scholarly journals LEFTYA Activates the Epithelial Na+ Channel (ENaC) in Endometrial Cells via Serum and Glucocorticoid Inducible Kinase SGK1

2016 ◽  
Vol 39 (4) ◽  
pp. 1295-1306 ◽  
Author(s):  
Madhuri S. Salker ◽  
Zohreh Hosseinzadeh ◽  
Nour Alowayed ◽  
Ni Zeng ◽  
Anja T. Umbach ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Ussing Chamber ◽  
Endometrial Adenocarcinoma ◽  
Negative Regulator ◽  
Na Channel ◽  
Uterine Receptivity ◽  
Endometrial Cells ◽  
Ishikawa Cells ◽  
Epithelial Na Channel ◽  
Endometrial Epithelial Cells

Background: Serum & glucocorticoid inducible kinase (SGK1) regulates several ion channels, including amiloride sensitive epithelial Na+ channel (ENaC). SGK1 and ENaC in the luminal endometrium epithelium, are critically involved in embryo implantation, although little is known about their regulation. The present study explored whether SGK1 and ENaC are modulated by LEFTYA, a negative regulator of uterine receptivity. Methods: Expression levels were determined by qRT-PCR and Western blotting, ENaC channel activity by whole cell patch clamp and transepithelial current by Ussing chamber experiments. Results: Treatment of Ishikawa cells, an endometrial adenocarcinoma model cell line of endometrial epithelial cells, with LEFTYA rapidly up-regulated SGK1 and ENaC transcript and protein levels. Induction of ENaC in response to LEFTYA was blunted upon co-treatment with the SGK1 inhibitor EMD638683. ENaC levels also significantly upregulated upon expression of a constitutively active, but not a kinase dead, SGK1 mutant in Ishikawa cells. LEFTYA increased amiloride sensitive Na+-currents in Ishikawa cells and amiloride sensitive transepithelial current across the murine endometrium. Furthermore, LEFTYA induced the expression of ENaC in the endometrium of wild-type but not of Sgk1-deficient mice. Conclusions: LEFTYA regulates the expression and activity of ENaC in endometrial epithelial cells via SGK1. Aberrant regulation of SGK1 and ENaC by LEFTYA could contribute to the pathogenesis of unexplained infertility.

scholarly journals miR-543 Inhibits the Occurrence and Development of Intrauterine Adhesion by Inhibiting the Proliferation, Migration, and Invasion of Endometrial Cells

2021 ◽  
Vol 2021 ◽  
pp. 1-9
Author(s):  
Xin Liu ◽  
Qian Xu ◽  
Chao Chen ◽  
Hua Duan
Keyword(s):  
Epithelial Cells ◽  
Protein Expression ◽  
Endometrial Adenocarcinoma ◽  
Luciferase Reporter ◽  
Control Group ◽  
Intrauterine Adhesions ◽  
Endometrial Cells ◽  
Intrauterine Adhesion ◽  
Adenocarcinoma Cells ◽  
Endometrial Epithelial Cells

Objective. To explore the function of miR-543 in endometrial cells and the possible mechanism of regulating the occurrence and development of intrauterine adhesion. Method. Endometrial epithelial cells and endometrial adenocarcinoma cells were transfected with miR-543 mimics and miR-543 inhibitor as the experimental group and were tested with the control group, using the CCK-8 method, scratch test, and Transwell assay, and flow cytometry was used to detect the proliferation, migration, invasion, and apoptosis of cells. RT-qPCR and Western blot were used to detect the expression of corresponding mRNA and protein. Results. After the overexpression of miR-543, endometrial epithelial cells and endometrial adenocarcinoma cells have reduced migratory, proliferative, and invasive capabilities, while the apoptosis rate has increased significantly. The mRNA expression of CDH2, COL16A1, vimentin, α-SMA and fibronectin decreased, and the protein expression of CDH2, vimentin, and α-SMA also decreased, while the mRNA and protein expression of CDH1 increased. The result after interfering with miR-543 is opposite, and luciferase reporter gene confirms that CDH2 is the target gene of miR-543. Conclusion. During the formation of intrauterine adhesions, the expression of CDH2, COL16A1, vimentin, and α-SMA may be inhibited by the high expression of miR-543, which may affect the degree of fibrosis and collagen content in the intrauterine adhesions, thereby inhibiting the occurrence and development of intrauterine adhesions.

