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.
Subject(s)
Aquaporin 5/metabolism , Cell Movement , Endometriosis/therapy , Stem Cell Transplantation , Stem Cells/metabolism , Adult , Animals , Aquaporin 5/genetics , Cell Proliferation , Cells, Cultured , Endometriosis/metabolism , Endometrium/cytology , Endometrium/metabolism , Estradiol/pharmacology , Female , Humans , Mice , Mice, SCID , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Response Elements , Second Messenger Systems , Stem Cells/drug effectsABSTRACT
Ezrin is a member of the ezrin-radixin-moesin (ERM) family of membrane-cytoskeletal linkage proteins. It is important for maintenance of cell shape, adhesion, migration and division. The overexpression of ezrin in some tumours is associated with increased cell migration that is mediated by the Rho/ROCK family of small GTPases. To investigate the role of ezrin in the migration of ectopic endometrial cells in endometriosis, we conducted real-time quantitative RT-PCR analysis of the eutopic and ectopic endometrium from women with endometriosis compared with those without the disease. RNAi, wound healing assays and western blot analysis of endometriotic cells were also included in this research. We found significantly higher levels of mRNA expression of ezrin (0.42 versus 0.27, P < 0.05), RhoA (0.99 versus 0.74, P < 0.05), RhoC (0.79 versus 0.43, P < 0.005) and ROCK1 (0.68 versus 0.38, P < 0.005) in the ectopic endometrial cells compared with the eutopic endometrial cells in endometriosis. Blocking ezrin with small-interfering RNA reduced the migration of ectopic endometrial cells with decreased expression of RhoA (42.68%), RhoC (58.42%) and ROCK1 (59.88%). Our results indicate that the over-expression of ezrin in endometriosis may play a significant role in the migration of endometrial cells of endometriosis, and the RhoC/Rock pathway may provide a promising treatment target.
