CMRL-1066 media (Invitrogen, Carlsbad, California, USA), supplemented with NaHCO3 (2.2 mg/ml; SigmaeAldrich, St. Louis, MO, USA), antibioticseantimycotics (Invitrogen), insulin (1 mg/ml; Sigma), retinol acetate (1 mg/ml; Sigma), L-glutamine (100 mg/ml; Sigma) and fetal bovine serum (5%; Wisent Inc., St. Bruno, Quebec, Canada) as previously described [21]. We and others [21,22] have shown that in placental explant cultures, the syncytiotrophoblast sheds within the first 2 days of culture and regenerates by days 3e4, using both measures of
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syncytiotrophoblast function and histological analysis of syncytiotrophoblast integrity. Therefore, placental explants were cultured for 4 days (20% O2, 5% CO2, 37 C) as previously described [21,22], which allowed syncytiotrophoblast shedding and regeneration to take place. On day 4, oxygen tension was changed to 3% (5% CO2, 37 C) or maintained at 20% for a further 24 and 48 h. Culture media was changed every 24 h during the culture period and care was taken throughout the culture period for explants to remain out of the incubators for the same length of time. Lactate dehydrogenase (LDH) and human chorionic gonadotropin (hCG) were measured in the media to assess explant viability and syncytiotrophoblast function, respectively [21,22].
2.2. Lactate dehydrogenase (LDH) and human chorionic gonadotropin (hCG) LDH and hCG were measured as described previously [21]. Briefly, LDH was quantified using the Cytotoxicity Detection kit (Roche Applied Science, Indianapolis, USA) according to the manufacturer’s instructions [19,21]. A standard curve for the LDH assay was generated using L-lactic dehydrogenase from rabbit muscle (Sigma) [19,21]. hCG was measured by ELISA (DRG Diagnostics, Germany) according to the manufactures’ instructions [21].
2.3. Quantitative real time PCR Total RNA was extracted from the placental explants using TRIzol reagent (Invitrogen) as described in the manufacturer’s protocol. Contaminating genomic DNA was removed from RNA samples using a DNA-free kit (Ambion, Austin, Texas, USA). RNA purity (A260/A280 ratio of 1.8e2.0) and concentration were assessed using spectrophotometric analysis, and RNA integrity was verified by agarose gel electrophoresis. RNA was reverse-transcribed to cDNA using High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Carlsbad, California, USA) according to the manufacturer’s instructions and stored (80 C). Levels of ABCB1, ABCG2, and VEGFA mRNA were measured by real time RT-PCR using SsoFast EvaGreen Supermix (Bio-Rad Laboratories, Hercules, CA, USA) and the CFX 96 Real-Time system C1000 Thermal Cycler (Bio-Rad). Relative gene expression was measured using [DDc(t)] method and normalized to the geometric mean of three housekeeping genes: glyceraldehyde 3-phosphate dehydrogenase (GAPDH), TATA-binding protein (TBP), and zeta polypeptide (YWHAZ) [19]. Expression of these genes was stable during the experiments (data not shown). Data was analyzed using CFX Manager Software (Bio-Rad). Primer sequences for the genes analyzed are listed in Table 1.
2.4. Western blotting Western blotting was performed as described previously [23]. Briefly, placental explants were homogenized in RIPA lysis buffer (Cell Signaling Technology, Inc., Danvers, MA), centrifuged (10,000 g, 10 min, 4 C), and the supernatant collected. Protein (40 mg) separation using SDS-PAGE was performed using polyacrylamide gels (7%) and transferred to nitrocellulose membranes using iBlot transfer apparatus (Invitrogen). Membranes were blocked in BSA (5% w/v of Tris buffered saline [TBS] with Tween 20: TBS-T, 1 h, 25 C). Membranes were then incubated with primary antibody overnight (4 C). The primary antibodies used were: mouse anti-P-gp (1:5000; clone 5A12.2; EMD Millipore Corporation, Billerica, MA, USA), mouse anti-BCRP (1:500; clone BXP-21; EMD Millipore Corporation), and rabbit anti-actin (1:10,000; Sigma). Membranes were washed and incubated with horseradish peroxidase conjugated secondary antibody (1:15,000; PerkinElmer, Waltham, MA) against the corresponding primary antibody, followed by incubation (1 min) in enhanced chemiluminescence substrate (Invitrogen) and exposed to chemiluminescence film (General Electric Healthcare, Baie d’Urfe, Quebec, Canada). The relative optical density (ROD) of the bands was measured using computerized image analysis and was standardized against the b-actin signal (MCID Core 7.0, Imaging Research Inc., Interfocus Imaging Ltd., Cambridge, England).
Table 1 Primers used for real-time PCR. Gene symbol
Sequence
References
ABCB1
Forward: AGC AGA GGC CGC TGT TCG TT Reverse: CCA TTC CGA CCT CGC GCT CC Forward: TGG AAT CCA GAA CAG AGC TGG GGT Reverse: AGA GTT CCA CGG CTG AAA CAC TGC Forward: CGG GCC TCC GAAA CCA TGA ACT T Reverse: CCC TCC TCC TTC TGC CAT GGG T Forward: CAC GAA CCA CGG CAC TGA TT Reverse: TTT TCT TGC TGC CAG TCT GGA C Forward: AGA TCA TCA GCA ATG CCT CC Reverse: CAT GAG TCC TCC CAC GAT AC Forward: ACT TTT GGT ACA TTG TGG CTT CAA Reverse: CCG CCA GGA CAA ACC AGT AT
[19]
ABCG2 VEGFA TBP GAPDH YWHAZ
[19] [19] [19] [19] [19]
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2.5. Immunofluorescence Placental explants were fixed in neutral buffered formalin (10%; Sigma), washed with PBS and cryosectioned (10 mm). Sections were mounted onto microscope slides and stored (80 C). Prior to immunofluorescence, slides were brought to room temperature and washed with PBS. Antigen retrieval was performed using boiling sodium citrate (10 mM, 10 min). Sections were cooled on ice (10 min) and washed with PBS before blocking in normal goat serum (5%, 1 h; Invitrogen). Slides were incubated overnight with mouse anti-P-gp primary antibody (1:50, D-11, Santa Cruz Biotechnology, Santa Cruz, CA, USA) [19], then washed and incubated (1 h, 25 C) with Alexa Fluor 488-conjugated goat secondary antibody (1:500; Invitrogen). Negative controls were stained by substitution of primary antibody with nonimmune mouse IgG (Dako, Burlington, ON, Canada). After final washes, slides were mounted and cover-slipped using mounting media with 40 ,6-diamidino-2phenylindole (Vector Laboratories, Burlingame, CA). Imaging was performed using Zeiss spinning disk confocal microscope (Zeiss Observer.Z1). 2.6. Deglycosylation P-gp deglycosylation was carried out using Peptide-N-Glycosidase F (PNGase F; Sigma) according to the manufacturer’s instructions. Briefly, denatured and nondenatured protein (30 mg) from fresh placental tissue was incubated with PNGase F (4 h, 37 C). Both denaturing and non-denaturing conditions were used because some proteins require denaturation prior to PNGase F digestion. Laemmli sample buffer (Bio-Rad) was added, and samples boiled (5 min) to inactivate the PNGase F. Western-blot analysis was performed as described above. 2.7. Statistical analysis Data are expressed as mean SEM and all statistical analyses were performed using Prism (GraphPad Software, Inc., San Diego, CA). hCG and LDH concentrations were log transformed and analyzed using one-way repeated measures ANOVA with NewmaneKeuls post-hoc test. Differences in mRNA and protein expression over time of culture and oxygen tension were assessed by two-way ANOVA with Bonferroni post-hoc test. Protein and mRNA expression were also analyzed using oneway ANOVA, followed by Dunnett post-test for comparisons against the control group. Statistical significance was set at p < 0.05.