scholarly journals Proteomic analysis of extracellular vesicles secreted by primary human epithelial endometrial cells reveals key proteins related to embryo implantation

2022 ◽  
Vol 20 (1) ◽  
Author(s):  
Marina Segura-Benítez ◽  
María Cristina Carbajo-García ◽  
Ana Corachán ◽  
Amparo Faus ◽  
Antonio Pellicer ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Proteomic Analysis ◽  
Embryo Implantation ◽  
Endometrial Receptivity ◽  
Biological Processes ◽  
Endometrial Cells ◽  
Isolation Methods ◽  
Ishikawa Cells ◽  
Endometrial Epithelial Cells ◽  
Implantation Success

Abstract Background Successful implantation is dependent on coordination between maternal endometrium and embryo, and the role of EVs in the required cross-talk cell-to-cell has been recently established. In this regard, it has been reported that EVs secreted by the maternal endometrium can be internalized by human trophoblastic cells transferring their contents and enhancing their adhesive and invasive capacity. This is the first study to comprehensively evaluate three EV isolation methods on human endometrial epithelial cells in culture and to describe the proteomic content of EVs secreted by pHEECs from fertile women. Methods Ishikawa cells and pHEECs were in vitro cultured and hormonally treated; subsequently, conditioned medium was collected and EVs isolated. Ishikawa cells were used for the comparison of EVs isolation methods ultracentrifugation, ExoQuick-TC and Norgen Cell Culture Media Exosome Purification Kit (n = 3 replicates/isolation method). pHEECs were isolated from endometrial biopsies (n = 8/replicate; 3 replicates) collected from healthy oocyte donors with confirmed fertility, and protein content of EVs isolated by the most efficient methodology was analysed using liquid chromatography–tandem mass spectrometry. EV concentration and size were analyzed by nanoparticle tracking analysis, EV morphology visualized by transmission electron microscopy and protein marker expression was determined by Western blotting. Results Ultracentrifugation was the most efficient methodology for EV isolation from medium of endometrial epithelial cells. EVs secreted by pHEECs and isolated by ultracentrifugation were heterogeneous in size and expressed EV protein markers HSP70, TSG101, CD9, and CD81. Proteomic analysis identified 218 proteins contained in these EVs enriched in biological processes involved in embryo implantation, including cell adhesion, differentiation, communication, migration, extracellular matrix organization, vasculature development, and reproductive processes. From these proteins, 82 were selected based on their functional relevance in implantation success as possible implantation biomarkers. Conclusions EV protein cargos are implicated in biological processes related to endometrial receptivity, embryo implantation, and early embryo development, supporting the concept of a communication system between the embryo and the maternal endometrium via EVs. Identified proteins may define new biomarkers of endometrial receptivity and implantation success.

scholarly journals Mouse double minute homologue 2 (MDM2) downregulation by miR-661 impairs human endometrial epithelial cell adhesive capacity

2018 ◽  
Vol 30 (3) ◽  
pp. 477 ◽  
Author(s):  
Amy Winship ◽  
Amanda Ton ◽  
Michelle Van Sinderen ◽  
Ellen Menkhorst ◽  
Katarzyna Rainczuk ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Adhesion ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Endometrial Cells ◽  
Endometrial Epithelial Cell ◽  
Mouse Double Minute ◽  
Double Minute ◽  
Ishikawa Cells ◽  
Endometrial Epithelial Cells

Human blastocysts that fail to implant following IVF secrete elevated levels of miR-661, which is taken up by primary human endometrial epithelial cells (HEECs) and impairs their adhesive capability. MicroRNA miR-661 downregulates mouse double minute homologue 2 (MDM2) and MDM4 in other epithelial cell types to activate p53; however, this has not been examined in the endometrium. In this study MDM2 protein was detected in the luminal epithelium of the endometrium, the site of blastocyst attachment, during the mid secretory receptive phase of the menstrual cycle. The effects of miR-661 on gene expression in and adhesion of endometrial cells was also examined. MiR-661 overexpression consistently downregulated MDM2 but not MDM4 or p53 gene expression in the Ishikawa endometrial epithelial cell line and primary HEEC. Adhesion assays were performed on the real-time monitoring xCELLigence system and by co-culture using Ishikawa cells and HEECs with HTR8/SVneo trophoblast spheroids. Targeted siRNA-mediated knockdown of MDM2 in endometrial epithelial cells reduced Ishikawa cell adhesion (P < 0.001) and also reduced HTR8/SVneo trophoblast spheroid adhesion to Ishikawa cells (P < 0.05) and HEECs (P < 0.05). MDM2 overexpression using recombinant protein treatment resulted in enhanced HTR8/SVneo trophoblast spheroid adhesion to Ishikawa cells (P < 0.01) and HEECs (P < 0.05). This study highlights a potential new mechanism by which human blastocyst-secreted miR-661 reduces endometrial epithelial cell adhesion; via downregulation of MDM2. These findings suggest that MDM2 contributes to endometrial–blastocyst adhesion, implantation and infertility in women.

