AUTOIMMUNE, CHOLESTATIC AND BILIARY DISEASE
Knockdown of Ezrin Causes Intrahepatic Cholestasis by the Dysregulation of Bile Fluidity in the Bile Duct Epithelium in Mice Ryo Hatano,1 Kaori Akiyama,1 Atsushi Tamura,2 Shigekuni Hosogi,3 Yoshinori Marunaka,3 Michael J. Caplan,4 Yoshiyuki Ueno,5 Sachiko Tsukita,2 and Shinji Asano1 Cholangiopathies share common features, including bile duct proliferation, periportal fibrosis, and intrahepatic cholestasis. Damage of biliary epithelium by autoimunne disorder, virus infection, toxic compounds, and developmental abnormalities causes severe progressive hepatic disorders responsible for high mortality. However, the etiologies of these cholestatic diseases remain unclear because useful models to study the pathogenic mechanisms are not available. In the present study, we have found that ezrin knockdown (Vil2kd/kd) mice develop severe intrahepatic cholestasis characterized by extensive bile duct proliferation, periductular fibrosis, and intrahepatic bile acid accumulation without developmental defects of bile duct morphology and infiltration of inflammatory cells. Ezrin is a membrane cytoskeletal crosslinker protein, which is known to interact with transporters, scaffold proteins, and actin cytoskeleton at the plasma membrane. We found that the normal apical membrane localizations of several transport proteins including cystic fibrosis transmembrane conductance regulator (CFTR), anion exchanger 2 (AE-2), aquaporin 1 (AQP1), and Na1/H1 exchanger regulatory factor were disturbed in bile ducts of Vil2kd/kd mice. Stable expression of a dominant negative form of ezrin in immortalized mouse cholangiocytes also led to the reduction of the surface expression of CFTR, AE-2, and AQP1. Reduced surface expression of these transport proteins was accompanied by reduced functional expression, as evidenced by the fact these cells exhibited decreased CFTR-mediated Cl2 efflux activity. Furthermore, bile flow and biliary HCO32 concentration were also significantly reduced in Vil2kd/kd mice. Conclusion: Dysfunction of ezrin mimics important aspects of the pathological mechanisms responsible for cholangiopathies. The Vil2kd/kd mouse may be a useful model to exploit in the development and testing of potential therapies for cholangiopathies. (HEPATOLOGY 2015;61:1660-1671) See Editorial on Page 1467
C
holangiocytes are epithelial cells that line hepatic bile ducts. They constitute only 3%5% of liver cells. The intrahepatic bile ducts
play important roles in modulating the fluidity and alkalinity of canalicular bile by secreting electrolytes, primarily Cl2 and HCO32.1 Osmotic gradients increase the flow of bile by inducing the secondary secretion of water.1 Several membrane transport proteins and channels have been identified and characterized in
Abbreviations: Abs, antibodies; AE-2, anion exchanger 2; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AMA, antimitochondrial antibody; AQP1, aquaporin 1; AST, aspartate aminotransferase; cAMP, cyclic adenosine monophosphate; CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; CK19, cytokeratin 19; dbcAMP, dibutylyl-cyclic AMP; EIA, enzyme immunoassay; ERM, ezrin-radixin-moesin; H&E, hematoxylin-eosin; HSCs, hepatic stellate cells; IBDUs, isolated bile duct units; IF, immunofluorescence; IL-1b, interleukin 1b; MIP-2a, macrophage inflammatory protein 2 alpha; MQAE, N(ethoxycarbonyl methyl)-6-methoxy quinolinium; mRNA, messenger RNA; NHE3, sodium-hydrogen exchanger 3; NHERF, Na1/H1 exchanger regulatory factor; NMC, normal mouse cholangiocyte; p-ANCA, perinuclear antineutrophil cytoplasmic antibody; PBC, primary biliary cirrhosis; PBS, phosphate-buffered saline; PDZ, PSD-95/Discs-large/ZO-1; PKA, protein kinase A; Procol1, procollagen type I; Procol3, procollagen type III; PSC, primary sclerosing cholangitis; RT-PCR, reverse-transcriptase polymerase chain reaction; TGF-b, transforming growth factor beta; TNF-a, tumor necrosis factor alpha; WT, wild type. From the 1Department of Molecular Physiology, College of Pharmaceutical Sciences, Ritsumeikan University, Shiga, Japan; 2Laboratory of Biological Science, Graduate School of Frontier Biosciences and Graduate School of Medicine, Osaka University, Osaka, Japan; 3Department of Molecular Cell Physiology and BioIonomics, Kyoto Prefectural University of Medicine, Kyoto, Japan; 4Department of Cellular and Molecular Physiology, Yale School of Medicine, New Haven, CT; and 5Department of Gastroenterology, Yamagata University School of Medicine, Yamagata, Japan Received May 19, 2014; accepted October 10, 2014. Additional Supporting Information may be found at onlinelibrary.wiley.com/doi/10.1002/hep.27565/suppinfo. 1660
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mammalian bile duct epithelium1 and are thought to participate in modifying the composition of canalicular bile. Cholangiocytes transport HCO32 into the bile, which is mediated by the Na1-independent Cl2/HCO32 exchanger, anion exchanger 2 (AE-2). AE-2 functions as a secondary active transporter, which is driven by the high lumen concentrations of Cl- that are in turn generated by secretion mediated by a cyclic adenosine monophosphate (cAMP)-stimulated low conductance Cl2 channel, cystic fibrosis transmembrane conductance regulator (CFTR).13 Furthermore, in response to osmotic gradients, the water-channel protein, aquaporin 1 (AQP1), mediates water movement from cholangiocyte into bile.2,3 The coordinated functions of these transporters are extremely important for regulation of bile fluidity and alkalinity. These processes are dynamically regulated by a gastrointestinal hormone, secretin.2 Recent studies suggest that these transporters are stored together in the same intracellular vesicles within cholangiocytes, and that they are dynamically trafficked to the apical membrane in response to secretin stimulation.4,5 Defects in cholangiocyte function appear to be closely related to the pathogenesis of chronic cholestatic disorders.6 Under basal conditions, the biliary epithelium plays an important role in bile acid recirculation by absorbing and metabolizing bile acids. Bile duct dysfunction may cause defects in metabolism of toxic bile acids and lead to intrahepatic accumulation of bile acids.6 Although all of the cholangiopathies are commonly characterized by cholangiocyte proliferation, fibrosis, cholestasis, and portal inflammation,6 it remains unclear how the cholangiopathies arise and what triggers these shared pathological features. Dysfunctions in bile duct epithelial apical transport processes are considered to be prominent candidate factors that could contribute to the pathogenesis of cholestatic diseases. In the liver, CFTR protein is exclusively expressed in the cholangiocyte. The role of altered Cl2 transport in the pathogenesis of cholangiopathies is demonstrated by the hepatic phenotype that is associated with cycstic fibrosis (CF).7 In CF patients, reduced bile hydration precipitates a series of events, such as retention of toxic bile acids, that damages the biliary epithelium.6 In addition, defective expression of AE-2 and reduced levels of immunodetectable AE-2 protein have also been reported in patients with primary biliary cirrhosis (PBC).8
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Recent studies suggest that the physiological functions of transporters are dynamically regulated by interaction with scaffolding proteins.9,10 CFTR has a PDZ-binding motif at the C-terminal and interacts with Na1/H1 exchanger regulatory factor 1 (NHERF1), which is a scaffold protein possessing PSD-95/Discs-large/ZO-1 (PDZ)-binding domains and ERM (ezrin-radixin-moesin)-binding domain.9,10 In the liver, ezrin is exclusively expressed in the bile duct epithelium, but its physiological roles in cholangiocytes are still unknown. Both ezrin and NHERF1 coordinately regulate apical surface expression of CFTR, and ezrin also serves as a scaffold protein of protein kinase A (PKA), which regulates CFTR function.9,10 Therefore, it seems likely that ezrin could play important roles in the regulation of CFTR-mediated ion transport function in cholangiocytes. Ezrin knockdown (Vil2kd/kd) mice were previously generated to investigate physiological roles of ezrin in adult mice given that ezrin null mice died within 1.5 weeks after birth before weaning.11 But, Vil2kd/kd mice still exhibit severe growth retardation and high mortality up to the weaning period. Only 20% of Vil2kd/kd mice survived beyond the weaning period in our experiments. Tamura et al. reported that ezrin expression level in Vil2kd/kd mice was reduced by approximately 5% of wild-type (WT) mice. In previous reports, several pathological features, including achlorhydria, hypophosphatemia, and osteomalacia, as gastric and renal phenotypes, were found in Vil2kd/kd mice without gross defects in gut morphology. The reason for high mortality found in these mice is still undetermined.11,12 In the present study, we found that Vil2kd/kd mice exhibited intrahepatic cholestasis together with bile duct proliferation and periductular fibrosis, but without apparent infiltration of inflammatory cells and autoantibodies. These observations suggest the possibility that dysfunction in the apical ion transports in the bile duct epithelium can serve as primary causes of bile duct injury and thus of the consequent proliferative and fibrogenic response in the liver.
Address reprint requests to: Ryo Hatano, Ph.D., Department of Molecular Physiology, College of Pharmaceutical Sciences, Ritsumeikan University, 1-1-1 NojiHigashi, Kusatsu, Shiga 525-8577, Japan. E-mail: [email protected]; fax: 181-77-561-5908. C 2014 by the American Association for the Study of Liver Diseases. Copyright V View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.27565 Potential conflict of interest: Nothing to report.
