Inactivation of p120 catenin in mice disturbs intrahepatic bile duct development and aggravates liver carcinogenesis

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Research paper

Inactivation of p120 catenin in mice disturbs intrahepatic bile duct development and aggravates liver carcinogenesis Jolanda van Hengel (Ph.D) a,b,c,∗∗ , Celine Van den Broeke a,b , Tim Pieters a,b,d , Louis Libbrecht e , Ilse Hofmann f,g , Frans van Roy (Ph.D) a,b,∗ a

Molecular Cell Biology Unit, Inflammation Research Center, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Faculty of Sciences, Ghent University, Ghent, Belgium c Department of Basic Medical Sciences, Faculty of Medicine and Health Sciences, Ghent University, Ghent, Belgium d Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium e Department of Pathology, Ghent University Hospital, Ghent, Belgium f Division of Vascular Oncology and Metastasis, German Cancer Research Center, DKFZ-ZMBH Alliance, Heidelberg, Germany g Department of Vascular Biology and Tumor Angiogenesis (CBTM), Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany b

a r t i c l e

i n f o

Article history: Received 25 August 2016 Received in revised form 6 October 2016 Accepted 9 October 2016 Keywords: Cadherin Armadillo Hepatoblast Hepato-biliary cells Ductal plate malformation Mouse model Liver tumor

a b s t r a c t p120 catenin (p120ctn) is required for the stability of classic cadherins at the cell surface and is thought to play a central role in modulating cell–cell adhesion. Cytoplasmic p120ctn promotes cell motility, and probably other activities, by modulating the activities of RhoA, Rac and Cdc42. E-cadherin is expressed in periportal but not in perivenous hepatocytes. In contrast, all hepatocytes of normal mouse liver express N-cadherin. Cholangiocytes express exclusively E-cadherin. Mice with p120ctn ablation in hepatocytes and cholangiocytes (p120LiKO mice) were generated by Cre-loxP technology. Livers were examined by histological, immunohistochemical, ultrastructural and serum analysis to determine the effect of the p120ctn ablation on liver structure and function. Mouse hepatocyte differentiation and homeostasis were not impaired. However, hepatoblasts differentiated abnormally into hybrid hepato-biliary cells, ductal plate structures were irregular in p120LiKO newborns, and further development of intrahepatic bile ducts was severely impaired. In adults, enrichment of ductular structures was accompanied by portal inflammation and fibrosis. p120LiKO mice did not spontaneously develop hepatocellular carcinoma but initiation of hepatocarcinogenesis by diethylnitrosamine was accelerated. In summary: p120ctn has a critical role in biliary differentiation and is a potent suppressor of liver tumor growth. © 2016 Elsevier GmbH. All rights reserved.

1. Introduction The liver consists of different cell populations including hepatocytes, which are the major parenchymal cells in the liver, and a variety of non-parenchymal resident cells, including Kupffer, stellate and endothelial cells, as well as cholangiocytes (bile duct

Abbreviations: BECs, bile duct epithelial cells; DEN, diethylnitrosamine; DPM, ductal plate malformation; HCC, hepatocellular carcinoma; IHBD, intrahepatic bile duct; p120LiKO, with knockout of p120 catenin in liver-specific hepatoblasts and cholangiocytes. ∗ Corresponding author at: Molecular Cell Biology Unit, Inflammation Research Center, VIB, Ghent, Belgium ∗∗ Corresponding author at: Department of Basic Medical Sciences, Faculty of Medicine and Health Sciences, Ghent University, De Pintelaan 185, Building B, B9000 Ghent, Belgium. E-mail addresses: [email protected] (J. van Hengel), [email protected] (F. van Roy).

epithelial cells, BECs). Hepatocytes and cholangiocytes differentiate from bipotential hepatoblasts. Mouse hepatoblasts start to differentiate into hepatocytes on E13.5 and into cholangiocytes on E14.5. By E17, hepatocytes adopt their characteristic cuboidal shape and prepare for transition from an hematopoietic supporting role to specialized metabolic functions. Intrahepatic bile duct (IHBD) development is initiated by alignment of biliary precursor cells around the branches of the portal vein to form a single-layered ring called the ductal plate. By E17.5, the ductal plate becomes bilayered at focal areas, which leads to the formation of bile ducts, while the remainder of the ductal plate disappears (Lemaigre, 2003). Specialized junctional structures are critical for specific cell–cell adhesion during normal epithelial homeostasis. These structures include adherens junctions, desmosomes and tight junctions. Proper functioning of the adherens junctions requires interaction between the cytoplasmic tails of classic cadherins (e.g. E-cadherin) and ␤-catenin. Association of p120-catenin (p120ctn) with the jux-

http://dx.doi.org/10.1016/j.ejcb.2016.10.003 0171-9335/© 2016 Elsevier GmbH. All rights reserved.

