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

Abstract

Objective: Na⁺ /H⁺ exchange regulatory factor-1 (NHERF-1) is a class I PDZ (PSD95/Discs-large/ZO-1) binding protein involved in cell-surface expression and stabilization of transporter proteins, including permeability-glycoprotein (P-gp) in various cell types. P-gp, expressed in placental trophoblasts, is an efflux transporter protein that influences the pharmacokinetics of various drugs used during pregnancy. Previously we have reported that NHERF-1 regulates fetal membrane inflammation. However, the role of NHERF-1 in regulating P-gp in the fetal membrane during drug transportation remains unclear. This study determined the interplay between NHERF-1 and P-gp in human fetal membrane cells.

Methods: Fetal membranes from normal, term cesareans were screened for P-gp by immunohistochemistry (IHC). Chorionic trophoblast (CTC), with the highest expression of P-gp among fetal membrane cells, was further used to test interactive properties between NHERF-1 and P-gp. BeWo (placental trophoblast cell line) cells were used as a control. Immunoprecipitation (IP) of CTC lysates using the P-gp antibody followed by western blot determined co-precipitation of NHERF-1. Silencing NHERF-1 using small interfering RNA further tested the relevance of NHERF-1 in P-gp expression and function in CTC and BeWo cells. NHERF-1 regulation of P-gp's efflux function (drug resistance) was further tested using the ENZO^TM efflux dye kit.

Results: Immunohistochemistry localized, and western blot confirmed P-gp in human fetal membranes, primarily in the CTC with limited expression in the amnion epithelial layer. P-gp expression in the membranes was similar to that seen in the placenta. IP data showed P-gp co-precipitating with NHERF1. Silencing of NHERF-1 resulted in significant drug resistance suggesting P-gp function mediated through NHERF1 in CTCs.

Conclusion: Proinflammatory mediator NHERF-1 regulates P-gp and control drug transportation across the fetal membranes. Our data suggest a novel functional role for fetal membranes during pregnancy. Besides the placenta, fetal membranes may also regulate efflux of materials at the feto-maternal interface and control drug transport during pregnancy.

Keywords: NHERF-1; P-gp; drug transport; fetal membrane.

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

(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.

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

(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.

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

(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.

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

(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

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

(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.