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Materials and Methods Animal Experiments. Vil2kd/kd mice were generated as described previously.11 Three- to twelve-weekold sex-matched WT and Vil2kd/kd mice were used in this study. All work with animals was performed with approval from the animal ethics committee of Ritsumeikan University (Shiga, Japan). Blood was obtained by heart puncture under appropriate anesthesia, and plasma was separated by centrifugation at 1,000g for 10 minutes at 4 C. Concentrations of plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were determined using DRI-CHEM 4000i (Fujifilm, Tokyo, Japan). Bile flow was measured by cannulation of the gallbladder and gravimetrically collection of bile, and intrahepatic bile acid contents were measured by enzyme immunoassay (EIA; see the Supporting Information for details). Real-Time Reverse-Transcriptase Polymerase Chain Reaction and Immunoblotting. Real time reverse-transcriptase polymerase chain reaction (RTPCR) and immunoblotting was performed as described previously12 (see the Supporting Information). Cell-Surface Biotinylation. Normal mouse cholangiocytes (NMCs) immortalized through the introduction of the Simian virus 40 large T antigen gene were previously established.13 See the Supporting Information for details on cell culture. Cell-surface proteins of untransfected or Flag-tagged-WT-ezrin and Flag-tagged-DN-ezrin stably transfected NMCs (control, WT-NMC, and DN-NMC, respectively) were biotinylated with sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate (sulfo-NHS-SS-Biotin). Briefly, NMC cells grown in 100-mm dishes were incubated at 37 C until they became 100% confluent. Cells were rinsed with ice-cold phosphate-buffered saline (PBS; 137 mM of NaCl, 2.7 mM of KCl, 4.3 mM of Na2HPO4, and 1.4 mM of KH2PO4; pH 7.4) containing 0.1 mM of CaCl2 and 1 mM of MgCl2 and incubated with 1 mg/mL of EZ-Link Sulfo-NHS-SS-biotin (Pierce Biotechnology, Inc., Rockford, IL) in PBS (pH adjusted to 8.0) for 30 minutes. Cell monolayers were rinsed with cold PBS, and unreacted biotin molecules were quenched with PBS containing 1% bovine serum albumin for 10 minutes before cell lysis and streptavidin-agarose capture. The captured proteins were eluted by the sodium dodecyl sulfate sample buffer and used for immunoblotting analysis. Measurement of Cl2 Efflux by MQAE Assay. Cl2 efflux was evaluated by using the Cl2 indicator, N-(ethoxycarbonyl methyl)-6-methoxy quinolinium
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(MQAE; Dojin Chemical, Inc., Kumamoto, Japan) and the cell volume marker, Calcein-AM (Dojin Chemical). Both indicators (5 mM of MQAE and 0.1 lg/lL of Calcein-AM) were loaded to NMCs, and the intensity of MQAE and Calcein was measured by FV10i (Olympus, Tokyo, Japan) (see the Supporting Information for details). Statistical Analysis. Statistical analyses were performed using Student t test in GraphPad Prism 6.0 (San Diego, CA) with P < 0.05 and P < 0.01 being considered statistically significant.
Results Vil2kd/kd Mice Exhibit Intrahepatic Cholestasis. Ezrin, radixin, and moesin were found to be expressed in different cell types in the liver: Ezrin is exclusively detected in bile ducts, radixin is detected in bile canaliculi, and moesin is primarily detected in blood vessels (Supporting Fig. 1). Ezrin expression colocalizes extensively with that of cytokeratin 19 (CK19), which is a bile-duct-specific intermediate filament in the liver. Ezrin localizes to the apical membranes of bile duct epithelial cells (Fig. 1A). Little or no staining for ezrin was observed in bile ducts of Vil2kd/kd mouse livers (Fig. 1B). Immunoblotting also revealed significant reductions in the levels of ezrin expression in IBDUs (isolated bile duct units) from Vil2kd/kd mice to approximately 9% of WT mice without any concomitant up-regulation of radixin and moesin expression in liver of Vil2kd/kd mice (Fig. 1C). Biochemical analysis of mouse plasma revealed marked differences between WT and Vil2kd/kd mice in levels of markers associated with hepatobiliary diseases. In 4-week-old mice, plasma concentrations of AST, ALT, alkaline phosphatase (ALP), and bile acids were significantly elevated in Vil2kd/kd mice, compared to WT mice (Table 1). To confirm whether the observed morphological abnormalities in the bile ducts are also related to the hepatic dysfunction in Vil2kd/kd mice, we performed electron microscopic analysis of livers obtained from WT and Vil2kd/kd mice at a young age (4 weeks of age). However, there was no apparent difference in bile duct morphology between WT and Vil2kd/kd mice at this early time point (Fig. 1D,E). The numbers and lengths of microvilli were not significantly different between WT and Vil2kd/kd mouse cholangiocytes (Fig. 1F). In hepatocytes, there was also no difference in the morphology of bile canaliculi between WT and Vil2kd/kd mice, but massive dilation of the endoplasmic reticulum was found in Vil2kd/kd mice hepatocytes. This dilation may be the result of
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Fig. 1. Immunostaining and structural analyses of bile ducts in WT and Vil2kd/kd livers. (A) Coimmunostaining of ezrin (green) and cholangiocyte marker CK19 (red). pv, portal vein. (B) IF analysis of ezrin expression in WT and Vil2kd/kd mouse livers. (C) Immunoblotting for ERM proteins in the total liver and IBDU lysates of WT and Vil2kd/kd mice. Intensity of ezrin expression was much lower than those of radixin and moesin, given that bile ducts constitute only 3%-5% of total liver cells. Ezrin expression level was greatly decreased in IBDU of Vil2kd/kd mice. Intensity was quantified, and data are represented in right panel. N 5 5, respectively. Electron microscopic analysis was performed in WT and Vil2kd/kd mouse livers at 4 weeks of age. (D) Low magnification (1,8703) exhibited no apparent morphological abnormality in Vil2kd/kd mouse bile ducts. (E) High magnifications (18,2003) of cholangiocytes are shown. (F) The number of microvilli at the apical membranes of cholangiocytes and the length of microvilli were measured.
the impairment of protein secretion secondary to cholestasis (Supporting Fig. 2). We also investigated the primary cilia formation by immunostaining because morphological defect in primary cilia causes cholangiopathy. However, no apparent alteration was found in Vil2kd/kd mouse bile ducts (Supporting Fig. 3). These results suggest that the hepatic disorder in Vil2kd/kd mice was not the result of any primary intrahepatocyte pathological process, but rather was attributed to functional abnormalities in cholangiocytes that were caused by loss of ezrin.
In adult Vil2kd/kd mice, plasma concentrations of AST, ALT, ALP, and bile acids were still significantly higher than WT mice (Table 1). Next, we measured bile flow in adult mice at 8-10 weeks of age because reductions of bile flow are also a common feature of cholangiopathies. A significant reduction of bile flow was found in Vil2kd/kd mice (Fig. 2A). We also measured the content of intrahepatic bile acids by EIA to confirm that cholestasis in liver of Vil2kd/kd mice is accompanied by reduced bile flow. Intrahepatic bile acid contents were significantly elevated in Vil2kd/kd
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Table 1. Biochemical Analysis for AST, ALT, ALP, Bile Acid, Total Bilirubin, and Direct Bilirubin in WT and Vil2kd/kd Mice Vil2kd/kd
WT
4 weeks (n 5 6, each)
6-7 weeks (n 5 6, each)
11-12 weeks (n 5 6, each)
AST (U/L) ALT (U/L) ALP (U/L) Bile acids (lmol/L) Total bilirubin (mg/dL) Direct bilirubin (mg/dL) AST (U/L) ALT (U/L) ALP (U/L) Bile acids (lmol/L) Total bilirubin (mg/dL) Direct bilirubin (mg/dL) AST (U/L) ALT (U/L) ALP (U/L) Bile acids (lmol/L) Total bilirubin (mg/dL) Direct bilirubin (mg/dL)
90.7 38.3 264 84.0 0.5 0.17 60.7 18.0 290 84.9 0.47 0.13 66.8 24.8 383 35.5 0.2 0.15
6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6
6.7 4.4 40 21.3 0.1 0.03 11.5 3.5 14 25.7 0.07 0.02 7.24 1.1 22 9.9 0.03 0.02
384 95.7 828 645.3 0.57 0.20 351 317 1,100 339.7 0.58 0.14 246 206 1242 166.2 0.45 0.20
6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6
119* 32.9* 299* 145.8* 0.08 0.0 45* 60* 49* 52.5* 0.08 0.04 48* 37* 199* 24.1* 0.08* 0.0
Data represent mean 6 standard error of the mean. *P < 0.05 versus WT mice.
Fig. 2. Bile flow and hepatic bile contents were measured in WT and Vil2kd/kd mice. (A) Bile flow was measured in both WT and Vil2kd/kd mice at 6-8 weeks of age (n 5 6, respectively). *P < 0.05 versus WT mice. (B) Intrahepatic total bile acid contents of WT and Vil2kd/kd mice at 4 and 6 weeks age were measured by EIA. *P < 0.05; **P < 0.01. (C) H&E staining was performed in WT and Vil2kd/kd mouse livers (12 weeks of age). Abnormally proliferating bile ducts and structural disruption of hepatic lobules with indistinct lobular septa were found in Vil2kd/kd mouse liver. (D) Sirius Red staining of Vil2kd/kd mouse liver. (E) Proliferating cholangiocytes were detected by coimmunostaining with Ki67 and CK19 (arrow).