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tamembrane domain of E-cadherin stabilizes E-cadherin at cell–cell contacts (Ireton et al., 2002). All hepatocytes of normal mouse liver express N-cadherin. Hepatocytes in the periphery of the lobules coexpress E-cadherin, but hepatocytes around the central veins do not (Doi et al., 2007; Kozyraki et al., 1996; Straub et al., 2011). Moreover, E-cadherin is present in the lateral membranes of mouse BECs, while N-cadherin is not. Mice lacking E-cadherin in both hepatocytes and BECs develop periportal inflammation via an impaired intrahepatic biliary network (Nakagawa et al., 2014). The roles of ␤-catenin and plakoglobin in liver morphogenesis, regeneration and carcinogenesis have been examined using conditional ␤-catenin– or plakoglobin- knockout (KO) mice. Specific deletion of ␤-catenin in the foregut endoderm and in mouse liver as early as E8 to E8.5 (utilizing Foxa3-Cre transgenic mice) was lethal for E17 embryos, which were overall deficient in parenchymal hepatocytes (Tan et al., 2008). However, when ␤-catenin was conditionally knocked out in hepatoblasts (E10) using the same Alb-Cre transgene as in our present study, liver mass was reduced by only 10–20% (Sekine et al., 2006; Tan et al., 2006). Loss of plakoglobin in the liver using the same Alb-Cre transgene results in a lack of an overt phenotype (Zhou 2015). The hepatic role of the related p120ctn has not been reported, though it is important in the embryogenesis of several tissues (Davis and Reynolds, 2006; Elia et al., 2006; Oas et al., 2010; Perez-Moreno et al., 2006; Smalley-Freed et al., 2010). The p120ctn family consists of four proteins: p120ctn, ARVCF, and the more distantly related ␦-catenin and p0071 (McCrea and Park, 2007). In vitro, both ␦-catenin and ARVCF can functionally substitute for p120ctn in stabilizing cell surface E-cadherin (Davis et al., 2003). p120ctn can stabilize many cadherins, including Eand N-cadherin (Reynolds, 2007). Numerous studies demonstrate that p120ctn can also intersect with a range of signal transduction molecules, including Rho-family GTPases and the transcriptional repressor Kaiso (Anastasiadis et al., 2000; Daniel and Reynolds, 1999). Phenotypes associated with p120ctn ablation in vivo are largely tissue-dependent and therefore poorly predictable (Bartlett et al., 2010; Davis and Reynolds, 2006; Elia et al., 2006; Hendley et al., 2015; Kurley et al., 2012; Marciano et al., 2011; Oas et al., 2010; Perez-Moreno et al., 2006; Schackmann et al., 2013; Smalley-Freed et al., 2010, 2011; Stairs et al., 2011; Tian et al., 2012). We analyzed the functions of p120ctn in hepatocytes and BECs in vivo by ablating p120ctn early in development using AlbCre in p120fl/fl mice. These mice exhibited ductal plate malformation (DPM) and were more sensitive to development of diethylnitrosamine (DEN)-induced hepatocellular carcinoma (HCC). This is the first description of the phenotype of p120LiKO mice.

26–50 weeks of age to determine tumor incidence and size. Hepatic tissue was prepared for histology.

2.3. RNA isolation and quantitative RT-PCR Total RNA was extracted from whole livers using the RNeasy Mini Kit (Qiagen, Venlo, the Netherlands), and 400 ng were used to synthesize cDNA using the iScript Kit (Bio-rad, Hercules, CA). To detect ARVCF, p0071 and ␦-catenin transcripts, primer pairs were designed so that amplicons were intron spanning. The resulting RTPCR products were cloned in pGEMTeasy (Promega, Madison, WI) and sequenced to confirm their specificity. RT-PCR was performed with SYBR Green Master Mix Reagent (Applied Biosystems, Foster City, CA) using an ABI Light Cycler 480. Transcript quantities were determined using the method of Ct and values were normalized to Hprt.

2.4. Histology and immunohistochemistry Paraffin sections were contrasted with hematoxylin and eosin. The following antibodies were used: anti-E-cadherin (BD Transduction Labs, San Jose, CA), anti-p120ctn and FITC-conjugated anti-p120ctn (BD Transduction Labs), anti-␤-catenin (BD Transduction Labs and Sigma, St Louis, MO), anti-ZO-1 (Zymed, St Francisco, CA), anti-N-cadherin (Zymed), anti-connexin 32 (Zymed), anti-Dsp (R&D systems, Minneapolis, MN), anti-glutamine synthetase (GS; BD, Transduction Labs); anti-BrdU-POD (Roche, Basel, Switzerland), anti-p0071 (Hofmann et al., 2009), anti-ARVCF (Walter et al., 2008), anti-␦-catenin (BD Transduction Labs) and Troma-3, an anti-CK19 antibody (generously provided by Rolf Kemler, Freiburg, Germany). For detection of cadherins and catenins, rehydrated paraffin sections were pretreated with H2 O2 (0.3% in methanol) for 45 min and with citrate buffer in a 2100 Retriever (PickCell Laboratories, Amsterdam, The Netherlands). For detection of CK19 the rehydrated paraffin sections were pretreated with H2 O2 followed by incubation for 30 min in EDTA-buffer (pH 9) at 98 ◦ C. Labeling with 5-bromo-2 -deoxyuridine (BrdU, Roche) was by injecting mice two hours before sacrificing. BrdU-positive cells were detected with anti-BrdU-peroxidase (Roche). For electron microscopy analysis small fragments of liver tissue (6 mice of five days old and 2 mice of 17 weeks old) were immediately fixed in 2.5% glutaraldehyde and further prepared for routine electron microscopy. Ultrathin sections were examined in a Zeiss EM 900 electron microscope (Jena, Germany).

