Dual catenin loss in murine liver causes tight junctional deregulation and progressive intrahepatic cholestasis

Animals

Previously reported homozygous β-catenin–floxed mice and γ-catenin–floxed mice were interbred to generate double-floxed mice.6, 9 Conditional deletion of both β- and γ-catenin is described in the Supporting Information. All animal experiments and procedures were performed according to the National Institutes of Health guidelines under an animal protocol approved by the Institutional Animal Use and Care Committee at University of Pittsburgh.

Viruses and Infections

AAV8-TBG-Cre or AAV8-TBG-GFP were obtained from Penn Vector Core at the University of Pennsylvania. Four-week-old or older β- and γ-double–floxed mice were given a single intraperitoneal injection of 2.5 × 10¹¹ genome copies of AAV8-TBG-Cre or AAV8-TBG-GFP. At least three mice per time point ranging from 7 to 14 days were used for the studies.

EVAN'S BLUE VASCULAR PERMEABILITY ASSAY

Next, 0.5% sterile solution of Evans blue was prepared in phosphate-buffered saline. At 10 or 12 days postinjection, 200 μL of Evans blue was injected in the inferior vena cava of double wild-type (dWT) or DKO2 mice. Bile was collected from the gall bladder 15 minutes after inferior vena cava injection to analyze the presence of Evans blue dye using fluorometric measurement.12 All experiments were performed in triplicate.

TRANSMISSION ELECTRON MICROSCOPY

For transmission electron microscopy, whole liver was perfused fixed in glutaraldehyde. The surgical and perfusion techniques have been described previously.5, 9 Slides were examined in a JEM 1011 transmission electron microscope at 80 kV. For lanthanum staining, tissues were incubated with lanthanum hydroxide (pH 7.8) for 3 hours at room temperature.13, 14 Lanthanum hydroxide was prepared by slowly adding drops of 0.01N NaOH to 2% lanthanum nitrate until the solution reached a pH of 7.8.

INTRAVITAL IMAGING OF LIVER

Intravital imaging of liver is described in detail elsewhere.15 Briefly, mice were anesthetized and placed on a heated stage. A catheter was placed into the right carotid artery to enable intravenous delivery of intravascular fluorochromes. Gentle vacuum suction was applied to the abdominal imaging window device to immobilize. FITC dextran (MW:70,000) or Texas Red (TXR) dextran (MW:70,000) was used to visualize the blood flow through liver sinusoids, whereas carboxyfluorescein (MW:367.82) was used to visualize biliary canaliculi.16 After being internalized by hepatocytes, carboxyfluorescein is hydrolyzed by esterase into fluorogenic carboxyfluorescein (CF), which emits green fluorescence at 517 nm. In wild-type (WT) mice, carboxyfluorescein is taken up and hydrolyzed into CF in 1-5 minutes after injection. Videos and images were recorded at 5, 10, and 30 minutes postinjection). Microscopy was performed using a Nikon multiphoton excitation microscope at the imaging service of the Pittsburgh Liver Research Center.

TOTAL INTERNAL REFLECTION FLUORESCENCE IMAGING

The method of total internal reflection fluorescence (TIRF) imaging has been described elsewhere.17 Briefly, a frozen section of hepatocytes was fixed on a coverslip. Immunofluorescence (IF) assay was performed using E-cadherin and occludin antibodies as described previously.17 Coverslips were mounted with phosphate-buffered saline and vectashield. Microscopy was performed using a Nikon TIRF microscope.

Additional methods are available in the Supporting Information.

SIMULTANEOUS DELETION OF β-CATENIN AND γ-CATENIN IN THE LIVER DURING DEVELOPMENT RESULTS IN SIGNIFICANT MORTALITY AND MORBIDITY

Liver-specific β- and γ-catenin DKO mice were generated using Alb-Cre as described in the Materials and Methods section and confirmed by genomic DNA polymerase chain reaction (PCR) analysis (Fig. 1A). When compared with WT, increased mortality was evident in DKO with >80% dying by 30 days and no survivors beyond 2.5 months (Fig. 1B). Both male and female DKO mice were smaller in body size compared with WT mice (Fig. 1C). Grossly, livers of DKO mice were yellow, smaller and stiffer as compared with WT mice at 24 days, albeit the liver weight to body weight ratio (LW/BW) was comparable to WT (Fig. 1D,E). DKO mice that survived to 2.5 months had higher LW/BW, and showed yellow and stiffer livers along with splenomegaly, a sign of portal hypertension (Fig. 1D,E). Intriguingly, serum alanine aminotransferase (ALT) levels were unchanged, although ALP and total and direct bilirubin were significantly up-regulated in DKO mice (Fig. 1F,G). Western blot (WB) and immunohistochemical (IHC) assay confirmed loss of both β-catenin and γ-catenin in DKO livers compared with livers from WT mice (Figs. 1H and 2A).

