P-Glycoprotein Restricts Access of Cortisol and Dexamethasone to the ...

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Endocrinology 147(11):5147–5152 Copyright © 2006 by The Endocrine Society doi: 10.1210/en.2006-0633

P-Glycoprotein Restricts Access of Cortisol and Dexamethasone to the Glucocorticoid Receptor in Placental BeWo Cells Peter J. Mark and Brendan J. Waddell School of Anatomy and Human Biology, The University of Western Australia, Perth, Western Australia 6009, Australia Exposure of the fetus and placenta to maternal glucocorticoids is normally limited by the placental glucocorticoid barrier, which consists primarily of placental 11␤-hydroxysteroid dehydrogenase type 2-mediated conversion of cortisol to the biologically inactive cortisone. Studies in the rodent brain show that P-glycoprotein (P-gp) is also an important physiological regulator of glucocorticoid access to the glucocorticoid receptor (GR) in target cells because it exports cortisol back into peripheral circulation against a concentration gradient. Whether P-gp of placental origin also has this capacity is unknown. Therefore, we used the human placental choriocarcinoma cell line BeWo and its daughter cell line, BeWoMDR, virally transduced with P-gp, to assess whether placental P-gp regulates access of glucocorticoids to the GR. Quantitative PCR showed that BeWoMDR cells express approximately 10-fold higher levels of P-gp mRNA than BeWo

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NTRAUTERINE GROWTH RETARDATION increases the risk of neonatal morbidity and mortality (1) and is implicated in the programming of various adult-onset diseases including type 2 diabetes, cardiovascular disease, and obesity (2, 3). The regulation of fetal and placental growth and development is a balance between factors that maximize growth (e.g. IGF-II) and those that reduce cellular proliferation and impart functionality via differentiation (4). Glucocorticoids provide key signals for differentiation of fetal and placental tissues (principally via cortisol in humans and corticosterone in rodents); yet excess glucocorticoid exposure limits fetal and placental growth. In normal pregnancy placental and fetal glucocorticoid exposure is determined largely by the placental glucocorticoid barrier, which serves as a metabolic gatekeeper to limit access of maternal glucocorticoids to the placenta and fetus (5). Placental expression of the enzyme 11␤-hydroxysteroid dehydrogenase type 2 (11␤-HSD2), which catalyzes the conversion of active glucocorticoids (cortisol and corticosterone) to their inert 11keto products (cortisone and 11-dehydrocorticosterone, respectively) is well recognized as the major component of this barrier. Experimental disruption of the barrier (via 11␤HSD2 inhibition) has been shown to stimulate placental apFirst Published Online July 27, 2006 Abbreviations: CsA, Cyclosporin A; GR, glucocorticoid receptor; 11␤HSD2, 11␤-hydroxysteroid dehydrogenase type 2; LSD, least significant difference; P-gp, P-glycoprotein. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.

cells, and syncytialization increased P-gp mRNA by approximately 7-fold. Elevated P-gp expression in BeWoMDR cells reduced activation of the GR by dexamethasone and cortisol (10ⴚ9 to 10ⴚ6 M) to around 40% of that in BeWo cells. Inhibition of P-gp-mediated glucocorticoid efflux by cyclosporin A in BeWoMDR cells returned GR activation to levels similar to those in BeWo cells. Diffusion of dexamethasone across BeWoMDR monolayers occurred at a slower rate than that across BeWo monolayers, but this difference was eliminated by cyclosporin A. These data support the hypothesis that P-gp contributes to the placental glucocorticoid barrier. Thus, 11␤hydroxysteroid dehydrogenase type 2 and P-gp may act in unison to reduce fetal and placental exposure to maternal glucocorticoids and thereby minimize their growth inhibitory actions. (Endocrinology 147: 5147–5152, 2006)

