A N-terminal Deteted Protein Of β-catenin In Promoting Liver Development And Its Mechanism

The liver as the largest organ in mammals plays important roles in multiple functions such as metabolism,detoxification,synthesis,and storage.During liver development,two main epithelial cell types including hepatocytes and cholangiocytes originate from bi-potential progenitor cells and subsequently are responsible for major liver functions.The differentiation from progenitor cells to hepatocytes and cholangiocytes begins at the Embryonic 12.5-13.5 days of the murine embryonic period(E12.5-E13.5).The Wnt pathway is very important in liver development and maintenance of homeostasis,which also determines the differentiation fate of liver stem/progenitor cells.In the presence of Wnt ligands,β-catenin,as the core protein of the Wnt signals,enters into the nucleus and transcriptionally activates the expression of downstream Wnt target genes such as Lgr5 and Axin2.β-catenin accelerates cell cycle and promots cell proliferation.In the absence of Wnt ligands,the destruction complex formed by Axin and APC functions and recruits GSK3β to phosphorylate the N-terminus of β-catenin,resulting in its degradation by the proteasome.The CTNNB1 mutations occupying about 30.3%of HCC patients drive hepato-carcinogenesis.Most of CTNNB1 mutations locate at the N-terminal of β-catenin,which are the phosphorylation sites of GSK3β and CK1α.It indicates that the N-terminal of β-catenin plays an important role in liver regulation.Although the mechanisms and important roles of canonical Wnt signaling pathway have been elucidated,the structure of β-catenin and the roles of trunctated β-catenin proteins remain unclear.The latest research proves that the N-terminal or C-terminal trunctated proteins of β-catenin binds to different cofactors to determine different fates of intestinal stem cells.Our previous results showed that one GSK3β inhibitor ChIR99021,as an activator of Wnt pathway,is necessary to maintain the hepatocyte fate and inhibit the cholangiocyte fate in liver organoids,but the specific mechanism is still unclear.One preliminary research has proved that the existence of truncated form of β-catenin at N-terminal with 1-95 amino acid deletion(defined as ΔN95 in our study)during embryonic liver development[1].However,the appearance and disappearance of ΔN95,its temporal and spatial characteristics,its specific role in molecular regulation have not been well studied.Based on our previous work,ΔN95 appeared in the period of E14.5-E18.5,the differentiation period of hepatic progenitor cells to hepatocytes and cholangiocytes,suggesting that ΔN95 may regulate the differentiation and maturation of hepatic progenitor cells.Therefore,our study focused on the effect of ΔN95 and its role in liver cell differentiation and maturation.We aim to uncover the specific molecular mechanism of ΔN95.In this study,mouse models were used to initially explore the expression characteristics of ΔN95 during embryonic development,and fetal liver organoids with overexpression of trunctated protein ΔN95 and full-length β-catenin were constructed for transcriptic analysis by RNA-seq.A series of results were obstained as follows:Part One:The expression and role of ΔN95 in fetal liver development.1.ΔN95 is specifically expressed in the E14.5-E18.5 during liver development.In this study,fetal liver and adult liver tissues from E12.5,E14.5,E16.5 and E18.5 periods were collected.Compared with full-length β-catenin,ΔN95 starts to express in the cytoplasm at E14.5 and reachs the highest level at E16.5.ΔN95 does not express at all in the adult liver.It has been found that E-cadherin interacts with the full-length β-catenin.The expression of E-cadherin and Calpain1,one protein catalyzed by β-catenin was consistent with that of ΔN95.The expression of liver cell marker Alb and cholangiocyte marker Krt19 mRNA gradually increased during E14.5-E18.5,indicating that liver cells differentiated and matured during this period,and ΔN95 specifically appeared during the period of differentiation and maturation of hepatocytes and duct cells.2.