Effect of glucocorticoids on regulation of placental multidrug resistance phosphoglycoprotein (P-gp) in the mouse
Introduction
The multidrug resistance phosphoglycoprotein (P-gp; encoded by Abcb1 gene) was first discovered in multidrug resistant tumour cells, where it was shown to reduce cellular accumulation of chemotherapeutic agents [1]. Subsequently, murine multidrug resistance genes (Abcb1a and Abcb1b) were identified and together functionally resemble Abcb1 [2], [3], [4]. In addition to expression in cancer cells, P-gp is expressed in normal tissues where it serves an important role in limiting absorption and/or facilitating excretion of a wide range of substrates by actively transporting substrates from the inner to outer leaflet of the cell membrane [2], [3], [5], [6]. Tissues with specialized barrier functions such as the blood–brain, blood–testes and maternal–fetal barrier also express P-gp [7], [8], [9], [10], [11], although information in the human and murine pertaining to the regulation and functional significance of P-gp in these tissues is limited.
Solutes can cross the placenta by passive diffusion, occurring via a paracellular or transcellular route as well as via processes involving pinocytosis, filtration, facilitated diffusion and active transport [12], [13], [14]. Digoxin is a 780 MW lipophilic molecule which passively diffuses via the transcellular pathway [13]. Digoxin is a specific substrate for P-gp and once it has entered the plasma membrane lipid bilayer P-gp is activated to pump digoxin out of the cell [15], [16], [17]. However, depending on the concentration of digoxin and the presence of functional P-gp, some digoxin continues to diffuse across the syncitiotrophoblast, eventually entering fetal circulation. The functional importance of placental P-gp in limiting fetal digoxin accumulation has been verified on E15.5 with the use of Abcb1a/Abcb1b knockout mice [18].
In both the mouse and human, we and others have recently shown that placental P-gp, localized within the apical membrane of the syncitium, is highly expressed at mid-gestation but dramatically declines near term [8], [9], [19], corresponding to a significant increase in net transplacental transfer of [3H]digoxin (a robust pharmacological probe for assessing P-gp function) [20], [21]. Overall, this suggests a decrease in global protection provided by the placenta to the developing fetus with advancing gestation. Other studies in the mouse, have shown that P-gp plays a crucial role in preventing transplacental transfer of a number of exogenous substrates; thus protecting the developing fetus from their potential adverse effects [18], [21], [22]. Despite these dramatic changes in mouse and human placental P-gp expression and function in late gestation, little is known with regards to regulation. Steroids have previously been shown to regulate Abcb1 gene expression in other tissues in the rat and mouse. For example, progesterone and estrogen are important regulators of Abcb1 gene expression in uterine tissue [18], [23], [24], [25]. However, we have recently shown that progesterone at physiological concentrations does not appear to exert a regulatory role on mouse placental P-gp [20], a finding recently corroborated in the rat [26]. Maternal plasma glucocorticoid levels increase exponentially in late gestation in all mammals [27], [28], [29], [30], [31]. Further, placental sensitivity to glucocorticoids increases with advancing gestation [32], [33]. This increase coincides with the down-regulation of placental Abcb1a, Abcb1b mRNA and P-gp, suggesting a potential regulatory role [9]. Given the difference in placental sensitivity to glucocorticoids and the low levels of P-gp on E18.5, we predicted that glucocorticoid regulation of placental P-gp would change as a function of gestational age and that glucocorticoid effects may be limited near term. Synthetic glucocorticoids are administered to approximately 10% of all pregnant women during the management of threatened preterm labour [34]. This treatment is very effective in bringing about the premature maturation of the fetal lung [35], however, due to the difficulty in diagnosing preterm labour, many fetuses that go on to deliver at normal term are exposed to synthetic glucocorticoid [34].
Dexamethasone has been shown to regulate P-gp expression in other barrier tissues (blood–brain barrier) and in vitro [36], [37], [38], [39]. To date, there is virtually no information on the potential glucocorticoid regulation of placental P-gp, in vivo. A single study has investigated the regulatory role of dexamethasone on placental Abcb1 mRNA expression in the rat placenta [26]. However, in the rat, the ontogenic profile of placental Abcb1 mRNA and P-gp differs compared to that shown in the human and mouse placenta. Determining the regulation of placental P-gp would be valuable for the development of improved strategies for maternal and fetal therapies as well as improved fetal protection. In this study, we hypothesized that exposure to synthetic glucocorticoid (dexamethasone) will: 1) result in a down-regulation of both Abcb1 mRNA isoforms and P-gp within the placental barrier, 2) increase substrate accumulation in the fetal compartment and 3) that these regulatory effects are both gestational age and dose-dependent.
Section snippets
Materials and methods
Female FVB mice (Charles River, Germantown, NY) were bred in our colony. Pregnancy was defined after presence of vaginal plug and designated as E0.5 (average gestation period ∼ 19.5 days). All animals were identically housed and provided with standard chow ad libitum (Teklad 2018, Harlan Laboratories, Mississauga, Canada). These studies were performed using protocols approved by the Animal Care Committee at the University of Toronto and in accordance with the Canadian Council for Animal Care.
Effect of dexamethasone on placental and fetal weights
Daily injections of dexamethasone (1 mg/kg) during mid-gestation (E9.5–E15.5) resulted in a significant (P < 0.05) reduction in placental weight on E15.5 when compared to vehicle, while the lower dose of dexamethasone (0.1 mg/kg) did not significantly affect placental weight (Fig. 1A). Neither dose of dexamethasone significantly altered placental weight on embryonic day (E) 18.5 (Fig. 1B). One-Way ANOVA revealed profound effects of synthetic glucocorticoid exposure on fetal growth during
Discussion
The present study has, for the first time, examined the regulation of mouse placental Abcb1a and Abcb1b mRNA, P-gp protein expression and function in vivo by synthetic glucocorticoid. We have shown that synthetic glucocorticoid (dexamethasone) exposure alters placental Abcb1a mRNA expression during mid- and late gestation in an age-dependent manner. However, the effects of dexamethasone on Abcb1a mRNA and P-gp protein expression do not correlate with changes in placental P-gp mediated
Acknowledgements
This study was funded by the Canadian Institutes for Health Research (FRN-57746; to S.G.M. and W.G. and Doctoral Research Award to S.P.).
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