Sodium Hydrogen Exchanger Regulatory Factor-1 (NHERF1) Regulates Fetal Membrane Inflammation

Kammala, Ananth Kumar; Sheller‐Miller, Samantha; Radnaa, Enkhtuya; Kechichian, Talar; Subramanian, Hariharan; Menon, Ramkumar

doi:10.3390/ijms21207747

Cited by 10 publications

(17 citation statements)

References 79 publications

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“…The collection of placentas was approved by the Institutional Review Board (IRB) at the University of Texas Medical Branch (UTMB) at Galveston, TX (IRB approval number IRB16.0058, January 2020). The protocol allows for the collection of discarded placentas from term vaginal or term, not in labor, cesarean deliveries (TL and TNIL, respectively) from John Sealy Hospital according to our laboratory's inclusion and exclusion criteria as described in previous reports [13,14]. The protocol is exempt from subject consent as the specimens are non-identifiable and were intended to be discarded.…”

Section: Tissue Collection and Institutional Review Board Approvalmentioning

confidence: 99%

“…Our recent data showed that fetal membranes (FMs-amniochorion), along with the placenta, express various transport proteins; however, their localization within FM cells and functions have not yet been elucidated. Since human fetal membranes are involved in various protective mechanisms [11][12][13][14][15][16] during pregnancy, it is likely that transport protein functions may also be controlled by the FMs. Determining the role of FMs in drug transport is necessary and could lead to a greater understanding of pharmacokinetics in pregnancy.…”

Section: Introductionmentioning

confidence: 99%

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Fetal Membranes Contribute to Drug Transport across the Feto-Maternal Interface Utilizing the Breast Cancer Resistance Protein (BCRP)

Kammala

Benson

Ganguly

et al. 2022

Life

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During pregnancy, the placenta is established as a primary organ for drug transport at the maternal-fetal interface. The fetal membranes (FM) also form an interface with maternal tissues; however, their role in drug transport has not been previously investigated. Knowledge of drug transport across this feto-maternal interface along with the placenta can improve new drug development and testing for use during pregnancy. We also hypothesize that extracellular vesicles (exosomes 30–160 nm) released from the FM and placental cells may also contain drug transport proteins and might impact drug trafficking across the feto-maternal interfaces. The objectives were to (1) localize the breast cancer resistance protein (BCRP) in human FM; (2) determine the drug transport function of BCRP in chorion trophoblast cells (CTCs) of the FM; and (3) investigate the presence of BCRP in FM cell-derived exosomes, as a paracrine modifier of the tissue environment for transport functions. The gene and protein expressions of ABCG2/BCRP in FMs were determined by quantitative real-time PCR (qRT-PCR) and western blotting (WB) and were localized by immunohistochemistry (IHC). The surface expression of BCRP in FM cells was determined by flow cytometry. The functional role of BCRP was assessed by an EFFLUX dye multidrug resistance assay. The presence of BCRP in exosomes derived from CTCs and BeWo cells was examined using ExoView®. Data derived from CTCs are compared with placental trophoblast cells (BeWo). BCRP is expressed and localized in the fetal membrane, primarily in the chorion trophoblast cell layer and scarcely in the amnion epithelial layer (AEC), and primarily localized on both AEC and CTC cell surfaces. Efflux assay data showed that FM cells have similar drug resistance activity as BeWo cells, suggesting that FM also have drug transportation capabilities. BeWo- and CTC-derived exosomes expressed limited BCRP protein on the surface, so it was predominantly contained in the exosomal lumen. As far as we are aware, this is the first study to report BCRP expression in fetal membrane cells and as cargo in fetal membrane-derived exosomes. We report that fetal membrane cells are capable of drug transportation. Based on these results, investigational drug trials should include the FM and its exosomes as possible drug transportation routes in pregnancy.