2.5. Immunofluorescence analysis 2. Materials and methods 2.1. Generation of p120ctnfl/fl;AlbCre mice Transgenic p120ctnfl/fl mice (Davis and Reynolds, 2006) (courtesy of Albert Reynolds, Vanderbilt University, Nashville) were crossed with AlbCre mice expressing Cre recombinase under control of a rat albumin promoter (Postic et al., 1999). All the mice analyzed had a genetically mixed background of C57BL6, 129, and Swiss strainsMice were maintained in standard SPF housing according to the European rules on animal welfare.

2.2. DEN model of hepatocellular carcinoma A single injection of DEN (20 ␮g/g) was administered to 15-day old p120LiKO mice and control littermates. Mice were killed at

Livers were frozen in cryo-embedding compound. Sections were air-dried for 1 h and fixed in 4% paraformaldehyde. Cells were permeabilized with 0.2% Triton X-100 in PBS. The detection of the primary antibodies was done with Alexa coupled secondary antibodies (Molecular probes, Eugene, OR). Sections were embedded in Vectashield with DAPI (Vector, Burlingame, CA) and examined with a Zeiss Axiophot or a confocal microscope (Leica, Mannheim, Germany).

2.6. Biochemical analysis Serum samples of knockout and age matched control mice were analyzed at the clinical-chemical and hematological laboratory of the German Mouse Clinic (GMC) using an AU400 autoanalyzer (Olympus) and adapted reagents (Olympus) (van Hengel et al., 2008a).

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Table 1 Liver-specific serum markers in control and p120LiKO mice. Data are the mean (+/−SD) of either three control or three p120LiKO mice, 5–10 weeks of age. Serum markera

Total cholesterol (mM) ALT (U/L) AST (U/L) ALP (U/L) Triglycerides (mg/dl) Conjugated bilirubin (mg/dl) Total bilirubin (mg/dl)

Mouse genotype control

p120LiKO

63 (1.4) 10 (5.3) 31 (9.8) 97 (53) 87 (4) 0 (0) 0.2 (0)

64 (14.5) 19 (8.0) 38 (12.2) 244 (113) 100 (38.5) 1.5 (1.7) 2.4 (2.6)

a ALT, Alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase.

3. Results 3.1. Mice lacking p120ctn in hepatocytes and bile duct cells

Fig. 1. Targeted hepatoblast-specific disruption of p120ctn. (A) Cre-induced recombination of floxed Ctnnd1 alleles, assessed by p120ctn staining, occurs in hepatocytes and biliary epithelial cells of p120LiKO animals. Arrows indicate bile ducts. PV: portal vein. The images shown are representative of 10 control and 10 p120LiKO livers analyzed. Scale bars: 20 ␮m. (B) Ultrastructural analysis of hepatoctyes. Bile canaliculi (asterisks) in control and p120LiKO hepatocytes have regular, thin, apical microvilli and are sealed by junctional complexes at the lateral membranes: tight junctions (TJ), adherens junctions (AJ), desmosomes (DS) and gap junctions (GJ). GL, glycogen. Scale bars: 0.5 ␮m. (C) Compared with the control liver, the liver from a p120LiKO mouse is enlarged and yellow (both mice were 12 months old). Liver weight/body weight ratio at two and twelve months after birth. Values are means ± SD. n: number of mice per group. Statistical differences were evaluated by Wilcoxon rank sum test: * P < 0.05. (D) Sirius red staining of p120LiKO livers shows accumulation of collagen around the portal regions and in the connections between portal regions, in contrast to control livers. Mice were 2 months old. All images shown are representative of six livers analyzed. Scale bars: 80 ␮m. Scale bars: 40 ␮m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

