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. Author manuscript; available in PMC: 2023 Feb 1.
Published in final edited form as: Am J Reprod Immunol. 2021 Dec 15;87(2):e13515. doi: 10.1111/aji.13515

Functional role and regulation of permeability-glycoprotein (P-gp) in the Fetal membrane during drug transportation

Ananthkumar Kammala 1,†, Meagan Benson 1,†, Esha Ganguly 1, Lauren Richardson 1, Ramkumar Menon 1,*
PMCID: PMC8776608  NIHMSID: NIHMS1762514  PMID: 34873775

1. INTRODUCTION

Several drug transporter proteins, such as P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), multidrug resistance-associated proteins, organic anion transporter proteins and equilibrative nucleoside transporters have been involved in the transport of drugs across the placenta, ultimately affecting drug distribution in the fetus (Evseenko, Paxton, and Keelan 2006; Ni and Mao 2011; Tetro et al. 2018). Current knowledge in the literature supports the placenta as primarily responsible for the regulation of drug transportation (Kozłowska-Rup et al. 2014). Drugs were also metabolized by the fetus and excreted into the amniotic fluid that comprises the fetal environment (Basraon et al. 2012; Tarca et al. 2020; Underwood, Gilbert, and Sherman 2005). The fetal membranes (FMs) are the innermost tissue layers forming the intrauterine cavity and serve as a barrier between the feto-placental and maternal compartments (Afrouzian et al. 2018; Menon 2016; Menon and Moore 2020). The FMs are in direct contact with the amniotic fluid and protect the developing fetus (Menon and Moore 2020; Menon, Richardson, and Lappas 2019). However, their contribution in transport of drugs or expression of any transporter proteins is currently not tested. We hypothesized that FMs, along with the placenta, also play a significant role in drug transport mechanisms.

Our study focused on P-gp, an ATP-binding cassette (ABC) drug efflux transporter protein (Choi and Yu 2014) and its function in fetal membrane drug transport mechanism. It is widely accepted that the role of ABC transporters is to limit the intracellular concentration of xenobiotic substances by actively pumping out substances in an energy-dependent manner to protect the cell from damage (Staud et al. 2010). P-gp is a 170 kDa protein comprised of a single polypeptide of two homologous halves. Each half consists of six transmembrane domains with nucleotide-binding domains on the cytoplasmic side of a membrane. P-gp is involved in developing multidrug resistance in cancer treatment. Additionally, P-gp alters drug pharmacokinetics in multiple tissues in the human body, such as the brain, kidney, gastrointestinal tract, and heart. In addition, prior studies have confirmed the presence of P-gp in the differentiating primary trophoblast cells and the choriocarcinoma cell lines (BeWo and Jar) used extensively in various in vitro reproductive biological studies (Dunk et al. 2018; Leschziner et al. 2007; Nanovskaya et al. 2005; Ni and Mao 2011; Oostendorp et al. 2009; Staud, Cerveny, and Ceckova 2012; Aye et al. 2007; Resch et al. 2010). However, the role of the P-gp regulated efflux transport mechanism has not been previously reported in FMs.

P-gp is regulated by several binding proteins, including microtubules, ezrin, radixin, moesin (ERM), CD44, (Pokharel et al. 2016; Ravindranath et al. 2015) and Na+/H+ exchange regulatory factor-1 (NHERF-1). NHERF1, a class I PDZ (PSD95/Discs-large/ZO-1) domain ERM binding protein, is involved in cell surface stabilization and/or transporter protein expression, including P-gp in various cell types (Ardura and Friedman 2011; DU et al. 2016; Leslie et al. 2013; Vaquero et al. 2017). Kawase et al. demonstrated that NHERF1 can modulate efflux transporters in human liver cancer HepG2 cells (Kawase et al. 2021). We previously reported that NHERF1 is a constitutively expressed protein in human FMs and plays a significant role in developing FMs inflammation by activating NF-kB (Kammala et al. 2020). By promoting localized inflammation, NHERF1 is likely involved in maintaining tissue homeostasis during gestation and promoting the fetal inflammatory response during infection or in response to other adverse events. Based on these data, we hypothesized that NHERF1 in FMs controls P-gp function during pregnancy to regulate the transport of various substances. However, NHERF-1 role in drug transportation has not been studied in FMs tissues. In the present study, we determined the expression and function of P-gp in the FMs and elucidated its regulatory mechanism by knocking down NHERF-1.

