Liver-Specific b-Catenin Knockout Mice Have BileCanalicular Abnormalities, Bile Secretory Defect, and

Intrahepatic CholestasisTzu-Hsuan Yeh,1 Lindsay Krauland,1 Vijay Singh,1 Baobo Zou,1 Prathab Devaraj,1 Donna B. Stolz,2

Jonathan Franks,2 Satdarshan P. S. Monga,1,3 Eizaburo Sasatomi,3 and Jaideep Behari1

Beta-catenin plays important roles in liver physiology and hepatocarcinogenesis. Whilestudying the role of b-catenin in diet-induced steatohepatitis, we recently found that liver-specific b-catenin knockout (KO) mice exhibit intrahepatic cholestasis. This study wasundertaken to further characterize the role of b-catenin in biliary physiology. KO miceand wild-type (WT) littermates were fed standard chow or a diet supplemented with 0.5%cholic acid for 2 weeks. Chow-fed KO mice had higher serum and hepatic total bile acidlevels and lower bile flow rate than WT mice. Expression levels of bile acid biosyntheticgenes were lower and levels of major bile acid exporters were similar, which therefore couldnot explain the KO phenotype. Despite loss of the tight junction protein claudin-2, KOmice had preserved functional integrity of tight junctions. KO mice had bile canalicularmorphologic abnormalities as evidenced by staining for F-actin and zona occludens 1.Electron microscopy revealed dilated and tortuous bile canaliculi in KO livers along withdecreased canalicular and sinusoidal microvilli. KO mice on a cholic acid diet had higherhepatic and serum bile acid levels, bile ductular reaction, increased pericellular fibrosis,and dilated, misshapen bile canaliculi. Compensatory changes in expression levels of sev-eral bile acid transporters and regulatory genes were found in KO livers. Conclusion:Liver-specific loss of b-catenin leads to defective bile canalicular morphology, bile secre-tory defect, and intrahepatic cholestasis. Thus, our results establish a critical role for b-cat-enin in biliary physiology. (HEPATOLOGY 2010;52:1410-1419)

Beta-catenin, the primary effector of the canonicalWnt signaling pathway, plays critical roles inhepatocarcinogenesis and liver development.1-6

However, its role in adult liver physiology is not wellunderstood. Cytoplasmic levels and localization of b-catenin are tightly regulated (reviewed in MacDonaldet al.7). In the absence of Wnt signaling, b-catenin isbound in the cytoplasm by a multiprotein complex.Phosphorylation via the action of glycogen synthase ki-nase 3b and casein kinase 1 targets b-catenin for protea-somal degradation. In the presence of Wnt ligands, b-catenin remains unphosphorylated, translocates to thenucleus, and activates transcription of its target genes bybinding to T cell factor/lymphoid-enhancing factorfamily of transcriptional activators. Through its associa-tion with E-cadherin at the cell membrane, where itlinks cadherins to the actin cytoskeleton, b-catenin alsoplays an important role in the formation of adherensjunctions (reviewed in Hartsock and Nelson8).We recently showed that liver-specific b-catenin knock-

out (KO) mice have increased susceptibility to developing

Abbreviations: ABC, ATP-binding cassette; BSEP, bile salt export pump;CAR, constitutive androstane receptor; Cyp, cytochrome P450; FD-40, FITC-conjugated dextran; FITC, fluorescein isothiocyanate; FXR, farnesoid Xreceptor; H&E, hematoxylin and eosin; KO, knockout; MCD, methionine- andcholine-deficient; mdr, multiple drug resistance; MRP, multidrug resistanceprotein; PE, polyethylene; PPARa, peroxisome proliferators activated receptoralpha; PXR, pregnane X receptor; RT-PCR, real-time polymerase chainreaction; Shp, small heterodimer partner; Slco, solute carrier organic aniontransporter; TRITC, tetramethylrhodamine isothiocyanate; WT, wild type.From the 1Department of Medicine; 2Department of Cell Biology and

Physiology; and 3Department of Pathology, University of Pittsburgh, Pittsburgh,PA.Received February 8, 2010; accepted June 3, 2010.Grant Support: Funded by University of Pittsburgh Department of Medicine