To study how p120ctn contributes to morphogenesis and cell junction formation in polarized epithelia, we inactivated p120ctn in mouse hepatoblasts. Mice carrying floxed p120ctn alleles (Davis and Reynolds, 2006) were crossed with mice carrying the AlbCre transgene (Postic et al., 1999). F2 progenies were born at the expected Mendelian ratio and about 75% of the hepatoblast-specific p120ctn KO mice (p120ctnfl/fl ;AlbCre = p120LiKO) survived for up to 17 months. As controls, AlbCre-negative p120ctnfl/fl littermates were analyzed. All phenotypes described here were seen in four or more p120LiKO mice, although the phenotype could differ in strength. This was clear for the survival after 17 months (25% p120LiKO mice did not survive) but equally well for liver size, extent of fibrosis, degree of proliferation. This variation could be explained by varying ablation efficiency in the p120LiKO mice. We admit that the mixed genetic background of these mice is a potential source of variability and should be optimized in future use of this model. In adult liver, albumin is expressed exclusively in hepatocytes. Consequently, p120ctn protein was not detectable in hepatocytes isolated from eight-week-old p120LiKO mice (Figs. 1 A and 2 A ). However, embryonic hepatoblasts express albumin as early as E13.5 and before BECs begin to differentiate from periportal hepatoblasts (Shiojiri and Sugiyama, 2004). In mice, the process of bile duct development starts at about E14.5 and continues for two weeks after birth (Antoniou et al., 2009). Biliary tubulogenesis starts with the formation of asymmetric ductal structures lined by cholangiocytes on the portal side and by hepatoblasts on the parenchymal side (Antoniou et al., 2009). When the ducts develop, the hepatoblasts lining the asymmetric structures differentiate into cholangiocytes, thereby allowing formation of symmetrical ducts lined only by cholangiocytes. Thus, recombination of floxed alleles in mice expressing the AlbCre transgene also occurs in intrahepatic bile ducts. Heterogeneity may occur by asymmetric formation of primitive ductal structures, as described (Antoniou et al., 2009). In p120LiKO mice, liver parenchymal cells and most bile ducts were consistently p120ctn-negative, but hematopoietic cells and portal vein mesenchymal and endothelial cells remained positive (Figs. 1 A and 2 A). p120LiKO mice showed growth retardation, liver hypertrophy, and chronic jaundice. Their liver weight to body weight ratio was higher than that of control littermates (Fig. 1C). Comparison of serum alkaline phosphatase levels in control and p120LiKO mice indicated cholestasis in p120LiKO mice, but serum cholesterol and triglycerides were not significantly different (Table 1). Total and conjugated bilirubin were significantly higher in p120LiKO mice than in controls. Accumulation of conjugated bilirubin in p120LiKO mice implies a post-hepatocytic defect and impaired bile transport,

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Fig. 2. Expression of classic cadherins and catenins in p120LiKO livers. In all images the lobule centre is in the middle. All images shown are representative of eight livers analyzed. (A) Hepatocytes in p120LiKO mice lack p120ctn expression. CV: central vein. Scale bar: 40 ␮m. (B) In livers of adult control mice, hepatocytes in the periphery of lobules are immunopositive for E-cadherin, in contrast to perivenous hepatocytes. In p120LiKO mice, E-cadherin staining is attenuated but more uniformly expressed in all hepatocytes, including those in the lobule centers. Arrows point at ductular reactions at the portal area, with biliary epithelial cells expressing E-cadherin strongly. (C) All hepatocytes of control mice express N-cadherin, and p120LiKO hepatocytes show weaker N-cadherin expression.

possibly due to obstruction of the biliary tree. Sirius red staining of liver sections showed evidence for fibrosis in livers of two-weekold p120LiKO mice (not shown), and severe fibrosis in such livers at two months, while being absent in control mice (Fig. 1D). We observed no cirrhosis in p120LiKO livers up to the age of 17 months. 3.2. Hepatocyte differentiation is largely independent of p120ctn To determine whether loss of p120ctn impairs cell–cell contacts, we examined the distribution of cell junctions in livers. Control hepatocytes in the periphery of the lobules showed strong E-cadherin staining (Fig. 2B), as reported (Fujimoto et al., 1997). N-cadherin was observed in all hepatocytes (Fig. 2C). In p120LiKO mice, both E- and N-cadherin were strongly reduced at hepatocytic cell–cell contacts (Fig. 2B and C). Although in normal mice hepatocytes around the central veins express no E-cadherin, Ecadherin was present in the cytoplasm and plasma membrane of all p120LiKO hepatocytes, albeit at lower levels (Fig. 2B). ␤catenin and ␣-catenin in adherens junctions were slightly reduced in mutant livers compared to control livers (data not shown). Glutamine synthetase (GS) is a downstream target of ␤-catenin and is

expressed strongly in hepatocytes surrounding the central veins. Immunohistochemical staining for GS revealed no significant difference between the control and p120LiKO livers (data not show). We analyzed other cell junctions in the liver by comparing immunostainings of sections from adult control and p120LiKO mice. In both liver types, ZO-1 was present at the plasma membrane and enriched at the tight junctions (Supporting Fig. 1A). Desmosomes were present in a dotted pattern in both control and p120ctn-deficient hepatocytes (Supporting Fig. 1B). The gap junction proteins Cx32 and Cx26 were detected near the apical membrane of both wild-type and mutant hepatocytes, as exemplified for Cx32 (Supporting Fig. 1C). Thus, there were no major differences between p120ctn-deficient hepatocytes and control hepatocytes in the expression level or correct localization of components of tight, gap and desmosomal junctions. Finally, we compared by electron microscopy the ultrastructure of hepatocytes in control mice and p120LiKO mice. In both strains, the bile canaliculi showed thin regular apical microvilli and were sealed by junctional complexes at the lateral membranes (Fig. 1B). Mitochondria were numerous in the cytoplasm, but no differences were seen between control and p120LiKO livers.

