Gestational age‐dependent gene expression profiling of ATP‐binding cassette transporters in the healthy human placenta

Abstract The ATP‐binding cassette (ABC) transporters control placental transfer of several nutrients, steroids, immunological factors, chemicals, and drugs at the maternal‐fetal interface. We and others have demonstrated a gestational age‐dependent expression pattern of two ABC transporters, P‐glycoprotein and breast cancer resistance protein throughout pregnancy. However, no reports have comprehensively elucidated the expression pattern of all 50 ABC proteins, comparing first trimester and term human placentae. We hypothesized that placental ABC transporters are expressed in a gestational‐age dependent manner in normal human pregnancy. Using the TaqMan® Human ABC Transporter Array, we assessed the mRNA expression of all 50 ABC transporters in first (first trimester, n = 8) and third trimester (term, n = 12) human placentae and validated the resulting expression of selected ABC transporters using qPCR, Western blot and immunohistochemistry. A distinct gene expression profile of 30 ABC transporters was observed comparing first trimester vs. term placentae. Using individual qPCR in selected genes, we validated the increased expression of ABCA1 (P < 0.01), ABCA6 (P < 0.001), ABCA9 (P < 0.001) and ABCC3 (P < 0.001), as well as the decreased expression of ABCB11 (P < 0.001) and ABCG4 (P < 0.01) with advancing gestation. One important lipid transporter, ABCA6, was selected to correlate protein abundance and characterize tissue localization. ABCA6 exhibited increased protein expression towards term and was predominantly localized to syncytiotrophoblast cells. In conclusion, expression patterns of placental ABC transporters change as a function of gestational age. These changes are likely fundamental to a healthy pregnancy given the critical role that these transporters play in the regulation of steroidogenesis, immunological responses, and placental barrier function and integrity.


| INTRODUCTION
The ATP-binding cassette (ABC) superfamily comprise 50 proteins classified into seven sub-families ranging from ABCA through ABCG.
Although some of the superfamily members (ABCE and ABCF subfamilies) act as translation factors, 1,2 the majority of the ABC superfamily are transporters, involved in the efflux transport activity of specific substrates across biological barriers. [3][4][5] In the placenta, ABC transporters regulate fetal accumulation of numerous physiological compounds, chemicals, and drugs that may be present in the maternal circulation. 6 Endogenous substrates of the ABC transporters include steroid hormones (glucocorticoids, mineralocorticoids, estrogens, androgens, and progestogens), nutrients (lipids, cholesterol, and folate), metabolic products (oxysterols, bilirubin-and bile salts-conjugated compounds) and immunological factors (cytokines and chemokines). 5,7,8 Exogenous substrates include different environmental chemicals (bisphenol A, ivermectin, organochlorine, and organophosphorus pesticides) and xenobiotics (antiretrovirals, antidepressants, antibiotics, etc). 5,[9][10][11][12] Thus, placental ABC transporters control cellular metabolism, regulate local, and systemic immunological responses and orchestrate the biodistribution of numerous endogenous and exogenous substrates at the maternal-fetal interface. 5,13,14 The cellular localization of ABC transporters in the syncytiotrophoblast (eg apical vs. basolateral) is essential to determine the transfer orientation of their substrates in or out of the placental barrier. 5,14-16 P-glycoprotein (P-gp, encoded by ABCB1), breast cancer resistance protein (BCRP, ABCG2), and the multidrug resistanceassociated proteins (MRPs)-2 and 3 (ABCC2 and ABCC3, respectively) are localized to the apical membrane of syncytiotrophoblasts, indicating their extrusion activity occur from within the syncytium (and thus the fetal compartment) towards the maternal blood-enriched intervillous space of the placenta. Conversely, ABCG1, MRP-1, 3 and 5 (ABCC1, ABCC3, and ABCC5) are localized to the basolateral membrane, suggesting that extrusion activity occurs from the maternal to the fetal compartment. 5 In some cases, the localization of ABC transporters appears to change with advancing gestation. This is the case for the ABCA1 lipid transporter, which has been associated with altered steroidogenesis, placental malformation, reduced pregnancies, and offspring morbidity. [17][18][19][20][21] Considering that ABC transporters are also involved in the biodistribution of several drugs commonly prescribed during pregnancy (ie antiemetic agents, antibiotics, synthetic glucocorticoids, anti-inflammatory, antidepressants, antihypertensive, and antiretroviral drugs) 5  between these two modes of delivery, thus all term placental specimens were combined into the Term group. After analysis of relative expression, the resulting P-values were corrected for multiple testing by a false discovery rate (FDR) 33 of 5% using the R programming statistical software.
To validate the relative expression results obtained in the array, individual qPCR of selected ABC genes, ABCA1, ABCA6, ABCA9, ABCB1, ABCB11, ABCG4, and ABCC3, was assessed using the same Taqman ® probes present in the TLDA cards (ID: Hs00194045_m1, Hs00365329_m1, Hs00184824_m1, Hs00184491_m1, Hs00358656_m1, Hs00223446_m1 and Hs00329320_m1, respectively). Their relative expression was normalized using the gene POLR2A (Hs00172187_m1). qPCR reactions were using the Taqman ® Universal Master Mix II (Applied Biosystems) in triplicates in a CFX96 real-time PCR detection system (Bio-Rad). The cycling conditions were: 50°C for 2 minutes, 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 60 seconds. Changes in mRNA expression were calculated according to the 2 −ΔΔCT method. 32