Noncoordinated expression of alpha-, beta-, and gamma-subunit mRNAs of epithelial Na+ channel along rat respiratory tract

1997 ◽  
Vol 272 (1) ◽  
pp. C131-C141 ◽  
Author(s):  
N. Farman ◽  
C. R. Talbot ◽  
R. Boucher ◽  
M. Fay ◽  
C. Canessa ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Respiratory Tract ◽  
Mrna Level ◽  
Airway Epithelial Cells ◽  
Respiratory Physiology ◽  
Na Channel ◽  
Alveolar Cells ◽  
Cellular Expression ◽  
Alpha Beta ◽  
Epithelial Na Channel

Na+ reabsorption from the epithelial surface of the respiratory tract plays a fundamental role in respiratory physiology. As in the epithelia of the renal collecting tubule and distal colon, Na+ enters across the luminal surface of respiratory epithelial cells via a recently cloned amiloride-sensitive multisubunit (alpha, beta, gamma) epithelial Na+ channel. We have examined the cellular expression at the mRNA level of the alpha-, beta-, and gamma-subunits of rat epithelial Na+ channel (rENaC) in the rat lung and upper airway epithelial cells using in situ hybridization. A large prevalence of alpha- and gamma-rENaC subunit expression (over beta) was found in tracheal epithelium, in a subpopulation of alveolar cells, presumably type II pneumocytes, and in nasal and tracheal gland acini. In contrast, equivalent levels of expression of all three subunits were detected in bronchiolar epithelium and in rat nasal gland ducts. This diversity of expression may reflect cell-specific functions of the amiloride-sensitive Na+ channel along the respiratory tract.

scholarly journals Lipocalin 2 induces the epithelial–mesenchymal transition in stressed endometrial epithelial cells: possible correlation with endometriosis development in a mouse model

Reproduction ◽  
2014 ◽  
Vol 147 (2) ◽  
pp. 179-187 ◽  
Author(s):  
Chi-Jr Liao ◽  
Pei-Tzu Li ◽  
Ying-Chu Lee ◽  
Sheng-Hsiang Li ◽  
Sin Tak Chu
Keyword(s):  
Epithelial Cells ◽  
Mouse Model ◽  
Epithelial Mesenchymal Transition ◽  
Migration And Invasion ◽  
Lipocalin 2 ◽  
Mesenchymal Transition ◽  
Endometrial Cells ◽  
Cellular Microenvironments ◽  
And Migration ◽  
Endometrial Epithelial Cells

Lipocalin 2 (LCN2) is an induced stressor that promotes the epithelial–mesenchymal transition (EMT). We previously demonstrated that the development of endometriosis in mice correlates with the secretion of LCN2 in the uterus. Here, we sought to clarify the relationship between LCN2 and EMT in endometrial epithelial cells and to determine whether LCN2 plays a role in endometriosis. Antibodies that functionally inhibit LCN2 slowed the growth of ectopic endometrial tissue in a mouse model of endometriosis, suggesting that LCN2 promotes the formation of endometriotic lesions. Using nutrient deprivation as a stressor, LCN2 expression was induced in cultured primary endometrial epithelial cells. As LCN2 levels increased, the cells transitioned from a round to a spindle-like morphology and dispersed. Immunochemical analyses revealed decreased levels of cytokeratin and increased levels of fibronectin in these endometrial cells, adhesive changes that correlate with induction of cell migration and invasion.Lcn2knockdown also indicated that LCN2 promotes EMT and migration of endometrial epithelial cells. Our results suggest that stressful cellular microenvironments cause uterine tissues to secrete LCN2 and that this results in EMT of endometrial epithelial cells, which may correlate with the development of ectopic endometriosis. These findings shed light on the role of LCN2 in the pathology of endometrial disorders.