Startup Funds and National Institutes of Health Grant 1K08AA017622-01 toJ.B.Address reprint requests to: Jaideep Behari, M.D., Ph.D., Department of

Medicine, Division of Gastroenterology, Hepatology and Nutrition, Universityof Pittsburgh, 200 Lothrop Street, Pittsburgh, PA 15213. E-mail: beharij@upmc.edu; fax: 412-647-9268.CopyrightVC 2010 by the American Association for the Study of Liver Diseases.View this article online at wileyonlinelibrary.com.DOI 10.1002/hep.23801Potential conflict of interest: Nothing to report.Additional Supporting Information may be found in the online version of

this article.

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steatohepatitis on the experimental methionine- and chol-ine-deficient (MCD) diet.9 Surprisingly, KO mice werefound to have higher hepatic total bile acid levels on bothMCD and MCD-control diets, suggesting a defect in bileacid metabolism in addition to lipid metabolic abnormal-ities. Audard et al. reported recently that hepatocellularcarcinomas containing b-catenin mutations exhibitedstriking cholestasis.10 These findings raised the intriguingpossibility that b-catenin plays an important role in bileacid homeostasis. Therefore, this study was undertaken tofurther characterize the role of b-catenin in bile acid phys-iology in the liver.

Materials and Methods

Animals and Diets. Liver-specific b-catenin KOmice (Ctnnb1loxp/loxp;Albumin-Cre) in a C56BL/6 back-ground were generated as described.11 Age- and sex-matched littermates of KO mice with wild typeCtnnb1 alleles were used as WT controls. Genotypesof all mice were confirmed by polymerase chain reac-tion (PCR) using primers specific for b-catenin. Micewere given ad libitum access to food and water andwere maintained under a 12-hour light/dark cycle. Forexperiments on chow-diet, 2-month-old to 3-month-old female mice were used. Mice were fasted overnightbefore sacrifice and collection of serum and liver tissuesamples. For experiments on cholic acid diet, 2-

month-old to 5-month-old male mice were fed eitherchow diet (Teklad Global Diet 2018; Harlan Teklad,Madison, WI) or chow supplemented with 0.5% cho-lic acid (catalog no. TD.06026; Harlan Teklad). Micewere fasted for 4 hours before sacrifice. All animalexperiments were performed in accordance with theUniversity of Pittsburgh Animal Use and Care Com-mittee and National Institutes of Health guidelines.Histological Analysis. Formalin-fixed, paraffin-em-

bedded liver sections were stained with hematoxylinand eosin (H&E) or Reticulin by the University ofPittsburgh Research Histology services. An experiencedpathologist (E.S.) evaluated liver histology whileblinded to the treatment groups.Sections were stained for F-actin with TRITC-phalloi-

din (Sigma-Aldrich, St. Louis, MO) or claudin-2 antibody.Fluorescence (Eclipse E600, Nikon) or confocal (LSM-510) microscopes were used to capture digital images.Electron Microscopy. Liver tissue was fixed with

2.5% glutaraldehyde in phosphate-buffered saline byperfusion via the inferior vena cava. Transmission andscanning electron microscopy were performed asdescribed previously by Wack et al.12

Bile Flow Rate and Functional Analysis of He-patic Tight Junction Integrity. The common bileduct was ligated and the gallbladder was cannulated af-ter a midline laparotomy with a polyethylene (PE-10)tube. Bile was collected for 20 minutes to obtain theflow rate and for analysis of bile composition.

Fig. 1. Liver bile acid levels, serum bio-chemistry, and expression of bile acid biosyn-thetic genes in chow-fed female WT and KOmice. (A) Hepatic total bile acid (TBA) levels.(B) Serum TBA levels. (C) Serum total biliru-bin levels. (D) Serum direct bilirubin levels.(E) Expression levels of bile acid biosyntheticgenes by real-time PCR. All results areexpressed as means 6 standard error ofmean (SEM) for each group (5-7 mice pergroup). *P < 0.05.