optosis (6), restrict fetal growth, and lead to subsequent disturbances in the postnatal phenotype (7–9). Thus, the placental glucocorticoid barrier is potentially an important therapeutic target in certain cases of intrauterine growth retardation, and so understanding its nature and regulation is important. In addition to glucocorticoid inactivation by 11␤-HSD-2, a potential new player in the placental glucocorticoid barrier is P-glycoprotein (P-gp/ABCB1), a membrane-bound protein known to export both dexamethasone and cortisol against a concentration gradient (10 –13). Recent evidence in the human and rodent brain has implicated P-gp in restricting the penetration of glucocorticoids across blood-tissue barriers (11, 12) such that in the absence of P-gp (mdr1a⫺/⫺ mice), substantially more 3H-cortisol is able to penetrate the bloodbrain barrier (12). Expression of P-gp has been demonstrated at the apical surface of the syncytiotrophoblast membrane of human placenta (14 –17), in primary cultures of human trophoblasts (18, 19), and at low levels in cultured human placental choriocarcinoma epithelial cells (BeWo cells) (16, 20). The major role of placental P-gp is thought to be the reduction the transplacental passage of toxic xenobiotic compounds to the fetus (19, 21). But given the location of P-gp within the placenta, we hypothesized that P-gp also limits access of glucocorticoids to placental cells and the transplacental passage of maternal glucocorticoids to the fetus. To test this hypothesis, we used a placental choriocarcinoma cell line (BeWo) and a virally transduced daughter cell line (BeWoMDR) that expresses elevated levels of P-gp (16)

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Endocrinology, November 2006, 147(11):5147–5152

Mark and Waddell • P-gp and the Placental Glucocorticoid Barrier

to investigate the influence of P-gp expression on access of glucocorticoids to the glucocorticoid receptor (GR) and their transcellular passage across BeWo monolayers. Materials and Methods Materials BeWo cells were obtained from American Type Culture Collection (Manassas, VA) and a virally transduced daughter cell line that overexpress P-gp, BeWoMDR cells (16), were a gift from Dr. Leslie Fairbairn (Paterson Institute of Cancer Research, Manchester, UK). Reagents for cell culture were supplied by Invitrogen Life Technologies (Sydney, Australia) except for charcoal-stripped fetal bovine serum from Trace Biosciences (Sydney, Australia), steroids and forskolin from SigmaAldrich (Sydney, Australia), and primers for PCR were synthesized by Geneworks (Adelaide, Australia). Cyclosporin A was obtained from Novartis (Basel, Switzerland) and RU486 from Roussel Uclaf (Paris, France).

and cortisol) and 50 ng pCMV-Ren (to control for transfection efficiency) per well using FuGene 6.0 transfection reagent (Roche Biochemicals, Sydney, Australia) according to the manufacturer’s protocols. Additionally, due to the low level of endogenous GR expressed in BeWo cells in comparison with placental cytotrophoblast and syncytiotrophoblast (25), 500 ng pRSVhGR, an expression vector for the human GR (26), was also included in the transfections.

Cell treatments Transfected cells were cultured overnight in 50:50 (vol/vol) DMEM/ Ham F-12K nutrient mixture (Kaighn’s modification) supplemented with 5% charcoal-stripped fetal bovine serum and 1% penicillin/streptomycin. The following day cells were dosed with either vehicle (0.1% ethanol) or appropriate doses of dexamethasone, cortisol, 10 ␮m cyclosporin A, or 5 ␮m RU486 according to the experimental design and cultured for a further 24 h before being harvested in 200 ␮l passive lysis buffer (Promega, Madison, WI) and 10 ␮l of cell lysate was assayed in a BMG LABTECH POLARstar luminometer (Offenberg, Germany) using the dual luciferase reporter assay kit (Promega).

Cell culture Cells were maintained in 75-cm2 flasks (Sarstedt, Adelaide, Australia) at 37 C in a humidified incubator with 5% CO2 air atmosphere in 50:50 (vol/vol) DMEM/Ham F-12K nutrient mixture (Kaighn’s modification) supplemented with 10% fetal bovine serum and 1% penicillin/ streptomycin.

P-gp expression in normal and syncytialized BeWo and BeWoMDR cells To assess the effects of syncytialization [induced by forskolin treatment (22)] on expression of P-gp, BeWo, and BeWoMDR cells (5 ⫻ 105) were seeded onto 35-mm-diameter 6-well plates, cultured overnight, and then treated with forskolin (20 ␮m) or vehicle for 72 h. The cells were then washed with cold PBS and total RNA extracted using TriReagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer’s instructions. Total RNA (2 ␮g) was reverse transcribed using murine Maloney leukemia virus reverse transcriptase RNase H minus point mutant and random hexamer primers (Promega, Madison, WI) at 42 C for 1 h according to the manufacturer’s protocol. After reverse transcription, the resulting cDNA was purified using the UltraClean PCR cleanup kit (Mo Bio Laboratories, Solana Beach, CA). Purified cDNAs (2 ␮l) were used as templates for real-time PCR quantitation using primers specific for human P-gp [designed using Primer3 software (23)] and the internal control ribosomal protein L19 mRNA (RPL19) (24). The specific primers and conditions used are summarized in Table 1, and both primer sets spanned an intron so as to not amplify from genomic DNA. Amplifications for P-gp and RPL19 were performed in the Rotorgene 3000 (Corbett Industries, Sydney, Australia) using 0.5 ␮m primers, 3 mm MgCl2, and Immolase Taq DNA polymerase (Bioline, Alexandria, New South Wales, Australia) according to the manufacturer’s instructions. The threshold cycle number of detection for each sample was compared against a standard curve constructed using 10-fold serial dilutions of corresponding standards specific for each transcript. Both P-gp and RPL19 standards were constructed using serial dilutions of the gel-extracted (QIAEX II, QIAGEN, Melbourne, Australia) conventional PCR product as a template.