2. MATERIALS AND METHODS

2.1. Tissue collection and Institutional Review Board approval

The collection of placentas for this study was approved by the Institutional Review Board (IRB) at the University of Texas Medical Branch (UTMB) at Galveston, TX, under IRB approval number IRB16.0058 (January 2020). The protocol allowed the collection of discarded placentas from the term, not-in-labor, and cesarean-delivered (TNIL) as well as term vaginally delivery following labor (TL) from John Sealy Hospital according to our laboratory’s inclusion and exclusion criteria. The tissue collection protocol was exempt from subject recruitment and consent, as the specimens were non-identifiable and were meant to be discarded.

2.3. Tissue processing and preparation

Placenta and FMs (FM) were processed according to our laboratory’s standard protocol for tissue collection that has previously been described (Lavu et al. 2020; Richardson et al. 2020; Lavu et al. 2019). Briefly, the FMs were dissected from the placenta, and 6 mm biopsies were performed from the midzone of the FMs for explant preparation for western blotting (WB). Placenta and FMs were fixed for 48 h in 4% paraformaldehyde (PFA) and embedded in paraffin for IHC.

2.2. Quantitative real-time PCR for P-gp

A high-speed homogenizer was used to dissociate FMs and the placenta in Trizol reagent (Life Technologies, Carlsbad, CA, USA). RNA was isolated using a RNeasy Kit (QIAGEN, Hilden, Germany) and treated with RNase-free DNase (QIAGEN). According to the manufacturer’s instructions, RNA (2 ug) was used to synthesize cDNA using a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher, Waltman, MA, USA). According to the manufacturer’s instructions, real-time qRT-PCR was performed using validated TaqMan gene expression P-gp primer (assay ID: Hs00184500_m1, gene symbol: ABCB1) probe sets. qRT-PCR reactions were carried out using Bio-Rad CFX PCR Instruments (Bio-Rad, Hercules, CA, USA). Results were analyzed using CFX Maestro software. mRNA expression was calculated using the 2−ΔCt method with GAPDH (assay ID: Hs02786624_g1, gene symbol: GAPDH) as an internal reference control.

2.4. Immunohistochemistry for P-gp localization on the FMs

Immunohistochemistry (IHC) for P-gp was performed on the paraffin-embedded placenta, and FM blocks were prepared as described in section 2.3. Placental tissue served as a positive control, as P-gp is known to be expressed in the placenta (Mathias, Hitti, and Unadkat 2005; Ni and Mao 2011). FMs without the application of the P-gp primary antibody functioned as the negative control.

Tissue sections (5 μm thickness) were cut and adhered to positively charged slides. After deparaffinizing with xylene, sections were rehydrated with 100, 95, and 70% ethanol. An IHC kit from Abcam (ab64264) was used, and the manufacturer’s instructions were followed for staining. For immunostaining, placenta and FMs sections were incubated with the P-gp primary antibody (Abcam [ab261736]; 1:1000 dilution) at 4°C overnight. For the negative control, FMs sections were incubated overnight in 3% BSA TBS-T without the primary antibody. The IHC antirabbit antibody (1:500, Vector Laboratories, CA, USA) was added, and samples were incubated for 10 minutes at room temperature. Subsequently, DAB as a chromogen and hematoxylin as a counter stain were added for color development. Slides were examined with bright field microscopy for the presence and localization of P-gp; images were taken at 20× and 40× magnification.

