The Role Of FKBP51 In Decidualization Of Endometrial Stromal Cells And Its Pathogenic Mechanism

Infertility is becoming a health problem that covers all countries and regions around the world.According to statistics,about 10%-15%of couples of child-bearing age are affected.Nowadays,the increasing pressure on people's work and life,the continual delay in the childbearing age,coupled with changes in dietary habits and the deterioration of the ecological environment have made the causes of declining fertility more complicated.The in vitro fertilization and embryo transfer(IVF-ET)technology,as a new scientific and technological means,has brought fertility to many infertile couples with fertility claims,and thus has been applied worldwide.More extensive.At present,IVF-ET technology is generally considered to rely on ovulation induction,in vitro fertilization and embryo transfer,and can help couples who suffer from infertility due to difficulties in ovulation of the woman,fallopian tube factors,and weak sperm and oligospermia.Patients with failed or repeated abortions have limited applications.The average embryo implantation success rate of IVF-ET technology is only about 25%,and this data has not been significantly improved in the recent 30 years.The reduction of endometrial receptivity is considered to be responsible for about 2/3 of the implant failure events.During normal pregnancy,the development of the embryo and the development of endometrial receptivity are independent of each other,and they are coordinated to complete implantation on time.The establishment of endometrial receptivity depends on the joint action of ovarian sex hormones and a variety of related factors,so that the uterine endometrium undergoes re-plastic changes in morphology and biochemistry to become a state that allows embryo implantation.These remodeling changes are mainly characterized by changes in endometrial epithelium(EEC)secretion,endometrial stromal cells(ESC)differentiation into capsular-like cells,recruitment of lymphocytes,and cells.Reconstruction of extrastromal matrix and reconstruction of vascular proliferation.Among them,mesenchymal cells differentiate into capsular-like cells under the action of hormones,and a series of morphological and functional changes are called Decidualization.Disruption of the decidualization process is the main reason for the low endometrial receptivity.The mesenchymal cells that have undergone decidualization gradually change from a slender fibroblast-like shape to a large,round polygon,with enlarged nuclei,increased nucleoli complexity,increased cytoplasmic endocrine-like substances,and typical decidua-like changes;Syntheses of many capsular proteins,such as insulin-like growth factor-binding protein-1(IGFBP-1)and prolactin(PRL),have increased.The investigators used the primary endometrial stromal cells with Medroxyprogesterone acetate(MPA)and 8-Bromoadenosine-3',5'-cyclic monophosphate(8-bromoadenosine cAMP,8-Br-cAMP)Co-culture in vitro induces decidualization in vitro.The morphological changes in the decidualized mesenchymal cells and the spontaneously decidualized mesenchymal cells are very small,and the expression of related genes is basically the same,which can be used as a good model to study the decidualization process in vitro.The decidualization process is mainly regulated by ovarian sex hormones.In addition,there are a variety of signal transduction factors,cytokines,transcription factors,and certain immune cells involved.The regulatory mechanism is a complex and sophisticated regulatory network,involving a series of Biological events and multiple regulatory pathways.Protein Kinase B(AKT)is a key kinase in PI3K-AKT-mTOR,a classical cell signaling pathway.AKT can be phosphorylated under the upstream signal.Activated AKT can activate many kinds of enzymes and transcription factors in cells,and then play a role in the regulation of biological functions of cells.This signaling pathway is a classic intracellular signaling pathway and is involved in a variety of tissue cells in vivo.It is involved in many important life activities such as energy metabolism,cell proliferation and apoptosis,cell cycle regulation,and protein synthesis.The AKT pathway is also closely linked to the decidualization process.Phosphorylation of AKT was significantly reduced during decidualization in vitro and in vivo,and AKT phosphorylation was abnormally elevated in a variety of intimal diseases that affected endometrial receptivity.Studies have confirmed that the down-regulation of AKT phosphorylation level can directly lead to the increase of the downstream product FOXOIA protein,promote the expression of decidualization marker genes IGFBP-1 and PRL,induce cell cycle arrest,inhibit proliferation,promote differentiation,and promote ?.Membrane process development.However,existing studies have not clarified the upstream mechanism leading to a decrease in AKT phosphorylation during decidualization.The 51 kDs FK506 binding protein(FKBP51)belongs to the family of FKBPs proteins,which are named for their ability to bind to the immunosuppressant FK506.FKBP51 has a variety of biological functions and is involved in the physiological and pathological processes such as microtubule formation,tumor regulation,mental stress,autophagy,and