3. Results 3.1. Tissue viability and syncytiotrophoblast function LDH release from placental explants across the 6-day culture period was measured to assess tissue viability [21,22]. LDH levels in the media decreased gradually, reaching a stable phase on day 4 (Fig. 1A). In order to monitor syncytial shedding and regeneration, hCG secretion was measured, a marker of syncytiotrophoblast endocrine function [21,22]. Between day 1 and day 2 there was a steep decline in hCG release into the media (p < 0.05) (Fig. 1B). From day 2 to day 3 there was a rapid increase in hCG levels (p < 0.05). The elevated hCG levels were sustained throughout the rest of the culture period. This hCG pattern was indicative of syncytiotrophoblast shedding and regeneration [21,22]. Therefore, placental explants were cultured for 4 days in 20% oxygen, which allowed syncytiotrophoblast shedding and regeneration to take
place. The oxygen tension was then reduced to 3% or maintained at 20% for 24 and 48 h. Day 4 was considered as the control group. 3.2. Effects of oxygen tension on ABCB1, ABCG2, and VEGFA mRNA expression ABCB1 and ABCG2 mRNA expression was measured in the control (day 4 of culture), 24 h (day 5), and 48 h (day 6) samples from the same placentae using qRT-PCR. There was a significant effect of oxygen tension on ABCB1 expression, assessed by two-way ANOVA (p < 0.05) (Fig. 2A), with no effects of time in culture or interaction of the two variables. There was a significant increase in ABCB1 expression after 48 h in 3% oxygen compared to the control group (p < 0.05) (Fig. 2A). Oxygen tension had no effect on ABCG2 expression, assessed by two-way ANOVA (Fig. 2B), with no effects of time in culture or interaction of the two variables. VEGFA mRNA expression was measured to assess activation of hypoxic pathways [19]. There was a significant increase in VEGFA expression in the hypoxic condition (3% O2) (p < 0.05) (Fig. 2C), with no effects of time in culture or interaction of the two variables. VEGFA expression was also significantly increased at both 24 h and 48 h hypoxic conditions compared to the control group. 3.3. Effects of oxygen on P-gp and BCRP expression There was no significant overall effect of oxygen tension on P-gp protein levels, assessed by two-way ANOVA (Fig. 3C), with no interaction between the two variables. However, there was a significant effect of time in culture on P-gp levels (p < 0.05), such that there was an increase in P-gp levels with increased time in culture. Further analysis using repeated measures one-way ANOVA revealed a significant increase in P-gp levels after 48 h of culture in 3% oxygen compared to control (p < 0.05) (Fig. 3C). Oxygen tension had no effect on BCRP protein levels, assessed by two-way ANOVA (Fig. 3D), with no effects of time in culture or interaction of the two variables. 3.4. Effect of tissue culture on P-gp glycosylation and localization Western blot analysis performed using fresh placental tissue, showed a predominant 150 kDa band for P-gp (Fig. 4A). In contrast, P-gp in cultured placental tissue was approximately 120 kDa (Fig. 4A). To assess whether the difference in the molecular weight of P-gp was due to deglycosylation, protein from fresh placental tissue was treated with PNGase F (this cleaves the link between asparagine and N-acetylglucosamines). PNGase F treatment resulted in a shift in the molecular weight of P-gp from 150 kDa to
Fig. 1. Placental explant viability and syncytiotrophoblast function. (A) Lactate dehydrogenase (LDH) and (B) human chorionic gonadotropin (hCG) released from explants into the culture media over the 6-day culture period. Results shown as mean S.E.M. Three separate tissues assayed in triplicate. hCG and LDH concentrations were log transformed and analyzed using one-way repeated measures ANOVA with NewmaneKeuls post-hoc test. # represents significant difference from day 1 (p < 0.05). * represents significant difference from all other days (p < 0.05).
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120 kDa (Fig. 4B). Changes in oxygen tension had no effect on the glycosylation state of P-gp (data not shown). To assess whether deglycosylation altered P-gp localization in the tissue, fresh and cultured explants were analyzed using immunofluorescent staining. In both instances P-gp was localized to the syncytiotrophoblast (Fig. 5) (n ¼ 4). Furthermore, decreasing oxygen tension did not alter P-gp localization to the syncytiotrophoblast (Fig. 5). 4. Discussion
Fig. 2. Effects of oxygen tension on ABCB1, ABCG2, and VEGF expression. (A) ABCB1, (B) ABCG2, (C) VEGFA mRNA expression in term placental explants normalized to the geometric mean of three housekeeping genes. Placental explants were cultured for 4 days in 20% oxygen, which allowed syncytiotrophoblast shedding and regeneration to take place. The oxygen tension was then reduced to 3% or continued at 20% for 24 and 48 h. Day 4 was considered as the control group. There was a significant effect of oxygen tension on ABCB1 and VEGFA expression. Solid bars represent 3% oxygen tension, while open bars represent 20% oxygen tension. * represents significant difference from control (p < 0.05). Results shown as mean S.E.M. (n ¼ 7).
We have shown, for the first time, that changes in oxygen tension in the term human placenta have significant but different effects on P-gp and BCRP expression. Decreased oxygen tension resulted in an increase in ABCB1/P-gp mRNA and protein expression in term placental explants but had no effect on ABCG2/BCRP mRNA or protein expression. We also report the novel finding that while P-gp is glycosylated in fresh placental tissue, it becomes fully deglycosylated in cultured placental explants. This de-glycosylation had no effect on the localization of P-gp to the syncytiotrophoblast. This novel observation has important implications for our understanding of the post-translational regulation of P-gp in the placenta and warrants further detailed investigation. In the human placenta, ABCB1/P-gp mRNA and protein expression decrease with advancing gestation [7], which correlates closely with changes in oxygen tension throughout pregnancy. In the present study, we found that 48 h exposure to low oxygen tension (3%) resulted in a significant increase in ABCB1/P-gp mRNA and protein levels, compared to the control group (20% O2). This increase in ABCB1 and P-gp expression in response to hypoxia is consistent with studies performed using other cell types [17,18]. Furthermore, the responsiveness of P-gp expression to a decrease in oxygen tension provides a possible explanation for the high P-gp levels in the low oxygen tension environment of the first trimester placenta compared to term placenta. Studies investigating the expression pattern of BCRP in the human placenta throughout gestation are inconsistent. We have previously shown that ABCG2 mRNA expression does not change throughout pregnancy, while BCRP protein levels increase towards term [8]. On the other hand, Meyer zu Schwabedissen et al. demonstrated that both ABCG2 mRNA and BCRP protein expression increase with advancing gestation [24] but Mathias et al. found that ABCG2 mRNA and BCRP protein expression did not change with gestational age [25]. The reason for the discrepancy in BCRP expression between studies may result from variability in sampling, as BCRP is present in multiple placental cell-types. In the present study oxygen tension had no effect on ABCG2 mRNA or BCRP protein expression. This would be consistent with the stable ABCG2/ BCRP expression pattern during pregnancy, despite the increase in oxygen tension that occurs with advancing gestation. However, a steady level of BCRP expression does not necessarily indicate no change in BCRP function, as no study has measured BCRP activity throughout gestation. In fact, there are numerous studies illustrating posttranslational modifications, such as glycosylation and phosphorylation, changing BCRP function [26e28]. The same is true for P-gp [29-31], as Thews et al. have shown a more than doubling of P-gp activity in prostate carcinoma cells in acidic environments with no change in P-gp expression [31]. Future studies are required to measure BCRP and P-gp activity in the placenta with advancing gestation. Compared to the third trimester, the placenta in the first trimester is exposed to lower oxygen levels [12]. Early in the first trimester the extravillous trophoblast (EVT) invade and occlude the terminal portion of spiral arteries, inhibiting maternal blood from entering the intervillous space and resulting in a low oxygen
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Fig. 3. Effects of oxygen tension on P-gp and BCRP protein expression. Representative Western blot of (A) P-gp and (B) BCRP protein from a single placenta. Protein expression of (C) P-gp and (D) BCRP in term placental explants normalized to b-actin. Placental explants were cultured for 4 days in 20% oxygen, which allowed syncytiotrophoblast shedding and regeneration to take place. The oxygen tension was then reduced to 3% or continued at 20% for 24 and 48 h. Day 4 was considered as the control group. There was a significant effect of oxygen tension on P-gp expression. Solid bars represent 3% oxygen tension, while open bars represent 20% oxygen tension. * represents significant difference from control (p < 0.05). Results shown as mean S.E.M. of relative optical density (ROD). (n ¼ 7).
