The Role Of AQP2 In The Pathogenesis Of Endometriosis

Endometriosis is a common gynecologic disease affecting approximately 3-10% of women of reproductive age,5-20%women with pelvic pain, and somewhere 20-40% of infertile women. It is manifested by symptoms such as dysmenorrhea, dyspareunia, pelvic pain, and infertility. Although endometriosis is a benign disease defined by the presence of ectopic endometrial glands and stroma, it is prone to progression and recurrence, exhibiting some characteristics of malignancy, such as cellular adhesion, invasion, metastasis, angiogenesis, and so on. It is well known that endometriosis is due to the tubal reflux of menstrual debris into the peritoneal cavity, then the endometrial tissue attaches, implants and proliferates at ectopic site of the peritoneum and the pelvic organs with angiogenesis. However, retrograde menstruation is a common phenomenon so it is still unknown why endometriosis affects no more than about 10%of the female population in their reproductive years. Accumulated evidences suggest that the intrinsic defect in endometriosis may locate in the eutopic endometrium. Some studies demonstrated that abnormal expression of cytokines, angiogenic factors as well as specific cancer-related genes occur in eutopic endometrium with endometriosis, which is believed to contribute to the establishment and maintenance of this diseaseThe aquaporins (AQPs) are a family of small integral membranes proteins distributing widely in various tissues throughout the body and play a major role in transcellular and transepithelial water movement. Since the initial discovery of AQP1, many other members of AQPs have been discovered in human being. Our previous studies showed that AQP1 and AQP2 were expressed in human endometrium, with AQP2 expressed mainly in luminal and glandular epithelial cells of endometrium, and AQP1 located in the endothelia of the capillaries and small blood vessels. Furthermore, we had found that cyclic expression of endometrial AQP1/AQP2 associated with steroid hormone levels might be essential to normal endometrial function. Since AQP1 is important to the angiogenesis and structurally modifications of microvessels, it might be involved in the pathogenesis of idiopathic menhorrhagia. And decreased AQP1/AQP2 expression in endometrial vessels or epithelium may be involved in the occurrence of anovulatory uterine bleeding. In addition, the overexpression of AQP1 in the microvessels and small vessels of endometroid adenocarcinoma is related to poor histological grade, surgical-pathologic stage, myometrial invasion, and extrauterine metastasis. Recently, several other studies have reported that AQPs are abnormally expressed in a variety of human tumors and correlated with metastasis and tumor grade. It is even proposed that the expression of AQPs in tumors has diagnostic and prognostic values. All these findings imply that AQP-dependent cell migration may be a general phenomenon, independent of AQP and cell type.In the present study, we verified the regulation of AQP2 by E2 in endometrium cell. To explore whether AQP2 is regulated directly by E2 through the ligand-dependent transcriptional factors, we examined the existence of estrogen response elements (EREs) in the promoter of AQP2. In addition, we investigated the role of AQP2 on the cell proliferation, cell adhesion, cell migration and cell invasion in vitro. Furthermore we explored the mechanism of AQP2 on the cell adhesion, cell migration and cell invasion. We hypothesized that E2 would increase the expression of AQP2, and the up-regulation of AQP2 would increase the cell adhesion, cell migration and cell invasion through the re-organization of cytoskeleton. we confirmed the hypothesis by endometriosis, which is an E2-dependent complex disease defined by the presence of ectopic endometrial glands and stroma. Part One:The expression ofAQP2 in the eutopic endometrium of endometriosisObjective:To confirm whether AQP2 was abnormally expressed in eutopic endometrium of endometriosis patients compared with normal women.Methods:qRT-PCR and Western blot assay were used to analyse the expression of AQP2 in human endometrium derived from 81 patients(44 cases, endometriosis patients and 37 cases, normal women). Immunohistochemical staining was used to locate aqp2 in human endometrium.Results:1. qRT-PCR and Western blotting analysis showed that the expression of AQP2 in eutopic endometrium from endometriosis patients was significantly higher than that from normal women.2. AQP2 Immunohistochemical staining was present in both luminal and glandular epithelial cells in human endometrium.Conclusion:the expression of AQP2 in eutopic endometrium from endometriosis patients was significantly higher than that that from normal women.Part Two:17-βestrodione up-regulated the expression of AQP2 through the putative EREObjective:To investigate the role of E2 on expression of AQP2 and its mechanism.Methods:1. In Vitro, endometrial cells were treated by E2 and/or estrogen receptor antangonist ICI 182780; qRT-PCR and Western blot assay were applied to analyse the expression of AQP2.2. Plasmids encoding AQP2 promoters were constructed, and then the plasmids were transfected to the human endometrial cells, luciferase activities in cell lysates were measured.Results:1.Real-time PCR and western blot examination showed that E2 up-regulated the expression of AQP2 in a dose-dependent manner, and the increasing effects of E2 would be blocked by ICI182780.2. the results from the luciferase reporter systems, there maybe exist a functional ERE-like motif in the promoter fragment 2 region of AQP2 which mediated the estrogen-dependent AQP2 expression.Conclusion:E2 regulates AQP2 expression via ERE in AQP2 promoter.Part Three:E2 increased the cell migration, invasion and adhesion in human endometrial cells by up-regulation of AQP2Objective:To investigate the role of AQP2 in the cell proliferation, cell adhesion, cell migration and cell invasion.Methods:To examine the role of AQP2 in cell proliferation, cell adhesion, cell migration and cell invasion, RNAi experiments were performed in this study,Results:AQP2-specific siRNAs significantly reduced the cell migration, invasion, adhesion but not the cell proliferation.Conclusion:E2 enhanced the cell migration, invasion, adhesion of human endometrial cells through the up-regulation of AQP2. Part Four:AQP2 knock-down altered the morphological features of endometrium cell by decreasing the expression of annexin 2 and F-actinObjective:To explore the relationship between the expression of Annexin2, F-actin and the cell migration after up-regulation or down-regulation of AQP2.Methods:The morphogram of the cells were examined by Scanning electron microscopy; the confocal image was applied to obsere the arrangement of F-actin; western blotting were used to explore the expression of F-actin and annexin-2.Results:1.E2 increased the expression levels of F-actin by nearly 1.3-fold and annexin-2 by 1.5-fold in human endometrial cells. AQP2 knock-down significantly reduced the expression levels of F-actin and annexin-2 human endometrial cells pretreated with E2.2.The confocal image showed that F-actin arranged from the nucleus to the periphery of the cytoplasm in a radial pattern in control cell. When cells were treated with E2, F-actin aligned in a bunchy way, and extended into the lamillipodia.3. Scanning electron microscopic analysis showed that human EC cells had long and slender lamellipodia in cellular membrane. Compared with the control, treatment of the cells with E2 induced more and larger lamellipodia in the membrane. In AQP2 knock-down EC cells, lamellipodia was reduced and displaced with thin and short pseudopod-like structure.Conclusion:Upregulation of AQP2 by E2 increases the cell migration, invasion and adhesion through increased annexin-2 and F-actin which is respond for the actin remolding and rearrangement.