Transfections Cells (1 ⫻ 105) were plated down in 35-mm-diameter six-well plates and cultured overnight. The following day the cells were transfected with 500 ng pGRE-Luc (to assess activation of the GR by dexamethasone

Steroid transport across monolayers BeWo (1 ⫻ 105) and BeWoMDR (0.75 ⫻ 105) cells were cultured on 1.0 ␮m pore polyethylene terephthalate membrane tissue culture inserts (BD Biosciences, Franklin Lakes, NJ) for 3 d to form a monolayer (27). Integrity of the monolayer was assessed both visually and by transepithelial electrical resistance (28) using the EVOM epithelial volt ohmmeter with STX2 electrode (World Precision Instruments, Sarasota, FL). Preliminary investigations using BeWo monolayers in our system determined that transepithelial electrical resistance measurements of greater than 300 ⍀ were consistent with complete monolayer formation. At confluence, the media in the top chamber were replaced with fresh medium containing approximately 600,000 cpm/ml 3H-dexamethasone and 1 ␮m cold dexamethasone with either vehicle (0.1% ethanol) or 10 ␮m cyclosporin A (CsA) for 6 h. Aliquots (50 ␮l) were taken from the basal chamber hourly and replaced with fresh medium to keep the volumes constant. The aliquots were counted by liquid scintillation in 2 ml of Emulsifier-safe scintillation fluid (Packard Biosciences, Meriden, CT) using a 1500 TRI-CARB liquid scintillation analyzer (Packard Biosciences).

Statistical analysis The effects of syncytialization on P-gp mRNA levels were assessed by one-way ANOVA using Genstat 7.0 software (Hemel Hempstead, UK). Transfection studies and steroid transport assays across monolayers were assessed by two-way ANOVA with cell type and treatment as sources of variation. Where the F test reached statistical significance (P ⬍ 0.05), specific differences were assessed by least significant difference (LSD) tests (29).

Results P-gp expression in normal and syncytialized BeWo and BeWoMDR cells

The previously reported (16) transduction of BeWo cells with an expression construct for the MDR1 gene (BeWoMDR cells) increased (P ⬍ 0.01) the levels of P-gp mRNA by approximately 10-fold, compared with endogenous P-gp expression in the parental BeWo cell line (Fig. 1). Syncytialization of BeWo cells with 20 mm forskolin for 72 h resulted

TABLE 1. Primer sequences and PCR conditions for quantitative PCR Transcript

MDR1 RPL19

Sequence (5⬘–3⬘)

Forward, Reverse, Forward, Reverse,

GTT TGC CTG GGA

TGA CAT AAG CAG

TGT TGA GTC AGT

GCA CTG AAA CTT

CGA AAA GGG GAT

TGT GAA AAT GAT

TG CA GTG CTC

Anneal temperature (C)

Size (bp)

61

110

52

195


FIG. 1. Relative levels of P-gp mRNA in normal and syncytialized BeWo and BeWoMDR cells. RNA from either control or forskolin (20 ␮M) stimulated BeWo and BeWoMDR cells was reverse transcribed and quantitated for P-gp transcript. Values are mean ⫾ SEM (n ⫽ 3 independent experiments). *, P ⬍ 0.01, compared with control; #, P ⬍ 0.01, compared with corresponding BeWo cells (two-way ANOVA, LSD test).

in a 7-fold increase (P ⬍ 0.01) in P-gp mRNA expression. Interestingly, forskolin treatment similarly elevated the augmented P-gp mRNA levels in the BeWoMDR cells (7-fold; P ⬍ 0.01).


