The Role Of PI3K/PTEN/AKT/Survivin Pathway And IκB/NFκB Pathway In Endometriosis

Endometriosis, a common disease in women of reproductive age, is characterized by the presence of functional endometrium-like glands and stromal tissues in sites outside of the uterine cavity. Endometriosis is an estrogen-dependent chronic disease with chronic pelvic pain and infertility are the main clinical features. Many studies have been reported about this disease; however, the pathogenesis of it is still unclear. In the present research, via tissular and cellular study, we investigated the presence and role of PI3K/PTEN/AKT/Survivin signaling and IκB/NFκB signaling in endometriosis.Object①To determine the presences and activities of proliferative signaling pathways:PI3K/PTEN/AKT pathway and IκB/NFκB pathway in endometriotic ectopic and eutopic endometria;②To decide the effect of 17βestrodial on the proliferation of endometriotic epithelial cells, and the role of these pathways in this effect;③To investigate the role of these pathways in the stromal-epithelial cell cross-talk in endometriosis.Methods①Normal endometrium and eutopic/ectopic endometrium from patients with endometriosis were obtained during operation. The tissues were divided into two parts:one for formalin fixation and immunohistochemistry, one for primary cell isolation and culture;②Determining the expression and activity of PI3K/PTEN/AKT pathway and IκB/NFκB pathway in normal and endometriotic eutopic endometria using immunohistochemistry analysis;③Cell isolation and primary culture:sterile normal and endometriotic endometria were obtained during operation. Tissues were treated with 0.25% collagenase IA. Normal and endometriotic eutopic and ectopic stromal cells and epithelial cells were isolated and cultured in vitro. Stromal cells and epithelial cells were characterized by immunocytochemistry analysis using vimentin and keratin antibodies;④Determining AKT and ERKl/2 phosphorylation to decide the activities of PI3K/PTEN/AKT pathway and MAPKs/ERK1/2 pathway in normal and endometriotic epithelial cells using Western Blot analysis; determining the DNA-binding activity of NFκB to decide the activity of IκB/NFκB pathway using electrophoretic mobility shift assay (EMSA);⑤Establishing stromal cell-epithelial cell co-culture systems:mixing stromal cells and epithelial cells in a ratio of 1:1 directly to establish contact-co-culture system; using conditioned medium, cell lysates, and Millipore transwell system to establish non-contact-co-culture system; using contact and non-contact co-culture systems to establish normal stromal cell-epithelial cells co-culture system, endometriotic stromal cell-epithelial cell co-culture system, and normal stromal cell-endometriotic epithelial cell co-culture system, endometriotic stromal cell-normal epithelial cell co-culture system;⑥Detecting the expression and location of Survivin, a member of inhibitor of apoptosis (IAP) family, in normal and endometriotic eutopic endometria using immunohistochemistry analysis; determining the expression and subcellular location of Survivin in primarily cultured normal and endometriotic eutopic epithelial cells using immunocytochemistry analysis and Western Blot analysis;⑦Determining the expression and secretion of matrix metalloproteinases 2/9 (MMP2/9) and tissue inhibitors of metalloproteinases 1/2 (TIMP1/2) using Western Blot analysis; detecting the activity of MMP2/9 in cell culture supernatants using gelatinase zymography;⑧Deciding the proliferation of all kinds of cells under different treatments using MTT test.Results①Significantly higher AKT phosphorylation and lower PTEN expression were present in endometriotic eutopic endometria;③Significantly higher AKT and ERK1/2 phosphorylation, lower PTEN expression, and higher NFκB DNA-binding activity were present in primarily cultured endometriotic eutopic and ectopic epithelial cells; higher MMP2/9 expression and secretion were also present in primarily cultured endometriotic eutopic and ectopic epithelial cells, while only weak MMP2/9 expression and secretion were present in normal and endometriotic stromal cells;③17βestrodial significantly enhanced AKT and ERK phosphorylation, NFκB DNA-biding activity in normal and endometriotic epithelial cells; also,17βestrodial decreased PTEN protein