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Fig. 3. Hepatoblast-specific ablation of p120ctn results in abnormal intrahepatic bile duct development. All images in panels A to C are representative of at least 8 livers analyzed; images in panel D are representative of 4 livers analyzed. (A) Hematoxylin-eosin staining of livers and (B) CK19 immunostaining of bile ducts, both from mice at two months of age. Compared to control livers, the parenchyma of p120LiKO livers is normal but the biliary system is highly disorganized. In the portal regions (PR) or portal vein (PV), the normal appearance of CK19-positive bile ducts (A and B, arrows in left images) is replaced in mutant mice by accumulation of biliary epithelial cells (A and B, right images). C) At the age of four months, the fibrous septa of p120LiKO livers contain bile ducts and separate hepatocyte nodules. E-cadherin immunostaining reveals the massive presence of bile ducts. (D) Anti-CD45 immunostaining reveals massive leukocyte infiltration in p120LiKO livers. Scale bars in A, B, D: 40 ␮m; in C: 80 ␮m.

3.3. Early postnatal IHBD development is impaired in p120LiKO mice Ablation of p120ctn resulted in disorganization of the biliary system (Fig. 3A and B). In all adult p120LiKO mice examined, portal and periportal areas and interlobular septa displayed multi-

ple, irregularly arborizing, E-cadherin-positive ductular structures extending far into the hepatic lobe (Fig. 3C). The portal areas showed distortion of mature bile ducts and mild portal inflammation (Fig. 3D). In contrast, livers of control mice showed at most three bile ducts in portal areas (Fig. 3B).

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Fig. 4. Hepatoblast-specific ablation of p120ctn results in abnormal intrahepatic bile duct development status, as assessed by ultrastructural analysis of bile ducts. (A) A bile duct (BD) from a control mouse at P5 (left panels) shows a regular architecture and a small lumen. Biliary epithelial cells (BEC) are surrounded by a basement membrane (BM) and contain junctional complexes on the lateral membranes. Some apical microvilli and a single cilium (C) are seen. H, hepatocyte; PV, portal vein. A portal tract of a p120LiKO mouse at P5 (right panels) shows interconnecting tubular structures resembling the ductal plate (DPT) surrounding the PV. Mesenchymal cells (MC) are present in the portal tract. The tubular structures are composed of BECs and hybrid hepatobiliary cells (HC) with more voluminous cytoplasm. ECM, extracellular matrix. (B) In a p120LiKO mouse aged for four months, interconnecting DPT and bile ductular structures are still present. Asterisks indicate some ductal plate remnants. Scale bars: A (upper images) and B, 7 ␮m; lower images in A, 2.5 ␮m. Table 2 Incidence of hepatocellular carcinoma (HCC) in p120LiKO mice treated with DEN, according to age, genotype and gender. 40 weeks

n

HCCa

Died

>50 weeks

n

HCCa

Died

control (M) KO (M) control (F) KO (F)

12 8 5 17

33% (3/10) 100% (6/6) 0% (0/5) 92% (12/13)

2 2 0 4

control (M) KO (M) control (F) KO (F)

4 3 9 10

100% (4/4) 100% (1/1) 63% (5/8) 100% (6/6)

0 2 1 4

M: male; F: female. a Fraction of live animals with HC

To detect differences in IHBD differentiation and morphogenesis between control and p120LiKO mice, we analyzed mice on postnatal day 5 (P5). In control mice, typical ductal plate remodeling at P5

was evident from the presence of only a few E-cadherin-positive ductal plate remnants in the portal mesenchyme, whereas the nontubular part was largely absent (Supporting Fig. 2A and B). Ductal

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Fig. 5. E-cadherin is expressed in biliary epithelial cells (BECs) of p120LiKO livers. In P16 livers of control (A) and p120LiKO (B) mice, BECs in the portal regions express E-cadherin also when p120ctn is ablated. The tubular structures in p120LiKO mice appear to be composed of cells with and without p120ctn expression. * Lumina of bile ducts. All images shown are representative of five livers analyzed. Scale bar: 10 ␮m.

plate cells were also detected in p120LiKO animals at P5. However, BECs in mutant livers were mostly arranged irregularly around the portal veins and very rarely formed typical tubular structures (Supporting Fig. 2B). Ductal cells in these structures were consistently E-cadherin-positive but generally p120ctn-negative (Supporting Fig. 2C). Most portal tracts did not contain differentiated bile ducts. Instead, ductal plate remnants and BECs were abundant in the periportal area. In summary, hepatoblast-specific disruption of p120ctn impaired IHBD development resulting in multiple irregular duct-

like structures, probably mainly due to impaired morphogenesis of the biliary tree. Ultrastructural analysis demonstrated that the mutant duct-like structures at P5 had irregular lumina and increased extracellular matrix deposition (Fig. 4A). Moreover, multiple branched ducts sharing a single layer of BECs were present. These structures are composed of BECs and hybrid hepato-biliary cells (Fig. 4A), as described by Clotman et al. (Clotman et al., 2005). These hybrid cells are characterized by apical microvilli, a round nucleus, voluminous cytoplasm with many mitochondria, ER cisternae,

