Dataset Information

Aquaporin 5 Plays a Role in Estrogen-Induced Ectopic Implantation of Endometrial Stromal Cells in Endometriosis.

ABSTRACT: Aquaporin 5 (AQP5) participates in the migration of endometrial cells. Elucidation of the molecular mechanisms associated with AQP5-mediated, migration of endometrial cells may contribute to a better understanding of endometriosis. Our objectives included identifying the estrogen-response element (ERE) in the promoter region of the AQP5 gene, and, investigating the effects of AQP5 on ectopic implantation of endometrial cells. Luciferase reporter assays and electrophoretic mobility shift assay (EMSA) identified the ERE-like motif in the promoter region of the AQP5 gene. After blocking and up-regulating estradiol (E2) levels, we analysed the expression of AQP5 in endometrial stromal (ES) cells. After blocking E2 /or phosphatidylinositol 3 kinase(PI3K), we analysed the role of AQP5 in signaling pathways. We constructed an AQP5, shRNA, lentiviral vector to knock out the AQP5 gene in ES cells. After knock-out of the AQP5 gene, we studied the role of AQP5 in cell invasion, proliferation, and the formation of ectopic endometrial implants in female mice. We identified an estrogen-response element in the promoter region of the AQP5 gene. Estradiol (E2) increased AQP5 expression in a dose-dependent fashion, that was blocked by ICI182,780(an estrogen receptor inhibitor). E2 activated PI3K /protein kinase B(AKT) pathway (PI3K/AKT), that, in turn, increased AQP5 expression. LY294002(PI3K inhibitor) attenuated estrogen-enhanced, AQP5 expression. Knock-out of the AQP5 gene with AQP5 shRNA lentiviral vector significantly inhibited E2-enhanced invasion, proliferation of ES cells and formation of ectopic implants. Estrogen induces AQP5 expression by activating ERE in the promoter region of the AQP5gene, activates the PI3K/AKT pathway, and, promotes endometrial cell invasion and proliferation. These results provide new insights into some of the mechanisms that may underpin the development of deposits of ectopic endometrium.

SUBMITTER: Jiang XX

PROVIDER: S-EPMC4682985 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Aquaporin 5 Plays a Role in Estrogen-Induced Ectopic Implantation of Endometrial Stromal Cells in Endometriosis.

Jiang Xiu Xiu XX Fei Xiang Wei XW Zhao Li L Ye Xiao Lei XL Xin Liao Bin LB Qu Yang Y Xu Kai Hong KH Wu Rui Jin RJ Lin Jun J

PloS one 20151217 12

Aquaporin 5 (AQP5) participates in the migration of endometrial cells. Elucidation of the molecular mechanisms associated with AQP5-mediated, migration of endometrial cells may contribute to a better understanding of endometriosis. Our objectives included identifying the estrogen-response element (ERE) in the promoter region of the AQP5 gene, and, investigating the effects of AQP5 on ectopic implantation of endometrial cells. Luciferase reporter assays and electrophoretic mobility shift assay (E ...[more]