2.5. Culture of immortalized human FMs cells

Maternal decidual and FM cells (amnion epithelial cells [AECs], amnion mesenchymal cells [AMCs], chorionic trophoblast cells [CTCs]) were isolated from term, not-in-labor, and caesarean-delivered FM and immortalized by the standard HPV16 E6E7 retrovirus protocol(Halbert, Demers, and Galloway 1991). Maternal decidual and FM cells were cultured as previously described (Menon et al. 2020; Kammala, et al. 2020; Hadley et al. 2018). The cells used for all experiments were within 10 passages.

2.6. BeWo cell culture

BeWo cells (from ATCC) were used to represent placental cytotrophoblasts. BeWo cells were plated in T75 flasks and maintained in DMEM and Ham’s F12 media with the addition of HI-FBS, amphotericin, and penicillin/streptomycin. The flasks were incubated at 37°C, 5% CO2, and 95% air humidity. The cells were grown to 75–80% confluence and passed every 48–72 hours. Cell culture media was changed every 48 hours.

2.7. Protein extraction and western blotting

Placental and FMs explant tissues and immortalized maternal decidual cells and FMs cells (AEC, AMC, CTC) were lysed with radioimmunoprecipitation lysis buffer [50 mM Tris (pH 8.0), 150 mM NaCl, 1% Triton X-100, and 1.0 mM EDTA (pH 8.0), and 0.1% SDS] supplemented with a protease and phosphatase inhibitor cocktail and phenyl methyl sulfonyl fluoride. Human FM were homogenized as previously described(Kammala, et al. 2020; Lavu, et al. 2019). After centrifugation at 12,000 × g for 20 minutes, the supernatant was collected, and protein concentrations were determined using a Pierce bicinchoninic acid kit (Thermo Scientific, Waltham, MA, USA)]. The protein samples were separated using SDS-polyacrylamide gel electrophoresis on a gradient (4–15%) Mini-PROTEAN TGX Precast Gel (Bio-Rad, Hercules, CA, USA) and transferred to the membrane using a Bio-Rad Gel Transfer Device (Bio-Rad). Membranes were blocked in 5% non-fat milk in 1× Tris-buffered saline-Tween 20 for a minimum of 1 hour at room temperature and then probed with the primary antibody overnight at 4°C. The membranes were then incubated with the secondary antibody conjugated with horseradish peroxidase, and immunoreactive proteins were visualized using a chemiluminescence reagent ECL WB detection system (Amersham Piscataway, NJ, USA). The following anti-human antibodies were used for western blot: P-gp (1:1000 for tissues and FMs cells, ab245246, Abcam, USA) and β-actin (1:15,000, Sigma-Aldrich, St. Louis, MO, USA). Protein band quantification was to β-actin expression in each lane, and expression was densitometrically determined using Bio-Rad Image Lab 6.0 software.

2.9. Immunoprecipitation

Cells were lysed with radioimmune precipitation assay buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 20 mm Tris-HCl, and 150 mm NaCl), and protein extracts were incubated overnight at 4°C with protein A/G beads (ab206996, Abcam, USA) with the anti-P-gp antibody. After centrifugation, the immunoprecipitated proteins were eluted with LDS sample buffer (Biorad) containing 10% β-mercaptoethanol. The resulting samples were then analyzed by western blot and probing with NHERF1 antibody (Abcam) at a concentration of 1 ug/mL.

2.10. Treatment of CTC and BeWo cells with the P-gp substrate (Rosuvastatin)

BeWo cells and CTCs were plated in six-well plates at 5 × 105 cells per well and incubated overnight at 37°C, 5% CO2, and 95% air humidity in their respective media, as described in section 2.5. Rosuvastatin (R-statin) at a dose of 20uM was used as the stimulus in time point stimulation. Culture media was removed at specific time points of 15, 30, 60, and 120 minutes, and the treatments were added. Untreated cells and fresh media were saved prior to incubation (time 0) and served as the control. To prepare cell lysates, cells were rinsed in cold PBS at specific time points after stimulation. The cells were treated with RIPA buffer, and protein samples were prepared.