environment (w3% O2) [10]. In late first trimester, the endovascular EVT causes physiological changes in placental spiral arteries, forming funnel-shaped flaccid arterioles with increased vascular compliance and circumference [10]. This leads to the flow of maternal blood into the intervillous space and a dramatic increase in oxygen levels for the remainder of pregnancy (w8% O2) [10]. However, improper or inadequate conversion of spiral arteries in the first trimester can lead to chronic placental ischemia and hypoxia later in gestation [10e13]. Moreover, embryogenesis in the first trimester already takes place under anaerobic conditions, and
Fig. 4. Deglycosylation of placental P-gp. (A) Representative Western blot comparing Pgp expression in fresh term placental tissue and cultured placental explants. (B) Deglycosylation of fresh placental tissue. (i) Fresh term placental tissue. (ii) Denatured protein from fresh placental tissue treated with PNGase F. (iii) Non-denatured protein from fresh placental tissue treated with PNGase F. (iv) cultured placental explant on Day 6.
in the second and third trimester oxygen becomes more important for normal fetal organogenesis and growth [32]. Protection from hypoxia at this stage of development is established via upregulation of various genes, including superoxide dismutase, insulin-like growth factor (IGF)-2, and heat shock protein 70 [33e 35]. Our data indicate that ABCB1 may be one of the protective genes activated in response to hypoxia in the third trimester placenta. In fact, P-gp has been suggested to play a critical role in cell protection by extruding toxins and harmful products of oxidative stress [36]. Therefore, P-gp may also play a role in protecting the fetus from toxic products of oxidative stress in third trimester hypoxic placentas. We have recently shown that in the first trimester placenta, ABCG2 mRNA expression increased significantly in hyperoxic (20%) conditions after 48 h in culture with no change in ABCB1 mRNA expression [19]. At the protein level, hypoxia resulted in increased immunoreactive BCRP in cytotrophoblasts and in the microvillous membrane of the syncytium, and increased P-gp immunoreactivity in proliferating cytotrophoblasts. This contrasts with the results of the current study and suggests that the placenta in the different stages of pregnancy responds quite differently to changes in oxygen tension. This is understandable, as oxygen tension that is physiological in the first trimester would be considered hypoxic at term. Interestingly, we found that cultured placental tissue contained P-gp that was less glycosylated compared to fresh placental tissue. Post-translational modifications, such as glycosylation, have been shown to alter P-gp activity in various ways, depending on the cell line examined [29]. In general, glycosylation can affect a transporter’s activity, folding, stability, subcellular trafficking, localization, and turnover [37,38]. P-gp is glycosylated on three recognition sites (Asn 91, 94, and 99) in the first extracellular loop [37]. Deglycosylation of P-gp in fresh placenta decreased the molecular weight from approximately 150 kDae120 kDa. This 30 kDa decrease is in agreement with previous studies [39,40]. Since the effect of deglycosylation on P-gp localization is cell type specific [30,37,40,41], we examined whether deglycosylation affects P-gp localization in term placental tissue. Using immunofluorescent staining, the glycosylation state of P-gp was not found to affect P-gp localization in the explants. Furthermore, oxygen tension had no effect on P-gp glycosylation or its localization to the syncytiotrophoblast. Since the impact of glycosylation on P-gp activity is cell-specific [29,30,41], future studies are required to determine the effects of deglycosylation on P-gp activity in the placenta. It is also important to highlight
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Fig. 5. Representative immunofluorescent images showing P-gp (green) localization to the syncytiotrophoblast in placental explants exposed to different oxygen tensions. Nuclei were stained by 40 ,6-diamidino-2-phenylindole (blue) (n ¼ 4). Negative controls were stained by substitution of primary antibody with non-immune mouse IgG. Arrows point to syncytiotrophoblast.
the potential limitation of the present study. For example, in the immunofluorescent staining we were unable to definitively distinguish between the syncytiotrophoblast and unfused cytotrophoblasts in the outer trophoblast layer. Also, it is possible that molecular changes in syncytiotrophoblast function occur in pathways that indirectly modulate multidrug resistance, following long term explant cultures. Future studies are required to determine the precise molecular pathways involved in P-gp and BCRP regulation. In conclusion, oxygen tension affects multidrug resistance in term human placenta. These changes could alter fetal exposure to drugs, xenobiotic and endogenous substrates. Notably, improper or
inadequate conversion of spiral arteries in the first trimester can lead to chronic placental ischemia and hypoxia later in gestation, which can cause deleterious outcomes such as intrauterine growth restriction (IUGR) and preeclampsia. Glycosylation is an important regulatory mechanism of P-gp function and expression, and should be considered so that we can better understand the various roles of P-gp both in normal pregnancy progression and in disease states. Moreover, future studies should focus on the expression profile and function of drug transporters in oxygen-related placental pathologies since changes in the activity of these transporters could have a profound impact on fetal drug exposure.
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Conflict of interest statement The authors declare that there are no conflicts of interest. Disclosure summary Authors have nothing to declare. Acknowledgments This study was funded by the Canadian Institutes of Health Research (grant: FRN-57746; to S.G.M. and W.G.). References [1] Iqbal M, Audette MC, Petropoulos S, Gibb W, Matthews SG. Placental drug transporters and their role in fetal protection. Placenta 2012;33(3):137e42. [2] Vahakangas K, Myllynen P. Drug transporters in the human blood-placental barrier. Br J Pharmacol 2009;158(3):665e78. [3] Lankas GR, Wise LD, Cartwright ME, Pippert T, Umbenhauer DR. Placental Pglycoprotein deficiency enhances susceptibility to chemically induced birth defects in mice. Reprod Toxicol 1998;12(4):457e63. [4] Smit JW, Huisman MT, van Tellingen O, Wiltshire HR, Schinkel AH. Absence or pharmacological blocking of placental P-glycoprotein profoundly increases fetal drug exposure. J Clin Invest 1999;104(10):1441e7. [5] Zhang Y, Wang H, Unadkat JD, Mao Q. Breast cancer resistance protein 1 limits fetal distribution of nitrofurantoin in the pregnant mouse. Drug Metab Dispos 2007;35(12):2154e8. [6] Prouillac C, Lecoeur S. The role of the placenta in fetal exposure to xenobiotics: importance of membrane transporters and human models for transfer studies. Drug Metab Dispos 2010;38(10):1623e35. [7] Sun M, Kingdom J, Baczyk D, Lye SJ, Matthews SG, Gibb W. Expression of the multidrug resistance P-glycoprotein, (ABCB1 glycoprotein) in the human placenta decreases with advancing gestation. Placenta 2006;27(6e7):602e9. [8] Yeboah D, Sun M, Kingdom J, Baczyk D, Lye SJ, Matthews SG, et al. Expression of breast cancer resistance protein (BCRP/ABCG2) in human placenta throughout gestation and at term before and after labor. Can J Physiol Pharmacol 2006;84(12):1251e8. [9] Schneider H. Oxygenation of the placental-fetal unit in humans. Respir Physiol Neurobiol 2011;178(1):51e8. [10] Patel J, Landers K, Mortimer RH, Richard K. Regulation of hypoxia inducible factors (HIF) in hypoxia and normoxia during placental development. Placenta 2010;31(11):951e7. [11] Tuuli MG, Longtine MS, Nelson DM. Review: oxygen and trophoblast biologye a source of controversy. Placenta 2011;32(Suppl. 2):S109e18. [12] Murray AJ. Oxygen delivery and fetal-placental growth: beyond a question of supply and demand? Placenta 2012;33(Suppl. 2):e16e22. [13] Burton GJ, Jauniaux E. Placental oxidative stress: from miscarriage to preeclampsia. J Soc Gynecol Investig 2004;11(6):342e52. [14] Mason CW, Buhimschi IA, Buhimschi CS, Dong Y, Weiner CP, Swaan PW. ATPbinding cassette transporter expression in human placenta as a function of pregnancy condition. Drug Metab Dispos 2011;39(6):1000e7. [15] Krishnamurthy P, Ross DD, Nakanishi T, Bailey-Dell K, Zhou S, Mercer KE, et al. The stem cell marker Bcrp/ABCG2 enhances hypoxic cell survival through interactions with heme. J Biol Chem 2004;279(23):24218e25. [16] Vaupel P, Mayer A. Hypoxia in cancer: significance and impact on clinical outcome. Cancer Metastasis Rev 2007;26(2):225e39. [17] Callaghan R, Crowley E, Potter S, Kerr ID. P-glycoprotein: so many ways to turn it on. J Clin Pharmacol 2008;48(3):365e78. [18] Comerford KM, Wallace TJ, Karhausen J, Louis NA, Montalto MC, Colgan SP. Hypoxia-inducible factor-1-dependent regulation of the multidrug resistance (MDR1) gene. Cancer Res 2002;62(12):3387e94. [19] Lye P, Bloise E, Dunk C, Javam M, Gibb W, Lye SJ, et al. Effect of oxygen on multidrug resistance in the first trimester human placenta. Placenta 2013;34(9):817e23.