Mechanism Of Epithelial Na+ Channel (ENaC) Inhibition By Hypoxia In Alveolar Epithelial Cells

2012 ◽  
Author(s):  
Thomas Gille ◽  
Nadia Randrianarison ◽  
Arnaud Goolaerts ◽  
Yurdagül Uzunhan ◽  
Evelyne Ferrary ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Alveolar Epithelial Cells ◽  
Na Channel ◽  
Alveolar Epithelial ◽  
Epithelial Na Channel

scholarly journals Molecular Cloning and Characterization of Prostaglandin (PG) Transporter in Ovine Endometrium: Role for Multiple Cell Signaling Pathways in Transport of PGF2α

Endocrinology ◽  
2007 ◽  
Vol 149 (1) ◽  
pp. 219-231 ◽  
Author(s):  
S. K. Banu ◽  
J. Lee ◽  
M. C. Satterfield ◽  
T. E. Spencer ◽  
F. W. Bazer ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Epithelial Cells ◽  
Estrous Cycle ◽  
Molecular Mechanisms ◽  
Growth Factor Receptor ◽  
Prostaglandin F2α ◽  
Cellular Transport ◽  
Endometrial Cells ◽  
Amino Terminal ◽  
Endometrial Epithelial Cells

In ruminants, endometrial prostaglandin F2α (PGF2α) is the luteolytic hormone. Cellular transport of PGF2α in the uterine endometrium is critical for regulation of the estrous cycle. Molecular mechanisms responsible for control of PGF2α transport in endometrium during luteolysis are largely unknown. In the present study, we characterized the prostaglandin transporter (PGT) in ovine endometrium. Ovine PGT cDNA consists of 1935 nucleotides that encode 644 amino acids. In ovine endometria, PGT is highly expressed during the period of luteolysis, between d 14 and 16 of the estrous cycle, in luminal and glandular epithelia. Pharmacological and genomic inhibition of PGT indicates that it is responsible for influx and efflux of PGF2α in ovine endometrial epithelial cells. Inhibition of PGT during the period of luteolysis prevents the release of oxytocin-induced PGF2α pulses, and maintains functional corpus luteum and its secretion of progesterone. In ovine endometrial epithelial cells, protein kinase A and protein kinase C pathways are involved in regulating the influx of PGF2α, whereas epidermal growth factor receptor pathways are implicated in regulation of influx and efflux of PGF2α. The ERK1/2 pathway is associated with efflux of PGF2α, whereas Jun-amino-terminal kinase/stress-activated protein kinase pathways are involved in both efflux and influx of PGF2α. Phosphatidylinositol 3-kinase pathways are not involved in either influx or efflux of PGF2α in ovine endometrial epithelial cells. These are the first results to demonstrate a functional role for PGT in regulation of PGF2α efflux and influx in ovine endometrial cells that influence luteolytic mechanisms in ruminants.

Mechanisms of electrolyte transport across the endometrium. I. Regulation by PGF2 alpha and cAMP

1996 ◽  
Vol 270 (2) ◽  
pp. C663-C672 ◽  
Author(s):  
A. E. Vetter ◽  
S. M. O'Grady
Keyword(s):  
Epithelial Cells ◽  
Rank Order ◽  
Short Circuit ◽  
Coupled Processes ◽  
Na Channel ◽  
Selectivity Ratio ◽  
Ussing Chambers ◽  
Subsequent Decrease ◽  
The Mean ◽  
Endometrial Epithelial Cells

The purpose of this study was to characterize the transport mechanisms in endometrial epithelial cells that are responsible for regulation of Na and K concentrations in uterine luminal fluid. Porcine endometrial tissues were mounted in Ussing chambers and bathed in plasmalike Ringer solution. The mean basal short-circuit current (Isc) was 40 microA/cm2, and the mean tissue conductance was 3.6 mS/cm2. Addition of amiloride to the luminal solution inhibited 86% of the basal Isc. Concentration-response experiments using amiloride analogues showed a rank order of potency of benzamil > amiloride > 5-(N-methyl-N-isobutyl)-amiloride in blocking the Isc, with no response to ethylisopropylamiloride. Na channel immunoreactivity was localized to the apical membrane of surface epithelial cells. The Na-to-K selectivity ratio of the amiloride-sensitive Na channel was calculated to be 6.4:1. Prostaglandin (PG) F2 alpha or 8-(chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (CPT-cAMP) added to the luminal solution stimulated a twofold increase in Isc that was inhibited by pretreatment with amiloride. Experiments using both amphotericin B-permeabilized tissues and intact tissues showed that PGF2 alpha and cAMP increased Na absorption by activation of basolateral K channels. Treatment of the luminal solution with 4-aminopyridine produced an effect on Isc that was consistent with block of K secretion and a subsequent decrease in Na absorption. These experiments showed that Na and K transport are tightly coupled processes occurring under basal conditions in surface endometrial epithelial cells and that these processes are regulated by PGF2 alpha and cAMP.