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mouse liver (Fig. 2B), suggesting that functional bile modulations, such as fluid and/or electrolyte secretion, by cholangiocytes are disturbed in Vil2kd/kd mice. Histological analysis, employing hematoxylin-eosin (H&E) staining performed on the livers of WT and Vil2kd/kd mice at 12 weeks of age, revealed the presence of abnormally proliferating bile ducts and structural disruption of hepatic lobules with unclear lobular septa in Vil2kd/kd mice (Fig. 2C). Fibrotic disposition in the periductular area was also found in Vil2kd/kd mouse liver (Fig. 2D). The number of proliferating cholangiocytes (Ki67-positive cholangiocytes) was also significantly increased in Vil2kd/kd mouse livers (percentage of Ki67-positive cholangiocytes: WT, less than 0.01%; Vil2kd/kd, 6.31%; Fig. 2E). These characteristic changes are commonly found in cholangiopathies, suggesting that Vil2kd/kd mice exhibited the symptoms of severe cholangiopathy resulting from loss of ezrin in cholangiocytes. In cholestatic conditions, bile duct epithelium is capable of synthesizing and secreting numerous bioactive mediators, including transforming growth factor beta (TGF-b), tumor necrosis factor alpha (TNF-a), interleukin 1 beta (IL-1b), macrophage inflammatory protein 2 alpha (MIP2a), and procollagen types I (Procol 1) and III (Procol 3). Thus, we investigated messenger RNA (mRNA) expression of these mediators by real-time RT-PCR. In Vil2kd/kd mouse livers, mRNA expressions of these five genes were significantly increased (Fig. 3). Furthermore, to investigate the infiltration of inflammatory cells in the periportal ductural area, immunofluorescence (IF) analysis of Vil2kd/kd mouse liver tissue was performed using F4/80, which is a marker antibody (Ab) for mature mouse macrophages and blood monocytes, as well as Abs directed against CD4 and CD8. No apparent inflammatory cell infiltration was detected in Vil2kd/kd mouse liver tissue (Supporting Fig. 4). Dominant-Negative Ezrin Disturbed Apical Membrane Localization and cAMP-Dependent Phosphorylation of CFTR in Immortalized Cholangiocytes. To investigate how ezrin regulates the physiological function of cholangiocytes, immortalized mouse cholangiocytes were utilized. This cell line possesses the differentiated characteristics of mouse cholangiocytes and forms a polarized monolayer.13 We generated the vectors to drive the expression of full-length ezrin (WT-ezrin) and of a dominant negative form of ezrin (DN-ezrin), which lacks the C-terminal regions of full-length ezrin. Cell lines stably expressing empty vector or either of these constructs were established
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Fig. 3. Real time RT-PCR for profibrotic and inflammatory factor expression in WT and Vil2kd/kd mouse livers. Relative mRNA expression levels of (A) Procol-1, (B) Procol-3, (C) TGF-b, (D) TNF-a, (E) MIP2a, and (F) IL-1b in WT and Vil2kd/kd mice livers were measured (n 5 8, respectively).
(mock NMC, WT-ezrin NMC, and DN-ezrin NMC, respectively). We assessed the expressions of ion transport proteins, including CFTR, AE-2, and AQP1, in NMC cell lines. The total quantities of CFTR, AE-2, and AQP1 proteins were not different in any of the cell lines tested (Fig. 4A). Next, apical surface expressions of all of these proteins were examined by surface biotinylation, followed by immunoblotting. The apical surface expressions of these three proteins were significantly reduced in DN-ezrin NMC, whereas those in WT-ezrin NMC were at similar levels to those detected with mock-NMC cells (Fig. 4B,C), suggesting that ezrin plays an important role in ensuring or stabilizing the apical distributions of these proteins.
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Fig. 4. Protein expression levels of CFTR, AE-2, and AQP1 were analyzed by immunoblotting in NMCs. (A) Expression levels of CFTR, AE-2, AQP1, ezrin, NHERF-1, and GAPDH were examined in total cell lysates of mock-NMC-, WT-ezrin-, or DN-ezrin-expressing NMCs. (B) CFTR, AE-2, and AQP1 expression levels in the apical surface membrane of NMCs. Surface proteins were extracted by surface biotinylation, as described in Materials and Methods. (C) Intensities of each band were measured, and the relative apical surface expression levels were calculated. Values were normalized to the intensities of GAPDH (n 5 3). (D) Cl2 efflux activities were measured in the mock- and WT-ezrin or DN-ezrin stably expressing NMCs. *P < 0.05 versus mock; **P < 0.01 versus mock. Abbreviation: GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
Furthermore, we examined the Cl2 transport activity of NMC-cultured cholangiocytes by means of MQAE assay. After Cl2 free medium was substituted for normal medium, intensities of intracellular MQAE and Calcein fluorescence were measured. Although WTezrin NMC exhibited similar levels of Cl2 efflux activity to those measured in mock-NMC, Cl2 efflux activity in DN-ezrin NMC was significantly reduced, compared to mock- and WT-ezrin NMCs (Fig. 4D).
This result is consistent with the reduced surface expression of CFTR in DN-ezrin NMC cells, suggesting that impaired function of ezrin leads to a reduction of CFTR-mediated Cl2 transport, and, possibly, secondary HCO32 and water flux as well, by perturbing the apical membrane targeting or retention of the relevant transport proteins. We also examined the effects of PKA activation on surface expressions of these proteins in NMC cells,
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Fig. 5. cAMP-dependent regulation of apical surface transporters was investigated by immunoblotting in NMCs. (A) Total expression levels of CFTR, AE-2, and AQP1 before and after dbcAMP treatment were examined by immunoblotting. (B) Apical surface expression levels of CFTR, AE-2, and AQP1 before and after dbcAMP treatment were examined by immunoblotting. Arrow indicates putative phosphorylated band of CFTR. Arrowhead indicates nonphosphorylated form of CFTR. (C) Surface expression levels of CFTR, AE-2, and AQP1 in NMCs with or without dbcAMP treatment were quantified, and data are represented as the value relative to mock NMCs without dbcAMP treatment. N 5 3 or 4. *P < 0.05 versus dbcAMP (2) of same cell lines for respective genes; †P < 0.05 versus mock-dbcAMP (1) for respective genes. (D) Microsome fractions of normal NMCs (before and after dbcAMP treatment) were treated with calf ALP for 2 hours at 37 C. The bands were separated by phostag-acrylamide gel electrophoresis. Upper band (arrow) disappeared after ALP treatment. Arrowhead indicates nonphosphorylated form of CFTR. (E) Cl2 efflux activities were measured in the mock- and DN-ezrin NMCs before and after dbcAMP treatment. Abbreviation: GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
given that the apical membrane targeting of intracellular vesicles containing CFTR, AE-2, and AQP1 in cholangiocytes is activated by secretin-dependent PKA activation. We used 100 lM of dibutylyl-cAMP (dbcAMP), a membrane-permeable cAMP analog, to mimic secretin stimulation in this study. The total amounts of the transport proteins were not different before and after dbcAMP treatment (Fig. 5A). Surface expressions of CFTR, AE-2, and AQP1 were significantly increased in all NMCs by dbcAMP treatment (Fig. 5B,C). However, the effects of dbcAMP on sur-
face expressions of these proteins were reduced in DNezrin NMCs, as compared to mock- and WT-ezrin NMCs (Fig. 5B,C). Interestingly, we found that CFTR was detected as a double band in immunoblotting, and the intensity of the upper band was significantly increased in mock- and WT-ezrin NMC cells in response to dbcAMP stimulation (Fig. 5B). However, the intensity of the upper band was not substantially increased by dbcAMP in DN-ezrin NMC cells. Because the upper band was predicted to represent phosphorylated CFTR, we assessed this possibility by
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Table 2. Analysis for Biliary Bicarbonate Concentration and pH in WT and Vil2kd/kd Mice HCO32 (mM) pH
WT
Vil2kd/kd
27.2 6 2.0 7.96 6 0.04
20.1 6 1.7* 7.72 6 0.02*
Data represent mean 6 SEM. N 5 8 and 5 respectively (WT and Vil2kd/kd mice). *P < 0.05 versus WT mice.
performing Phos-tag acrylamide electrophoresis, which is an affinity-based electrophoretic method that enhances separation between phosphorylated and nonphosphorylated forms of proteins. We prepared the crude membrane fractions from dbcAMP-treated or untreated normal NMC cells and treated them with or without calf ALP. These samples were loaded on to a Phos-tag acrylamide gel and immunoblotted with an Ab directed against CFTR. The upper band disappeared in the ALP-treated fraction (Fig. 5D), indicating that it does indeed represent a phosphorylated form of CFTR. In dbcAMP-stimulated NMCs, Cl2 efflux activity was significantly increased in mockNMCs, whereas it was not significantly changed in DN-ezrin NMCs, indicating that less responsiveness of CFTR trafficking to dbcAMP stimulation causes substantially reduced Cl2 efflux activity in DN-ezrin NMCs (Fig. 5E). These results suggest that impaired function of ezrin reduced PKA-dependent phosphorylation of CFTR in NMC cholangiocytes. Furthermore, these in vitro experiments suggest that two PKAdependent pathways—the PKA-dependent apical surface targeting of intracellular vesicles and the PKA-dependent activation of CFTR—are impaired by perturbations in ezrin function in DN-ezrin NMCs. Apical Membrane Expression of CFTR and NHERF1 Was Impaired in Bile Ducts of Vil2kd/kd Mice. We investigated the subcellular localizations of CFTR, AE-2, and AQP1 by performing immunostaining on liver tissue from WT and Vil2kd/kd mice. These proteins colocalized with ezrin at the apical membranes of bile duct epithelial cells in WT mice (Supporting Fig. 5). Furthermore, strong positive staining for CFTR and NHERF1 was detected in apical membranes of intrahepatic bile ducts in WT mouse tissue (Fig. 6A). Intensities of apical localizations of these proteins were significantly reduced in Vil2kd/kd mouse bile ducts. In addition, intracellular vesicular pools of these proteins were found in Vil2kd/kd mouse bile ducts, whereas this pattern was not observed in WT tissue (Fig. 6A). AE-2 and AQP1 were also located at the apical membranes of bile duct epithelial cells in WT mice, and the apical localizations of these proteins
were also diminished in bile ducts of Vil2kd/kd mice (Fig. 6B,C). Furthermore, the biliary HCO32 concentration and pH were significantly reduced in bile from Vil2kd/kd mice (Table 2). These data suggest that the apical membrane targeting or stabilization of CFTR, AE-2, and AQP1 proteins were all perturbed when ezrin expression was impaired in vivo, which is consistent with the results of the in vitro experiments.