[20] Pringle KG, Kind KL, Sferruzzi-Perri AN, Thompson JG, Roberts CT. Beyond oxygen: complex regulation and activity of hypoxia inducible factors in pregnancy. Hum Reprod Update 2010;16(4):415e31. [21] Audette MC, Greenwood SL, Sibley CP, Jones CJ, Challis JR, Matthews SG, et al. Dexamethasone stimulates placental system A transport and trophoblast differentiation in term villous explants. Placenta 2010;31(2):97e105. [22] Siman CM, Sibley CP, Jones CJ, Turner MA, Greenwood SL. The functional regeneration of syncytiotrophoblast in cultured explants of term placenta. Am J Physiol Regul Integr Comp Physiol 2001;280(4):R1116e22. [23] Iqbal M, Gibb W, Matthews SG. Corticosteroid regulation of P-glycoprotein in the developing blood-brain barrier. Endocrinology 2011;152(3):1067e79. [24] Meyer zu Schwabedissen HE, Grube M, Dreisbach A, Jedlitschky G, Meissner K, Linnemann K, et al. Epidermal growth factor-mediated activation of the map kinase cascade results in altered expression and function of ABCG2 (BCRP). Drug Metab Dispos 2006;34(4):524e33. [25] Mathias AA, Hitti J, Unadkat JD. P-glycoprotein and breast cancer resistance protein expression in human placentae of various gestational ages. Am J Physiol Regul Integr Comp Physiol 2005;289(4):R963e9. [26] Xie Y, Xu K, Linn DE, Yang X, Guo Z, Shimelis H, et al. The 44-kDa Pim-1 kinase phosphorylates BCRP/ABCG2 and thereby promotes its multimerization and drug-resistant activity in human prostate cancer cells. J Biol Chem 2008;283(6):3349e56. [27] Sugiyama T, Shuto T, Suzuki S, Sato T, Koga T, Suico MA, et al. Posttranslational negative regulation of glycosylated and non-glycosylated BCRP expression by Derlin-1. Biochem Biophys Res Commun 2011;404(3):853e8. [28] Draheim V, Reichel A, Weitschies W, Moenning U. N-glycosylation of ABC transporters is associated with functional activity in sandwich-cultured rat hepatocytes. Eur J Pharm Sci 2010;41(2):201e9. [29] Wojtowicz K, Szaflarski W, Januchowski R, Zawierucha P, Nowicki M, Zabel M. Inhibitors of N-glycosylation as a potential tool for analysis of the mechanism of action and cellular localisation of glycoprotein P. Acta Biochim Pol 2012;59(4):445e50. [30] Gribar JJ, Ramachandra M, Hrycyna CA, Dey S, Ambudkar SV. Functional characterization of glycosylation-deficient human P-glycoprotein using a vaccinia virus expression system. J Membr Biol 2000;173(3):203e14. [31] Thews O, Gassner B, Kelleher DK, Schwerdt G, Gekle M. Impact of extracellular acidity on the activity of P-glycoprotein and the cytotoxicity of chemotherapeutic drugs. Neoplasia 2006;8(2):143e52. [32] Hutter D, Kingdom J, Jaeggi E. Causes and mechanisms of intrauterine hypoxia and its impact on the fetal cardiovascular system: a review. Int J Pediatr 2010;2010:401323. [33] Clifton VL, Vanderlelie J, Perkins AV. Increased anti-oxidant enzyme activity and biological oxidation in placentae of pregnancies complicated by maternal asthma. Placenta 2005;26(10):773e9. [34] Trollmann R, Klingmuller K, Schild RL, Rascher W, Dotsch J. Differential gene expression of somatotrophic and growth factors in response to in vivo hypoxia in human placenta. Am J Obstet Gynecol 2007;197(6):601.e1e6. [35] Richter HG, Camm EJ, Modi BN, Naeem F, Cross CM, Cindrova-Davies T, et al. Ascorbate prevents placental oxidative stress and enhances birth weight in hypoxic pregnancy in rats. J Physiol 2012;590(Pt 6):1377e87. [36] Huls M, Russel FG, Masereeuw R. The role of ATP binding cassette transporters in tissue defense and organ regeneration. J Pharmacol Exp Ther 2009;328(1): 3e9. [37] Greer DA, Ivey S. Distinct N-glycan glycosylation of P-glycoprotein isolated from the human uterine sarcoma cell line MES-SA/Dx5. Biochim Biophys Acta 2007;1770(9):1275e82. [38] Vagin O, Kraut JA, Sachs G. Role of N-glycosylation in trafficking of apical membrane proteins in epithelia. Am J Physiol Renal Physiol 2009;296(3): F459e69. [39] Beaulieu E, Demeule M, Ghitescu L, Beliveau R. P-glycoprotein is strongly expressed in the luminal membranes of the endothelium of blood vessels in the brain. Biochem J 1997;326(Pt 2):539e44. [40] Schinkel AH, Kemp S, Dolle M, Rudenko G, Wagenaar E. N-glycosylation and deletion mutants of the human MDR1 P-glycoprotein. J Biol Chem 1993;268(10):7474e81. [41] Seres M, Cholujova D, Bubencikova T, Breier A, Sulova Z. Tunicamycin depresses p-glycoprotein glycosylation without an effect on its membrane localization and drug efflux activity in l1210 cells. Int J Mol Sci 2011;12(11): 7772e84.