2.11. Small interfering RNA-mediated knockdown of NHERF1 in BeWo cells and CTCs

Small interfering RNA (siRNA) On-Target plus smart pool oligos (Dharmacon, Lafayette, CO, USA) directed against NHERF1 or nontarget scrambled (NT; control) siRNA was used to transfect FMs cells (AECs, AMCs, CMCs, and CTCs) using the RNAimax lipofectamine reagent as previously described (Kammala, et al. 2020). Twenty-four hours after transfection, the cells were used for subsequent assessment of protein expression and stimulation.

2.12. Efflux dye assay

The efflux dye assay (eFluxx-IDH Green Multi-Drug Resistance [MDR] Assay Kit, ENZO Life Sciences, Inc., Farmingdale, NY) was performed according to the manufacturer’s instructions (Lebedeva, Pande, and Patton 2011). Briefly, on the day of the assay, NHERF-1 siRNA (NH) and non-targeted siRNA (NT) treated CTCs and BeWo cells were collected, washed with PBS, and incubated with or without P-gp inhibitors (Verapamil, 300nmoles) in the presence of the eFluxx-dye for 30 min at 37°C and analyzed immediately by flow cytometry. eFluxx-dyes are xanthene-based small molecule dyes developed for the detection of MDR activity in living cells. To exclude dead cells in this assay, propidium iodide (PI) solution was added to the cells during the last 5 min of incubation in some experiments. After incubation, the cells were spun down (1 min, 200 × g) to remove the excess probe, resuspended in an ice-cold medium, and kept on ice until flow cytometry was performed. The fluorescence intensity of EFluxx-IDH Green dye (ex/em 490/514 nm) was measured in the FL1/FITC (530/30 filter) channel. The mean fluorescence intensity (MFI) values were analyzed using FlowJo software. The MDR activity factor (MAF) of P-gp in different cell types was calculated according to the formula: MAF = 100 × (MFI with inhibitor − MFI Control)/(MFI with Inhibitor).

2.13. Statistical analyses

Statistical analyses and significance were determined by using Prism 9 (Graph Pad, San Diego, CA). Statistical analyses for normally distributed data were performed using analysis of variance (ANOVA) with the Tukey multiple comparison test. Significance is shown as *p<0.05, **p<0.01 and ***p<0.001. All data are shown as the mean ± standard error of the mean (SEM).

3. RESULTS

3.1. Human FM expresses a P-gp transporter similar to the placenta

The expression levels of P-gp in human FM tissues were examined. P-gp (ABCB1) gene expression levels were assessed by quantitative real-time PCR (qRT-PCR) in FM tissues obtained from normal labor cesarean sections. P-gp protein localization was assessed by IHC. Interestingly, the gene expression levels of P-gp in FM tissues were similar to placental tissues (p = 0.3; Fig 1A). P-gp was localized in the chorion trophoblast layer of FM with limited expression in the amnion epithelial layer, as shown in Fig 1B. Consistent with qRT-PCR and IHC data, P-gp expression was confirmed using western blot, as shown in Fig 1C. Along with FM tissues, placental tissues were also analyzed for P-gp expression and considered as the control. P-gp expression in FMs suggests that, along with the placenta, membranes may also perform efflux functions.

Figure 1. Expression and localization of P-gp transporter protein in human fetal membranes (FM).

Figure 1.

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(A) Real time analysis of P-gp (ABCB1) mRNA. RT-PCR using mRNA from fetal membrane tissues show gene expression of P-gp. The expression levels of P-gp are similar to the gene expression levels compared with placental tissues (PLA). (B) Immunohistochemistry images of human fetal membranes stained for P-gp. P-gp was found localized in the 1. amnion epithelial layers and 2. chorion trophoblast layers of the fetal membranes. (C) Western blots of P-gp in human fetal membranes. Representative blots from n=6 for each tissues are shown. Data presented as mean ± SEM.