Discussion We demonstrate that loss of ezrin in bile ducts causes abnormal apical secretion of solutes and water, leading, in turn, to reduced bile flow and a severe cholestatic phenotype without inflammation in Vil2kd/kd mice. In cholangiocyte cell lines, the apical localization of transporters, including CFTR, AE-2, and AQP1, which are important for maintenance of bile fluidity and alkalinity, was disturbed under both basal and stimulated conditions when the function of ezrin was perturbed. Similar effects on the distributions of these transport proteins and of the scaffold protein, NHERF1, were observed in vivo in bile ducts of the Vil2kd/kd mice. These results suggest that defects of ezrin function in cholangiocytes cause abnormal regulation of the localization and activation of apical membrane transporters, leading to decreased bile flow and consequent bile duct injury and cholestasis. In the liver, radixin is found predominantly in bile canaliculi of hepatocytes, where it serves as a scaffold protein for multidrug resistance-associated protein 2.14 Moesin is mainly expressed in hepatic stellate cells (HSCs) and in endothelial cells of blood vessels and is related to injury-induced activation of HSCs.15 On the other hand, the physiological roles of ezrin in the liver have not been investigated thoroughly, given that the total expression level of ezrin in the liver is much lower than those of radixin and moesin.16 In the liver, ezrin is exclusively expressed in cholangiocytes, which make up only 3%-5% of liver cell mass.1 To date, the physiological importance of ezrin in the liver has been underestimated, compared to that of radixin and moesin. In the present study, we found that Vil2kd/kd mice showed severe hepatic disorder. Owing to the expression pattern in the liver, we postulated that the functional abnormalities of bile ducts are responsible for the cholestatic liver injury observed in Vil2kd/kd mice, given that morphological abnormalities were not found in Vil2kd/kd bile ducts. Intrahepatic bile ducts play important roles in the modulation of bile flow through secretion of electrolytes and water. Derangements in physiological function of bile ducts can produce chronic cholestasis.
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Fig. 6. IF analyses of bile ducts in WT and Vil2kd/kd mice. (A) Coimmunostaining of CFTR, NHERF1, and ZO-1 was performed using WT and Vil2kd/kd mouse liver sections. Colocalization of CFTR and NHERF1 was found at the apical membranes of bile ducts in WT mice, but such apical localizations were significantly reduced in Vil2kd/kd mouse bile ducts. Most of the positive staining for CFTR and NHERF1 was found in the cytoplasmic compartments. (B) Coimmunostaining of AE-2 and ZO-1 in bile duct was performed using WT and Vil2kd/kd mice liver sections. Apical surface expression level of AE-2 was significantly decreased in Vil2kd/kd mouse bile duct. (C) Coimmunostaining of AQP-1 and ZO-1 in bile duct was performed using WT and Vil2kd/kd mouse liver sections. Apical surface expression level of AQP-1 was significantly decreased in Vil2kd/kd mouse bile ducts. Abbreviation: ZO-1, zonula occludens 1.
Chronic cholangiopathies are classified into several groups based upon their etiology,6 whereas the pathogenetic mechanisms responsible for the common phenotype in these cholangiopathies still remain unclear. Antimitochondrial antibodies (AMAs) and perinuclear antineutrophil cytoplasmic antibody (p-ANCA) are often observed in serum of almost all PBC and PSC (primary sclerosing cholangitis) patients, respectively.17 However, in our study, no apparent production of autoantibodies, such as AMA and p-ANCA, was found in Vil2kd/kd mice (data not shown), suggesting that the pathogenesis of cholangiopathy in Vil2kd/kd mice is not the result of immunological abnormalities. On the other hand, the role of altered ion transport systems in the
pathogenesis of cholangiopathies is demonstrated in CF, a common genetic disease in which mutations in CFTR lead to pulmonary, pancreatic, and liver disease.7 Roughly 5%-10% of CF patients develop rapidly progressive biliary fibrosis resembling PSC.7 Thus, loss of CFTR ion channel function leads to onset of liver disease in humans. Likewise, reduced gene expression of AE-2 in the bile duct is also related to PBC.8 These transporters are exclusively expressed in the apical membrane of bile duct in the liver, and their being coupled ion transport is essential for bile flow regulation. These results suggest that dysfunction of their coupled ion transport in the bile duct is etiologically associated with cholangiopathies.
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Tietz et al.4 reported that CFTR, AE-2, and the AQP1 water channel are coordinately trafficked to the apical membrane in response to hormonal stimulation by secretin. They also suggested that the interaction between vesicles containing these proteins and cytoskeletal and motor proteins is essential for their apical membrane targeting.5 Disturbed apical membrane localizations of CFTR, AE-2, and AQP1 were found in Vil2kd/kd mice and in mouse cholangiocytes expressing dominant negative ezrin. Thus, ezrin may mediate the interaction between cytoskeletal or motor proteins and vesicles containing these proteins to ensure their appropriate membrane targeting or stabilization. Recently, Ramel et al.18 reported that in Drosophila, moesin, which is another member of the ERM family of proteins, interacts with the small GTPase, Rab11, which plays important roles in vesicle trafficking. Thus, ezrin may play a pivotal role in formation of the complex necessary for trafficking of transportercontaining vesicles. Further experiments will be required to explore this possibility. Ezrin also serves as a scaffold for PKA to regulate the activities of apical membrane proteins, such as CFTR and sodium-hydrogen exchanger 3 9,10,19 2 (NHE3). The Cl transport activity of CFTR is activated by the PKA-dependent phosphorylation of this protein’s intracellular R-domain, whereas the ion transport activity of NHE3 is suppressed by the PKAdependent phosphorylation of serine residues at the NHE3 C-terminus.9,10,19 Recently, Hayashi et al.20 reported that loss of ezrin expression reduced responsiveness to the PKA-dependent inactivation of NHE3 without affecting its apical membrane expression in the small intestine of Vil2kd/kd mice. Thus, loss of ezrin could be related to the defect in PKA-dependent membrane protein regulation. In cholangiocytes, ezrin colocalizes with CFTR at the apical membrane. Possibly, the interaction between ezrin and CFTR is mediated by NHERF1. Loss of ezrin may thus lead to reduced activation of CFTR by secretin-dependent PKA phosphorylation. In our in vitro experiments, the levels of phosphorylated CFTR were significantly reduced in cholangiocytes expressing dominant negative ezrin, even when those cells were treated with dbcAMP, suggesting that ezrin plays an important role in the regulation of CFTR activity in cholangiocytes. In liver of Vil2kd/kd mice, levels of mRNA expression of inflammatory and profibrotic markers were significantly increased, although there were no apparent invasions of inflammatory cells in the periductural area. This absence of inflammatory cell infiltration suggests the possibility that the observed enhanced
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mRNA signals may be derived from cholangiocytes themselves or from other hepatic cells, such as HSCs, although the origin of these inflammatory cytokines is not fully revealed in the present study. In humans, most cholangiopathies are associated with significant quantities of inflammatory infiltrate in the portal spaces, which is believed to be a trigger for periductular fibrosis and hepatocyte injuries, ultimately leading to liver cirrhosis.6,21 However, few studies have addressed the pattern of cytokines produced in the liver in the course of immune-mediated cholangiopathies. Thus, our results suggest that defective bile duct function may itself cause epithelial damage through accumulation of toxic bile components, leading to the release of inflammatory mediators from intrahepatic cells without apparent contributions from inflammatory cell infiltration. Furthermore, increased production of these inflammatory cytokines might secondarily cause the biliary proliferation that is observed in Vil2kd/kd mice. mRNA of these inflammatory cytokines was also expressed in our cultured cholangiocytes, but functional inhibition of ezrin by DN-ezrin itself did not induce the up-regulation of these gene expressions in NMCs (data not shown). We still need further investigation for the source of these inflammatory cytokines by consideration of the secondary effects by intrahepatic bile acid accumulation to these cells. In summary, our results suggest that the defective function of coupled ion transport at the apical membrane of bile duct epithelial cells is the primary cause of intrahepatic cholestasis in Vil2kd/kd mice. Loss of ezrin in cholangiocytes leads to further progression of the characteristic symptoms of cholangiopathies, including bile duct proliferation and periductural fibrosis. These phenotypes in Vil2kd/kd mice resemble some parts of the liver diseases observed in human conditions, including CF, PBC, and PSC. Ezrin and its interacting proteins, such as NHERF1 and CFTR, possibly play a critical role in the regulation of bile duct physiological functions. These mice will be a new and useful animal model to investigate the pathogenesis of these liver diseases and may be utilized to develop and test new therapeutic agents in the future.