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Effect of oxygen on multidrug resistance in term human placenta M. Javam a, M.C. Audette a, M. Iqbal a, E. Bloise a, W. Gibb b, c, S.G. Matthews a, d, e, f, * a
Department of Physiology, University of Toronto, Toronto, Canada Dept Ob-Gyn, University of Ottawa, Ottawa, Canada c Dept Cellular & Molecular Medicine, University of Ottawa, Ottawa, Canada d Department of Ob-Gyn, University of Toronto, Toronto, Canada e Department of Medicine, University of Toronto, Toronto, Canada f Fraser Mustard Institute for Human Development, University of Toronto, Toronto, Canada b
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 23 February 2014
Introduction: The placenta contains efflux transporters, including P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), that limit the passage of xenobiotics, certain hormones and nutrients from the maternal to the fetal circulation. The expression of these transporters changes with gestational age, yet the mechanisms involved remain unknown. However, the changes in P-gp and BCRP transporter expression coincide with those of oxygen tension in the placenta, and oxygen tension has been shown to modulate P-gp and BCRP expression in other tissues. The objective of this study was to investigate the effects of oxygen tension on P-gp and BCRP expression in the term human placenta. Methods: Following equilibration in culture (96 h), term placental explants (n ¼ 7) were cultured in 3% or 20% oxygen for 24 and 48 h. Culture medium was collected every 24 h to measure lactate dehydrogenase (LDH; explant viability) and human chorionic gonadotropin (hCG; syncytiotrophoblast function). P-gp (encoded by ABCB1) and BCRP (encoded by ABCG2) protein and mRNA, as well as VEGFA mRNA were measured using western blot and qRT-PCR. P-gp localization was determined using immunofluorescence. Results: Oxygen tension had a significant effect on P-gp expression, with ABCB1/P-gp mRNA and protein levels increased in the hypoxic condition (3% O2) after 48 h (p < 0.05). VEGFA mRNA was elevated by hypoxia at both 24 and 48 h (p < 0.05). In contrast, placental ABCG2/BCRP mRNA and protein expression were stable with changes in oxygen tension. We identified profound differences in the glycosylation of Pgp between cultured and non-cultured placental tissue, with cultured explants expressing deglycosylated P-gp. Conclusions: These findings demonstrate that, at term, the expression of placental P-gp, is regulated by oxygen tension. This suggests that changes in oxygenation of the placenta in the third trimester may alter levels of placental P-gp, and in doing so alter fetal exposure to P-gp substrates, including xenobiotics and certain hormones. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: P-glycoprotein BCRP Third-trimester placenta Hypoxia Multidrug resistance
1. Introduction One of the many roles of the placenta throughout gestation is to act as a protective barrier. In addition to acting as a barrier that separates maternal blood from fetal blood, the placenta also contains a variety of efflux transporters that are localized to the syncytiotrophoblast and fetal capillary endothelium, which limit the passage of molecules from the maternal to the fetal circulation [1].
* Corresponding author. Department of Physiology, Faculty of Medicine, Medical Sciences Building, Rm 3302, University of Toronto, 1 King’s College Circle, Toronto, ON M5S 1A8, Canada. Tel.: þ1 416 978 2025; fax: þ1 416 978 4940. E-mail address: [email protected] (S.G. Matthews). http://dx.doi.org/10.1016/j.placenta.2014.02.010 0143-4004/Ó 2014 Elsevier Ltd. All rights reserved.
Many of these efflux transporters belong to the ATP-binding cassette (ABC) family of transporters, which decrease intracellular substrate concentrations through an active, energy-dependent mechanism [2]. Two of these important ABC efflux transporters are P-glycoprotein (P-gp encoded by ABCB1) and breast cancer resistance protein (BCRP encoded by ABCG2) [1]. Prior studies have shown these transporters play a critical role in fetal protection throughout pregnancy [3e6]. For example, all the offspring of mice deficient in placental P-gp showed the cleft palate phenotype when exposed to Avermectin, a known substrate of P-gp, whereas the offspring from wild type animals was normal [3]. In BCRP-deficient pregnant mice there was a five-fold increase in fetal concentrations of the BCRP substrate, nitrofurantoin, compared with wild type animals [5].
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We have previously shown that the expression of these transporters in the human placenta changes throughout pregnancy [7,8]. ABCB1 mRNA and P-gp protein expression decrease with advancing gestation [7,8]. This decrease in P-gp levels has a potential to leave the fetus susceptible to teratogens that may be present in the maternal circulation. The mechanism behind this decrease in P-gp expression remains unknown. However, the decrease in P-gp expression coincides with changes in oxygen tension in the placenta. In the first trimester, the fetus is highly sensitive to molecular oxygen and a low oxygen environment helps to protect against oxidative stress [9]. At this early stage of pregnancy, the partial pressure of oxygen is about 20 mmHg (w3% O2) due to the occlusion of the terminal portion of maternal spiral arteries by the invading extravillous trophoblast (EVT) [9]. In the early second trimester, the de-plugging of the spiral arteries allows maternal blood to enter the intervillous space, causing an increase in oxygen tension to about 60 mmHg (w8% O2) [9]. However, improper or inadequate conversion of spiral arteries in the first trimester can lead to chronic placental ischemia and hypoxia later in gestation, which can cause deleterious outcomes such as intrauterine growth restriction (IUGR), preeclampsia, unexplained miscarriage, and preterm labor [10e13]. Furthermore, these hypoxic placentas may have altered transporter expression patterns compared to placentas from normal pregnancies [14]. Studies in other cell types, especially cancer cells, have provided valuable insight into the relationship between oxygen tension and transporter expression. Both P-gp and BCRP are highly expressed in a number of cancer cells and expression has been shown to increase under conditions of low oxygen tension [15e18]. This increase in expression is mediated through the binding of hypoxia inducible factor (HIF-1a) to the HIF responsive element (HRE) in the ABCB1 and ABCG2 promoters [15,18]. We have recently shown that oxygen tension affects P-gp and BCRP transporter expression in first trimester human placental explants [19]. An increase in oxygen tension resulted in an increase in ABCG2 expression with no change in ABCB1 mRNA expression. At the protein level, hypoxia induced Pgp and BCRP expression in proliferating cytotrophoblasts [19]. Term placenta is subjected to higher oxygen tension than first trimester placenta and it is not known whether oxygen tension regulates P-gp and BCRP expression at term. Moreover, uteroplacental hypoxia, as a result of occlusion or inadequate trophoblast invasion of maternal spiral arteries, can lead to chronic placental ischemia and hypoxia later in gestation [20]. Altered P-gp and BCRP expression in response to hypoxia would impact fetal exposure to xenobiotics and hormones present in the maternal circulation. The purpose of the present study was to examine the effect of oxygen on the expression of these transporters in term placenta using an in vitro model of intact placental explants. We hypothesized that a decrease in oxygen tension would result in an increase in P-gp and BCRP levels. 2. Methods 2.1. Placental collection and tissue culture Term C-section placentas (n ¼ 7) were collected within 30 min of delivery from the Research Centre for Women’s and Infants’ Health (RCWIH) BioBank program at Mount Sinai Hospital. Written informed consent was obtained from all subjects and ethical approval was obtained from the Mount Sinai Hospital and the University of Toronto Research Ethics Board. Placental tissue biopsies (1 cm3) were further dissected in the laboratory into approximately 8 mm3 fragments. Villous explants were then cultured in individual Costar Netwells (Corning, New York) in CMRL-1066 media (Invitrogen, Carlsbad, California, USA), supplemented with NaHCO3 (2.2 mg/ml; SigmaeAldrich, St. Louis, MO, USA), antibioticseantimycotics (Invitrogen), insulin (1 mg/ml; Sigma), retinol acetate (1 mg/ml; Sigma), L-glutamine (100 mg/ml; Sigma) and fetal bovine serum (5%; Wisent Inc., St. Bruno, Quebec, Canada) as previously described [21]. We and others [21,22] have shown that in placental explant cultures, the syncytiotrophoblast sheds within the first 2 days of culture and regenerates by days 3e4, using both measures of
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syncytiotrophoblast function and histological analysis of syncytiotrophoblast integrity. Therefore, placental explants were cultured for 4 days (20% O2, 5% CO2, 37 C) as previously described [21,22], which allowed syncytiotrophoblast shedding and regeneration to take place. On day 4, oxygen tension was changed to 3% (5% CO2, 37 C) or maintained at 20% for a further 24 and 48 h. Culture media was changed every 24 h during the culture period and care was taken throughout the culture period for explants to remain out of the incubators for the same length of time. Lactate dehydrogenase (LDH) and human chorionic gonadotropin (hCG) were measured in the media to assess explant viability and syncytiotrophoblast function, respectively [21,22].