DKO MICE SHOW EXTENSIVE DUCTULAR REACTION, LIVER FIBROSIS, INFLAMMATION, TUMORIGENESIS, AND LOSS OF E-CADHERIN

To further characterize the DKO phenotype displaying notable morbidity in neonatal stages, we assessed histology and performed IHC analysis for injury and inflammation markers in DKO livers 24 days after birth. A notable increase in the presence of smaller ductular hepatocytes, especially in the periportal region, were evident on hematoxylin and eosin staining in DKO livers (Fig. 2B). Enhanced ductular reaction was also evident as seen by a notable increase in the numbers of ductules positive for epithelial cell adhesion molecule and CK-19 (Fig. 2C,D). A profound increase in numbers of ductular hepatocytes was also verified by sox-9 staining, as is often seen in CLD (Fig. 2D). Accompanying the ductular reaction, a noteworthy increase in hepatic fibrosis as seen by Sirius red staining along with increased presence of activated myofibroblasts as evident by α-smooth muscle actin (α-SMA) IHC analysis was also observed in DKO at 24 days (Fig. 2E).

Because 10% of mice survived to around 2.5 months, we assessed the phenotype of DKO livers at this stage. A notable increase in CD45-positive inflammatory cell infiltrate was evident in DKO at this time (Supporting Fig. S1A). Sirius red and α-SMA staining showed extensive collagen deposition and activated myofibroblasts, respectively, in DKO mice (Supporting Fig. S1A). Enhanced fibrosis was also confirmed by increased expression of profibrogenic markers like col1a1, Pdgfra, and Tgfb1 by reverse-transcription PCR (RT-PCR) analysis (Supporting Fig. S1B).

Any compensatory changes in ductular and hepatocyte proliferation were assessed next by IHC analysis for Ki-67 at 24 days and 2.5 months (Supporting Fig. S2A,B). Careful quantification showed greater baseline Ki-67 staining in hepatocytes in WT livers at 24 days due to known postnatal hepatic growth; however, DKO mice showed significantly fewer Ki-67–positive hepatocytes (Supporting Fig. S2C). Intriguingly, a significant increase in Ki-67–positive ductular cells was evident in DKO mice (Supporting Fig. 2C). At 2.5 months, both hepatocytes and ductular cells showed significantly more Ki-67 positivity in DKO mice compared with WT mice (Supporting Fig. S2D).

As a result of the chronic injury and regeneration, dysplastic nodules were apparent especially at 2.5 months in DKO mice (Supporting Fig. S1A). A subset of such nodules progressed to frank hepatocellular carcinoma (Supporting Fig. S2E).

LOSS OF β- AND γ-CATENIN LEADS TO IMPAIRED BILE FLOW AND ALTERED EXPRESSION OF SEVERAL JUNCTIONAL PROTEINS AND BILIARY TRANSPORTERS

Elevated serum ALP and bilirubin, extensive ductular reaction and increased biliary markers in hepatocytes, all indicated development of CLD in DKO mice. Indeed, in WT mice where gall bladders were intact and filled with bile, in DKO these structures were involuted and without bile (Fig. 3A). We next assessed whether cholestasis is leading to altered levels of BA in the liver and blood to also validate the presence of cholemia. DKO showed around three-fold higher hepatic BA levels and almost 300-fold higher serum BA levels (Fig. 3B,C). Associated with increased BA levels, we observed notable ductular reaction at this stage (Supporting Fig. S3A). Also, increased TUNEL-positive apoptotic nuclei were evident in the livers of DKO mice (Supporting Fig. S3B).