References 1. Esteller A. Physiology of bile secretion. World J Gastroenterol 2008;14: 5641-5649. 2. Banales JM, Prieto J, Medina JF. Cholangiocyte anion exchange and biliary bicarbonate excretion. World J Gastroenterol 2006;12:3496-3511. 3. Frizzell RA, Hanrahan JW. Physiology of epithelial chloride and fluid secretion. Cold Spring Harb Perspect Med 2012;2:a009563. 4. Tietz PS, Marinelli RA, Chen XM, Huang B, Cohn J, Kole J, et al. Agonist-induced coordinated trafficking of functionally related
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9. 10.
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transport proteins for water and ions in cholangiocytes. J Biol Chem 2003;278:20413-20419. Tietz PS, McNiven MA, Splinter PL, Huang BQ, Larusso NF. Cytoskeletal and motor proteins facilitate trafficking of AQP1-containing vesicles in cholangiocytes. Biol Cell 2006;98:43-52. Strazzabosco M, Spirlı C, Okolicsanyi L. Pathophysiology of the intrahepatic biliary epithelium. J Gastroenterol Hepatol 2000;15:244-253. Herrmann U, Dockter G, Lammert F. Cystic fibrosis-associated liver disease. Best Pract Res Clin Gastroenterol 2010;24:585-592. Salas JT, Banales JM, Sarvide S, Recalde S, Ferrer A, Uriarte I, et al. Ae2a,b-deficient mice develop antimitochondrial antibodies and other features resembling primary biliary cirrhosis. Gastroenterology 2008; 134:1482-1493. Guggino WB, Stanton BA. New insights into cystic fibrosis: molecular switches that regulate CFTR. Nat Rev Mol Cell Biol 2006;7:426-436. Naren AP, Cobb B, Li C, Roy K, Nelson D, Heda GD, et al. A macromolecular complex of beta 2 adrenergic receptor, CFTR, and ezrin/ radixin/moesin-binding phosphoprotein 50 is regulated by PKA. Proc Natl Acad Sci U S A 2003;100:342-346. Tamura A, Kikuchi S, Hata M, Katsuno T, Matsui T, Hayashi H, et al. Achlorhydria by ezrin knockdown: defects in the formation /expansion of apical canaliculi in gastric parietal cells. J Cell Biol 2005;169:21-28. Hatano R, Fujii E, Segawa H, Mukaisho K, Matsubara M, Miyamoto K, et al. Ezrin, a membrane cytoskeletal cross-linker, is essential for the regulation of phosphate and calcium homeostasis. Kidney Int 2013;83:41-49. Mano Y, Ishii M, Kisara N, Kobayashi Y, Ueno Y, Kobayashi K, et al. Duct formation by immortalized mouse cholangiocytes: an in vitro model for cholangiopathies. Lab Invest 1998;78:1467-1468. Kikuchi S, Hata M, Fukumoto K, Yamane Y, Matsui T, Tamura A, et al. Radixin deficiency causes conjugated hyperbilirubinemia with loss of Mrp2 from bile canalicular membranes. Nat Genet 2002;31: 320-325.
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15. Okayama T, Kikuchi S, Ochiai T, Ikoma H, Kubota T, Ichikawa D, et al. Attenuated response to liver injury in moesin-deficient mice: impaired stellate cell migration and decreased fibrosis. Biochim Biophys Acta 2008;1782:542-548. 16. Sato N, Funayama N, Nagafuchi A, Yonemura S, Tsukita S, Tsukita S. A gene family consisting of ezrin, radixin and moesin. Its specific localization at actin filament/plasma membrane association sites. J Cell Sci 1992;103:131-143. 17. Boonstra K, Beuers U, Ponsioen CY. Epidemiology of primary sclerosing cholangitis and primary biliary cirrhosis: a systematic review. J Hepatol 2012;56:1181-1188. 18. Ramel D, Wang X, Laflamme C, Montell DJ, Emery G. Rab11 regulates cell-cell communication during collective cell movements. Nat Cell Biol 2013;15:317-324. 19. Dransfield DT, Bradford AJ, Smith J, Martin M, Roy C, Mangeat PH, et al. Ezrin is a cyclic AMP-dependent protein kinase anchoring protein. EMBO J 1997;16:35-43. 20. Hayashi H, Tamura A, Krishnan D, Tsukita S, Suzuki Y, Kocinsky HS, et al. Ezrin is required for the functional regulation of the epithelial sodium proton exchanger, NHE3. PLoS One 2013;8:e55623. 21. Karlsen TH, Boberg KM. Update on primary sclerosing cholangitis. J Hepatol 2013;59:571-582.
Author names in bold designate shared co-first authorship.
Supporting Information Additional Supporting Information may be found at onlinelibrary.wiley.com/doi/10.1002/hep.27565/suppinfo.
Knockdown of Ezrin Causes Intrahepatic Cholestasis by the Dysregulation of Bile Fluidity in the Bile Duct Epithelium in Mice Ryo Hatano,1 Kaori Akiyama,1 Atsushi Tamura,2 Shigekuni Hosogi,3 Yoshinori Marunaka,3 Michael J. Caplan,4 Yoshiyuki Ueno,5 Sachiko Tsukita,2 and Shinji Asano1 Cholangiopathies share common features, including bile duct proliferation, periportal fibrosis, and intrahepatic cholestasis. Damage of biliary epithelium by autoimunne disorder, virus infection, toxic compounds, and developmental abnormalities causes severe progressive hepatic disorders responsible for high mortality. However, the etiologies of these cholestatic diseases remain unclear because useful models to study the pathogenic mechanisms are not available. In the present study, we have found that ezrin knockdown (Vil2kd/kd) mice develop severe intrahepatic cholestasis characterized by extensive bile duct proliferation, periductular fibrosis, and intrahepatic bile acid accumulation without developmental defects of bile duct morphology and infiltration of inflammatory cells. Ezrin is a membrane cytoskeletal crosslinker protein, which is known to interact with transporters, scaffold proteins, and actin cytoskeleton at the plasma membrane. We found that the normal apical membrane localizations of several transport proteins including cystic fibrosis transmembrane conductance regulator (CFTR), anion exchanger 2 (AE-2), aquaporin 1 (AQP1), and Na1/H1 exchanger regulatory factor were disturbed in bile ducts of Vil2kd/kd mice. Stable expression of a dominant negative form of ezrin in immortalized mouse cholangiocytes also led to the reduction of the surface expression of CFTR, AE-2, and AQP1. Reduced surface expression of these transport proteins was accompanied by reduced functional expression, as evidenced by the fact these cells exhibited decreased CFTR-mediated Cl2 efflux activity. Furthermore, bile flow and biliary HCO32 concentration were also significantly reduced in Vil2kd/kd mice. Conclusion: Dysfunction of ezrin mimics important aspects of the pathological mechanisms responsible for cholangiopathies. The Vil2kd/kd mouse may be a useful model to exploit in the development and testing of potential therapies for cholangiopathies. (HEPATOLOGY 2015;61:1660-1671) See Editorial on Page 1467
C
holangiocytes are epithelial cells that line hepatic bile ducts. They constitute only 3%5% of liver cells. The intrahepatic bile ducts
play important roles in modulating the fluidity and alkalinity of canalicular bile by secreting electrolytes, primarily Cl2 and HCO32.1 Osmotic gradients increase the flow of bile by inducing the secondary secretion of water.1 Several membrane transport proteins and channels have been identified and characterized in
Abbreviations: Abs, antibodies; AE-2, anion exchanger 2; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AMA, antimitochondrial antibody; AQP1, aquaporin 1; AST, aspartate aminotransferase; cAMP, cyclic adenosine monophosphate; CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; CK19, cytokeratin 19; dbcAMP, dibutylyl-cyclic AMP; EIA, enzyme immunoassay; ERM, ezrin-radixin-moesin; H&E, hematoxylin-eosin; HSCs, hepatic stellate cells; IBDUs, isolated bile duct units; IF, immunofluorescence; IL-1b, interleukin 1b; MIP-2a, macrophage inflammatory protein 2 alpha; MQAE, N(ethoxycarbonyl methyl)-6-methoxy quinolinium; mRNA, messenger RNA; NHE3, sodium-hydrogen exchanger 3; NHERF, Na1/H1 exchanger regulatory factor; NMC, normal mouse cholangiocyte; p-ANCA, perinuclear antineutrophil cytoplasmic antibody; PBC, primary biliary cirrhosis; PBS, phosphate-buffered saline; PDZ, PSD-95/Discs-large/ZO-1; PKA, protein kinase A; Procol1, procollagen type I; Procol3, procollagen type III; PSC, primary sclerosing cholangitis; RT-PCR, reverse-transcriptase polymerase chain reaction; TGF-b, transforming growth factor beta; TNF-a, tumor necrosis factor alpha; WT, wild type. From the 1Department of Molecular Physiology, College of Pharmaceutical Sciences, Ritsumeikan University, Shiga, Japan; 2Laboratory of Biological Science, Graduate School of Frontier Biosciences and Graduate School of Medicine, Osaka University, Osaka, Japan; 3Department of Molecular Cell Physiology and BioIonomics, Kyoto Prefectural University of Medicine, Kyoto, Japan; 4Department of Cellular and Molecular Physiology, Yale School of Medicine, New Haven, CT; and 5Department of Gastroenterology, Yamagata University School of Medicine, Yamagata, Japan Received May 19, 2014; accepted October 10, 2014. Additional Supporting Information may be found at onlinelibrary.wiley.com/doi/10.1002/hep.27565/suppinfo. 1660
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mammalian bile duct epithelium1 and are thought to participate in modifying the composition of canalicular bile. Cholangiocytes transport HCO32 into the bile, which is mediated by the Na1-independent Cl2/HCO32 exchanger, anion exchanger 2 (AE-2). AE-2 functions as a secondary active transporter, which is driven by the high lumen concentrations of Cl- that are in turn generated by secretion mediated by a cyclic adenosine monophosphate (cAMP)-stimulated low conductance Cl2 channel, cystic fibrosis transmembrane conductance regulator (CFTR).13 Furthermore, in response to osmotic gradients, the water-channel protein, aquaporin 1 (AQP1), mediates water movement from cholangiocyte into bile.2,3 The coordinated functions of these transporters are extremely important for regulation of bile fluidity and alkalinity. These processes are dynamically regulated by a gastrointestinal hormone, secretin.2 Recent studies suggest that these transporters are stored together in the same intracellular vesicles within cholangiocytes, and that they are dynamically trafficked to the apical membrane in response to secretin stimulation.4,5 Defects in cholangiocyte function appear to be closely related to the pathogenesis of chronic cholestatic disorders.6 Under basal conditions, the biliary epithelium plays an important role in bile acid recirculation by absorbing and metabolizing bile acids. Bile duct dysfunction may cause defects in metabolism of toxic bile acids and lead to intrahepatic accumulation of bile acids.6 Although all of the cholangiopathies are commonly characterized by cholangiocyte proliferation, fibrosis, cholestasis, and portal inflammation,6 it remains unclear how the cholangiopathies arise and what triggers these shared pathological features. Dysfunctions in bile duct epithelial apical transport processes are considered to be prominent candidate factors that could contribute to the pathogenesis of cholestatic diseases. In the liver, CFTR protein is exclusively expressed in the cholangiocyte. The role of altered Cl2 transport in the pathogenesis of cholangiopathies is demonstrated by the hepatic phenotype that is associated with cycstic fibrosis (CF).7 In CF patients, reduced bile hydration precipitates a series of events, such as retention of toxic bile acids, that damages the biliary epithelium.6 In addition, defective expression of AE-2 and reduced levels of immunodetectable AE-2 protein have also been reported in patients with primary biliary cirrhosis (PBC).8
