功能性作用和调节胎盘药物转运中的多药耐药相关蛋白（P-gp）。
Functional role and regulation of permeability-glycoprotein (P-gp) in the fetal membrane during drug transportation.
机构信息
Department of Obstetrics & Gynecology, Division of Basic and Translational Research, The University of Texas Medical Branch at Galveston, Galveston, Texas, USA.
出版信息
Am J Reprod Immunol. 2022 Feb;87(2):e13515. doi: 10.1111/aji.13515. Epub 2021 Dec 15.
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 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.
目的
Na+/H 交换调节因子-1（NHERF-1）是一种 I 类 PDZ（PSD95/Discs-large/ZO-1）结合蛋白，参与多种细胞中转运蛋白的细胞表面表达和稳定，包括胎盘滋养层中的多药外排泵蛋白 P-糖蛋白（P-gp）。P-gp 是一种外排转运蛋白，影响妊娠期间使用的各种药物的药代动力学。先前我们报道 NHERF-1 调节胎儿膜炎症。然而，在药物转运过程中 NHERF-1 调节胎儿膜中 P-gp 的作用尚不清楚。本研究旨在确定人胎儿膜细胞中 NHERF-1 和 P-gp 之间的相互作用。
方法
通过免疫组织化学（IHC）筛选来自正常足月剖宫产的胎儿膜中 P-gp 的表达。绒毛膜滋养层（CTC）是胎儿膜细胞中 P-gp 表达最高的细胞，进一步用于测试 NHERF-1 和 P-gp 之间的相互作用特性。BeWo（胎盘滋养层细胞系）细胞用作对照。使用 P-gp 抗体对 CTC 裂解物进行免疫沉淀（IP），然后进行 Western blot 确定 NHERF-1 的共沉淀。使用小干扰 RNA 沉默 NHERF-1 进一步测试了 NHERF-1 在 CTC 和 BeWo 细胞中 P-gp 表达和功能的相关性。使用 ENZO 外排染料试剂盒进一步测试了 NHERF-1 对 P-gp 外排功能（耐药性）的调节作用。
结果
免疫组织化学定位和 Western blot 证实 P-gp 存在于人胎儿膜中，主要存在于 CTC 中，在羊膜上皮层中的表达有限。膜中的 P-gp 表达与胎盘中的表达相似。IP 数据显示 P-gp 与 NHERF1 共沉淀。沉默 NHERF-1 导致明显的耐药性，表明 CTC 中的 P-gp 功能通过 NHERF1 介导。
结论
促炎介质 NHERF-1 调节 P-gp 并控制胎儿膜的药物转运。我们的数据表明，胎儿膜在妊娠期间具有新的功能作用。除了胎盘，胎儿膜还可能调节胎-母界面物质的外排并控制妊娠期间的药物转运。
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