3.2. P-gp expression in FMs cell lines and its co-existence with NHERF-1

The different layers of FM tissues are depicted in Fig 2A. Understanding specific cell types expressing P-gp is essential to determine the role of different cellular layers of FM in drug transport mechanisms. P-gp expression was observed in all cells of FM tissue and in BeWo cell lines, as shown in Fig 2B. To detect the direct interaction between P-gp and NHERF-1 proteins, immunoprecipitation of NHERF-1 with P-gp was performed in FM cells. As shown in Fig 2C, the pulldown of cell lysates using the P-gp antibody led to coprecipitation of NHERF-1 in all FM cell lines. Conversely, samples containing protein A-conjugated beads alone showed a lack of NHERF1, confirming the specificity of the reaction.

Figure 2. Expression and co-immunoprecipitation of P-gp with NHERF-1 in the human fetal membranes.

Figure 2.

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(A) Illustration of fetal membrane structure with different cell layers. (B) WBs of human fetal membrane cell lines for expression of P-gp. (C) NHERF1 was co-immunoprecipitated with P-gp (IP P-gp) from FM cell lines. No signal was detected when blots were run using protein A-conjugated beads alone (IP Control). Representative blots from n=3 for were shown.

3.4. Expression of P-gp and NHERF1 in CTC and BeWo cells after stimulation with the substrate

ERM binding proteins, such as NHERF-1, regulate efflux mechanisms in a variety of cells by interacting with P-gp (Kawase, et al. 2021; Bushau-Sprinkle and Lederer 2020; Walsh, Nolin, and Friedman 2015). This suggests that NHERF-1 could regulate P-gp expression and efflux mechanisms in FMs cells. To determine the regulation of P-gp by NHERF-1, CTCs and BeWo cells were treated with rosuvastatin (one of the statin drugs and a substrate to P-gp) at different points (Fig 3A and 3B). P-gp transports rosuvastatin in hepatic and intestinal transport systems (Deng et al. 2021; Goard et al. 2010). As anticipated, both CTCs and BeWo cells showed expression patterns similar to NHERF-1 and P-gp at different time points after substrate stimulation (Fig 3C and 3D).

Figure 3. Expression of P-gp and NHERF-1 after rosuvastatin stimulation at different time points in BeWo and CTC cells.

Figure 3.

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(A and B) Representative western blots of P-gp and NHERF-1 expression in BeWo and CTC cells in response to rosuvastatin at different timepoints. (C and D) Correlation of NHERF-1 and P-gp expression in BeWo and CTC cells. Representative blots from n=3 for were shown. Data presented as mean ± SEM.

3.5. NHERF-1 regulates P-gp expression in FM cells

To evaluate the potential dependency between P-gp and NHERF-1 after substrate stimulation, we silenced the expression of NHERF-1 in BeWo cells and CTCs and stimulated cells with rosuvastatin. As shown in Fig 4A and C, NHERF-1 expression was significantly (P<0.01) knocked down in BeWo cells and CTCs. Compared to BeWo cells, knocking down NHERF-1 decreased the response of P-gp to the substrate in CTCs (Fig 4B and D). Deficiency of NHERF-1 expression impacts the CTCs more than BeWo cells upon P-gp substrate stimulation. Thus, the interaction of NHERF-1 with P-gp was strongly expressed in CTCs.

Figure 4. Silencing of NHERF-1 expression and functional response of BeWo and CTCs.

Figure 4.

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(A) BeWo and CTCs were transiently transduced with NHERF-1 small interfering RNA (siRNA) and nontargeted (NT) siRNA as control. Representative blots of NHERF-1 knockdown are shown. (B) NHERF-1 KD cells were stimulated with substrate (Rosuvastatin 20uM) and expression of levels of P-gp were determined. Data presented as the mean ± SD from 3 independent experiments (n = 3; **p<0.01). Representative blots from n=3 for were shown

3.6. NHERF-1 regulates the P-gp efflux mechanism at the feto maternal interface

To further confirm NHERF-1 regulates P-gp efflux mechanism at the feto maternal interface, the ENZO™ EFFLUX dye experiment was performed. CTCs and BeWo cells were silenced with siRNA NHERF-1 and knock down cells were tested for efflux assay. As per the experiment principle, efflux dye will diffuse into the cell and effluxed by P-gp transporter proteins. Cells having functional P-gp will efflux the dye and treating with P-gp inhibitors (Verapamil) will trapped the dye inside cells. Due to accumulation of dye inside cells, they will show higher fluorescence.