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11-fold over basal levels, which was a 50% decrease (P ⬍ 0.01) in the dexamethasone-induced response that occurred in BeWo cells. The reduction of GR activation by dexamethasone, due to elevated P-gp expression in BeWoMDR cells, occurred across a broad concentration range from 10⫺9 to 10⫺6 m dexamethasone (Fig. 3A) when compared with the response in BeWo cells. The activation of the GR was maximal at 10⫺9 m dexamethasone for both cell lines, and this fell away slightly in both at higher concentrations of dexamethasone. At all concentrations of dexamethasone, however, responses in the BeWo cell line were greater (P ⬍ 0.01) than those in the BeWoMDR cells. Cortisol also activated the GR in BeWo cells by up to 10-fold from 10⫺9 to 10⫺6 m, reaching maximal activation at 10⫺8 m cortisol (Fig. 3B). Unlike the response to dexamethasone, however, GR activation by cortisol did not decline at higher doses. The response of the high P-gp expressing BeWoMDR cells to cortisol was 40 –50% lower than that of BeWo cells for corresponding concentrations. Coincubation of the BeWoMDR cells with dexamethasone (10⫺6 m) and the P-gp efflux inhibitor, CsA (10 ␮m), increased (P ⬍ 0.01) the activation of the GR by 2-fold above that achieved by a corresponding concentration of dexamethasone (Fig. 4).

Effects of dexamethasone, RU486, and CsA on GR activation

Activation of transfected GR by maximal levels of dexamethasone (Fig. 2) resulted in a 22-fold increase in luciferase expression from the pGRE-Luc plasmid in BeWo cells. This activation was completely abrogated by coincubation with the antiglucocorticoid, RU486 (5 ␮m), which did not alter levels of luciferase expression in the absence of dexamethasone. Induction of luciferase activity in response to maximal levels of dexamethasone in BeWoMDR cells was increased

FIG. 2. GR activation by dexamethasone in BeWo and BeWoMDR cells. Cells were transfected with reporter construct and then dosed with 10⫺6 M dexamethasone or vehicle with or without 5 ⫻ 10⫺6 M RU486 for 24 h and assayed for luciferase activity. Values are mean ⫾ SEM (n ⫽ 3 independent experiments). *, P ⬍ 0.01, compared with BeWo cells (two-way ANOVA, LSD test).

FIG. 3. Dose response of BeWo and BeWoMDR cells to dexamethasone and cortisol. Cells were transfected with reporter construct, dosed with vehicle or dexamethasone (A) or cortisol (B) from 10⫺10 to 10⫺6 M for 24 h and assayed for luciferase activity. Values are mean ⫾ SEM (n ⫽ 6 –12 independent experiments). *, P ⬍ 0.05, **, P ⬍ 0.01, compared with corresponding dose in BeWo cells (two-way ANOVA, LSD test).

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dexamethasone passage across BeWoMDR monolayers relative to that across BeWo monolayers was evident as early as 2 h of incubation (P ⬍ 0.01) and remained lower thereafter. Coincubation with 10 ␮m CsA did not affect the rate of steroid transport across the BeWo monolayer at any time point, but this treatment accelerated the rate of dexamethasone transport across BeWoMDR monolayers from as early as 2 h (P ⬍ 0.01) such that it was comparable to that in the parental BeWo cell line. Discussion

FIG. 4. Effect of inhibition of P-gp-mediated efflux by CsA on GR response to dexamethasone. BeWoMDR cells were transfected with reporter construct and incubated in the presence or absence of 10 ␮M CsA, with or without 10⫺6 M dexamethasone. Values are mean ⫾ SEM (n ⫽ 3 independent experiments). *, P ⬍ 0.01, compared with cells in the absence of CsA (two-way ANOVA, LSD test).

Steroid transport across monolayers

In the absence of any cells, almost half of the 3H dexamethasone in the apical chamber diffused across the tissue culture insert into the basal chamber by 6 h. In the presence of a BeWo monolayer, however, only 18 ⫾ 1% of 3H dexamethasone crossed to the basal chamber, and this transfer was further decreased (P ⫽ 0.014) to only 15 ⫾ 1% across a monolayer of BeWoMDR cells (Fig. 5). This impedance of 3H

FIG. 5. Steroid transport across BeWo and BeWoMDR monolayers. Passage of 3H-dexamethasone from the apical to the basal chamber was measured across the BeWo and BeWoMDR monolayers in the presence or absence of 10 ␮M CsA over a 6-h period. Values represent mean ⫾ SEM (n ⫽ 4 independent experiments). *, P ⬍ 0.01, compared with BeWo cells and CsA-treated BeWoMDR cells in the presence of CsA (two-way ANOVA and LSD test at each time point).