It was intriguing that despite increased hepatocyte injury, serum ALT levels remained unchanged. We hypothesized this to be due to inappropriate hepatocyte maturation since DKO livers showed several sox-9–positive ductular hepatocytes (Fig. 2D). Indeed, RT-PCR for targets of a major hepatocyte transcriptional regulator HNF4α18 showed significant decreases in its positive targets, including Acot3, Ces3, Ugt2b1, and Dio1, and increased expression of negative targets, including Akr1b7, Ect2, Egr1, and Myc, in DKO livers (Supporting Fig. S3C). Expression of Foxa2, another early hepatic transcription factor was also reduced (Supporting Fig. S3D). Likewise, expression of the ALT gene was significantly reduced in DKO livers (Supporting Fig. S3E), suggesting lack of increased serum ALT despite increased hepatocyte injury is due to improper hepatocyte maturation.

We next assessed localization of biliary canalicular markers to address the structure and viability of intrahepatic biliary canaliculi. IHC and IF verified decreased levels and mislocalization of CD10, occludin, claudin-2, and ZO-1 in DKO livers, suggesting perturbations in biliary canaliculi (Fig. 3D).

To further address any molecular aberrations that may be contributing to cholestasis and cholemia, expression of various biliary transporters, BA metabolism genes, and TJ proteins were assessed. WB from whole liver lysates showed decreases in several junctional proteins such as occludin, claudin-2, JAM-A, and E-cadherin in DKO mice, whereas ZO-2 (TJP2) expression was increased (Fig. 3E). E-cadherin decrease was also verified by way of IHC analysis in DKO (Supporting Fig. S4A). Bile salt export pump (BSEP) protein levels were decreased (Fig. 3E) while its messenger RNA (mRNA) expression was unchanged (Supporting Fig.4B). RT-PCR also revealed decreases in the mRNA expression of NTCP, Cyp7a1, and MRP2 in DKO (Supporting Fig. S4B).

Thus overall, DKO mice exhibit a phenotype that is truly reminiscent of PFIC patients showing a plethora of aberrations including defects in expression of biliary transporter BSEP and deregulation of a host of hepatic junctional proteins.

SPECIFIC DELETION OF β- AND γ-CATENIN ONLY FROM HEPATOCYTES LEADS TO TIME-DEPENDENT CHOLEMIA

To identify the primary molecular basis and sequence of the phenotype evident in DKO mice, we conditionally deleted β- and γ-catenin from the hepatocytes only by injecting AAV8-TBG-Cre in 4-week-old β- and γ-catenin double-floxed mice (DKO2) and sacrificing at 7-14 days for evaluation (Supporting Fig. S5A).19 As a control, β- and γ-catenin double-floxed mice were injected with AAV8-TBG-GFP (dWT). To show the robustness and hepatocyte selectivity, IHC for GFP was performed at 7 days postinjection of AAV8-TBG-GFP. Almost 100% of hepatocytes and no cholangiocytes were GFP-positive (Supporting Fig. S5B). Efficacy of AAV8-TBG-Cre was also evident by a significant and sustained decrease in the protein and mRNA expression both catenins in DKO2 livers at 7-14 days postinjection compared with dWT (Supporting Fig. S5C-E). Because we posit that loss of both catenins simultaneously from hepatocytes affects junctional stability, we assessed serum from DKO2 mice at various times. Although serum bilirubin (both total and direct) and ALP were unremarkable at day 7 and day 10, a significant and progressive increase in their levels was evident at days 12-14 (Supporting Fig. S5F). An increase in ductular reaction was also observed at day 14 in DKO2 livers (Supporting Fig. S5G). Thus, an acute and hepatocyte-specific loss of β- and γ-catenin resulted in an acute onset of cholemia similar to DKO and provided a unique opportunity to examine sequentially both cellular and molecular basis of the observed pathology.

UNIQUE PARA-HEPATOCYTE LEAKS LEAD TO ADMIXING OF BLOOD AND BILE AND CHANNELING OF BLOOD FROM ONE SINUSOID TO ANOTHER IN DKO LIVERS AS SHOWN BY MULTIPHOTON INTRAVITAL MICROSCOPY

To investigate the structural basis of observed cholemia at the earliest time in DKO2 mice, we used multiphoton excitation–enabled in vivo real-time fluorescence microscopy of intact liver in live mice as described previously.15 FITC-dextran was intravascularly administered to visualize the blood flow in liver sinusoids. Blood flow (green) was restricted and linear within sinusoids in dWT mice (Fig. 4A; Supporting Movies S1 and S2). However, thin conduits reminiscent of para-hepatocyte leaks carrying cell-free plasma (green) were observed connecting adjacent sinusoids outlining single hepatocytes in DKO2 livers at day 12 (Fig. 4A; Supporting Movies S3 and S4).