ΔN95 promotes the differentiation of progenitor cells into cholangiocytes,while full-length β-catenin promotes the differentiation of progenitor cells into hepatocytes.Subsequently,we isolated fetal livers from E12.5 mouse and cultured them into organoids.The cell fate of the stable ethmoid organoids was examined after the establishment of stable ethmoid organoids with overexpression of full-length β-catenin and ΔN95 and knockdown of Ctnnb1.It was found that hepatocyte markers such as Alb,Cyp3a4 and HNF4αwere increased in fetal liver organoids overexpressing full-length β-catenin,and cholangiocyte markers such as Krt19 were decreased.Meanwhile,ΔN95-overexpressed organoids showed decreased hepatocyte markers such as Alb,and increased cholangiocyte markers such as Krt19.Both full-length and truncated ΔN95 caused the increase of cell proliferation marker Pcna.Knockdown of Ctnnb1 significantly decreased the liver cell marker Alb and inhibited the proliferation of fetal liver organoids.The results showed thatΔN95 inhibited the differentiation of hepatic progenitor cells to the fate of hepatocytes and promoted the differentiation of progenitor cells to the fate of cholangiocytes.In addition,both full-length and truncation promoted fetal liver organoid proliferation.3.Transcriptomics analysis of full-length β-catenin and ΔN95 overexpressed organoids.We performed RNA sequencing and transcriptomic analysis on the full-length β-catenin and ΔN95 overexpressed fetal liver organoids.The results showed that overexpression ofΔN95 caused 2441 genes to be up-regulated and 2743 genes to be down-regulated.Full-lengthβ-catenin caused 2229 genes to be up-regulated and 4287 genes to be down-regulated.Heat map analysis verified that the genes Aqp5,Agt,Hnf1β related to hepatocytes were up-regulated after overexpression of β-catenin,and the expression of cholangiocyte function-related genes Krt7,Ggt1,Epcam was up-regulated after overexpression of ΔN95.The results of glycogen staining(hepatocytes)and KRT19(cholangiocytes)immunofluorescence staining proved that overexpression of β-catenin increased the number of hepatocytes,and overexpression of ΔN95 increased the number of cholangiocytes.Therefore,ΔN95 is specifically expressed at E14.5-E18.5 in the early stage of liver development,and has the same role as full-length β-catenin in promoting proliferation,but has a different role in promoting progenitor cell differentiation.The expression of ΔN95 promoted the differentiation of progenitor cells into cholangiocytes and inhibited the differentiation of progenitor cells into hepatocytes.Part Two:Exploration of the molecular mechanism of ΔN95 in promoting the differentiation of progenitor cells into cholangiocytes.1.ΔN95 promotes the differentiation of cholangiocytes by activating the TGF-β/BMP pathway.By KEGG analysis,it was found that a variety of significant enrichments including TGF-β/BMP signaling pathway in ΔN95 overexpressed organoids.TGF-β/BMP downstream target genes including Id1,Id3 were significantly upregulated in ΔN95 overexpression organoids,but not in full-length β-catenin overexpressed organoids,suggesting that ΔN95 promotes cholangiocyte differentiation by activating TGF-β/BMP.2.ΔN95 specifically binds to ALK1 to activate TGF-β/BMP pathway.In this study,the differential proteins binding to ΔN95 and full-length β-catenin were identified by Co-IP binding mass spectrometry.We found that the ALK1,the receptor of TGF-β/BMP pathway,bind to ΔN95 instead of β-catenin.Thus,ΔN95 regulates the TGF-β/BMP signaling pathway by specifically binding to ALK1.3.With the addition of ALK1 inhibitor,the phenotype of ΔN95 in promoting cholangiocyte differentiation was rescued.We constructed ΔN95-overexpression HepaRG cells,and investigated the differentiation fate of hepatic progenitor cells by adding the inhibitor LDN193189 specific for ALK1.Real-time quantitative results showed that the effect of overexpression of ΔN95 was significantly rescued and cholangiocarcinoma differentiation was inhibited after addition of ALK1 inhibitor.4.The function of ΔN95 involved in the regulation of liver cell differentiation is not dependent on E-cadherinE-cadherin combines with full-length β-catenin to participate in liver development.To verify whether ΔN95 promoted cholangiocyte differentiation was affected by E-cadherin,we knocked down the Cdh1 gene in fetal liver organoids and found that its function is inconsistent with that of ΔN95,suggesting that it does not affect ΔN95 is involved in the regulation of liver cell differentiation.In summary,our study reveals the expression characteristics and functions of ΔN95 in the liver during mouse embryonic development and preliminarily clarifies that ΔN95 activates TGF-β/BMP signaling through binding ALK1 pathway,promote the expression of downstream target genes Id1 and Id3,and promote the differentiation of hepatic progenitor cells into cholangiocytes.This study provides a new mechanism for the regulation of liver development,and also provides new clues for the study of liver diseases and tumors.