regulation of steroid hormone receptor activity.Studies at home and abroad have found that in many cell lines,the expression level of FKBP51 can be rapidly up-regulated by progesterone and androgen.At the same time,FKBP51 can also bind to Hsp90 and participate in the formation of steroid receptor complexes to regulate receptor activity.FKBP51 can also be used as a bridging protein to associate AKT with PHLPP(PH domain and leucine rich repeat protein phosphatase),leading to the spatial proximity of the two,resulting in the specific dephosphorylation of AKT Ser473.The role of ovarian sex hormones and regulation of AKT signaling pathways are important regulatory mechanisms in the process of decidualization.The immunophilin FKBP51 is closely related to both,so we speculated that FKBP51 may play an important role in the decidualization process.ObjectivesIt is clear whether FKBP51 is an important regulatory protein in the decidualization process,and the mechanism of its regulation on the decidualization process is discussed.To study the interaction between FKBP51 expression and AKT signaling pathway during decidualization and its mechanism.Methods1 The expression of FKBP51 protein in normal endometrium on tissue microarray was detected by immunohistochemical technique.According to the different stages of menstrual cycle and the types of endometrial cells,the differences of FKBP51 expression levels among the groups were compared.2 The early pregnancy model was established in mice.The expression of FKBP51 was detected by immunohistochemistry during the early pregnancy.3 A total of 27 endometrial tissues from the Reproductive Hospital of Shandong University were collected.The average age of the collected objects was 31.4 years.The menstrual cycle was regular.The level of sex hormones was within the normal reference range and the nutritional status was normal.Primary endometrial cells were obtained according to the methods described in the literature,and the purity of the primary cells was evaluated by immunohistochemistry and Western blotting techniques.4 Primary cells were co-cultured with media containing MPA and 8-Br-cAMP for 4 days to construct a decidualization model in vitro.The morphological changes after decidualization of the cells were observed by phase contrast microscopy;the cell proliferation and cycle changes during decidualization were detected using the CCK8 kit and flow cytometry;and the decidua in cells were detected by Western blotting and RT-PCR.Changes in expression levels of related proteins.5 Use FKBP51 to down-regulate lentivirus to transfect endometrial stromal cells and observe its effect on decidualization of endometrial stromal cells.The efficiency of transfection was observed by fluorescence microscopy and flow cytometry.Western blotting was used to observe the effect of transfection and expression of intracellular protein.Observe changes in cell morphology,changes in proliferation and changes in cell cycle distribution.6 Using FKBP51-cDNA plasmid transiently transfected endometrial stromal cells FKBP51 down-regulated the stable cell line to observe its effect on decidualization of endometrial stromal cells.Western blotting was used to observe the effect of intracellular protein expression,and cell proliferation was observed.7 FKBP51 cDNA was used to transfect endometrial glandular epithelial cells.The effect of FKBP51 overexpression on glandular epithelial implantation ability was observed by embryo implantation model.8 AKT agonist SC79 was used to up-regulate AKT phosphorylation in mesenchymal cells,CCK8 kit and flow cytometry were used to detect changes in cell proliferation and cycle,Western blotting was used to detect decidualization related proteins and AKT pathway proteins in cells.Changes in expression levels,observed the effect of AKT phosphorylation upregulation on decidualization of endometrial stromal cells.9 Using AKT agonist SC79 to stimulate endometrial stromal cells with stable overexpression of FKBP51,and to observe its effect on decidualization of endometrial stromal cells.Western blotting was used to observe the effect of intracellular protein expression,and cell proliferation was observed.Results1 Increased expression of FKBP51 in human and mouse decidualizationThe results of human endometrial tissue microarray staining showed that the expression of FKBP51 in endometrial stromal cells was not statistically different among different age groups(P= 0.483),and the expression level of FKBP51 was significantly secreted during the menstrual cycle.Higher than the proliferative phase(P = 0.000);in the endometrial glandular epithelial cells,the expression level of FKBP51.was not statistically different in different age groups(P = 0.515),and its expression level was in the secretory phase of the menstrual cycle and There was also no significant difference in the proliferative phase(P = 0.521).In the early pregnancy model of mice,the expression level of FKBP51 was very low on the first day after fertilization;on the 5th day after fertilization,the decidualization process was basically completed on the 5th day after fertilization.At this time,the expression level of FKBP51 was significantly increased;From the 8th to the 11th day,the expression of FKBP51 in the mesenchymal cells gradually decreased as