HEPATOLOGY, Vol. 52, No. 4, 2010 YEH ET AL. 1411

Excretion rates of bile components were calculated bymeasuring the concentration of each component bybiochemical assay as described below and adjusting forbile flow rate and body weight.Functional integrity of hepatic tight junctions was

measured as described by Han et al. using fluoresceinisothiocyanate (FITC)-conjugated dextran, molecularweight 40 kDa (FD-40).13

Serum, Bile, and Tissue Biochemical Assays. Glu-tathione, phospholipids, total bile acids, and total cho-lesterol levels in bile were determined using commer-cially available kits from Biovision (Mountain View,CA), Wako Chemicals (Richmond, VA), Diazyme(Poway, CA), and Stanbio (Boerne, TX), respectively,according to the manufacturer’s instructions. Serumbilirubin, alkaline phosphatase, alanine aminotransfer-ase (ALT), and aspartate aminotransferase (AST) levelswere determined using automated methods in the Uni-versity of Pittsburgh clinical chemistry laboratory.

Hepatic total bile acid levels was determined by ho-mogenizing 100 mg minced frozen liver tissue in 0.5mL ice-cold phosphate-buffered saline with Halt prote-ase and phosphatase inhibitor cocktail (Pierce, Rock-ford, IL). Cellular debris was removed by centrifugingat 600g for 10 minutes at 4�C, and the supernatantcentrifuged at 105,000g at 4�C to recover purifiedcytosol. Cytosolic total bile acid levels were measuredusing a total bile acids kit from Diazyme and normal-ized to protein concentration for each sample.RNA Extraction and Real-Time PCR. RNA extrac-

tion and real-time PCR (RT-PCR) were performed aspreviously described using proprietary TaqMan primersfrom Applied Biosystems (Foster City, CA).14 The listof primers is provided in Supporting Table 1. Theresults are expressed as relative to that in WT mice onchow diet (set at 1).Protein Extraction and Western Blots. Crude liver

membrane extracts were prepared by homogenizing

Fig. 2. Bile flow rates and expression ofcanalicular bile acid transporters. (A) Bileflow rate in WT and KO mice (n ¼ 5 pergroup). (B) Excretion rates of major compo-nents of bile as indicated. Excretion rates ofbile constituents were calculated by meas-uring concentration in bile and correcting forthe bile flow rate (five mice per group). TBA,total bile acid. (C) Expression levels of Bsep(Abcb11) and Mrp2 (Abcc2) by real-time PCR(eight samples per group). Results areexpressed as means 6 SEM for each group.*P < 0.05. (D) Western blot analysis usingcrude membrane extracts for BSEP andMRP2. Actin is shown as the internal loadingcontrol.

1412 YEH ET AL. HEPATOLOGY, October 2010

minced tissue in 100 volumes of ice-cold homogeniz-ing buffer (0.25 M sucrose, 10 mM Tris-HCl [pH7.4]) containing protease and phosphatase inhibitorcocktail (Pierce). The homogenate was filtered througha gauze sponge and centrifuged at 100,000g for 1hour at 4�C. The resulting pellet was resuspended inresuspension buffer (0.25 M sucrose, 10 mM HEPES[4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid][pH 7.5]) containing a protease and phosphatase in-hibitor cocktail. Protein concentration was determinedby bicinchoninic acid protein assay. Western blot anal-ysis was performed as described.15 Antibodies used inthis study are listed in Supporting Table 2.Statistical Analysis. Results were analyzed using

one-way analysis of variance followed by pairwise com-parisons with the two-tailed Student t test. Values ofP < 0.05 were considered significant.

Results

Higher Serum and Hepatic Bile Acid Levels inKO Mice. Hepatic total bile acid levels were measuredin fasted chow-fed mice. Higher liver bile acid levelswere found in both female (Fig. 1A) and male (Fig.6D) KO mice. Serum bile acid levels were two-foldhigher in KO mice of both sexes (Figs. 1B and 6C).