Since NHERF-1 regulates P-gp, knockdown of NHERF-1 is expected to show a similar effect to that exhibited by a P-gp inhibitor, i.e., reduction of P-gp efflux activity and retention of efflux dye and increased fluorescence intensity. As shown in Fig 5A and B, NHERF-1 knockdown in CTCs showed increased fluorescence intensity of efflux dye similar to CTCs treated with the P-gp inhibitor. In contrast, BeWo cells with NHERF-1 knockdown showed a decrease in fluorescence intensity of the efflux dye, indicating that there was no significant effect of NHERF-1 on P-gp activity. The MDR activity was significantly (**p<0.01) higher in CTCs compared with BeWo cells when treated with P-gp inhibitor and similarly NHERF-1 KD CTCs have shown significantly (***p<0.001) higher MDR activity compared to NHERF-1 KD BeWo cells. These results strongly suggest that the impact of NHERF-1 in regulating P-gp is seen predominantly in FMs.

Figure 5. Efflux functional activity of CTCs and BeWo cells.

Figure 5.

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(A) Efflux function was measured by eFFlux ID Green dye assay. BeWo and CTCs (NT and NH cells) were incubated with eFFlux ID dye (Blue line), Dye + P-gp inhibitor (Verapamil, indicated as Red line) and Dye + NHERF-1 KD cells (Green line) for 40 min, allowing the cells to uptake and efflux this dye. Cells were suspended in cold PBS for flow cytometry analysis. Fluorescence of eFluxx ID dye was analyzed by flow cytometer and mean fluorescence intensity was calculated by FlowJo software. (B) The formula of calculation of multidrug resistance activity factor (MAF) as: MAFMRP= 100 × (FMRP-F0)/FMRP where FMRP corresponds to the fluorescence intensity in the presence of P-gp specific inhibitor and NHERF-1 KD to the fluorescence intensity in absence of inhibitor. Calculated MAF is presented as the mean ± SD from 3 independent experiments (n = 3, ** p<0.01, ***p<0.001). Representative flow charts from n=3 for were shown.

Discussion

P-gp is widely expressed in cells throughout the human body, and it is one of the most extensively studied drug transporter proteins. P-gp belongs to the ATP-binding cassette superfamily of efflux transporters. P-gp is highly expressed on the placental syncytiotrophoblast apical membrane and restricts fetal exposure to drugs and xenobiotics due to its expression and activity at the placental barrier. Although the placenta plays a significant role in drug transportation, a recent study suggested that placental P-gp expression decreases as gestation advances (Choi and Yu 2014; Daud et al. 2015; Han, Gao, and Mao 2018; Ni and Mao 2011; Oostendorp, et al. 2009). These reports led us to hypothesize that an alternate transport mechanism to excrete the drugs should exist to compensate for the loss of efflux function in the placenta mediated by P-gp. Previous reports have stated that human amnion membranes have shown expression of ATP binding cassette family of drug transporters (Resch, et al. 2010; Aye, et al. 2007). These data provided more credence to testing the hypothesis that P-gp expression and functions can likely exist in human FMs. FMs are extra-embryonic tissues that surround the fetus and form a feto-maternal interface (Afrouzian, et al. 2018; Menon and Richardson 2017; Menon, Richardson, and Lappas 2019). The amnion layer of the FM is in direct contact with amniotic fluid and maintains homeostasis through aquaporins and cyclic transition of cells (Ducza, Csányi, and Gáspár 2017). Although human FMs are not vascularized, they allow paracrine signaling between the maternal and fetal compartments via extracellular vesicles (Menon 2019; Sheller-Miller et al. 2019). FMs are capable of synthesizing and secreting a wide variety of chemokines, growth factors, and cytokines(Tantengco et al. 2021; Stalberg et al. 2017) and provide antimicrobial defense(Boldenow et al. 2015; Mao et al. 2017). FMs likely participate in the restriction of fetal exposure to chemical compounds and xenobiotics (Mittal et al. 2009; Murtha and Menon 2015). The current study showed that FMs are involved in the drug transport mechanism by P-gp, and NHERF-1 regulates its function. The principal findings of our studies are as follows: (1) P-gp is expressed and localized in human FMs; (2) P-gp is primarily expressed by CTCs but limited expression is seen in amnion epithelial and mesenchymal cells of the matrix; (3) P-gp expression is not impacted by rosuvastatin, a drug substrate, treatment in FMs cells; and (4) P-gp expression and function in FMs are likely mediated by NHERF-1. In summary, we reported a novel function for FMs during pregnancy. By expressing P-gp, FMs provide an efflux function for drugs and other toxic materials by the expression of P-gp and provide a barrier function at the maternal-fetal interface during pregnancy.