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Recent studies suggest that the physiological functions of transporters are dynamically regulated by interaction with scaffolding proteins.9,10 CFTR has a PDZ-binding motif at the C-terminal and interacts with Na1/H1 exchanger regulatory factor 1 (NHERF1), which is a scaffold protein possessing PSD-95/Discs-large/ZO-1 (PDZ)-binding domains and ERM (ezrin-radixin-moesin)-binding domain.9,10 In the liver, ezrin is exclusively expressed in the bile duct epithelium, but its physiological roles in cholangiocytes are still unknown. Both ezrin and NHERF1 coordinately regulate apical surface expression of CFTR, and ezrin also serves as a scaffold protein of protein kinase A (PKA), which regulates CFTR function.9,10 Therefore, it seems likely that ezrin could play important roles in the regulation of CFTR-mediated ion transport function in cholangiocytes. Ezrin knockdown (Vil2kd/kd) mice were previously generated to investigate physiological roles of ezrin in adult mice given that ezrin null mice died within 1.5 weeks after birth before weaning.11 But, Vil2kd/kd mice still exhibit severe growth retardation and high mortality up to the weaning period. Only 20% of Vil2kd/kd mice survived beyond the weaning period in our experiments. Tamura et al. reported that ezrin expression level in Vil2kd/kd mice was reduced by approximately 5% of wild-type (WT) mice. In previous reports, several pathological features, including achlorhydria, hypophosphatemia, and osteomalacia, as gastric and renal phenotypes, were found in Vil2kd/kd mice without gross defects in gut morphology. The reason for high mortality found in these mice is still undetermined.11,12 In the present study, we found that Vil2kd/kd mice exhibited intrahepatic cholestasis together with bile duct proliferation and periductular fibrosis, but without apparent infiltration of inflammatory cells and autoantibodies. These observations suggest the possibility that dysfunction in the apical ion transports in the bile duct epithelium can serve as primary causes of bile duct injury and thus of the consequent proliferative and fibrogenic response in the liver.
Address reprint requests to: Ryo Hatano, Ph.D., Department of Molecular Physiology, College of Pharmaceutical Sciences, Ritsumeikan University, 1-1-1 NojiHigashi, Kusatsu, Shiga 525-8577, Japan. E-mail: [email protected]; fax: 181-77-561-5908. C 2014 by the American Association for the Study of Liver Diseases. Copyright V View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.27565 Potential conflict of interest: Nothing to report.
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Materials and Methods Animal Experiments. Vil2kd/kd mice were generated as described previously.11 Three- to twelve-weekold sex-matched WT and Vil2kd/kd mice were used in this study. All work with animals was performed with approval from the animal ethics committee of Ritsumeikan University (Shiga, Japan). Blood was obtained by heart puncture under appropriate anesthesia, and plasma was separated by centrifugation at 1,000g for 10 minutes at 4 C. Concentrations of plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were determined using DRI-CHEM 4000i (Fujifilm, Tokyo, Japan). Bile flow was measured by cannulation of the gallbladder and gravimetrically collection of bile, and intrahepatic bile acid contents were measured by enzyme immunoassay (EIA; see the Supporting Information for details). Real-Time Reverse-Transcriptase Polymerase Chain Reaction and Immunoblotting. Real time reverse-transcriptase polymerase chain reaction (RTPCR) and immunoblotting was performed as described previously12 (see the Supporting Information). Cell-Surface Biotinylation. Normal mouse cholangiocytes (NMCs) immortalized through the introduction of the Simian virus 40 large T antigen gene were previously established.13 See the Supporting Information for details on cell culture. Cell-surface proteins of untransfected or Flag-tagged-WT-ezrin and Flag-tagged-DN-ezrin stably transfected NMCs (control, WT-NMC, and DN-NMC, respectively) were biotinylated with sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate (sulfo-NHS-SS-Biotin). Briefly, NMC cells grown in 100-mm dishes were incubated at 37 C until they became 100% confluent. Cells were rinsed with ice-cold phosphate-buffered saline (PBS; 137 mM of NaCl, 2.7 mM of KCl, 4.3 mM of Na2HPO4, and 1.4 mM of KH2PO4; pH 7.4) containing 0.1 mM of CaCl2 and 1 mM of MgCl2 and incubated with 1 mg/mL of EZ-Link Sulfo-NHS-SS-biotin (Pierce Biotechnology, Inc., Rockford, IL) in PBS (pH adjusted to 8.0) for 30 minutes. Cell monolayers were rinsed with cold PBS, and unreacted biotin molecules were quenched with PBS containing 1% bovine serum albumin for 10 minutes before cell lysis and streptavidin-agarose capture. The captured proteins were eluted by the sodium dodecyl sulfate sample buffer and used for immunoblotting analysis. Measurement of Cl2 Efflux by MQAE Assay. Cl2 efflux was evaluated by using the Cl2 indicator, N-(ethoxycarbonyl methyl)-6-methoxy quinolinium
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(MQAE; Dojin Chemical, Inc., Kumamoto, Japan) and the cell volume marker, Calcein-AM (Dojin Chemical). Both indicators (5 mM of MQAE and 0.1 lg/lL of Calcein-AM) were loaded to NMCs, and the intensity of MQAE and Calcein was measured by FV10i (Olympus, Tokyo, Japan) (see the Supporting Information for details). Statistical Analysis. Statistical analyses were performed using Student t test in GraphPad Prism 6.0 (San Diego, CA) with P < 0.05 and P < 0.01 being considered statistically significant.
Results Vil2kd/kd Mice Exhibit Intrahepatic Cholestasis. Ezrin, radixin, and moesin were found to be expressed in different cell types in the liver: Ezrin is exclusively detected in bile ducts, radixin is detected in bile canaliculi, and moesin is primarily detected in blood vessels (Supporting Fig. 1). Ezrin expression colocalizes extensively with that of cytokeratin 19 (CK19), which is a bile-duct-specific intermediate filament in the liver. Ezrin localizes to the apical membranes of bile duct epithelial cells (Fig. 1A). Little or no staining for ezrin was observed in bile ducts of Vil2kd/kd mouse livers (Fig. 1B). Immunoblotting also revealed significant reductions in the levels of ezrin expression in IBDUs (isolated bile duct units) from Vil2kd/kd mice to approximately 9% of WT mice without any concomitant up-regulation of radixin and moesin expression in liver of Vil2kd/kd mice (Fig. 1C). Biochemical analysis of mouse plasma revealed marked differences between WT and Vil2kd/kd mice in levels of markers associated with hepatobiliary diseases. In 4-week-old mice, plasma concentrations of AST, ALT, alkaline phosphatase (ALP), and bile acids were significantly elevated in Vil2kd/kd mice, compared to WT mice (Table 1). To confirm whether the observed morphological abnormalities in the bile ducts are also related to the hepatic dysfunction in Vil2kd/kd mice, we performed electron microscopic analysis of livers obtained from WT and Vil2kd/kd mice at a young age (4 weeks of age). However, there was no apparent difference in bile duct morphology between WT and Vil2kd/kd mice at this early time point (Fig. 1D,E). The numbers and lengths of microvilli were not significantly different between WT and Vil2kd/kd mouse cholangiocytes (Fig. 1F). In hepatocytes, there was also no difference in the morphology of bile canaliculi between WT and Vil2kd/kd mice, but massive dilation of the endoplasmic reticulum was found in Vil2kd/kd mice hepatocytes. This dilation may be the result of
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Fig. 1. Immunostaining and structural analyses of bile ducts in WT and Vil2kd/kd livers. (A) Coimmunostaining of ezrin (green) and cholangiocyte marker CK19 (red). pv, portal vein. (B) IF analysis of ezrin expression in WT and Vil2kd/kd mouse livers. (C) Immunoblotting for ERM proteins in the total liver and IBDU lysates of WT and Vil2kd/kd mice. Intensity of ezrin expression was much lower than those of radixin and moesin, given that bile ducts constitute only 3%-5% of total liver cells. Ezrin expression level was greatly decreased in IBDU of Vil2kd/kd mice. Intensity was quantified, and data are represented in right panel. N 5 5, respectively. Electron microscopic analysis was performed in WT and Vil2kd/kd mouse livers at 4 weeks of age. (D) Low magnification (1,8703) exhibited no apparent morphological abnormality in Vil2kd/kd mouse bile ducts. (E) High magnifications (18,2003) of cholangiocytes are shown. (F) The number of microvilli at the apical membranes of cholangiocytes and the length of microvilli were measured.