Serum total bilirubin levels were similar in the twostrains (Fig. 1C). Serum direct bilirubin (Fig. 1D) andalkaline phosphatase (data not shown) levels were notsignificantly different (P ¼ 0.07 for both).Lower Expression Levels of Bile Acid Biosynthetic

Enzymes in KO Mice. Higher bile acid levels in KOmice may result from increased bile acid synthesis. There-fore, we measured expression levels of bile acid biosyn-thetic genes by RTPCR (Fig. 1E). Cytochrome P450 7a1(Cyp7a1; cholesterol 7a-hydroxylase), the rate limitingenzyme in the classic pathway of bile acid synthesis, andcytochrome P450 27 (Cyp27; cholesterol 27-hydroxy-lase), the rate limiting enzyme of the alternate pathway ofbile acid, were significantly down-regulated in KO liversof both sexes. Expression of cytochrome P450 8b1(Cyp8b1; sterol 12a-hydroxylase), important in the classicpathway, was lower in female KO mice (Fig. 1E) but notin male KO mice (data not shown). We conclude fromthese data that higher bile acid levels in KO mice did notresult from increased bile acid biosynthesis.Lower Bile Flow Rate in KO Mice. To determine

whether KO mice had a bile secretory defect, we meas-ured bile flow rate after bile duct cannulation. KO micehad bile flow rate less than 50% of WT levels (P <0.001) after adjusting for either body weight (Fig. 2A),or liver weight (data not shown). Excretion rates of all

Fig. 3. Expression of claudins and evaluation of tight junction integrity. (A) Western blot analysis for claudin-1 to claudin-4 in crude membranefractions from livers of WT and KO mice. Actin was used as an internal loading control. (B) Expression levels of claudin-2 by RT-PCR (five sam-ples per group). Results are expressed as means 6 SEM for each group. *P < 0.05. (C) Evaluation of hepatic tight junction integrity. Bile sam-ples were collected in 2.5-minute fractions after intravenous injection of FD-40 and analyzed for immunofluorescence. Results are expressed inarbitrary fluorescence units. Results are expressed as means 6 SEM for each group (five mice per group). *P < 0.05. (D) Transmission electronmicrographs showing bile canaliculi (BC), tight junctions (arrowheads), and hepatocytes (H). Scale bar ¼ 500 nm.

HEPATOLOGY, Vol. 52, No. 4, 2010 YEH ET AL. 1413

four major components of bile, namely total bile acids,total cholesterol, phospholipids, and total glutathione,were significantly lower in KO mice (Fig. 2B).Expression levels of bile salt export pump (BSEP or

ABCB11), responsible for bile acid-dependent bileflow, and ATP-binding cassette subfamily C member 2(ABCC2 or MRP2), responsible for bile acid-inde-pendent bile flow, were similar in WT and KO by

RTPCR (Fig. 2C). Western blot analysis showed nodifference in BSEP and modestly higher MRP2 levelsin KO mice (Fig. 2D).Down-Regulation of Claudin-2 Expression in KO

Mice. Liver tumors with activating b-catenin mutationsexhibit cholestasis and increased expression of the he-patic tight junction protein claudin-2.9,10 We measuredhepatic levels of claudin proteins by western blot

Fig. 4. Bile canalicular morphol-ogy in chow-fed WT and KO mice.Liver sections were stained for F-actin with TRITC-phalloidin. (A) WTliver showing an interconnected lat-tice of bile canaliculi (magnifica-tion ¼ �1000). A magnified viewof a bile canaliculus within thewhite square is shown in (B). (B)Magnified view of a bile canalicu-lus from a WT liver. Note the paral-lel walls and ‘‘train-track’’appearance of the canaliculus. (C)KO liver showing distorted bilecanaliculi and areas of dilatationinterspersed with short segments ofnormal-appearing canaliculi (mag-nification ¼ �1000). A magnifiedview of the area within the whitesquare is shown in (D). (D) Magni-fied view of a canaliculus from aKO liver showing an example of anabnormal ‘‘corkscrew’’-shapedcanaliculus. (E,F) Two more exam-ples of abnormal bile canalicularstructures seen in KO livers.