To visualize both blood and bile flow using multiphoton excitation–enabled intravital microscopy, we next used TXR-dextran (red) to label blood flow through liver sinusoids and CF (green) to envision bile flow through biliary canaliculi.20 In dWT mice, the uptake of CF in hepatocytes occurred within 1-5 minutes after injection and CF appeared as a linear green streak highlighting its secretion into biliary canaliculi (Fig. 4B; Supporting Movie S5). Red fluorescence indicated normal blood flow in sinusoids. At 10 minutes, CF prominently localized to biliary canaliculi (Fig. 4B; Supporting Movie S6). By 30 minutes, CF decreased considerably in biliary canaliculi, suggesting its clearance from the intrahepatic biliary compartment (Fig. 4B; Supporting Movie S7). DKO2 at day 12 showed a peculiar and unique phenotype in which TXR-dextran–positive red spots along with CF were found in punctate patterns within sinusoids along with absence of green linear biliary canalicular presence at 5, 10, and 30 minutes (Fig. 4B; Supporting Movies S8-S10). Furthermore, thin conduits reminiscent of para-hepatocyte leaks, outlining hepatocytes and interconnecting sinusoids through which CF and TXR-dextran flowed freely, were evident in DKO2 mice at all times (Supporting Movies S8-S10).

In vivo vascular permeability assay using Evans blue vascular dye was performed next.12 Complete absence of Evans blue in the bile was observed after a single intravenous injection of Evans blue to DKO2 mice at day 10 (Fig. 4C). However, at day 12, Evans blue was significantly increased in bile in DKO2 but not dWT mice (Fig. 4C).

Altogether, these observations allowed a direct visualization of structural perturbations in the form of para-hepatocyte leaks connecting adjacent sinusoids and sinusoids to biliary canaliculi, demonstrating the basis of cholemia in DKO2 mice at 12 days.

DUAL LOSS OF β- AND γ-CATENIN LEADS TO THE DISRUPTION OF HEPATOCYTE JUNCTIONS

Because β-catenin—and, in its stead, γ-catenin—is a structural component of AJ, to understand the primary mechanism that led to disruption of the blood–biliary barrier following the loss of both, we next assessed the structure of AJ and the barrier forming TJ in DKO2 and dWT livers. Transmission electron microscopy of TJ and AJ in dWT liver sections at any stage (shown for day 7) revealed TJ as electron-dense structures in the space between cells near the bile canaliculi abutted by AJ, which is evident as thin double-stranded structure of defined diameter (Fig. 5A). In DKO2 livers, the electron dense TJ structure appeared normal at day 7; however, at days 10 and 14, TJ contained less electron-dense material, resulting in a dilated structure (Fig. 5A). AJ in DKO2 livers showed increased intercellular distance at day 7, which were further dilated by day 10, and their structure was lost by day 14 (Fig. 5A).

TJ integrity was tested next using lanthanum hydroxide permeability.13 Intact TJs surrounding biliary canaliculi shown by lanthanum hydroxide exclusion from TJ showing only its extracellular distribution signified an intact barrier in the dWT livers (Fig. 5B). In DKO2 livers, such exclusion was apparent at day 7; however, by day 10 and 14, lanthanum was seen accumulating in the TJ, indicating loss of structural integrity (Fig. 5B).

DUAL LOSS OF β- AND γ-CATENIN LEADS TO DISTORTION OF BILIARY CANALICULAR NETWORK

Biliary canalicular networks, which are lined by the apical surfaces of hepatocytes and flanked by TJ proteins, allow unidirectional flow of bile secreted by hepatocytes toward the portal triad. Because the simultaneous loss of the two catenins disrupted TJ, we next investigated whether this results in any disruption of the structural integrity and patency of biliary canalicular networks. We stained liver sections from DKO2 and dWT mice with TRITC-phalloidin to visualize F-actin, which normally marks the pericanalicular outline of hepatocytes. dWT livers at d12 exhibited interconnected, evenly spaced “train track” bile canalicular structures (Fig. 5C). In contrast, DKO2 livers at day 12 showed pronounced distortion of bile canaliculi observed as misshapen, collapsed, and incomplete network structures (Fig. 5C).