the impairment of protein secretion secondary to cholestasis (Supporting Fig. 2). We also investigated the primary cilia formation by immunostaining because morphological defect in primary cilia causes cholangiopathy. However, no apparent alteration was found in Vil2kd/kd mouse bile ducts (Supporting Fig. 3). These results suggest that the hepatic disorder in Vil2kd/kd mice was not the result of any primary intrahepatocyte pathological process, but rather was attributed to functional abnormalities in cholangiocytes that were caused by loss of ezrin.
In adult Vil2kd/kd mice, plasma concentrations of AST, ALT, ALP, and bile acids were still significantly higher than WT mice (Table 1). Next, we measured bile flow in adult mice at 8-10 weeks of age because reductions of bile flow are also a common feature of cholangiopathies. A significant reduction of bile flow was found in Vil2kd/kd mice (Fig. 2A). We also measured the content of intrahepatic bile acids by EIA to confirm that cholestasis in liver of Vil2kd/kd mice is accompanied by reduced bile flow. Intrahepatic bile acid contents were significantly elevated in Vil2kd/kd
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Table 1. Biochemical Analysis for AST, ALT, ALP, Bile Acid, Total Bilirubin, and Direct Bilirubin in WT and Vil2kd/kd Mice Vil2kd/kd
WT
4 weeks (n 5 6, each)
6-7 weeks (n 5 6, each)
11-12 weeks (n 5 6, each)
AST (U/L) ALT (U/L) ALP (U/L) Bile acids (lmol/L) Total bilirubin (mg/dL) Direct bilirubin (mg/dL) AST (U/L) ALT (U/L) ALP (U/L) Bile acids (lmol/L) Total bilirubin (mg/dL) Direct bilirubin (mg/dL) AST (U/L) ALT (U/L) ALP (U/L) Bile acids (lmol/L) Total bilirubin (mg/dL) Direct bilirubin (mg/dL)
90.7 38.3 264 84.0 0.5 0.17 60.7 18.0 290 84.9 0.47 0.13 66.8 24.8 383 35.5 0.2 0.15
6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6
6.7 4.4 40 21.3 0.1 0.03 11.5 3.5 14 25.7 0.07 0.02 7.24 1.1 22 9.9 0.03 0.02
384 95.7 828 645.3 0.57 0.20 351 317 1,100 339.7 0.58 0.14 246 206 1242 166.2 0.45 0.20
6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6
119* 32.9* 299* 145.8* 0.08 0.0 45* 60* 49* 52.5* 0.08 0.04 48* 37* 199* 24.1* 0.08* 0.0
Data represent mean 6 standard error of the mean. *P < 0.05 versus WT mice.
Fig. 2. Bile flow and hepatic bile contents were measured in WT and Vil2kd/kd mice. (A) Bile flow was measured in both WT and Vil2kd/kd mice at 6-8 weeks of age (n 5 6, respectively). *P < 0.05 versus WT mice. (B) Intrahepatic total bile acid contents of WT and Vil2kd/kd mice at 4 and 6 weeks age were measured by EIA. *P < 0.05; **P < 0.01. (C) H&E staining was performed in WT and Vil2kd/kd mouse livers (12 weeks of age). Abnormally proliferating bile ducts and structural disruption of hepatic lobules with indistinct lobular septa were found in Vil2kd/kd mouse liver. (D) Sirius Red staining of Vil2kd/kd mouse liver. (E) Proliferating cholangiocytes were detected by coimmunostaining with Ki67 and CK19 (arrow).
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mouse liver (Fig. 2B), suggesting that functional bile modulations, such as fluid and/or electrolyte secretion, by cholangiocytes are disturbed in Vil2kd/kd mice. Histological analysis, employing hematoxylin-eosin (H&E) staining performed on the livers of WT and Vil2kd/kd mice at 12 weeks of age, revealed the presence of abnormally proliferating bile ducts and structural disruption of hepatic lobules with unclear lobular septa in Vil2kd/kd mice (Fig. 2C). Fibrotic disposition in the periductular area was also found in Vil2kd/kd mouse liver (Fig. 2D). The number of proliferating cholangiocytes (Ki67-positive cholangiocytes) was also significantly increased in Vil2kd/kd mouse livers (percentage of Ki67-positive cholangiocytes: WT, less than 0.01%; Vil2kd/kd, 6.31%; Fig. 2E). These characteristic changes are commonly found in cholangiopathies, suggesting that Vil2kd/kd mice exhibited the symptoms of severe cholangiopathy resulting from loss of ezrin in cholangiocytes. In cholestatic conditions, bile duct epithelium is capable of synthesizing and secreting numerous bioactive mediators, including transforming growth factor beta (TGF-b), tumor necrosis factor alpha (TNF-a), interleukin 1 beta (IL-1b), macrophage inflammatory protein 2 alpha (MIP2a), and procollagen types I (Procol 1) and III (Procol 3). Thus, we investigated messenger RNA (mRNA) expression of these mediators by real-time RT-PCR. In Vil2kd/kd mouse livers, mRNA expressions of these five genes were significantly increased (Fig. 3). Furthermore, to investigate the infiltration of inflammatory cells in the periportal ductural area, immunofluorescence (IF) analysis of Vil2kd/kd mouse liver tissue was performed using F4/80, which is a marker antibody (Ab) for mature mouse macrophages and blood monocytes, as well as Abs directed against CD4 and CD8. No apparent inflammatory cell infiltration was detected in Vil2kd/kd mouse liver tissue (Supporting Fig. 4). Dominant-Negative Ezrin Disturbed Apical Membrane Localization and cAMP-Dependent Phosphorylation of CFTR in Immortalized Cholangiocytes. To investigate how ezrin regulates the physiological function of cholangiocytes, immortalized mouse cholangiocytes were utilized. This cell line possesses the differentiated characteristics of mouse cholangiocytes and forms a polarized monolayer.13 We generated the vectors to drive the expression of full-length ezrin (WT-ezrin) and of a dominant negative form of ezrin (DN-ezrin), which lacks the C-terminal regions of full-length ezrin. Cell lines stably expressing empty vector or either of these constructs were established
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Fig. 3. Real time RT-PCR for profibrotic and inflammatory factor expression in WT and Vil2kd/kd mouse livers. Relative mRNA expression levels of (A) Procol-1, (B) Procol-3, (C) TGF-b, (D) TNF-a, (E) MIP2a, and (F) IL-1b in WT and Vil2kd/kd mice livers were measured (n 5 8, respectively).
(mock NMC, WT-ezrin NMC, and DN-ezrin NMC, respectively). We assessed the expressions of ion transport proteins, including CFTR, AE-2, and AQP1, in NMC cell lines. The total quantities of CFTR, AE-2, and AQP1 proteins were not different in any of the cell lines tested (Fig. 4A). Next, apical surface expressions of all of these proteins were examined by surface biotinylation, followed by immunoblotting. The apical surface expressions of these three proteins were significantly reduced in DN-ezrin NMC, whereas those in WT-ezrin NMC were at similar levels to those detected with mock-NMC cells (Fig. 4B,C), suggesting that ezrin plays an important role in ensuring or stabilizing the apical distributions of these proteins.
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Fig. 4. Protein expression levels of CFTR, AE-2, and AQP1 were analyzed by immunoblotting in NMCs. (A) Expression levels of CFTR, AE-2, AQP1, ezrin, NHERF-1, and GAPDH were examined in total cell lysates of mock-NMC-, WT-ezrin-, or DN-ezrin-expressing NMCs. (B) CFTR, AE-2, and AQP1 expression levels in the apical surface membrane of NMCs. Surface proteins were extracted by surface biotinylation, as described in Materials and Methods. (C) Intensities of each band were measured, and the relative apical surface expression levels were calculated. Values were normalized to the intensities of GAPDH (n 5 3). (D) Cl2 efflux activities were measured in the mock- and WT-ezrin or DN-ezrin stably expressing NMCs. *P < 0.05 versus mock; **P < 0.01 versus mock. Abbreviation: GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
Furthermore, we examined the Cl2 transport activity of NMC-cultured cholangiocytes by means of MQAE assay. After Cl2 free medium was substituted for normal medium, intensities of intracellular MQAE and Calcein fluorescence were measured. Although WTezrin NMC exhibited similar levels of Cl2 efflux activity to those measured in mock-NMC, Cl2 efflux activity in DN-ezrin NMC was significantly reduced, compared to mock- and WT-ezrin NMCs (Fig. 4D).