P-gp is expressed in the capillary endothelial cells of the brain, intestine, kidney, liver, testes, and placenta (Bloise et al. 2013; Staud, et al. 2010). These tissues are considered very important in determining the pharmacokinetics of drugs, as they are a rich source of drug transporter proteins. The present study has shown the gene and protein expression of P-gp in human FM and placenta. The expression levels of P-gp in FMs were similar to those seen in the placenta, suggesting that FMs, along with the placenta, also play a role in the drug transport mechanism. The placenta expresses P-gp on the apical membranes of the syncytiotrophoblast. Interestingly, there is differential expression of P-gp in FMs. Among different layers of FMs, the CTC layer has shown strong expression of P-gp, suggesting that the chorion trophoblast layer of FMs might play a significant role in the efflux drug transport mechanism and restrict fetal exposure to drugs and xenobiotics. Although the expression levels of P-gp in the amnion epithelial layer are minimal, it is hardly seen in the mesenchymal layer. This might be due to the loss of P-gp expression during epithelial-mesenchymal transition that occurs during gestation, which is required for FM remodeling (Richardson, Taylor, and Menon 2020). This finding indirectly supports a prior report by Richardson et al. that amnion mesenchymal cells are transitioned epithelial cells and not necessarily permanent constituents (Omere et al. 2020; de Castro Silva et al. 2020; Richardson, Taylor, and Menon 2020). They are seen in the extracellular matrix during their transient state to become epithelial cells again (Dunk, et al. 2018). During this state of cyclic transition, cells do not express markers that are expressed by primary cells (i.e., amnion epithelial cells) and do not perform similar functions. Besides undergoing several physical and biochemical changes during pregnancy to maintain homeostasis, human FMs also protect the fetus by regulating drug transport mechanisms.

Previous reports have suggested that ERM binding proteins are essential for the stability and functionality of P-gp in normal tissues and cancer cells (Georgescu et al. 2008; Georgescu et al. 2018; Kawase, et al. 2021; Shibata et al. 2003). NHERF-1 possesses two PDZ domains (PDZ1 and PDZ2) and an ERM-binding domain (Ardura and Friedman 2011; Broadbent et al. 2017; Singh et al. 2009). NHERF1 is a major PDZ-containing adapter protein that facilitates multi-protein complex formation, an essential step required for phosphorylation and regulation of various transporters, channels, and receptors (Ardura and Friedman 2011; Vaquero, et al. 2017; Voltz et al. 2007; Wheeler et al. 2011; Zhang et al. 2019). Kano et al. suggested that ERM binding proteins are transcriptional regulators of P-gp (Kano et al. 2011). In support, we also observed similarities in the expression of both P-gp and NHERF-1 when the cells were treated with a substrate. The interaction between P-gp and NHERF-1 indicates that NHERF-1 can anchor and stabilize P-gp in the FM. P-gp function is also regulated by other molecules, including CD44 and Tubulin, but these were not studied in this report. Although not reported in human FMs, there may be a role for these molecules in FMs P-gp expression and function.