DUAL LOSS OF β- AND γ-CATENIN LEADS TO DECREASED LEVELS OF KEY JUNCTIONAL PROTEINS

To address the temporal deregulation of various junctional proteins following loss of β- and γ-catenin, we next investigated the protein and mRNA expression of these proteins in DKO2 and dWT livers. Decrease in E-cadherin protein was observed at days 10, 12, and 14 (Fig. 6A). This was also visible by IF, which not only displayed E-cadherin decrease at hepatocyte membrane but also showed its redistribution as punctate cytoplasmic staining in the DKO2 livers at day 12, suggestive of proteasomal degradation (Fig. 6B). Indeed, mRNA expression of E-cadherin was unchanged until day 14 (Fig. 6C). Next, we assessed claudins, which are tetraspanins and key components of TJ in DKO2 liver lysates. Claudin-3 and -5, which are plaque-forming, were decreased at day 12-14 (Fig. 6A). IF staining verified a notable decrease in surface localization of claudin-3 and -5 at hepatocyte surface at day 12 as well (Fig. 6D). Claudin-2, a known Wnt/β-catenin target in the liver, was significantly decreased at day 7-14 in DKO2 livers by both WB and RT-PCR (Fig. 6A,C). Occludin, another TJ tetraspanin protein, was also decreased at days 12 and 14, whereas its mRNA expression was unaffected until day 14 (Fig. 6A,C). Occludin staining at the TJ in hepatocytes was decreased in DKO2 livers compared with dWT at day 12, whereas ZO-1 remained unchanged (data not shown).

To unequivocally demonstrate the lack of localization of E-cadherin and occludin at cell surface in the DKO2 livers, we used TIRF microscopy. TIRF revealed E-cadherin and occludin localizing at hepatocyte membrane in dWT livers, which was completely lost in DKO2 livers at 12d (Fig. 6E).

Thus, dual loss of catenins triggers deregulation of E-cadherin, along with of key TJ proteins including occludin and claudin-2, -3, and -5 in the DKO2 livers.

ABSENCE OF β- AND γ-CATENIN LEADS TO ENHANCED DEGRADATION OF E-CADHERIN AND KEY TJ PROTEINS, WHICH IS ALLEVIATED BY PROTEASOME INHIBITOR WHEREAS CLAUDIN-2 LOSS IS UNAFFECTED

Next, we addressed the basis of decrease in E-cadherin, occluding, and claudins following loss of both catenins in vitro and in vivo. First, we used human Hep3B cells to determine whether TJ proteins such as occludin coprecipitate with AJ proteins. Immunoprecipitation (IP) studies in scrambled small interfering RNA (siRNA) (scrambled [Sc])-transfected Hep3B cells showed association of occludin with E-cadherin, β-catenin, and γ-catenin (Fig. 7A). Dual silencing of β- and γ-catenin (SiBG) led to abrogation of this association (Fig. 7A). SiBG transfection in Hep3B cells also led to notable decreases in total E-cadherin, occludin, claudin-2, and claudin-3 (Fig. 7B,C). Blocking proteasomal degradation by MG132 in SiBG-transfected cultures rescued E-cadherin, occluding, and claudin-3 but not claudin-2 (Fig. 7B,C). Similarly, increased E-cadherin ubiquitination was observed upon SiBG transfection in Hep3B cells (Fig. 7D). Blocking lysosomal degradation by cyclohexamide did not influence stabilization of any of these proteins, and no advantage of combining MG132 and cyclohexamide was observed (Fig. 7B).

Because a decrease in AJ component E-cadherin due to proteasomal degradation, preceded a loss of TJ proteins both in vitro and in vivo, we next silenced E-cadherin in Hep3B cells. SiBG transfection in Hep3B cells led to notable decreases in occludin and claudin-3 but did not affect β- or γ-catenin levels (Fig. 7E).

To confirm that the mechanism of E-cadherin and occludin loss in vivo in DKO2 livers was indeed evidence of proteasomal degradation, we immunoprecipitated E-cadherin or occludin and performed WB analysis for poly- and mono-ubiquitin. A notable and time-dependent increase in ubiquitination of both proteins was observed at days 10-12 in DKO2 livers (Fig. 7F,G).