| Western blot
To investigate protein expression of the selected ABC transporter, total protein was extracted from placental tissue (~50 mg) using approaches described previously. 31 Briefly, nitrocellulose membranes to which protein had been transferred were incubated overnight at 4°C in the presence of a specific primary antibody for the proteins of interest: Anti-ABCA6 (ab180567; Abcam, Toronto, ON, Canada) in a 1:250 PBS dilution with 5% BSA; and anti-ERK (sc-7383; Santa Cruz Biotechnology, Dallas, TX, USA) as an internal control, diluted 1:3000 in PBS with 5% milk. The membrane was then washed, processed, and analysed as described previously. 31

| Immunohistochemistry
Mounted paraffin-embedded tissue sections (0.5 μm thickness) were processed as described previously. 31 (Table 1). ABCA13, ABCB4, ABCB9, ABCB11, ABCC2, and ABCG4 were the most decreased, while ABCA6, ABCA8, ABCA9, ABCA10, ABCC3, and ABCG1 are the most increased ABC genes ( Table 1). As previously observed, 31 expression of ABCG5 and ABCG8 was below detection limit and, therefore, not included in the analysis. ABCB5, ABCC8, ABCC12, and ABCC13 transcripts exhibited inconsistent amplification results likely because of very low levels of expression, and were not evaluated. Based on their potential physiological relevance and mRNA abundance (baseline mRNA expression), seven ABC transporter genes were selected for validation using individual qPCR. We confirmed the same pattern previously observed in the array, i.e., significantly increased expression of ABCA1 (P < 0.01), ABCA6 (P < 0.001), ABCA9 (P < 0.001) and ABCC3 (P < 0.001), no difference in ABCB1 and significantly decreased expression of ABCB11 (P < 0.001) and ABCG4 (P < 0.01), in first trimester compared to term placentae (Figure 2).

| ABCA6 placental protein expression increases
at term and is localized to the syncytiotrophoblast We next evaluated whether protein levels of a specific ABC transporter, would follow the same pattern as for gene expression. We selected an as yet uncharacterized ABC transporter, the ABCA6 lipid tranporter for analysis, based on its degree of change (fold change: 2.63, P < 0.001) and potential physiological relevance for the placenta. As shown in Figure 3A, ABCA6 exhibited increased (P < 0.05) total protein expression at term, demonstrating correspondence between increased transcription and translation of this tranporter with advancing gestation. Immunohistochemical analysis indicated that ABCA6 protein was highly localized to the cytoplasm of the syncytiotrophoblast. Variable ABCA6 immunoreactivity in the microvillous membrane of the syncytiotrophoblast was also detected, though at a much lower level compared to the cytoplasmic staining.
Additionally, some cells of interstitial villi of both first trimester and term placentae were also positive for ABCA6 ( Figure 3B,C).