and mRNA expression and promoted cell proliferation; specific PI3K/PTEN/AKT pathway inhibitor LY294002, specific MAPKs/ERK1/2 pathway inhibitor U0126, and specific IκB/NFκB inhibitor PDTC significantly inhibited the background and estrogen-stimulated AKT and ERK1/2 phosphorylation, NFκB DNA-binding activity in normal and endometriotic epithelial cells; and also decreased the background and estrogen-induced cell proliferation; meanwhile, PDTC significantly abolished the PTEN-decrease by 17βestrodial while LY294002 and U0126 could not; also, LY294002 significantly inhibited the background and estrogen-induced NFκB DNA-binding activity; in converse, IκB/NFκB pathway inhibitor PDTC significantly inhibited the background and estrogen-induced AKT phosphorylation; MAPKs/ERK1/2 pathway inhibitor U0126 could only weakly inhibit estrogen-induced AKT phosphorylation and NFκB DNA-binding activity; also, LY294002 and PDTC could only inhibit estrogen-induced ERK1/2 phosphorylation;④The expression of Survivin existed in normal and endometriotic epithelial cells, and endometriotic epithelial cells showed higher Survivin expression compared with normal epithelial cells; PI3K/PTEN/AKT pathway inhibitor LY294002 could significantly inhibit Survivin protein expression; in the co-culture system, conditioned medium by normal and endometriotic eutopic stromal cells could inhibit AKT phosphorylation, Survivin expression, and cell proliferation in normal and endometriotic eutopic epithelial cells, and progesterone could enhance this inhibition; conditioned medium by endometriotic ectopic stromal cells could not inhibit AKT phosphorylation, Survivin expression, and cell proliferation in normal or endometriotic ectopic epithelial cells; in vitro, the proliferation of epithelial cells from endometriotic eutopic endometria＞ectopic endometria＞normal endometria;⑤In the co-culture system, conditioned medium and cell lysates from endometriotic eutopic and ectopic epithelial cells could induce higher MMP2/9 expression and secretion in normal stromal cells; and when the activity of IκB/NFκB pathway in epithelial cells was inhibited using PDTC, the MMP2/9 expression and secretion in stromal cells could still be induced; however, when the activity of IκB/NFκB pathway in stromal cells was inhibited using PDTC, the MMP2/9 expression and secretion could not be induced.Conclusion ①Significantly higher activities of PI3K/PTEN/AKT pathway, IκB/NFκB pathway, and MAPKs/ERK1/2 pathway were present in endometriotic eutopic and ectopic epithelial cells;②Loss of PTEN protein and mRNA expression existed in endometriotic eutopic and ectopic epithelial cells; in vitro,17βestrodial could decrease PTEN protein and mRNA expression in normal and endometriotic eutopic and ectopic epithelial cells via IκB/NFκB pathway;③In vitro,170 estrodial could promote cells proliferation in normal and endometriotic eutopic and ectopic epithelial cells through PI3K/PTEN/AKT pathway, IκB/NFκB pathway, and MAPKs/ERK1/2 pathway;④In normal and endometriotic epithelial cells, PI3K/PTEN/AKTsignaling pathway and IκB/NFκB signaling pathway could be positively regulated by each other, and both of the pathways were upstream regulator of MAPKs/ERKl/2 signaling pathway;⑤Proliferative positive feedback loop:higher estrogen level→higher PI3K/PTEN/AKT pathway activity→higher IκB/NFκB pathway activity→decreased PTEN expression→higher PI3K/PTEN/AKT pathway activity, was present in endometriotic eutopic and ectopic epithelial cells;⑥Higher Survivin expression was present in endometriotic epithelial cells;⑦In normal and endometriotic ectopic endometria, stromal cells could regulated cell proliferation in epithelial cells through PI3K/PTEN/AKT/Survivin pathway, which was lost in endometriotic ectopic endometria;⑧Higher MMP2/9 expression and secretion were present in endmetriotic epithelial cells;⑨Endometriotic eutopic and ectopic epithelial cells induced MMP2/9 expression and secretion in normal stromal cells via IκB/NFκB pathway. In the present studies, we described the role of PI3K/PTEN/AKT pathway, IκB/NFκB pathway, and MAPKs/ERKl/2 pathway in endometriosis, which will bring new sight on studying the pathogenesis of this disease, will provide new marker for disease diagnosis and evaluation, and will provide new therapy target for the disease.