PMID: 26679484

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Project description:BackgroundAs a dual-function metabolite, succinate has emerged in cell function and plays a key signaling role in linking mitochondrial function to other cellular functions. Succinate accumulation in the cytoplasm is commonly associated with hypoxia in the microenvironment and immune cell activation. Extracellular succinate released into the microenvironment is considered an inflammatory alarm that can be sensed by its membrane receptor SUCNR1, which boosts proinflammatory responses and acts akin to classical hormones and cytokines. Succinate plays an important role in the development of inflammatory diseases. Whether succinate facilitates the progression of endometriosis (EMs), characterized by chronic inflammation and peritoneal adhesion, is worth exploring.ObjectiveWe mimicked the ectopic milieu in vitro and in vivo to evaluate the main source and potential role of succinate in endometriosis. We assessed the molecular and functional effects of succinate on macrophages and peritoneal mesothelial cells in peritoneal cavity. The effect of succinate/SUCNR1 signaling on ectopic endometrial stromal cells (ESCs) was further explored in this study.MethodsIn this study, we used targeted organic acid metabolomics analysis and in vitro assays to assess the potential accumulation of succinate in the peritoneal fluid of EMs patients. We examined its correlation with disease severity, Visual Analogue Scale, and the Endometriosis Fertility Index. Flow cytometry, enzyme linked immunosorbent assay, western blot assay, quantitative real-time PCR, and other molecular biology techniques were used to explore the potential mechanisms.ResultsBy mimicking the ectopic milieu, we constructed an in vitro co-culture system and found that M1 polarized macrophages and that the peritoneal mesothelial cell line (HMrSV5) mainly released succinate into their microenvironment and activated the succinate receptor (SUCNR1) signal, which further polarized the macrophages and significantly enhanced the invasive survival of ESCs, and the adhesion to the peritoneum. We further investigated the pathological effects of extracellular succinate in vivo using a xenograft mouse models of endometriosis.ConclusionsSuccinate-SUCNR1 signaling facilitates the creation of inflammatory cells and plays a vital role in EMs progression and peritoneal adhesion. Our work on the molecular mechanisms underlying succinate accumulation and function will help elucidate the phenotypic mysteries of pain and infertility in EMs. Video Abstract.

Project description:Endometriosis is an estrogen-dependent disease. Studies have shown that miR-139-5p is significantly upregulated in endometriosis lesions, but its specific role and molecule mechanism in endometriosis has not yet been reported. The malignant biological behavior of ectopic endometrial stromal cells (ESCs) is similar to that of malignant cancer cells. BBC3 (BCL2 binding component 3) is a known apoptosis inducer and it serves key roles in the regulation of cell behavior. However, the role of BBC3 in ectopic ESCs remains unknown. The present study aimed to investigate the role of miR-139-5p in the progression of endometriosis and to determine its underlying molecular mechanism of action. Ectopic, non-ectopic and normal endometrial stromal cells (ESCs) were extracted from endometrial samples, and reverse transcription-quantitative PCR was performed to determine microRNA (miR)-139-5p and Bcl-2 binding component 3 (BBC3) mRNA expression levels in endometrial tissue samples and ESCs. The target gene of miR-139-5p was predicted using TargetScan software and verified using a dual luciferase reporter assay. Western blotting was performed to determine BBC3 protein expression levels. Flow cytometry analysis, and MTT and Transwell assays were performed to assess cell apoptosis, viability, and migration and invasion of the cells transfected with inhibitor control, miR-139-5p inhibitor, miR-139-5p inhibitor + control-small interfering (si)RNA or miR-139-5p inhibitor + BBC3-siRNA, respectively. The results demonstrated that miR-139-5p expression levels were upregulated in ectopic endometrial samples and ESCs compared with the respective control groups. Furthermore, it was verified that BBC3 was a direct target of miR-139-5p, and both BBC3 mRNA and protein expression levels were downregulated in ectopic endometrial samples and ESCs. Both transfection with the miR-139-5p inhibitor and BBC3-small interfering (si)RNA markedly downregulated miR-139-5p and BBC3 expression levels in ectopic ESCs, respectively. miR-139-5p inhibitor-induced upregulated BBC3 expression was reversed following transfection with BBC3-siRNA. Furthermore, the miR-139-5p inhibitor significantly decreased viability, migration and invasion, while inducing apoptosis in ectopic ESCs compared with the inhibitor control group. Notably, the aforementioned effects were reversed by knocking down BBC3 expression. In conclusion, the results of the present study suggested that miR-139-5p may play a key role in the progression of endometriosis by regulating the viability of ESCs and directly targeting BBC3.

Dataset Information

Aquaporin 5 Plays a Role in Estrogen-Induced Ectopic Implantation of Endometrial Stromal Cells in Endometriosis.

Publications

Aquaporin 5 Plays a Role in Estrogen-Induced Ectopic Implantation of Endometrial Stromal Cells in Endometriosis.

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