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Fig. 5. Electron micrographs showingbile canalicular and sinusoidal morphol-ogy. (A) Low-power scanning electronmicrograph from WT liver showing uniformand narrow bile canaliculi (arrows). (B)Low-power view from KO liver showingdilated canaliculi (arrows). (C) High-powerview from WT liver showing numerous mi-crovilli within the canaliculus (arrow-heads). Also note the abundant microvilliat the sinusoidal surfaces of hepatocytes(asterisks). (D) High-power scanning elec-tron micrographs from KO liver showingdilated and tortuous canaliculi. Note thedecreased microvilli within the canaliculus(black arrowhead) and areas devoid ofmicrovilli within the lumen (white arrow-heads). Also note the relative paucity ofmicrovilli at the sinusoidal surface of thehepatocytes at the bottom left (asterisk).(E) Transmission electron micrograph fromWT liver showing abundant microvilli(arrowheads) at the sinusoidal surface.(F) KO liver showing decreased microvilliat the sinusoidal surface (arrowheads).Ec, endothelial cell; RBC, red blood cell;S, sinusoid.

Fig. 6. Effect of cholic acid feeding. (A)Growth curves on chow or cholic acid diet formale WT and KO mice. *P < 0.05 for bodyweight difference between WT and KO miceon chow diet. (B) Liver weight:body weight ra-tio. (C) Hepatic total bile acid (TBA) levels.(D) Serum total bile acid levels. Results areexpressed as means 6 SEM for each group.*P < 0.05 for WT versus KO mice on thesame diet; #P < 0.05 for chow and cholicacid–fed mice of same genotype (N ¼ 5-7mice per group for all experiments). CA, cho-lic acid; CTL, control.

HEPATOLOGY, Vol. 52, No. 4, 2010 YEH ET AL. 1415

analysis. Although no differences were found in claudin1 and 3 levels, claudin-2 was nearly undetectable in KOlivers (Fig. 3A). Bands for claudin-4, which is notexpressed in the liver, and claudin-5, which is expressedonly in endothelial junctions,16 were not detected (Fig.3A and data not shown). RTPCR analysis showed thatexpression of the claudin-2 gene was 10-fold lower inKO mice, suggesting its regulation at the transcriptionallevel by b-catenin (Fig. 3B). Immunostaining for clau-din-2 showed prominent zone 3 staining in WT butnot in KO livers (Supporting Fig. 2).

Hepatic tight junctions form the blood–bile barrierthat keeps sinusoidal blood spatially separated fromcanalicular bile. We asked whether disruption of he-patic tight junction integrity from loss of claudin-2could account for the bile secretory defect in KOmice. We tested the functional integrity of hepatictight junctions in KO mice by assaying for biliary FD-40 excretion after intravenous injection (Fig. 3C). Dis-ruption of tight junctions causes an early peak in bilefluorescence.13 No such early peak in fluorescence wasdetected in KO mice to suggest defective tight junc-tion integrity. Instead, KO mice had a significant delayin FD-40 excretion in bile, likely resulting from theirlower bile flow rate. Evaluation by transmission elec-tron microscopy showed normal appearing tight junc-tions in KO mice (Fig. 3D).Bile Canalicular Abnormalities in KO Mice. To

further evaluate the cholestatic phenotype in KO mice, westained liver sections with TRITC-phalloidin for F-actin,which exhibits pericanalicular localization in hepatocytes.WT livers exhibited interconnected, evenly spaced ‘‘train-track’’ bile canaliculi (Fig. 4A,B). In contrast, KO liversshowed distorted bile canaliculi (Fig. 4C,D) with occa-sional grossly misshapen ‘‘corkscrew’’ shaped canaliculi(Fig. 4D-F). We confirmed that these abnormal structuresseen in KO livers on F-actin staining were bile canaliculiby double-labeling liver sections with TRITC-phalloidinand anti–zona occludens 1 antibody (Supporting Fig. 2).Bile canalicular morphology was also evaluated by scan-

ning electron microscopy (Fig. 5A-D). Canaliculi in KOlivers were dilated, with an average diameter of approxi-mately 1 lM, which was 25%-40% greater than in WTmice. Canaliculi in KO livers were tortuous and showedfrequent blind loops. Strikingly, there was a marked pau-city of microvilli within canaliculi in KO mice with corre-sponding bare areas within the canalicular lumina (Fig.5C,D). The subsinusoidal surface of KO hepatocytes alsoshowed a relative decrease in microvilli by both scanning(Fig. 5D) and transmission electron microscopy (Fig. 5F).The ultrastructure of bile ducts within portal triadsappeared normal in KO mice (data not shown).Effect of Cholic Acid–Containing Diet on Liver