| DISCUSSION
In the current study, we show for the first time, a gestational-age specific pattern of expression of 30 ABC transporters in the healthy human placenta, highlighting a potential role for these transporters in regulating placental development, metabolism, and intrauterine disposition of specific substrates throughout pregnancy. Importantly, the lipid transporter ABCA6 exhibited increased placental mRNA and protein expression towards term and was highly localized to the syncytiotrophoblast in first trimester and term placentae, suggesting that ABCA6 may have a relevant role supporting placental lipid metabolism and transport, particularly in late pregnancy, when protein levels are at their highest. Among all the regulated ABC genes, the ABCA subset exhibited the greatest degree of upregulation across pregnancy, particularly for ABCA6, ABCA8, ABCA9, and ABCA10. This is perhaps unsurprising, considering that this subfamily of transporters comprises the major lipid transporters responsible for lipid homeostasis, 34-36 lipid translocation and cell signalling. [37][38][39][40] In fact, ABCA transporters are of primary importance for cholesterol transport, which is paramount for normal fetal brain and overall body development. [41][42][43][44][45][46][47][48] Cholesterol is also the precursor of lipid metabolites such as oxysterols and steroid hormones. [49][50][51][52] Given that the human placenta lacks the cellular machinery required for cholesterol synthesis, 53 and the fact that F I G U R E 1 Heatmap indicating the time-dependent expression of the 30 ATP-binding cassette (ABC) genes that exhibited developmentally regulated expression in healthy human placentae. Relative quantities (Rq) of the ABC genes obtained from the Human ABC Transporters Taqman ® Array, comparing first trimester (n = 8) and term (n = 12) human placentae. Red represents increased expression, green indicates reduced expression T A B L E 1 Fold-change in mRNA expression of the ATP-binding cassette (ABC) transporters, obtained from the Human ABC Transporters Taqman ® Array, comparing term (n = 12) to first trimester (n = 8) human placentae Fold-change was calculated as the ratio between term and first trimester expression. Fold-change >1 indicates increased expression; <1 indicates decreased expression. *P < 0.05, **P < 0.01 and ***P < 0.001. ogy. Interestingly, we detected a 2.14-fold increased expression in ABCA1 mRNA at term compared to first trimester. This finding contrasts with previous published data demonstrating no changes in placental ABCA1 mRNA levels comparing early vs. late pregnancy placentae. 20 These differences may be due to patient selection criteria and or differing sensitivity of the techniques undertaken in both studies.
We selected the ABCA6 lipid transporter for further protein analysis. ABCA6 exhibited a marked gestational time-dependent regulation and to the best of our knowledge, has never been previously characterized in the placenta. Placental ABCA6 protein levels increased with advancing gestation, which paralleled increases in mRNA levels detected by the microfluid array and individual qPCR.
Furthermore, ABCA6 was highly localized to the syncytiotrophoblast cytoplasm regardless of the gestational age. Some sections exhibited variable localization in the microvillous membrane of the syncytiotrophoblast and in interstitial villi cells in both first trimester and term placentae. Considering that ABCA6 is expressed at Golgi complexes, 40 our results suggest that ABCA6 may contribute to lipid homeostasis in the syncytiotrophoblast, probably by transferring cholesterol and oxysterols across the Golgi complex membrane. Furthermore, the variable presence of ABCA6 in the microvillous membrane of the syncytiotrophoblast suggests that it may also contribute to lipid transport in the placental barrier across pregnancy. However, these hypotheses require further investigation.
Levels of mRNA expression of ABCG genes involved in lipid transport and homeostasis were also developmentally regulated. Of importance, ABCG1 was highly upregulated in term placentae.
ABCG1 has been demonstrated to synergize with ABCA1-which also exhibited higher gene transcript levels in term placentae, to efflux cholesterol and generate HDL particles. 56 In contrast to the ABCA subset of transporters, the ABCB transporters exhibited the greatest decreases of mRNA throughout pregnancy. Nine out 11 ABCB transporters were decreased at term, in particular, ABCB2, ABCB4, and ABCB9 mRNA. The ABCB subset comprise transporters known to elicit multidrug resistance to neoplastic cells. 57 In the placenta, ABCB transporters confer embryo/fetal protection against xenobiotics and environmental toxins that may present in the maternal circulation. 5,14 The best well-characterized placental ABCB transporters are ABCB1, which encodes P-gp and ABCB4, the MRP-3 encoding gene. 5 Previously, we have demonstrated a distinct pattern of time-dependent expression of P-gp/ ABCB1 in the human placenta. P-gp is highly expressed in the human placenta and is primarily localized to the apical membrane of the syncytiotrophoblast. P-gp functions as a major efflux transporter that protects the fetus from accumulation of several obstetric-relevant drugs and environmental toxins 5 and is an important transporter that modulates extravillous trophoblast invasion in early pregnancy. 58 In our previous studies, syncytiotrophoblast P-gp staining and placental ABCB1 expression decreased towards term, suggesting a higher protection of the fetus from exposure to P-gp substrates in the first trimester, a period in which the developing conceptus is most vulnerable to teratogenicity. 23 Gene expression of the intracellular ABC proteins, ABCE1 and ABCF1-3, was also decreased in term placentae. While their placental function are not known, our data provide evidence these ABC genes may developmentally regulate intracellular processes in trophoblast cells, which may include ribosomal cycling and protein synthesis 74 and inflammatory responses to cytosolic DNA (DNA sensing) 75 and chorioamnionitis. 31 ABCG2, which encodes the multidrug resistance transporter BCRP, did not show changes in gene expression throughout pregnancy. Like P-gp, BCRP is highly expressed in the apical membrane of the syncytiotrophoblast barrier to confer fetal protection against harmful substances present in the maternal blood; but with an additional role, to regulate cytotrophoblast fusion into syncytiotrophoblasts. 76 Stable levels of ABCG2 across pregnancy are consistent with our previous studies. 23,27 BCRP staining however, is increased in the syncytiotrophoblast towards the end of pregnancy, indicating an increment of fetal protection against BCRP substrates as gestation proceeds. 23,27 IMPERIO ET AL.

| 615
In conclusion, placental development is associated with a very specific expression pattern of 30 ABC transporter genes. We have also demonstrated a gestational age-dependent pattern of ABCA6 mRNA and protein expression and its abundant localization to the syncytiotrophoblast. Our findings suggest that ABCA6 is likely to exert yet unexplored gestational-age dependent actions in placental lipid homeostasis and transport. Our data also highlight the need for further studies exploring the role of other yet uncharacterized developmentally-regulated ABC transporters in the placenta, which likely exert important actions throughout pregnancy. Considering their crucial role in regulating steroidogenesis, placental nutrient transfer, barrier efficiency and integrity, as well as diverse intracellular processes, the ABC transporters in the placenta likely play critical roles in normal, and pathological pregnancies. This represents a critical area for future research.