2.2. Lactate dehydrogenase (LDH) and human chorionic gonadotropin (hCG) LDH and hCG were measured as described previously [21]. Briefly, LDH was quantified using the Cytotoxicity Detection kit (Roche Applied Science, Indianapolis, USA) according to the manufacturer’s instructions [19,21]. A standard curve for the LDH assay was generated using L-lactic dehydrogenase from rabbit muscle (Sigma) [19,21]. hCG was measured by ELISA (DRG Diagnostics, Germany) according to the manufactures’ instructions [21].
2.3. Quantitative real time PCR Total RNA was extracted from the placental explants using TRIzol reagent (Invitrogen) as described in the manufacturer’s protocol. Contaminating genomic DNA was removed from RNA samples using a DNA-free kit (Ambion, Austin, Texas, USA). RNA purity (A260/A280 ratio of 1.8e2.0) and concentration were assessed using spectrophotometric analysis, and RNA integrity was verified by agarose gel electrophoresis. RNA was reverse-transcribed to cDNA using High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Carlsbad, California, USA) according to the manufacturer’s instructions and stored (80 C). Levels of ABCB1, ABCG2, and VEGFA mRNA were measured by real time RT-PCR using SsoFast EvaGreen Supermix (Bio-Rad Laboratories, Hercules, CA, USA) and the CFX 96 Real-Time system C1000 Thermal Cycler (Bio-Rad). Relative gene expression was measured using [DDc(t)] method and normalized to the geometric mean of three housekeeping genes: glyceraldehyde 3-phosphate dehydrogenase (GAPDH), TATA-binding protein (TBP), and zeta polypeptide (YWHAZ) [19]. Expression of these genes was stable during the experiments (data not shown). Data was analyzed using CFX Manager Software (Bio-Rad). Primer sequences for the genes analyzed are listed in Table 1.
2.4. Western blotting Western blotting was performed as described previously [23]. Briefly, placental explants were homogenized in RIPA lysis buffer (Cell Signaling Technology, Inc., Danvers, MA), centrifuged (10,000 g, 10 min, 4 C), and the supernatant collected. Protein (40 mg) separation using SDS-PAGE was performed using polyacrylamide gels (7%) and transferred to nitrocellulose membranes using iBlot transfer apparatus (Invitrogen). Membranes were blocked in BSA (5% w/v of Tris buffered saline [TBS] with Tween 20: TBS-T, 1 h, 25 C). Membranes were then incubated with primary antibody overnight (4 C). The primary antibodies used were: mouse anti-P-gp (1:5000; clone 5A12.2; EMD Millipore Corporation, Billerica, MA, USA), mouse anti-BCRP (1:500; clone BXP-21; EMD Millipore Corporation), and rabbit anti-actin (1:10,000; Sigma). Membranes were washed and incubated with horseradish peroxidase conjugated secondary antibody (1:15,000; PerkinElmer, Waltham, MA) against the corresponding primary antibody, followed by incubation (1 min) in enhanced chemiluminescence substrate (Invitrogen) and exposed to chemiluminescence film (General Electric Healthcare, Baie d’Urfe, Quebec, Canada). The relative optical density (ROD) of the bands was measured using computerized image analysis and was standardized against the b-actin signal (MCID Core 7.0, Imaging Research Inc., Interfocus Imaging Ltd., Cambridge, England).
Table 1 Primers used for real-time PCR. Gene symbol
Sequence
References
ABCB1
Forward: AGC AGA GGC CGC TGT TCG TT Reverse: CCA TTC CGA CCT CGC GCT CC Forward: TGG AAT CCA GAA CAG AGC TGG GGT Reverse: AGA GTT CCA CGG CTG AAA CAC TGC Forward: CGG GCC TCC GAAA CCA TGA ACT T Reverse: CCC TCC TCC TTC TGC CAT GGG T Forward: CAC GAA CCA CGG CAC TGA TT Reverse: TTT TCT TGC TGC CAG TCT GGA C Forward: AGA TCA TCA GCA ATG CCT CC Reverse: CAT GAG TCC TCC CAC GAT AC Forward: ACT TTT GGT ACA TTG TGG CTT CAA Reverse: CCG CCA GGA CAA ACC AGT AT
[19]
ABCG2 VEGFA TBP GAPDH YWHAZ
[19] [19] [19] [19] [19]
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2.5. Immunofluorescence Placental explants were fixed in neutral buffered formalin (10%; Sigma), washed with PBS and cryosectioned (10 mm). Sections were mounted onto microscope slides and stored (80 C). Prior to immunofluorescence, slides were brought to room temperature and washed with PBS. Antigen retrieval was performed using boiling sodium citrate (10 mM, 10 min). Sections were cooled on ice (10 min) and washed with PBS before blocking in normal goat serum (5%, 1 h; Invitrogen). Slides were incubated overnight with mouse anti-P-gp primary antibody (1:50, D-11, Santa Cruz Biotechnology, Santa Cruz, CA, USA) [19], then washed and incubated (1 h, 25 C) with Alexa Fluor 488-conjugated goat secondary antibody (1:500; Invitrogen). Negative controls were stained by substitution of primary antibody with nonimmune mouse IgG (Dako, Burlington, ON, Canada). After final washes, slides were mounted and cover-slipped using mounting media with 40 ,6-diamidino-2phenylindole (Vector Laboratories, Burlingame, CA). Imaging was performed using Zeiss spinning disk confocal microscope (Zeiss Observer.Z1). 2.6. Deglycosylation P-gp deglycosylation was carried out using Peptide-N-Glycosidase F (PNGase F; Sigma) according to the manufacturer’s instructions. Briefly, denatured and nondenatured protein (30 mg) from fresh placental tissue was incubated with PNGase F (4 h, 37 C). Both denaturing and non-denaturing conditions were used because some proteins require denaturation prior to PNGase F digestion. Laemmli sample buffer (Bio-Rad) was added, and samples boiled (5 min) to inactivate the PNGase F. Western-blot analysis was performed as described above. 2.7. Statistical analysis Data are expressed as mean SEM and all statistical analyses were performed using Prism (GraphPad Software, Inc., San Diego, CA). hCG and LDH concentrations were log transformed and analyzed using one-way repeated measures ANOVA with NewmaneKeuls post-hoc test. Differences in mRNA and protein expression over time of culture and oxygen tension were assessed by two-way ANOVA with Bonferroni post-hoc test. Protein and mRNA expression were also analyzed using oneway ANOVA, followed by Dunnett post-test for comparisons against the control group. Statistical significance was set at p < 0.05.
