The Involvement Of MRP4 In Signal Transduction During Embryo Implantation

Background:The implantation of the blastocyst into the uterus is one of the most critical steps in human reproduction.Nearly 75%of pregnancy losses in humans are believed to be due to implantation failure.Despite the advances in IVF and embryo transfer for assisted reproduction technology(ART),the pregnancy rate of ART remains low,which is largely due to implantation failure.In addition,defective implantation can affect the later course of pregnancy resulting in poor outcomes or diseases.Successful implantation requires the coordination of embryo development into an activated blastocyst and uterine transition into the receptive state.As the maternal component,the uterus comprises mainly epithelial and stromal tissues that constitute the endometrium,and the outlying muscles known as the myometrium.The endometrium plays essential roles in embryo implantation and is only receptive for the blastocyst to implant for a limited period of time,the so-called 'receptive window'.Although endometrial receptivity is not yet fully understood,it is well documented that a series of physiological events along with morphological changes and an altered gene expression profile in the endometrium occur during the preimplantation period,which are believed to ensure the success of embryo implantation.For instance,upon attachment of the blastocyst to the endometrial epithelial cells,the underneath stromal cells begin a differentiation process into large,round decidua cells,the so called decidualization.Decidua cells provide nutrition for early embryo development and is essential to placentation and fetal development during the later stage of gestation.Aberrant decidualization can result in severe outcomes such as preeclampsia,preterm labor and even fetal death.Although a variety of signaling molecules associated with implantation have been identified,including leukaemia inhibitory factor(Lif),peroxisome proliferator-activated receptor gamma(PPARy),homeboxA 10(HoxA 10),insulin-like growth factor 2,(IgF2)and Claudin 4,the mechanisms underlying implantation remain largely unclear.Notably,the Wnt/beta-catenin pathway,which is known for its multiple roles in various physiological and pathological events such as development and cancer,has been recently demonstrated to be activated at embryo implantation.However,the Wnt/beta-catenin pathway is activated at embryo implantation remains elusive.Among the implantation associated signaling pathways,prostaglandins(PGs)related pathway is the most well recognized one to be essential for embryo implantation.Generally,PGs is one kind of unsaturated fatty acids with biological activity involved in the physiological process,such as inflammation,immune response,muscle contraction,cell proliferation and differentiation.They are synthesizes by cyclooxygenases(COXs)from phospholipase A(PLA)-derived arachidonic acid.Deletion of Pla-2 and Cox-2 genes results in multiple problems in the female reproductive system leading to female infertility.In humans,women who are exposed to those COX-inhibiting non-steriod anti-inflammatory drugs(NSAID)during pregnancy have been reported to have 80%increased risk of miscarriage as compared to those without NSAID exposure at pregnancy.Moreover,reduced PGs synthesis was detected in women with repeated ART failure trails.Among the PGs,COX-2 derived PGE2,plays a key role in the process of implantation,especially decidualization.The activation of PGE2 could induce a series of decidualization related genes and molecules expression through the activation of prostaglandin receptor(EP)in the stromal cell membrane and increased the intracellular cAMP levels.Although PGE2 is believed to be abundantly produced by the endometrial epithelial cells upon blastocyst attachment,how such a signal is initiated had not been clear until our recent discovery in the epithelial sodium channel(ENaC)mediated pathway at implantation.ENaC,also known as amiloride-sensitive sodium channel(ASSC)is well known for its role in epithelial fluid absorption especially in the lung and kidney.ENaC is also expressed in the endometrium and highly unregulated at embryo implantation.We have demonstrated that ENaC in the endometrial epithelial cells are activated by signals from the embryo,which results in membrane depolarization,Ca2+ influx,CREB activation,COX-2 upregulation and eventually the increased production and release of PGE2 from the epithelial cells to the stromal cells.The importance of ENaC-mediated pathway at implantation is also evident by the finding that women with IVF trail failure showed significantly lower expression level of ENaC in the endometrium.Since PGs have been identified to be inefficiently diffused across cell membranes,a question remains that how PGE2 is released or transported by the endometrial epithelial cells to the stromal cells at implantation.A couple of PGs transporter have been identified,among which multidrug resistance associated protein 4(MRP4),one of the MRP/ABCC subfamily of ATP-binding cassette transporters,is believed to one of the most important PGs transporters.Generally,MRP4 is a transmembrane protein and is capable of pumping a wide