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Section: Tissue Collection and Institutional Review Board Approvalmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Fetal Membranes Contribute to Drug Transport across the Feto-Maternal Interface Utilizing the Breast Cancer Resistance Protein (BCRP)

Kammala

Benson

Ganguly

et al. 2022

Life

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“…Immunohistochemistry (IHC) for OATP2B1 was performed on FM tissues using methods previously described by our laboratory (Kammala et al, 2020). Briefly, FM sections were fixed in 4% paraformaldehyde for 48 h and embedded in paraffin.…”

Section: Immunohistochemistry For Oatp2b1mentioning

confidence: 99%

Organic Anion Transporting Polypeptide 2B1 in Human Fetal Membranes: A Novel Gatekeeper for Drug Transport During Pregnancy?

Ganguly¹,

Kammala²,

Benson³

et al. 2021

Front. Pharmacol.

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Current intervention strategies have not been successful in reducing the risks of adverse pregnancy complications nor maternal and fetal morbidities associated with pregnancy complications. Improving pregnancy and neonatal outcomes requires a better understanding of drug transport mechanisms at the feto-maternal interfaces, specifically the placenta and fetal membrane (FM). The role of several solute carrier uptake transporter proteins (TPs), such as the organic anion transporting polypeptide 2B1 (OATP2B1) in transporting drug across the placenta, is well-established. However, the mechanistic role of FMs in this drug transport has not yet been elucidated. We hypothesize that human FMs express OATP2B1 and functions as an alternate gatekeeper for drug transport at the feto-maternal interface. We determined the expression of OATP2B1 in term, not-in-labor, FM tissues and human FM cells [amnion epithelial cell (AEC), chorion trophoblast cell (CTC), and mesenchymal cells] using western blot analyses and their localization using immunohistochemistry. Changes in OATP2B1 expression was determined for up to 48 h after stimulation with cigarette smoke extract (CSE), an inducer of oxidative stress. The functional role of OATP2B1 was determined by flow cytometry using a zombie violet dye substrate assay. After OATP2B1 gene silencing, its functional relevance in drug transport through the feto-maternal interface was tested using a recently developed feto-maternal interface organ-on-a-chip (OOC) system that contained both FM and maternal decidual cells. Propagation of a drug (Rosuvastatin, that can be transported by OATP2B1) within the feto-maternal interface OOC system was determined by mass spectrometry. FMs express OATP2B1 in the CTC and AEC layers. In FM explants, OATP2B1 expression was not impacted by oxidative stress. Uptake of the zombie violet dye within AECs and CTCs showed OATP2B1 is functionally active. Silencing OATP2B1 in CTCs reduced Rosuvastatin propagation from the decidua to the fetal AEC layer within the feto-maternal interface-OOC model. Our data suggest that TPs in FMs may function as a drug transport system at the feto-maternal interface, a function that was previously thought to be performed exclusively by the placenta. This new knowledge will help improve drug delivery testing during pregnancy and contribute to designing drug delivery strategies to treat adverse pregnancy outcomes.

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“…33 Maternal decidual and FM cells were cultured as previously described. 34,28,35 The cells used for all experiments were within 10 passages.…”

Section: Maternal Decidual and Fm Cells (Amnion Epithelial Cells [Aecs] Amnion Mesenchymal Cells [Amcs] Chorionic Trophoblast Cells [Ctcsmentioning

confidence: 99%

“…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. 28 Twenty-four hours after transfection, the cells were used for subsequent assessment of protein expression and stimulation.…”

Section: 10mentioning

confidence: 99%

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

Kammala

Benson

Ganguly

et al. 2021

American J Rep Immunol

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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

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Sodium Hydrogen Exchanger Regulatory Factor-1 (NHERF1) Regulates Fetal Membrane Inflammation

Cited by 10 publications

References 79 publications

Fetal Membranes Contribute to Drug Transport across the Feto-Maternal Interface Utilizing the Breast Cancer Resistance Protein (BCRP)

Fetal Membranes Contribute to Drug Transport across the Feto-Maternal Interface Utilizing the Breast Cancer Resistance Protein (BCRP)

Organic Anion Transporting Polypeptide 2B1 in Human Fetal Membranes: A Novel Gatekeeper for Drug Transport During Pregnancy?

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

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