3. Results 3.1. Tissue viability and syncytiotrophoblast function LDH release from placental explants across the 6-day culture period was measured to assess tissue viability [21,22]. LDH levels in the media decreased gradually, reaching a stable phase on day 4 (Fig. 1A). In order to monitor syncytial shedding and regeneration, hCG secretion was measured, a marker of syncytiotrophoblast endocrine function [21,22]. Between day 1 and day 2 there was a steep decline in hCG release into the media (p < 0.05) (Fig. 1B). From day 2 to day 3 there was a rapid increase in hCG levels (p < 0.05). The elevated hCG levels were sustained throughout the rest of the culture period. This hCG pattern was indicative of syncytiotrophoblast shedding and regeneration [21,22]. Therefore, placental explants were cultured for 4 days in 20% oxygen, which allowed syncytiotrophoblast shedding and regeneration to take
place. The oxygen tension was then reduced to 3% or maintained at 20% for 24 and 48 h. Day 4 was considered as the control group. 3.2. Effects of oxygen tension on ABCB1, ABCG2, and VEGFA mRNA expression ABCB1 and ABCG2 mRNA expression was measured in the control (day 4 of culture), 24 h (day 5), and 48 h (day 6) samples from the same placentae using qRT-PCR. There was a significant effect of oxygen tension on ABCB1 expression, assessed by two-way ANOVA (p < 0.05) (Fig. 2A), with no effects of time in culture or interaction of the two variables. There was a significant increase in ABCB1 expression after 48 h in 3% oxygen compared to the control group (p < 0.05) (Fig. 2A). Oxygen tension had no effect on ABCG2 expression, assessed by two-way ANOVA (Fig. 2B), with no effects of time in culture or interaction of the two variables. VEGFA mRNA expression was measured to assess activation of hypoxic pathways [19]. There was a significant increase in VEGFA expression in the hypoxic condition (3% O2) (p < 0.05) (Fig. 2C), with no effects of time in culture or interaction of the two variables. VEGFA expression was also significantly increased at both 24 h and 48 h hypoxic conditions compared to the control group. 3.3. Effects of oxygen on P-gp and BCRP expression There was no significant overall effect of oxygen tension on P-gp protein levels, assessed by two-way ANOVA (Fig. 3C), with no interaction between the two variables. However, there was a significant effect of time in culture on P-gp levels (p < 0.05), such that there was an increase in P-gp levels with increased time in culture. Further analysis using repeated measures one-way ANOVA revealed a significant increase in P-gp levels after 48 h of culture in 3% oxygen compared to control (p < 0.05) (Fig. 3C). Oxygen tension had no effect on BCRP protein levels, assessed by two-way ANOVA (Fig. 3D), with no effects of time in culture or interaction of the two variables. 3.4. Effect of tissue culture on P-gp glycosylation and localization Western blot analysis performed using fresh placental tissue, showed a predominant 150 kDa band for P-gp (Fig. 4A). In contrast, P-gp in cultured placental tissue was approximately 120 kDa (Fig. 4A). To assess whether the difference in the molecular weight of P-gp was due to deglycosylation, protein from fresh placental tissue was treated with PNGase F (this cleaves the link between asparagine and N-acetylglucosamines). PNGase F treatment resulted in a shift in the molecular weight of P-gp from 150 kDa to
Fig. 1. Placental explant viability and syncytiotrophoblast function. (A) Lactate dehydrogenase (LDH) and (B) human chorionic gonadotropin (hCG) released from explants into the culture media over the 6-day culture period. Results shown as mean S.E.M. Three separate tissues assayed in triplicate. hCG and LDH concentrations were log transformed and analyzed using one-way repeated measures ANOVA with NewmaneKeuls post-hoc test. # represents significant difference from day 1 (p < 0.05). * represents significant difference from all other days (p < 0.05).
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120 kDa (Fig. 4B). Changes in oxygen tension had no effect on the glycosylation state of P-gp (data not shown). To assess whether deglycosylation altered P-gp localization in the tissue, fresh and cultured explants were analyzed using immunofluorescent staining. In both instances P-gp was localized to the syncytiotrophoblast (Fig. 5) (n ¼ 4). Furthermore, decreasing oxygen tension did not alter P-gp localization to the syncytiotrophoblast (Fig. 5). 4. Discussion
Fig. 2. Effects of oxygen tension on ABCB1, ABCG2, and VEGF expression. (A) ABCB1, (B) ABCG2, (C) VEGFA mRNA expression in term placental explants normalized to the geometric mean of three housekeeping genes. Placental explants were cultured for 4 days in 20% oxygen, which allowed syncytiotrophoblast shedding and regeneration to take place. The oxygen tension was then reduced to 3% or continued at 20% for 24 and 48 h. Day 4 was considered as the control group. There was a significant effect of oxygen tension on ABCB1 and VEGFA expression. Solid bars represent 3% oxygen tension, while open bars represent 20% oxygen tension. * represents significant difference from control (p < 0.05). Results shown as mean S.E.M. (n ¼ 7).
We have shown, for the first time, that changes in oxygen tension in the term human placenta have significant but different effects on P-gp and BCRP expression. Decreased oxygen tension resulted in an increase in ABCB1/P-gp mRNA and protein expression in term placental explants but had no effect on ABCG2/BCRP mRNA or protein expression. We also report the novel finding that while P-gp is glycosylated in fresh placental tissue, it becomes fully deglycosylated in cultured placental explants. This de-glycosylation had no effect on the localization of P-gp to the syncytiotrophoblast. This novel observation has important implications for our understanding of the post-translational regulation of P-gp in the placenta and warrants further detailed investigation. In the human placenta, ABCB1/P-gp mRNA and protein expression decrease with advancing gestation [7], which correlates closely with changes in oxygen tension throughout pregnancy. In the present study, we found that 48 h exposure to low oxygen tension (3%) resulted in a significant increase in ABCB1/P-gp mRNA and protein levels, compared to the control group (20% O2). This increase in ABCB1 and P-gp expression in response to hypoxia is consistent with studies performed using other cell types [17,18]. Furthermore, the responsiveness of P-gp expression to a decrease in oxygen tension provides a possible explanation for the high P-gp levels in the low oxygen tension environment of the first trimester placenta compared to term placenta. Studies investigating the expression pattern of BCRP in the human placenta throughout gestation are inconsistent. We have previously shown that ABCG2 mRNA expression does not change throughout pregnancy, while BCRP protein levels increase towards term [8]. On the other hand, Meyer zu Schwabedissen et al. demonstrated that both ABCG2 mRNA and BCRP protein expression increase with advancing gestation [24] but Mathias et al. found that ABCG2 mRNA and BCRP protein expression did not change with gestational age [25]. The reason for the discrepancy in BCRP expression between studies may result from variability in sampling, as BCRP is present in multiple placental cell-types. In the present study oxygen tension had no effect on ABCG2 mRNA or BCRP protein expression. This would be consistent with the stable ABCG2/ BCRP expression pattern during pregnancy, despite the increase in oxygen tension that occurs with advancing gestation. However, a steady level of BCRP expression does not necessarily indicate no change in BCRP function, as no study has measured BCRP activity throughout gestation. In fact, there are numerous studies illustrating posttranslational modifications, such as glycosylation and phosphorylation, changing BCRP function [26e28]. The same is true for P-gp [29-31], as Thews et al. have shown a more than doubling of P-gp activity in prostate carcinoma cells in acidic environments with no change in P-gp expression [31]. Future studies are required to measure BCRP and P-gp activity in the placenta with advancing gestation. Compared to the third trimester, the placenta in the first trimester is exposed to lower oxygen levels [12]. Early in the first trimester the extravillous trophoblast (EVT) invade and occlude the terminal portion of spiral arteries, inhibiting maternal blood from entering the intervillous space and resulting in a low oxygen
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Fig. 3. Effects of oxygen tension on P-gp and BCRP protein expression. Representative Western blot of (A) P-gp and (B) BCRP protein from a single placenta. Protein expression of (C) P-gp and (D) BCRP in term placental explants normalized to b-actin. Placental explants were cultured for 4 days in 20% oxygen, which allowed syncytiotrophoblast shedding and regeneration to take place. The oxygen tension was then reduced to 3% or continued at 20% for 24 and 48 h. Day 4 was considered as the control group. There was a significant effect of oxygen tension on P-gp expression. Solid bars represent 3% oxygen tension, while open bars represent 20% oxygen tension. * represents significant difference from control (p < 0.05). Results shown as mean S.E.M. of relative optical density (ROD). (n ¼ 7).
environment (w3% O2) [10]. In late first trimester, the endovascular EVT causes physiological changes in placental spiral arteries, forming funnel-shaped flaccid arterioles with increased vascular compliance and circumference [10]. This leads to the flow of maternal blood into the intervillous space and a dramatic increase in oxygen levels for the remainder of pregnancy (w8% O2) [10]. However, improper or inadequate conversion of spiral arteries in the first trimester can lead to chronic placental ischemia and hypoxia later in gestation [10e13]. Moreover, embryogenesis in the first trimester already takes place under anaerobic conditions, and
Fig. 4. Deglycosylation of placental P-gp. (A) Representative Western blot comparing Pgp expression in fresh term placental tissue and cultured placental explants. (B) Deglycosylation of fresh placental tissue. (i) Fresh term placental tissue. (ii) Denatured protein from fresh placental tissue treated with PNGase F. (iii) Non-denatured protein from fresh placental tissue treated with PNGase F. (iv) cultured placental explant on Day 6.