the decidualization reaction subsided.2 The increase of FKBP51 expression is synchronized with the progress of decidualization.Incubation of endometrial stromal cells with media containing MPA and 8-Br-cAMP can successfully induce decidualization in vitro.A typical decidualization of the mesenchymal cell morphology can be observed.At the same time,in the process of decidualization of mesenchymal cells,the proliferation of mesenchymal cells decreased,and the cell cycle arrested at G0/G1 and S phases.In the process of decidualization of stromal cells,the expression level of FKBP51 was gradually increased,which was consistent with the increase of the expression level of the decidualized marker protein.3 Progesterone induces expression of FKBP51 in ESCProgesterone induces an increase in the expression of FKBP51 in stromal cells,and this induction has a progesterone dose-dependent manner.After RU486 is added to block progesterone,the expression of FKBP51 is elevated and IGFBP-1 is synthesized.All were inhibited(P<0.05).4 FKBP51 down-regulation inhibits decidualizationTransfection of primary endometrial stromal cells with shRNA lentivirus downregulated FKBP51 expression levels was observed to be inhibited by decidualization:typical morphological changes in the decidualization of mesenchymal cells disappeared,and decidualization was associated with mesenchymal cell proliferation.The inhibition of the ability and cell cycle disappeared,and the expression of two classical marker proteins,IGFBP-1 and PRL,in the decidualization process was also inhibited.In the recovery experiment,transfection of FKBP51 cDNA corrected the inhibition of decidualization by shRNA.5 FKBP51 does not affect implantation functions of EECAfter transient transfection of FKBP51 cDNA into glandular epithelial cells,we examined the ability of glandular epithelial cells to adhere to the blastocyst via embryo implantation model.The results showed that there was no significant difference in the function of glandular epithelial cells transfected with FKBP51 cDNA and transfected with empty plasmid(P=0.083).This shows that FKBP51 does not affect the implantation function of glandular epithelial cells.6 Phosphorylation of AKT Ser473 decreases in decidualizationAfter MPA+cAMP stimulation,the phosphorylation level of AKT Ser473 was significantly decreased in endometrial stromal cells and glandular epithelial cells,while the phosphorylation level at Thr308 was not significantly changed and was associated with decidualization.The downstream protein FOXO1 A at Ser 473 showed a gradual increase with the development of decidualization(P<0.05).This shows that AKT is involved in decidualization by decreasing the phosphorylation level of Ser473.7 Increased phosphorylation of AKT Ser473 inhibits decidualizationAfter stimulating interstitial cells with different concentrations of SC79,decidualization was induced with MPA and 8-Br-cAMP.The results showed that under the action of SC79,the phosphorylation level of AKT Ser 473 in decidualization was significantly higher than that in the absence of SC79(P<0.05).In the SC79-treated group,IGFBP-1 and PRL,which are phasic membrane proteins,were significantly inhibited after MPA+cAMP induction compared with the control group(P<0.05).Increased AKT phosphorylation at the Ser473 site caused by SC79 inhibited decidualization of mesenchymal cells.8 FKBP51 inhibits phosphorylation of AKT Ser473The up-regulation and down-regulation of lentivirus transfected with FKBP51 into mesenchymal cells transfected with lentivirus demonstrated that the down-regulation of FKBP51 expression resulted in elevated phosphorylation of Ser 473 at the AKT site and downstream degradation of FOXO1 A protein;Up-regulation of levels significantly inhibited the phosphorylation of AKT S473 and led to an increase in downstream FOXO1A protein levels(P<0.05).9 FKBP51 regulates decidualization by regulating AKT phosphorylationInhibition of decidualization by SC79 can be corrected by overexpression of FKBP51.SC79 significantly increased the phosphorylation level of AKT Ser473 and inhibited the synthesis of capsularized proteins.In FKBP51 overexpressing cells,phosphorylation of AKT S473 was inhibited by overexpression of FKBP51,and phosphorylation of AKT by SC79 was observed.The level of elevation was no longer significant,and the inhibitory effect on the synthesis of decidualized marker proteins disappeared(P<0.05).SC79 could lead to the disappearance of the inhibitory effect of MPA+cAMP on the proliferation of mesenchymal cells,but overexpression of FKBP51 could make the inhibition of MPA+cAMP reappear.It was suggested that during the decidualization of the endometrium,the change of FKBP51 expression level regulates the decidualization process by regulating the phosphorylation level of AKT Ser473.ConclusionFKBP51 protein is an important protein that regulates decidualization.During decidualization,progesterone induces an increase in the expression of FKBP51 in the mesenchymal cells,thereby inhibiting the phosphorylation level of AKT Ser473,reducing the degradation of FOXOIA,and promoting the secretion of the IFPBP-1 and PRL proteins,which are the eptiform proteins.The proliferation of stromal cells promotes cell differentiation and promotes the progression of decidualization.