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ribosomes and polyribosomes, and some dense lysosomes, but no glycogen particles. The ultrastructural abnormalities remained unchanged after four months (Fig. 4B). Structures resembling ductal plates and composed of BECs and hybrid hepatobiliary cells were present, but no progenitor cells as in chronic hepatitis (Rita De Vos, unpublished data). 3.4. E-cadherin expression is not disturbed in p120ctn mutant BECs At P16, all cholangiocytes in control livers coexpressed p120ctn and E-cadherin (Fig. 5A). Though most BECs in p120LiKO livers lacked p120ctn expression, there were also seemingly normal bile ducts with p120ctn-expressing BECs. Surprisingly, p120ctnnegative cholangiocytes expressed E-cadherin at their lateral membranes (Fig. 5B). Some tubular structures contained both p120ctn-positive and -negative cells. The p120ctn-negative cells probably correspond to the hybrid cells identified by ultrastructural analysis (Fig. 4A). Expression of E-cadherin in p120ctn-negative tubular structures continued during adulthood (Fig. 3C). 3.5. Mechanisms of weight increase of p120ctn mutant livers Compared to control livers, p120LiKO livers at all ages had more hepatocytes containing mitotic figures. We determined the number of S-phase hepatocytes by measuring BrdU incorporation in livers of mutant and control mice at various ages. BrdU incorporation was invariably higher in p120LiKO livers than in controls (illustrated in Supporting Fig. 3). Hepatocytes and cells in duct-like structures proliferated all over the liver. Another reason for the increased liver weight of p120LiKO mice might have been the increased deposit of extracellular matrix, revealed by EM (Fig. 1B) and Sirius red staining (Fig. 1D). 3.6. Expression of p120ctn family members Compensatory expression of p120ctn family members might explain the rather normal liver development and function in p120LiKO mice. To test this, we performed RT-PCR experiments using mRNA extracted from livers of control and p120LiKO mice. Expression of p0071 and ARVCF transcripts was indeed stronger in p120LiKO livers than in control livers (data not shown). ␦-catenin mRNA expression levels were similar in control and p120LiKO livers. We used immunohistochemistry to further test the hypothesis that p120ctn-deficient hepatocytes show few abnormalities because of compensation by p120ctn family members. p0071 was not expressed in control hepatocytes but in p120LiKO hepatocytes it was expressed at the borders between hepatocytes (Fig. 6A). In control hepatocytes some ARVCF localized at basal sites, while in p120LiKO hepatocytes it also localized at the lateral borders (Fig. 6B). In both control and p120LiKO hepatocytes, ␦-catenin was seen at the basal side of hepatocytes, most probably in endothelial cells lining the hepatocytes (Fig. 6C). In contrast to hepatocytes, control bile ducts were positive for both p0071 and ARVCF. Their expression persisted when p120ctn was knocked out (shown for p0071 in Fig. 6D). The loss of p120ctn resulted in formation of BEC strings even though p0071 and ARVCF

Fig. 6. Expression of p120ctn family members in p120LiKO hepatocytes and BECs. In all cases, representative confocal sections are shown. (A) In livers of adult control mice, perivenous hepatocytes do not express p0071, whereas p120-negative perivenous hepatocytes show p0071 staining at their lateral membranes. (B) Expression of ARVCF in livers of adult mice.

In comparison to control perivenous hepatocytes, p120LiKO perivenous hepatocytes express more ARVCF at the lateral and basal membranes. (C) In portal areas of both control and p120LiKO adult mice, cholangiocytes (identified by strong ␤-catenin staining at their cell contacts) do not express ␦-catenin. Some positive staining is visible at the basal side of hepatocytes in control and in p120LiKO livers. (D) BECs in control and p120LiKO portal areas of two-month-old mice express p0071. Cell contacts are identified by staining for E-cadherin; strong staining is seen for bile ducts in control livers (asterisks) and in BEC strings in p120LiKO livers. All images shown are representative of four livers analyzed. Scale bars: 20 ␮m.

Please cite this article in press as: van Hengel, J., et al., Inactivation of p120 catenin in mice disturbs intrahepatic bile duct development and aggravates liver carcinogenesis. Eur. J. Cell Biol. (2016), http://dx.doi.org/10.1016/j.ejcb.2016.10.003