in the second and third trimester oxygen becomes more important for normal fetal organogenesis and growth [32]. Protection from hypoxia at this stage of development is established via upregulation of various genes, including superoxide dismutase, insulin-like growth factor (IGF)-2, and heat shock protein 70 [33e 35]. Our data indicate that ABCB1 may be one of the protective genes activated in response to hypoxia in the third trimester placenta. In fact, P-gp has been suggested to play a critical role in cell protection by extruding toxins and harmful products of oxidative stress [36]. Therefore, P-gp may also play a role in protecting the fetus from toxic products of oxidative stress in third trimester hypoxic placentas. We have recently shown that in the first trimester placenta, ABCG2 mRNA expression increased significantly in hyperoxic (20%) conditions after 48 h in culture with no change in ABCB1 mRNA expression [19]. At the protein level, hypoxia resulted in increased immunoreactive BCRP in cytotrophoblasts and in the microvillous membrane of the syncytium, and increased P-gp immunoreactivity in proliferating cytotrophoblasts. This contrasts with the results of the current study and suggests that the placenta in the different stages of pregnancy responds quite differently to changes in oxygen tension. This is understandable, as oxygen tension that is physiological in the first trimester would be considered hypoxic at term. Interestingly, we found that cultured placental tissue contained P-gp that was less glycosylated compared to fresh placental tissue. Post-translational modifications, such as glycosylation, have been shown to alter P-gp activity in various ways, depending on the cell line examined [29]. In general, glycosylation can affect a transporter’s activity, folding, stability, subcellular trafficking, localization, and turnover [37,38]. P-gp is glycosylated on three recognition sites (Asn 91, 94, and 99) in the first extracellular loop [37]. Deglycosylation of P-gp in fresh placenta decreased the molecular weight from approximately 150 kDae120 kDa. This 30 kDa decrease is in agreement with previous studies [39,40]. Since the effect of deglycosylation on P-gp localization is cell type specific [30,37,40,41], we examined whether deglycosylation affects P-gp localization in term placental tissue. Using immunofluorescent staining, the glycosylation state of P-gp was not found to affect P-gp localization in the explants. Furthermore, oxygen tension had no effect on P-gp glycosylation or its localization to the syncytiotrophoblast. Since the impact of glycosylation on P-gp activity is cell-specific [29,30,41], future studies are required to determine the effects of deglycosylation on P-gp activity in the placenta. It is also important to highlight
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Fig. 5. Representative immunofluorescent images showing P-gp (green) localization to the syncytiotrophoblast in placental explants exposed to different oxygen tensions. Nuclei were stained by 40 ,6-diamidino-2-phenylindole (blue) (n ¼ 4). Negative controls were stained by substitution of primary antibody with non-immune mouse IgG. Arrows point to syncytiotrophoblast.
the potential limitation of the present study. For example, in the immunofluorescent staining we were unable to definitively distinguish between the syncytiotrophoblast and unfused cytotrophoblasts in the outer trophoblast layer. Also, it is possible that molecular changes in syncytiotrophoblast function occur in pathways that indirectly modulate multidrug resistance, following long term explant cultures. Future studies are required to determine the precise molecular pathways involved in P-gp and BCRP regulation. In conclusion, oxygen tension affects multidrug resistance in term human placenta. These changes could alter fetal exposure to drugs, xenobiotic and endogenous substrates. Notably, improper or
inadequate conversion of spiral arteries in the first trimester can lead to chronic placental ischemia and hypoxia later in gestation, which can cause deleterious outcomes such as intrauterine growth restriction (IUGR) and preeclampsia. Glycosylation is an important regulatory mechanism of P-gp function and expression, and should be considered so that we can better understand the various roles of P-gp both in normal pregnancy progression and in disease states. Moreover, future studies should focus on the expression profile and function of drug transporters in oxygen-related placental pathologies since changes in the activity of these transporters could have a profound impact on fetal drug exposure.
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Conflict of interest statement The authors declare that there are no conflicts of interest. Disclosure summary Authors have nothing to declare. Acknowledgments This study was funded by the Canadian Institutes of Health Research (grant: FRN-57746; to S.G.M. and W.G.). References [1] Iqbal M, Audette MC, Petropoulos S, Gibb W, Matthews SG. Placental drug transporters and their role in fetal protection. Placenta 2012;33(3):137e42. [2] Vahakangas K, Myllynen P. Drug transporters in the human blood-placental barrier. Br J Pharmacol 2009;158(3):665e78. [3] Lankas GR, Wise LD, Cartwright ME, Pippert T, Umbenhauer DR. Placental Pglycoprotein deficiency enhances susceptibility to chemically induced birth defects in mice. Reprod Toxicol 1998;12(4):457e63. [4] Smit JW, Huisman MT, van Tellingen O, Wiltshire HR, Schinkel AH. Absence or pharmacological blocking of placental P-glycoprotein profoundly increases fetal drug exposure. J Clin Invest 1999;104(10):1441e7. [5] Zhang Y, Wang H, Unadkat JD, Mao Q. Breast cancer resistance protein 1 limits fetal distribution of nitrofurantoin in the pregnant mouse. Drug Metab Dispos 2007;35(12):2154e8. [6] Prouillac C, Lecoeur S. The role of the placenta in fetal exposure to xenobiotics: importance of membrane transporters and human models for transfer studies. Drug Metab Dispos 2010;38(10):1623e35. [7] Sun M, Kingdom J, Baczyk D, Lye SJ, Matthews SG, Gibb W. Expression of the multidrug resistance P-glycoprotein, (ABCB1 glycoprotein) in the human placenta decreases with advancing gestation. Placenta 2006;27(6e7):602e9. [8] Yeboah D, Sun M, Kingdom J, Baczyk D, Lye SJ, Matthews SG, et al. Expression of breast cancer resistance protein (BCRP/ABCG2) in human placenta throughout gestation and at term before and after labor. Can J Physiol Pharmacol 2006;84(12):1251e8. [9] Schneider H. Oxygenation of the placental-fetal unit in humans. Respir Physiol Neurobiol 2011;178(1):51e8. [10] Patel J, Landers K, Mortimer RH, Richard K. Regulation of hypoxia inducible factors (HIF) in hypoxia and normoxia during placental development. Placenta 2010;31(11):951e7. [11] Tuuli MG, Longtine MS, Nelson DM. Review: oxygen and trophoblast biologye a source of controversy. Placenta 2011;32(Suppl. 2):S109e18. [12] Murray AJ. Oxygen delivery and fetal-placental growth: beyond a question of supply and demand? Placenta 2012;33(Suppl. 2):e16e22. [13] Burton GJ, Jauniaux E. Placental oxidative stress: from miscarriage to preeclampsia. J Soc Gynecol Investig 2004;11(6):342e52. [14] Mason CW, Buhimschi IA, Buhimschi CS, Dong Y, Weiner CP, Swaan PW. ATPbinding cassette transporter expression in human placenta as a function of pregnancy condition. Drug Metab Dispos 2011;39(6):1000e7. [15] Krishnamurthy P, Ross DD, Nakanishi T, Bailey-Dell K, Zhou S, Mercer KE, et al. The stem cell marker Bcrp/ABCG2 enhances hypoxic cell survival through interactions with heme. J Biol Chem 2004;279(23):24218e25. [16] Vaupel P, Mayer A. Hypoxia in cancer: significance and impact on clinical outcome. Cancer Metastasis Rev 2007;26(2):225e39. [17] Callaghan R, Crowley E, Potter S, Kerr ID. P-glycoprotein: so many ways to turn it on. J Clin Pharmacol 2008;48(3):365e78. [18] Comerford KM, Wallace TJ, Karhausen J, Louis NA, Montalto MC, Colgan SP. Hypoxia-inducible factor-1-dependent regulation of the multidrug resistance (MDR1) gene. Cancer Res 2002;62(12):3387e94. [19] Lye P, Bloise E, Dunk C, Javam M, Gibb W, Lye SJ, et al. Effect of oxygen on multidrug resistance in the first trimester human placenta. Placenta 2013;34(9):817e23.
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