Histology in KO Mice. Cholic acid feeding has beenused to study bile acid-mediated liver toxicity.17 Todetermine the effect of cholic acid feeding, mice werefed either chow or chow supplemented with 0.5%cholic acid for 2 weeks. Both strains of mice had bodyweight loss on the cholic acid diet (Fig. 6A) butincrease in liver weight and liver-to-body weight ratio(Fig. 6B). The latter finding is consistent with theresults of Huang et al. showing bile acid–dependentliver growth and regeneration.18

Fig. 7. Analysis of expression of hepatic bile acid transporters andbile acid regulatory genes in chow and cholic acid–fed WT and KOmice by RT-PCR. (A) Basolateral transporters. (B) Canalicular transport-ers. (C) Genes involved in regulation of bile acid biosynthesis. #P <0.05 between chow-fed and cholic acid–fed groups; *P < 0.05between WT and KO groups (4-5 mice per group).

1416 YEH ET AL. HEPATOLOGY, October 2010

Serum ALT and AST levels were higher in both cho-lic acid–fed groups, but not different between WT andKO mice (data not shown). However, serum total bileacid levels in cholic acid–fed KO mice were approxi-mately five-fold higher and hepatic bile acid levels three-fold higher than in WTmice, suggesting defective abilityto regulate serum and liver bile acid levels (Fig. 6C,D).Gene Expression Changes on Cholic Acid Diet. Ex-

pression of the sinusoidal bile acid uptake transportersNtcp, Slco1a1, and Slco1b2 were lower in both cholicacid–fed groups (Fig. 7A). Expression of the latter twogenes was also lower in chow-fed KO mice. Expressionof the basolateral efflux transporters Abcc3 (Mrp3)and Abcc4 (Mrp4) was not different, although therewas a trend toward higher Abcc4 levels in KO mice.Among the canalicular transporters, expression of Bsep

(Abcb11) was lower in cholic acid–fed KO mice,whereas Abcb4 (Mdr2) was up-regulated in both cho-lic acid–fed groups (Fig. 7B). Expression levels ofother sinusoidal transporters were similar between thecholic acid–fed groups.Of the bile acid regulatory genes, expression of farne-

soid X receptor (FXR) was lower in both cholic acid–fedgroups. However, Shp-1 expression, a downstream targetof FXR, was up-regulated in both WTand KO mice on acholic acid diet (Fig. 7C). Fgf4r levels were similar in allfour groups. Expression of constitutive androstane recep-tor (CAR) was lower in both cholic acid–fed groups andin chow-fed KO mice. However, despite lower expressionlevels of CAR, KO mice had higher expression of thedownstream target of CAR, Cyp2b10. This result suggeststhat activation of CAR, possibly by a post-transcriptional

Fig. 8. Liver histology in chow-fed and cholic acid–fed WT and KO mice. (A,E,I,M): WT/chow-fed; (B,F,J,N): WT/cholic acid–fed; (C,G,K,O): KO/chow-fed; (D,H,L,P): KO/cholic acid–fed. (A-D) H&E stained liver sections showing increased lobular inflammation in both cholic acid–fed groups.Magnification ¼ �100. (E-H) High-power view of H&E-stained sections showing portal triads. Note the bile ductular reaction in KO/cholic acidgroup in (H). Magnification ¼ �400. (I-L) Reticulin staining for fibrosis. Note the increased pericellular fibrosis in the KO/cholic acid group in(L). Magnification ¼ �400. (M-P) TRITC-phalloidin staining for F-actin showing bile canalicular morphology. Note the dilated but uniformly con-toured ‘‘train-track’’ canaliculi in cholic acid–fed WT mice (N), areas of irregular F-actin staining and focal dilation in the KO/chow-fed group (O),and the dilated, tortuous bile canaliculi in the KO/cholic acid–fed group (P). Magnification ¼ �1000.