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were expressed at cell–cell contacts. Therefore, ARVCF and p0071 do not functionally compensate for loss of p120ctn in cholangiocytes. BECs expressed no ␦-catenin (Fig. 6C). 3.7. Acceleration of DEN-initiated hepatocarcinogenesis in p120ctn mutant livers As described above, p120LiKO hepatocytes proliferate actively (Supporting Fig. 3). In older animals we observed pericentral and pericellular fibrosis characterized by wire-like bands of collagen radiating from the terminal hepatic venule along the space of Disse (Fig. 1D). Abnormal hepatocytes with enlarged nuclei or abnormal mitotic figures were seen in some p120LiKO livers (data not shown). At 10–16 months, enlarged livers with multiple neoplastic foci were evident in all p120LiKO mice. These findings indicate that loss of p120ctn results in hepatomegaly but not in spontaneous HCC. We also observed no cholangiocarcinoma in untreated p120LiKO mice. p120LiKO and control mice were injected at P15 with 20 mg DEN/g body weight. After 40 weeks, none of the female control mice had developed tumors, but 33% of their male counterparts did (Table 2). In p120LiKO mice, tumors developed in 92% of females and 100% of males. Over time, macroscopically visible tumors developed in most surviving controls and in all surviving p120LiKO mice. In most p120LiKO animals, individual tumors could not be counted because the entire liver was involved, in contrast to control mice (Fig. 7A). Tumors were positive for p120ctn in control mice and negative in p120LiKO mice (Fig. 7B). No differences in tumor histology could be observed. Next we examined expression of ␤catenin and GS. None of the tumors in either WT or p120LiKO mice were positive for Glutamine Synthetase or nuclear ␤-catenin (data not shown). Lung metastases in DEN-injected mice were macroscopically visible in 5% of mice of >50 weeks old, but p120LiKO tumors displayed no evidence of increased metastatic potential. 4. Discussion Prenatal loss of p120ctn in the liver leads to DPM. In p120LiKO mice, hepatocytes and most BECs do not express p120ctn because both cell types are differentiated from bipotential hepatoblasts, in which the Alb-driven Cre is already active. The complex between Ecadherin and p120ctn is critical for the formation and maintenance of the adherens junctions (Ireton et al., 2002). Disruption of this complex is a hallmark of cancers derived from epithelia (Berx and van Roy, 2009). In particular, p120ctn loss or mislocalization has been shown in vitro to destabilize E-cadherin (Pieters et al., 2012). In vivo, the situation seems more complicated. We found that loss of p120ctn in hepatocytes does not induce major changes in hepatocytic functions. We observed a reduction of cadherin levels at the lateral membranes of hepatocytes but this seems to have no effect on the cell–cell adhesion capacity or on other cell-cell adhesion junctions. At later stages, proliferation of p120ctn-negative hepatocytes increased. As p0071 and ARVCF were expressed at the lateral membranes in p120LiKO hepatocytes but not in control hepatocytes, these related armadillo proteins might largely compensate for the loss of p120ctn in hepatocytes. Normal BECs express p120ctn, p0071 and ARVCF at their lateral membranes. In p120LiKO livers, most BECs did not express p120ctn, but biliary tubuli containing both p120ctn-positive and −negative cells were also present. Surprisingly, loss of p120ctn had no apparent effect on E-cadherin expression at the cell–cell junctions of BECs during development and in adulthood. Despite preservation of E-cadherin in p120LiKO BECs, p120ctn ablation impaired BEC differentiation and increased proliferation. Our data reveal that some cell types (BECs in this study) are more sensitive than others (e.g. hepatocytes) to ablation of p120ctn. This

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difference might depend on at least three parameters: the types of cadherins expressed, the levels of their expression and the presence or induction of related p120ctn family members. Our study demonstrates such differences between BECs and hepatocytes, and is in line with similar findings in other tissues. In the liver, the levels of E-cadherin at the plasma membrane seem to be much higher in BECs than in hepatocytes, and ablation of p120ctn does not cause a major loss of E-cadherin in BECs. In the developing kidney, several cadherins are differentially expressed within tissue subdomains and cadherin-6 was found to be less sensitive to p120ctn ablation than E-cadherin, cadherin-4 or cadherin-11 (Marciano et al., 2011). Another study showed that p120ctn is essential for mammary terminal end bud morphogenesis (Kurley et al., 2012). Dysfunction of the terminal bud is likely due to E-cadherin loss affecting cell–cell adhesion. Because the other three p120ctn family members were expressed in normal mammary epithelium and were not altered upon p120ctn ablation, the authors concluded that p120ctn has a non-redundant function in cell–cell adhesion of mammary ductal cells (Kurley et al., 2012). Partial retention of E-cadherin and adherens junctions, potentially stabilized by substitution of ARVCF, ␦-catenin, or p0071 for p120ctn, has also been reported in case of p120ctn ablation in pancreatic progenitor cells (Hendley et al., 2015). However, E-cadherin is not retained in adult mice of this pancreatic ablation model. In p120LiKO BECs, the unchanged expression of p0071 and ARVCF at the plasma membrane did not prevent an increase in proliferation capacity, whereas the induction of p0071 and ARVCF in p120LiKO hepatocytes appeared to mediate homeostasis of the latter cell type. The involvement of the three above mentioned parameters and their mutual complex interactions make simple predictions of the consequences of p120ctn ablation in different cell types quite unrealistic. Prenatal loss of p120ctn in the liver leads to DPM. In Hnf6−/− and other mutant mice (Clotman et al., 2005; Raynaud et al., 2011), DPM is characterized by the presence of hybrid cells expressing both hepatocytic and biliary markers and reflecting abnormal differentiation of hepatoblasts into ductal plate cells. In these studies, identification of this hybrid cell type was mainly based on immunofluorescent labeling for HNF-4a (hepatoblasts and hepatocytes) and cytokeratin (biliary cells); phenotyping by electron microscopy was minimal. We performed a more detailed ultrastructural examination of these hybrid cells to confirm their mixed biliary-hepatocytic phenotype. Importantly, the ultrastructural features of hybrid cells are clearly different from those of the reactive bile ductular cells seen in adult liver diseases (De Vos and Desmet, 1992). Recently, a distinct population of cells residing at the periportal region of the healthy liver was described to have high regeneration potential (Font-Burgada et al., 2015). Because these cells express several bile-duct-enriched genes, they were named hybrid hepatocytes (HybHP). Extensive studies with several markers are necessary to study the different hybrid cells in detail. p120ctn abnormalities are common in different tumors: p120ctn can either stimulate or inhibit cell proliferation and tumor growth, depending on its subcellular localization and molecular interaction partners (Pieters et al., 2012). Targeted disruption p120ctn in the salivary gland of mice results in dysplasia (Davis and Reynolds, 2006), whereas its targeted disruption in the epidermis results in hyperplasia and inflammation (Perez-Moreno et al., 2006). Half of p120ctnfl/fl ;VillinCre-ERT2 mice develop tumors within 18 months of tamoxifen-induced p120ctn ablation (Smalley-Freed et al., 2011). Other data showed that p120ctn can function as a bona fide tumor suppressor in the oral cavity, esophagus and squamous forestomach (Stairs et al., 2011). Indeed, p120ctn ablation in p120ctnfl/fl ;L2Cre mice led to squamous cell carcinoma accompanied by autocrine production of monocyte/macrophage attractants, thus promoting a proinvasive tumor microenvironment (Stairs et al., 2011). Moreover, loss of p120ctn in a conditional