The current study’s exciting and surprising finding was differential regulation of the efflux transport mechanism between CTCs and BeWo cells by NHERF-1 after substrate stimulation. Silencing NHERF-1 in CTCs diminished the expression and efflux transport activity of P-gp, whereas in BeWo cells, it was not significantly affected. We used BeWo cells to represent cytotrophoblast cells. Although these cells are extensively used for this purpose and several reports have indicated similarities between primary trophoblast functions, these choriocarcinoma derived cell lines may differ in their function compared to primary trophoblast cells. Future experiments should test and compare P-gp in primary trophoblast cells. Multiple reports state that P-gp substrates are transported at a 100-fold greater efficiency from the fetal to the maternal direction compared to the maternal to fetal direction (Bloise, et al. 2013; Mayer et al. 1996; Schinkel et al. 1997; Michelsen et al. 2019). Our study substantiates these reports, as we also observed a similar pattern. In our study, FM CTCs have shown a significant efflux mechanism compared to placental cells, reaffirming the membrane’s prominent role in the efflux of specific substances.

Regulation of P-gp by NHERF1 provides new insights to consider when determining drug pharmacokinetics during pregnancy. As reported earlier, NHERF1 is increased in FMs in response to an infectious stimulus. Previously, we have reported that NHERF-1 is involved in the activation of NF-κB, and silencing NHERF-1 reduced inflammation and increased the production of the anti-inflammatory cytokine interleukin-10 (Kammala, et al. 2020; Li et al. 2015). A plurifunctional molecule like NHERF1 can increase inflammation as well as regulate efflux mechanisms in the feto-maternal interface. This knowledge should be considered when designing drugs to treat infection/inflammation associated adverse pregnancy outcomes. Several small molecules have been designed to treat inflammation, but they were not successful during pregnancy because either the drug is not reaching the fetus at therapeutic concentrations, or it may have teratogenic effects. Besides having teratogenic effects, another possible mechanism might be the efflux of therapeutically beneficial drugs from the fetal to maternal side. It would be possible to decrease the drug resistance at the fetal side and enhance the pharmacological benefit of novel drugs when combined with NHERF-1 inhibitors. Moreover, the possibility of diminishing P-gp functional activity, at least in specific situations, can be advantageous for the pharmacotherapy of pregnant women. For example, anti-HIV treatment to protect the child during pregnancy may benefit from combining anti-HIV drugs with NHERF-1 inhibitors. Penetration of the drug to reach the fetus will likely be enhanced by this approach (Ham et al. 2009; Kim et al. 1998; Leschziner, et al. 2007; McConville, Boyd, and Major 2014; Staud, Cerveny, and Ceckova 2012). Obviously, the feasibility, safety, and efficacy of these drug delivery methods need to be tested in preclinical and clinical trials, but this could be a possible mechanism to treat various pregnancy-related disorders.

Conclusion

In conclusion, we have shown that FM also expresses the functional efflux transporter protein P-gp similar to placenta and is regulated by the NHERF-1 adapter molecule. To our knowledge, this study thus uncovers a novel mechanism for the regulation of drug transportation during pregnancy at the feto-maternal interface. This knowledge of FMs transport mechanisms can be exploited further when developing new therapies for pregnancy-related disorders.

Funding:

This study was supported by funding from the National Institutes of Health / Eunice Kennedy Shriver National Institute of Child Health and Human Development (NIH/ NICHD) grant #R01 5R01HD100729-03 supplement and National Center for Advancing Translational Sciences (NCATS) grant # 1UG3TR003283 to R. Menon and A. Han

Footnotes

Conflict of interest statement: The authors state no conflict of interest regarding this study.

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