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mechanism, occurs in KO mice and may represent a pro-tective mechanism to down-regulate bile acid biosyntheticgenes. PPARa expression was higher in both cholic acidgroups whereas pregnane X receptor expression was lowerin cholic acid–fed KO mice.Liver Histology and Bile Canalicular Morphology

on Cholic Acid Diet. After H&E staining, histologysamples from WT mice showed mildly increased lobu-lar inflammation on cholic acid diet (Fig. 8). On theother hand, cholic acid–fed KO mice had increasedductular reaction and greater lobular inflammation oncholic acid diet along with increased pericellular fibro-sis on reticulin staining.Staining for F-actin showed that WTmice on cholic

acid diet had dilated bile canaliculi, likely reflectingthe choleretic effect of bile acids (Fig. 8, bottompanel). However, the interconnected lattice-like struc-ture of canaliculi was preserved in WT livers on cholicacid diet. On the other hand, KO mice on cholic aciddiet had canaliculi that were not only dilated but alsotortuous and distorted (Fig. 8P).

Discussion

Formation and excretion of bile is one of the impor-tant functions of the liver. This task is achieved by thecoordinated action of multiple proteins and cellular or-ganelles. Disruption in canalicular transporter func-tion, loss of nuclear receptors, or disruption of tightjunction integrity can result in defective bile secretionand intrahepatic cholestasis.19-22 Results presented inthis article establish that the Wnt/b-catenin pathway isanother regulatory input in the complex process ofbile secretion and an important mediator of normalbile canalicular morphogenesis.The most significant finding of our study is that

loss of b-catenin causes bile canalicular abnormalitiescharacterized by dilatation, tortuosity, and loss of cana-licular microvilli. It is noteworthy that two down-stream targets of b-catenin, claudin-2 and senescencemarker protein-30 (SMP30), have been independentlyshown to be important in bile canalicular formation invitro and SMP30 has been implicated in the formationof microvilli in hepatoma cells.23,24 In further supportof a role of b-catenin in the process of canalicularmorphogenesis, Theard et al. have shown that deple-tion of b-catenin-E-cadherin based adherens junctionsleads to defective canalicular lumen remodeling, sug-gesting that b-catenin may be involved in bile canalic-ular morphogenesis via more than one mechanism.25

Because b-catenin plays an important role inanchoring the actin cytoskeleton to the cell membrane,

it is plausible that KO mice have defective bile canalic-ular contractility that leads to cholestasis and canalicu-lar dilatation. A similar mechanism has been suggestedfor connexin-32–deficient mice that exhibit canaliculardilatation and decrease in sympathetic nerve-stimulatedbile secretion.26 Furthermore, disruption of the actin-containing microfilament network by cytochalasinleads to decreased cytoplasmic contractile movementsand dilation of bile canalicular lumina.27 Furtherexperiments to test this hypothesis in b-catenin KOmice are currently ongoing.We propose the following model for the cholestatic

phenotype observed in b-catenin KO mice: Loss of b-catenin, either via alteration of the actin cytoskeleton-adherens junction interactions or via loss of claudin-2and SMP30, results in bile canalicular morphologicalabnormalities and bile secretory defect. Decreased bileflow contributes to the development of intrahepaticcholestasis. As a compensatory mechanism, expressionof bile acid biosynthetic enzymes is down-regulated,along with that of bile acid uptake transporters,Slco1a1 and Slco1b2. CAR may be functionally acti-vated and play a protective role in KO mice becauseexpression of its downstream target Cyp2b10 is signifi-cantly higher in KO mice. On being stressed with cho-lic acid, there is activation of additional protectivemechanisms, including down-regulation of the uptaketransporter, Ntcp, up-regulation of the efflux trans-porter Mdr2, and up-regulation of Shp-1, an FXR-tar-get gene that is a negative regulator of bile acid bio-synthesis. CAR and FXR/Shp-1 have previously beenshown to be activated by bile acids and negatively reg-ulate bile acid biosynthesis.28,29

In summary, b-catenin KO mice exhibit intrahepaticcholestasis, lower bile flow rates, and abnormalities inbile canalicular morphology. Thus, in addition to itsmultifaceted roles in liver biology, b-catenin plays animportant role in biliary physiology in the adult mam-malian liver.

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