variety of endogenous and xenobiotic organic anionic compounds out of the cell.The expression of MRP4 is widely detected in the whole body.MRP4 can be expressed either in the apical membrane or basolateral membrane of the polarised cell surface depending on the cell type,indicating that MRP4 has versatile transport function.In addition to the role of MRP4 in the body distribution and renal excretion of a wide variety of antiviral,cytostatic,antibiotic and cardiovascular drugs,MRP4 has the remarkable ability to transport molecules involved in cellular signaling.These molecules include cyclic nucleotides,eicosanoids,urate and conjugated steroids.The unique structure,regulation and dual localization of MRP4 in polarised cells could be connected with a key function in cellular protection and extracellular signaling pathways.Among the PGs,PGE2 is one of the high affinity transporter substrates of MRP4.MRP4 has been reported that may also be involved in the regulation of PGs synthesis,in addition to transport PGE2.Moreover,knockdown of MRP4 could inhibit the transcriptional level of COX-2 and decrease the expression of prostaglandin receptors,EP1 and EP2 in esophageal carcinoma cells.Although the molecular mechanism has not been clarified,these results suggest that MRP4 not only mediated the signal transduction through transportation of PGE2 between cells,but also has the potential capacity of regulation of intracellular signaling pathways directly.Given such capacity of MRP4 in both transporting PGE2 and regulating signaling pathways,potential roles of MRP4 in embryo implantation are suggested.However,whether and how MRP4 plays a role in implantation remains unknown.Based on our previous findings in ENaC-mediated and PGs-involved signaling transduction at embryo implantation and given the reported capacities of MRP4 in transporting PGE2 and regulating signaling pathways,we hypothesized that MRP4 have play an essential role at embryo implantation.Therefore,the present study was carried out investigate the involvement of MRP4 in embryo implantation,especially in ENaC-mediated and PGs-involved signaling transduction.Methods:We used human endometrial surface epithelial(HES)cells as in vitro model to study the MRP4 involvement in signaling transduction.The HES cells were cultured in DMEM supplemented with 10%FBS(v/v)in 5%CO2 incubators at 37?.We used siRNA-based gene-silencing assay to knockdown the expression of MRP4 in HES cells.The siRNA_MRP4 and siRNA_NC were transfected with Lipofectamine 2000 transfection reagent into HES cells.To measure the release of PGE2 from HES cell,cell-free supernatant with FBS-free DMEM of HES cells was collected and PGE2 content was measured using an PGE2 ELISA kit.RT-PCR and real-time PCR were performed to check the mRNA level of MRP4,COX-2 and implantation related genes.Western blot analysis were performed to check the protein level of MRP4,p-CREB,COX-2,beta-catenin,active beta-catenin,TCF1 and tubulin in the HES cells or uterus tissue.Moreover,we used an in vivo mouse model for the study on embryo implantation.Also,intrauterine siRNA injection was used to knockdown the expression of MRP4 in vivo.Intrauterine injection with drug and/or siRNA was performed to disturb the process of embryo implantation.The uterus was taken out for counting the implanted embryo numbers and further other experimental analyses.Immunofluorescent analysis detected the localization of MRP4 in the mice uterus.Results:Using RT PCR and real-time PCR analysis,we detected that the mRNA of MRP4 and COX-2 in HES cells was increased by the treatment with Trypsin(20 ug/mL),an activator of ENaCs which was abolished by pretreatment with amiloride(10 ?M,),suggesting the regulation of MRP4 transcription by ENaC activation.In line with this ELISA detected an nearly ten-fold increase in PGE2 release from HES cells in response to the treatment with trypsin(20 ug/mL),which was significantly reduced by pretreatment with either MK-571(10 ?M)or 24 h after the transfection with siRNA_MRP4(100 nM).Besices,western blot analysis showed COX-2 and phosphorylated CREB(p-CREB)were both significantly increased in HES cells after treatment with trypsin for 15 min,which was attenuated in the cells pre-transfected with siRNA_MRP4 as compared to cells transfected with siRNA_NC.In addition,p-CREB and COX-2 expression levels were also reduced in siRNA_MRP4 transfected cells at basal condition in absence of trypsin.It is suggested that not only contributing to PGE2 transport,MRP4 might also be involved in regulation of PGE2 synthesis pathways.Moreover,real-time PCR analysis revealed that expression of three implantation markers,Claudin-4,Lif and PPARy,were also remarkably reduced when MRP4 was knocked down in HES cells as compared to the control cells transfected with siRNA_NC.Next,we tested the involvement of MRP4 in embryo implantation in vivo by using a mouse implantation model.MRP4 protein expression levels in the mouse uteri during the peri-implantation period(days 2-6 after mating)were determined by western blot analysis,which showed that the expression of MRP4 in the uterus gradually increased with the peak reached in the midnight of day 4(embryo attachment),which persisted to