Please cite this article in press as: van Hengel, J., et al., Inactivation of p120 catenin in mice disturbs intrahepatic bile duct development and aggravates liver carcinogenesis. Eur. J. Cell Biol. (2016), http://dx.doi.org/10.1016/j.ejcb.2016.10.003

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Fig. 7. Enhanced liver tumorigenesis in p120LiKO mice exposed to DEN. (A) Representative images of tumor-bearing livers in DEN treated control and p120LiKO mice at the age of 30 wk (left panel) or 60 wk (right panel). (B) p120ctn immunohistochemistry in tumors of control and p120LiKO mice exposed to DEN. Tumors in control mice were positive for p120ctn; tumors in p120LiKO mice were negative for p120ctn. All images shown are representative of at least eight livers analyzed. Scale bars: 40 ␮m.

mouse model of noninvasive mammary carcinoma results in formation of highly metastatic tumors (Schackmann et al., 2013). p120ctn is suppressed in most HCC, and this suppression correlates with increased malignancy and poor survival (Zhai et al., 2008). One of the reasons we generated p120LiKO mice was to investigate the potential tumor suppressor role of p120ctn in the liver. We found that p120ctn deficiency by itself did not predispose to the formation of spontaneous HCC. However, it dramatically aggravated DEN-induced hepatocarcinogenesis. DEN treatment of mice is a frequently used model of HCC (Fausto and Campbell, 2010). DEN is metabolized in the centrilobular area (zone 3) into an alkylating agent that induces DNA damage as well as hepatocyte death. When surviving cells proliferate, some of the DNA adducts that are formed are fixed into permanent mutations that may cause activation of oncogenes. It was reported that about 30% of DEN-induced tumors harbor mutations in H-Ras (Aydinlik et al., 2001). Thus, the earlier onset of nodule formation and the higher HCC occurrence in DEN-treated p120ctn-negative livers might be explained by increased hepatocyte proliferation due to p120ctn ablation in combination with DEN activated oncogenes. Preliminary analysis determined no activation of Wnt signaling. A strong connection exists between inflammation and tumor progression (Grivennikov et al., 2010). In contrast to the “spontaneous” HCC we observed in Cdc42-AlbCre mice (van Hengel et al., 2008b), our present findings do not favor a model in which p120ctn loss disrupts tight junctions and cause oncogenic bile leakage. There might be a separate cell-autonomous consequence of p120ctn ablation leading to the production of cytokines that recruit inflammatory cells. While the precise nature of the influx of inflammatory cells may depend on the model system, several groups have reported that p120ctn ablation leads to inflammation

(Perez-Moreno et al., 2008; Schackmann et al., 2013; SmalleyFreed et al., 2010; Stairs et al., 2011). Thus, conditional p120ctn loss in the gastrointestinal tract, skin or mammary gland results in cytokine secretion and subsequent attraction of immune cells. A more inflammatory microenvironment due to p120ctn ablation might accelerate tumor initiation also in the DEN-HCC model. Indeed, a periportal inflammatory infiltrate containing CD45positive cells was seen in the p120LiKO livers. However, further research is needed to identify the mechanisms and biological consequences of this immune response. Apparently, the induction of BEC proliferation by p120ctn ablation is not sufficient to induce cholangiocarcinoma. In conclusion, our results underline the importance of p120ctnmediated junction stabilization and intracellular signaling for correct development of the ductal plate. Later in life, p120ctn expression protects hepatocytes from inflammation and HCC development. Conflict of interest The authors declare to have no conflict of interests. Acknowledgements This research was supported by the Research Foundation – Flanders (FWO), the Concerted Research Actions (GOA) of Ghent University, the Foundation against Cancer – Belgium, the Hercules Foundation – Flanders (grant AUGE/11/14) and the Belgian Science Policy (Interuniversity Attraction Pools – IAP7/07). Serum samples of mice were analyzed by Martina Klempt at the clinical chemical and hematological laboratory of the German Mouse Clinic (GMC).

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Please cite this article in press as: van Hengel, J., et al., Inactivation of p120 catenin in mice disturbs intrahepatic bile duct development and aggravates liver carcinogenesis. Eur. J. Cell Biol. (2016), http://dx.doi.org/10.1016/j.ejcb.2016.10.003