The presence of P-gp and a multitude of other ATP-driven efflux pumps within the human placenta is thought to facilitate the reduction of fetal exposure to xenobiotic compounds that are potentially harmful to the developing fetus (16, 21, 30). More recent evidence in the human and rodent brain has implicated P-gp in restricting the penetration of glucocorticoids across blood-tissue barriers (11, 12), giving rise to the possibility that placental P-gp decreases the transfer of maternal glucocorticoids into the placenta and across to the fetus. Consistent with this proposed function, we demonstrate here that elevated expression of P-gp (in placental BeWoMDR cells) does indeed reduce the access of glucocorticoids to their intracellular receptors, compared with the parental BeWo cell line, and decreases glucocorticoid passage across trophoblast cell monolayers. These data support the hypothesis that P-gp contributes to the placental glucocorticoid barrier and as such may impact the regulation of placental and fetal growth. In the current study, BeWoMDR cells consistently exhibited only 30 –50% activation of the GR by synthetic (dexamethasone) and endogenous (cortisol) glucocorticoids in comparison with BeWo cells across a large physiological range of concentrations (10⫺9 to 10⫺6 m). Inhibition of P-gpmediated glucocorticoid efflux with CsA restored dexamethasone-induced GR activation in BeWoMDR cells to levels comparable with parental BeWo cells. Additionally, diffusion of dexamethasone across BeWoMDR monolayers was considerably lower than that across BeWo monolayers at 6 h, but again this difference was eliminated by the presence of CsA. Numerous previous studies have shown CsA to be a functional inhibitor of P-gp efflux (16, 19, 20), and although it can also inhibit multidrug resistance protein 1 (31, 32) and activate intracellular cyclophilins (reviewed in Ref. 33), neither of these interactions appear to affect the current studies because treatment of the parental BeWo cell line with CsA did not result in increased GR activation by the glucocorticoids or alter the rate of diffusion across BeWo monolayers. Therefore, the observed differences between BeWo and BeWoMDR cells with respect to both GR activation and glucocorticoid passage across cell monolayers are likely due to the marked differences in expression of P-gp in the two cell lines. Within the human placenta, P-gp is localized to the syncytiotrophoblast layer but not the cytotrophoblast cells (14 – 17), a pattern consistent with our observation that P-gp expression increased after induction of the syncytialized phenotype in both BeWo and BeWoMDR cells. Although both cell lines normally exhibit a cytotrophoblast phenotype, viral transduction with MDR1 results in elevated P-gp ex-


pression in the BeWoMDR cells. This is particularly useful for steroid transport studies in vitro because, unlike syncytialized BeWo cells, untreated BeWoMDR cells form an intact monolayer that can be used to measure transcellular glucocorticoid passage in cell culture inserts. A further consideration in this context is that BeWo cells exhibit a polarized attachment to substratum (16, 20, 22, 27), with the apical surface being analogous to the maternal surface and the basal surface corresponding to the fetal side. Importantly, P-gp localization in BeWo cells appears to be consistent with this polarization, with expression primarily occurring on the apical surface, corresponding to the microvillous brush border membrane in the placenta, whereas it is not expressed on the basolateral side (16, 20). These properties suggest that, at least from a P-gp perspective, BeWo and BeWoMDR cells are both appropriate models to study transplacental passage of glucocorticoids. The reduced passage of 3H-dexamethasone across BeWoMDR cells, compared with that across BeWo cells, is consistent with P-gp-mediated efflux at the apical surface. The magnitude of this difference would likely have been even greater had the cells been syncytialized because this would also promote formation of microvilli (34) and thus P-gp localization at the apical surface. It is also noteworthy that BeWo cells express considerably less P-gp than primary cultures of human trophoblasts (16). Overall, therefore, it seems highly likely that the contribution of P-gp to glucocorticoid efflux at the placental interface in vivo is even higher than that observed in BeWoMDR monolayers in the present study. BeWo and BeWoMDR cells were supplemented during transfection studies with an expression plasmid for the human GR (pRSVhGR). Early investigations into the phenotype of BeWo cells determined that they express very low levels of GR in comparison with normal placental cells (25). Moreover, BeWo cells failed to respond to treatment with prednisolone under conditions that were adequate to stimulate GR activation in HeLa cells (25). Therefore, transfection of the GR expression construct produced a BeWo phenotype more comparable with normal placental cells, with respect to GR expression, and facilitated assessment of GR activation by glucocorticoids. In addition to limiting transplacental passage of maternal glucocorticoids to the fetus, placental P-gp is also likely to restrict their access to the GR within the placenta. This would be expected to prevent adverse effects of glucocorticoids on placental function that might otherwise lead to fetal growth retardation via placental insufficiency (35, 36). For example, exposure of the rat placenta to excess glucocorticoid increases trophoblast apoptosis (6), down-regulates expression of peroxisome proliferator-activated receptor ␥ (37) and inhibits that of vascular endothelial growth factor (Hewitt, D. P., P. J. Mark, and B. J. Waddell, unpublished observations), all of which are likely to impact negatively on placental and ultimately fetal growth. Furthermore, in pregnancies complicated by possible preterm delivery, routine clinical practice involves maternal administration of glucocorticoids, sometimes in repeated doses, to promote maturation of vital organs in preparation for birth (38). Our data suggest that placental P-gp is likely to impact on the pharmacodynamics of exogenous glucocorticoid access to both the fetus and placenta.