be high on day 5(initial decidualization),and went down afterwards to a low level on day 6.Also,immunofluorescence staining results showed that MRP4 was abundantly expressed in the basolateral membrane of the endometrial epithelial cells,where epithelial and stromal cells are interfaced.These results suggested that MRP4 might play an importantly role in embryo implantation,particular the epithelial-stromal crosstalk.To test whether MRP4 is indeed required for embryo implantation.We intrauterinely injected MK-571,a reported inhibitor of MRP4,in pregnant mice on day 3 before embryo implanation occurs,which showed that MK-571(0.5 mM,1 mM and 5 mM)significantly lowered the implantation rate when compared to the vehicle-treated control uterine horns.To further confirm the involvement of MRP4 in implantation,we used in vivo knockdown assay.SiRNA_MRP4 was injected into the mouse uteri on day 4 and siRNA_NC was used as negative control.We first tested the in vivo knock-down efficiency.By western blot analysis,the MRP4 expression in mice uteri on day 5 were significantly decreased in the siRNA_MRP4 transfected uterine horns compared with that in the control siRNA_NC.Immunofluorescence staining also showed MRP4 was decreased in the siRNA_MRP4 transfected uterine horns compared with that in the control siRNA_NC.Moreover,intrauterine injection with siRNA_MRP4(50 nM and 100 nM)reduced significantly in the number of implanted embryos in the siRNA_MRP4 transfected uterine horns compared with that in the control treated with siRNA_NC,indicating the requirement of MRP4 for embryo implantation.We next to determine the expression of four implantation related markers,Lif,HoxA10,PPAR? and LgF2 in mice uterine after knocked down MRP4,which showed that these four implantation related genes in mice uteri on day 4 were substantially reduced in the siRNA_MRP4 transfected uterine horns compared with that in the control siRNA_NC.Having established the effect of MRP4 on embryo implantation,we next test the possible involvement of MRP4 in regulation of Wnt/beta-catenin pathway at implantation.First of all,real-time PCR analysis revealed that the mRNA level of the Wnt/beta-catenin upstream receptors,Fzd family(Fzdl,Fzd2,Fzd4,Fzd5,Fzd6,Fzd7 and Fzd10)and LRP5 protein,were largely reduced when MRP4 was knocked down in HES cells as compared to the siRNA_NC treated cells.Next,western blot analysis showed that beta-catenin,active beta-catenin and TCF1 were significantly increased in HES cells after treatment with Wnt3a(100 ng/mL)or CHIR-99021(10?M)for 24 h,suggesting an activation of Wnt/beta-catenin pathway.In cells pre-transfected with siRNA_MRP4,the activation of Wnt/beta-catenin by both Wnt3a and CHIR-99021 was significantly attenuated.Also,in absence of these Wnt/beta-catenin pathway activator,the level of beta-catenin,active beta-catenin and TCF1 was also decreased in HES/siRNA_MRP4 transfected cells compared to the control cells,suggesting that MRP4 is also involved in the baseline activation of Wnt/beta-catenin.Given these results,we thought that activation of Wnt/beta-catenin by exogenous activators would be able to reverse the effect caused by MRP4 knock-down,if MRP4 plays a role in implantation through its regulation of the Wnt/beta-catenin.To test this,we performed intrauterine injection with CHIR-99021(100 ?M,10 ?M and 1 ?M),which showed that CHIR-99021 significantly induced a drastically increase in the number of implanted embryos in the siRNA_MRP4 transfected uterine horns compared with that in the siRNA_MRP4 uterine horns alone,indicating the CHIR-99021 could rescue the decrease the numbers of implanted embryos induced by knockdown of MRP4.This important results demonstrated the requirement of MRP4 for embryo implantation by regulating Wnt/beta-catenin pathway.Conclusion:The present study investigated the involvement of MRP4,a traditionally believed important drug target for cancer treatment,in embryo implantation.Our results have shown that MRP4 is highly expressed at the basolateral membrane of endometrial epithelial cells where implantation and decidualization occurs in mice.Also,the expression of MRP4 in endometrial epithelial cells in vitro is subjected to ENaC activation,the previously identified essential element for implantation.Moreover,MRP4 contributes to PGE2 transportation from the endometrial epithelial cells and ENaC regulation of CREB/COX-2 leading to PGE2 synthesis.Most importantly,the present study has identified that,in addition to contributing to PGE2 transport/synthesis,MRP4 also has the capacity in regulation of Wnt/beta-catenin,an important signaling pathway at implantation.Consistently,inhibition or knockdown of MRP4 resulted in significant decreases in implantation rate in mice.Taken together,the present results have shown that MRP4 is required for embryo implantation and therefore provided new insights into the mechanism underlying embryo implantation.In addition,given the multiple roles of Wnt/beta-catenin in other physiological or pathological events,the identified novel role of MRP4 in regulation of Wnt/beta-catenin in the present study may have implications in other aspects far beyond embryo implantation such as development and tumorgenesis.