| Background and ObjectionA contiguous layer of cells plays a pivotal role in the epithelial barrier function,self-renew and regeneration of intestinal epithelial cells(IECs)are mainly dependent on intestinal stem cells(ISCs)activation,proliferation and differentiation.Ulcerative colitis(UC)is a subcategory of inflammatory bowel disease(IBD)with high-risk of colitis-associated cancer(CAC).Two key pathophysiological features of this disease are the dysregulation of innate immune system and impaired epithelial regeneration.Mucosal healing has recently been regarded as a key treatment goal to UC.The Hippo pathway is particularly essential in control of organ size and tissue growth.YAP is a core component of the Hippo pathway and a candidate oncogene in humans.When Hippo is inactive,the LATS1/2 kinases of the Hippo pathway are unable to phosphorylate YAP,YAP binds to transcription factors(mainly TEAD family proteins)and acts as a transcriptional co-activator to initiate the transcription of target genes.In addition to YAP in epithelial cells,the Wnt signaling pathway is a critical regulator of stem cell maintenance,cell fate and proliferation.When activated,?-catenin escapes the regulation of the destruction complex and triggers transcription by subsequent nuclear accumulation and interaction with TCF/LEF transcription factors.The overlap between YAP and Wnt regulation in IEC proliferation imply that these factors do not work independently and might have a potential link.In this study,we demonstrate that YAP triggers Wnt/?-catenin signaling,which stimulates epithelial cell proliferation and not only facilitates enterocyte self-renewal and crypt regeneration after colitis,but also promotes CAC development through chronic inflammation and excessive regeneration.Materials and results1.YAP expression was decreased in human ulcerative colitis tissues We first examined the expression of proliferation-related genes in UC tissues and found that YAP was significantly reduced in 62.5%(45/72)of UC tissues,especially in active UC,and was undetectable in 18.1%(13/72)of these specimens.In addition,Spearman correlation analyses revealed that both YAP and Ki67 expression in epithelial cells correlated negatively with colitis severity(R=-0.674,p<0.0001;R=-0.481,p<0.0001).2.YAP expression was up-regulated during epithelium regeneration in murine colitis modelWe took advantage of the well-established DSS-induced colitis and repair model.Five days of 3%DSS exposure led to the dramatically decreased expression of YAP in crypts.However,after 3 days of DSS withdrawal,a tremendous restoration of YAP expression was detected,which extended throughout the whole crypt.By 5 days,YAP expression was detected in the crypt base again,at almost identical levels as those in normal colon tissue.3.YAP accelerated self-renewal,protected enterocytes from DSS-induced colitis and promoted epithelial regeneration after mucosal injuryWe constructed a YAP overexpression model through intraperitoneal injections of YAP-wild-type lentivirus in adult mice.Compared to mice injected with the empty vector(EV),YAPWT mice had an increased expression of proliferation-related proteins Brdu,PCNA,?-catenin and Lgr5.Next,we assessed the susceptibility of YAPWT and EV mice to acute DSS-induced colitis.Histological assessments demonstrated that YAPWT mouse crypts exhibited better structural integrity;in contrast,the control littermates presented with catastrophic crypt loss and widespread ulcers.More importantly,5 days after DSS withdrawal,the structure of crypts and the gut barrier function in YAPWT mice were almost restored.Moreover,colonic crypts of YAPWT mice contained more Brdu and PCNA-positive proliferating cells and fewer apoptotic cells than those in EV mice.4.YAP triggered Wnt/?-catenin signaling during epithelial cell inflammation and regenerationWe used DSS to induce epithelial cell injury in vitro,during the regeneration process,an abundant expression of cytoplasmic YAP rapidly occurred after termination of DSS administration and was gradually translocated into the nucleus;by 2 hours post DSS cessation,YAP expression reached the highest amount of nuclear accumulation,and by 4 hours,it became a weak immune-signal again.Meanwhile,?-catenin showed clear nuclear localization,which was accompanied by up-regulated total protein expression levels of ?-catenin,Lgr5 and cyclin D1 by 2 hours DSS withdrawn.We then investigated the interaction between YAP and ?-catenin in FHC clones stably overexpressing YAP(YAPWT mainly expressed in the nucleus)or empty vector(EV)control.The expression of Wnt-associated proteins including ?-catenin,Lgr5 and cyclin D1 was up-regulated in YAPWT cells compared to that in EV cells.Moreover,silencing YAP by siRNA caused a decrease in the expression of Lgr5 in HT29 cells.In addition,YAPWT increased wound healing and cell proliferation.However,silencing(3-catenin obviously impeded Wnt transcription and monolayer wound healing in YAPWT FHC cells.Furthermore,flow cytometry analysis indicated that YAPWT cells demonstrated lower cell apoptosis than EV cells(8.7%VS 15%).5.Nuclear YAP and ?-catenin/TCF4 formed a transcriptional complex;Lgr5 and cyclin D1 were targets of this complex in epithelial cellsWe then constructed ?-catenin-overexpressing cells by pc-DNA-?-catenin transfection,and by using siRNA,we transfected FHC P-catenin-overexpressing cells with YAP-specific siRNA.The result showed that ?-catenin overexpression enhanced "wound" closure,but silencing YAP in FHC-?-catenin(+)cells attenuated this effect.Moreover,endogenous co-immunoprecipitation(IP)analysis indicated that YAP directly interacted with ?-catenin in the cell nucleus,but not in the cytoplasm.Chromatin IP detection showed that Lgr5 and cyclin D1 expression was clearly increased in FHC-YAPWT cells.6.YAP promoted development of colitis associated carcinoma in AOM/DSS induced modelWe then constructed another stable clone in which YAP was phospho-mimetically mutated(S127D)in DLD1 cells.YAPS127D significantly depressed DLD1 clone formation and cell proliferation.Accordingly,immunoblotting analysis verified the substantial suppression of ?-catenin and Lgr5 protein expression in YAPS127D cells compared that in YAPWT or EV cells.In addition,the Wnt transcriptional targets Lgr5 and cyclin D1 were clearly down-regulated in YAPS127D cells.In vivo,we performed a classic CAC procedure in mice that were infected with YAPS112D(mouse S112D corresponding to S127D in humans)to further probe the effect of YAP on chronic inflammation.After the first cycle,compared with YAPWT mice,YAPS112D mice presented with worse intestinal barrier dysfunction,combined with more severe destruction including the disappearance of crypt cells and inflammatory cell infiltration in the mucosa.Finally,by AOM/DSS administration,we had successfully created a CAC model(figure 7A)a.Interestingly,mice infected with YAPS112D developed significantly smaller tumour areas and tumour numbers than YAPWT mice.In addition,histological assessments showed that the tumours in YAPWT mice were usually identified as having high-grade dysplasia,but the colonic mucosa of YAPS112D mice presented with low-grade dysplasia.Conclusion:1.YAP expression was decreased in human ulcerative colitis tissues2.YAP expression was up-regulated during epithelium regeneration in murine colitis model3.YAP accelerated self-renewal,protected enterocytes from DSS-induced colitis and promoted epithelial regeneration after mucosal injury4.YAP triggered Wnt/?-catenin signaling during epithelial cell inflammation and regeneration5.Nuclear YAP and ?-catenin/TCF4 formed a transcriptional complex;Lgr5 and cyclin D1 were targets of this complex in epithelial cells6.YAP promoted development of colitis associated cancer in AOM/DSS induced model... |
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