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The relative contributions of placental P-gp and 11␤-HSD2 to the placental glucocorticoid barrier are currently unknown, but it is of interest that expression of each appears to change over the course of gestation. Thus, P-gp expression in the human placenta decreases between early and late gestation, although there are still significant amounts of P-gp detected in the syncytial layer at term (17, 39). In contrast, placental 11␤-HSD2 expression increases after midgestation in baboons and humans (40 – 42) and then appears to fall just before term in both species (43, 44), possibly to facilitate late maturation of fetal organs. Thus, P-gp may be particularly important for limiting maternal glucocorticoid access to the placenta and fetus during early gestation, with the role of 11␤-HSD2 increasing toward term as P-gp expression declines. The rise is 11␤-HSD2 expression after midgestation is due in part to the stimulatory effects of rising estrogen (45, 46), but the factors regulating placental P-gp expression await further study. In relation to the present study, reduced GR activation in BeWoMDR, compared with BeWo cells, could partly reflect elevated 11␤-HSD2 activity in BeWoMDR cells and an associated reduction in glucocorticoid bioavailability. This is very unlikely, however, because quantitative RT-PCR analysis shows that BeWoMDR cells actually express considerably less 11␤-HSD2 mRNA than the parental BeWo cells (Mark, P. J., and B. J. Waddell, unpublished observations). Dexamethasone was around 10-fold more potent than cortisol in activating the GR, consistent with previous reports on relative potency of glucocorticoids (47), but for each dose, activation in BeWoMDR cells was consistently only 30 –50% of that in BeWo cells. Interestingly, GR activation by dexamethasone, but not cortisol, fell slightly at doses above 10⫺9 m, and this effect was evident in both cell lines. Although the mechanism underlying this response is unclear, a previous report suggested that such a response to dexamethasone may reflect transcriptional squelching at saturating concentrations (48). It was not, however, due to an up-regulation in P-gp expression and an associated increase in dexamethasone efflux because expression was unaffected by treatment with 10⫺6 m dexamethasone for 24 h (results not shown). In conclusion, the present study demonstrates that P-gp, virally overexpressed in placental BeWo cells, reduces access of both dexamethasone and cortisol to the GR, effectively reducing glucocorticoid-mediated activation of target genes. Additionally, P-gp reduced the rate at which dexamethasone is able to pass from the apical to the basal surface of BeWo cells in a monolayer. These observations support the hypothesis that placental P-gp augments the placental glucocorticoid barrier, serving in conjunction with 11␤-HSD2 to reduce placental and fetal exposure to elevated levels of maternal glucocorticoids and thus to minimize associated growth retardation. Acknowledgments We thank Professor Leslie Fairbairn and Dr. Diane Atkinson for their gift of the BeWoMDR cells, Dr. Thomas Ratajczak for CsA, and Dr. Jay Steer for RU486. Received May 11, 2006. Accepted July 10, 2006. Address all correspondence and requests for reprints to: Brendan J. Waddell, Ph.D., School of Anatomy and Human Biology, The University

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of Western Australia, 35 Stirling Highway, Crawley, Western Australia 6009, Australia. E-mail: [email protected]. This work was supported by Project Grant 403999 from the National Health and Medical Research Council of Australia. Disclosure statement: the authors have nothing to disclose.


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