Annexin A2 acts as an adherent molecule under the regulation of steroids during embryo implantation

2020 ◽  
Vol 26 (11) ◽  
pp. 825-836
Author(s):  
Bing Wang ◽  
Yan Shao
Keyword(s):  
Epithelial Cells ◽  
Outer Surface ◽  
Calcium Binding ◽  
Surface Membrane ◽  
Embryo Implantation ◽  
Annexin A2 ◽  
Endometrial Cells ◽  
Membrane Bound ◽  
Endometrial Epithelial Cells ◽  
Embryo Attachment

Abstract We previously showed that annexin A2 (Axna2) was transiently expressed at the embryo-uterine luminal epithelium interface during the window of implantation and was involved in mouse embryo implantation. At the same time, Axna2 was reported to be upregulated in human receptive endometrium, which was critical for embryo attachment as an intracellular molecule. Here, we identified Axna2 as a membrane-bound molecule on human endometrial epithelial cells and trophoblast cells, and the outer surface membrane-bound Axna2 was involved in human embryo attachment. In addition, physiological levels of estrogen and progesterone increased the expression of overall Axna2 as well as that in the extracellular surface membrane protein fraction in human endometrial cells. Furthermore, p11 (or S100A10, a member of the S100 EF-hand family protein, molecular weight 11 kDa) was involved in the translocation of Axna2 to the outer surface membrane of endometrial epithelial cells without affecting its overall expression. Finally, the surface relocation of Axna2 was also dependent on cell–cell contact and calcium binding. A better understanding of the function and regulation of Axna2 in human endometrium may help us to identify a potential therapeutic target for subfertile and infertile patients.

scholarly journals Isoflavones regulate secretion of leukemia inhibitory factor and transforming growth factor β and expression of glycodelin in human endometrial epithelial cells

2007 ◽  
Vol 196 (2) ◽  
pp. 425-433 ◽  
Author(s):  
Jin-Wen Xu ◽  
Naomi Yasui ◽  
Katsumi Ikeda ◽  
Wei-Jun Pan ◽  
June Watanabe ◽  
...  
Keyword(s):  
Growth Factor ◽  
Protein Kinase ◽  
Epithelial Cells ◽  
Leukemia Inhibitory Factor ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β ◽  
Mitogen Activated Protein Kinase ◽  
Mitogen Activated Protein ◽  
Ishikawa Cells ◽  
Endometrial Epithelial Cells

Isoflavones have attracted much attention due to their association with health benefits; however, comprehensive understanding of the beneficial impacts of isoflavones on uterine biology at the molecular level remains unexplored. In the present study, our data showed that isoflavones aglycones AglyMax, genistein, and equol, but not daidzein, within the range of plasma concentration, displayed bioavailability in regulating the secretion of leukemia inhibitory factor (LIF) and transforming growth factor β (TGF-β) in Ishikawa cells, which was blocked by an estrogen receptor antagonist ICI 182 780, mitogen-activated protein kinase kinase (MEK)1/2 inhibitor PD98059, and p38 mitogen-activated protein kinase inhibitor SB203580. We also found that AglyMax and genistein increased in cyclic AMP release and the expression of glycodelin protein in Ishikawa cells assayed using western blot and immunochemical staining. The MEK1/2 inhibitor PD98059 and the protein kinase A inhibitor H89, but not SB203580, attenuated this glycoprotein expression. Moreover, isoflavone aglycones AglyMax stimulated LIF, and TGF-β secretion, and glycodelin expression in separate primary endometrial epithelial cells in the follicular phase or luteal phase from healthy subject donors. Overall, our findings suggest that isoflavones may alter the uterine expression of estrogen-responsive genes.

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