This result is consistent with the reduced surface expression of CFTR in DN-ezrin NMC cells, suggesting that impaired function of ezrin leads to a reduction of CFTR-mediated Cl2 transport, and, possibly, secondary HCO32 and water flux as well, by perturbing the apical membrane targeting or retention of the relevant transport proteins. We also examined the effects of PKA activation on surface expressions of these proteins in NMC cells,
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Fig. 5. cAMP-dependent regulation of apical surface transporters was investigated by immunoblotting in NMCs. (A) Total expression levels of CFTR, AE-2, and AQP1 before and after dbcAMP treatment were examined by immunoblotting. (B) Apical surface expression levels of CFTR, AE-2, and AQP1 before and after dbcAMP treatment were examined by immunoblotting. Arrow indicates putative phosphorylated band of CFTR. Arrowhead indicates nonphosphorylated form of CFTR. (C) Surface expression levels of CFTR, AE-2, and AQP1 in NMCs with or without dbcAMP treatment were quantified, and data are represented as the value relative to mock NMCs without dbcAMP treatment. N 5 3 or 4. *P < 0.05 versus dbcAMP (2) of same cell lines for respective genes; †P < 0.05 versus mock-dbcAMP (1) for respective genes. (D) Microsome fractions of normal NMCs (before and after dbcAMP treatment) were treated with calf ALP for 2 hours at 37 C. The bands were separated by phostag-acrylamide gel electrophoresis. Upper band (arrow) disappeared after ALP treatment. Arrowhead indicates nonphosphorylated form of CFTR. (E) Cl2 efflux activities were measured in the mock- and DN-ezrin NMCs before and after dbcAMP treatment. Abbreviation: GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
given that the apical membrane targeting of intracellular vesicles containing CFTR, AE-2, and AQP1 in cholangiocytes is activated by secretin-dependent PKA activation. We used 100 lM of dibutylyl-cAMP (dbcAMP), a membrane-permeable cAMP analog, to mimic secretin stimulation in this study. The total amounts of the transport proteins were not different before and after dbcAMP treatment (Fig. 5A). Surface expressions of CFTR, AE-2, and AQP1 were significantly increased in all NMCs by dbcAMP treatment (Fig. 5B,C). However, the effects of dbcAMP on sur-
face expressions of these proteins were reduced in DNezrin NMCs, as compared to mock- and WT-ezrin NMCs (Fig. 5B,C). Interestingly, we found that CFTR was detected as a double band in immunoblotting, and the intensity of the upper band was significantly increased in mock- and WT-ezrin NMC cells in response to dbcAMP stimulation (Fig. 5B). However, the intensity of the upper band was not substantially increased by dbcAMP in DN-ezrin NMC cells. Because the upper band was predicted to represent phosphorylated CFTR, we assessed this possibility by
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Table 2. Analysis for Biliary Bicarbonate Concentration and pH in WT and Vil2kd/kd Mice HCO32 (mM) pH
WT
Vil2kd/kd
27.2 6 2.0 7.96 6 0.04
20.1 6 1.7* 7.72 6 0.02*
Data represent mean 6 SEM. N 5 8 and 5 respectively (WT and Vil2kd/kd mice). *P < 0.05 versus WT mice.
performing Phos-tag acrylamide electrophoresis, which is an affinity-based electrophoretic method that enhances separation between phosphorylated and nonphosphorylated forms of proteins. We prepared the crude membrane fractions from dbcAMP-treated or untreated normal NMC cells and treated them with or without calf ALP. These samples were loaded on to a Phos-tag acrylamide gel and immunoblotted with an Ab directed against CFTR. The upper band disappeared in the ALP-treated fraction (Fig. 5D), indicating that it does indeed represent a phosphorylated form of CFTR. In dbcAMP-stimulated NMCs, Cl2 efflux activity was significantly increased in mockNMCs, whereas it was not significantly changed in DN-ezrin NMCs, indicating that less responsiveness of CFTR trafficking to dbcAMP stimulation causes substantially reduced Cl2 efflux activity in DN-ezrin NMCs (Fig. 5E). These results suggest that impaired function of ezrin reduced PKA-dependent phosphorylation of CFTR in NMC cholangiocytes. Furthermore, these in vitro experiments suggest that two PKAdependent pathways—the PKA-dependent apical surface targeting of intracellular vesicles and the PKA-dependent activation of CFTR—are impaired by perturbations in ezrin function in DN-ezrin NMCs. Apical Membrane Expression of CFTR and NHERF1 Was Impaired in Bile Ducts of Vil2kd/kd Mice. We investigated the subcellular localizations of CFTR, AE-2, and AQP1 by performing immunostaining on liver tissue from WT and Vil2kd/kd mice. These proteins colocalized with ezrin at the apical membranes of bile duct epithelial cells in WT mice (Supporting Fig. 5). Furthermore, strong positive staining for CFTR and NHERF1 was detected in apical membranes of intrahepatic bile ducts in WT mouse tissue (Fig. 6A). Intensities of apical localizations of these proteins were significantly reduced in Vil2kd/kd mouse bile ducts. In addition, intracellular vesicular pools of these proteins were found in Vil2kd/kd mouse bile ducts, whereas this pattern was not observed in WT tissue (Fig. 6A). AE-2 and AQP1 were also located at the apical membranes of bile duct epithelial cells in WT mice, and the apical localizations of these proteins
were also diminished in bile ducts of Vil2kd/kd mice (Fig. 6B,C). Furthermore, the biliary HCO32 concentration and pH were significantly reduced in bile from Vil2kd/kd mice (Table 2). These data suggest that the apical membrane targeting or stabilization of CFTR, AE-2, and AQP1 proteins were all perturbed when ezrin expression was impaired in vivo, which is consistent with the results of the in vitro experiments.
Discussion We demonstrate that loss of ezrin in bile ducts causes abnormal apical secretion of solutes and water, leading, in turn, to reduced bile flow and a severe cholestatic phenotype without inflammation in Vil2kd/kd mice. In cholangiocyte cell lines, the apical localization of transporters, including CFTR, AE-2, and AQP1, which are important for maintenance of bile fluidity and alkalinity, was disturbed under both basal and stimulated conditions when the function of ezrin was perturbed. Similar effects on the distributions of these transport proteins and of the scaffold protein, NHERF1, were observed in vivo in bile ducts of the Vil2kd/kd mice. These results suggest that defects of ezrin function in cholangiocytes cause abnormal regulation of the localization and activation of apical membrane transporters, leading to decreased bile flow and consequent bile duct injury and cholestasis. In the liver, radixin is found predominantly in bile canaliculi of hepatocytes, where it serves as a scaffold protein for multidrug resistance-associated protein 2.14 Moesin is mainly expressed in hepatic stellate cells (HSCs) and in endothelial cells of blood vessels and is related to injury-induced activation of HSCs.15 On the other hand, the physiological roles of ezrin in the liver have not been investigated thoroughly, given that the total expression level of ezrin in the liver is much lower than those of radixin and moesin.16 In the liver, ezrin is exclusively expressed in cholangiocytes, which make up only 3%-5% of liver cell mass.1 To date, the physiological importance of ezrin in the liver has been underestimated, compared to that of radixin and moesin. In the present study, we found that Vil2kd/kd mice showed severe hepatic disorder. Owing to the expression pattern in the liver, we postulated that the functional abnormalities of bile ducts are responsible for the cholestatic liver injury observed in Vil2kd/kd mice, given that morphological abnormalities were not found in Vil2kd/kd bile ducts. Intrahepatic bile ducts play important roles in the modulation of bile flow through secretion of electrolytes and water. Derangements in physiological function of bile ducts can produce chronic cholestasis.
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Fig. 6. IF analyses of bile ducts in WT and Vil2kd/kd mice. (A) Coimmunostaining of CFTR, NHERF1, and ZO-1 was performed using WT and Vil2kd/kd mouse liver sections. Colocalization of CFTR and NHERF1 was found at the apical membranes of bile ducts in WT mice, but such apical localizations were significantly reduced in Vil2kd/kd mouse bile ducts. Most of the positive staining for CFTR and NHERF1 was found in the cytoplasmic compartments. (B) Coimmunostaining of AE-2 and ZO-1 in bile duct was performed using WT and Vil2kd/kd mice liver sections. Apical surface expression level of AE-2 was significantly decreased in Vil2kd/kd mouse bile duct. (C) Coimmunostaining of AQP-1 and ZO-1 in bile duct was performed using WT and Vil2kd/kd mouse liver sections. Apical surface expression level of AQP-1 was significantly decreased in Vil2kd/kd mouse bile ducts. Abbreviation: ZO-1, zonula occludens 1.
Chronic cholangiopathies are classified into several groups based upon their etiology,6 whereas the pathogenetic mechanisms responsible for the common phenotype in these cholangiopathies still remain unclear. Antimitochondrial antibodies (AMAs) and perinuclear antineutrophil cytoplasmic antibody (p-ANCA) are often observed in serum of almost all PBC and PSC (primary sclerosing cholangitis) patients, respectively.17 However, in our study, no apparent production of autoantibodies, such as AMA and p-ANCA, was found in Vil2kd/kd mice (data not shown), suggesting that the pathogenesis of cholangiopathy in Vil2kd/kd mice is not the result of immunological abnormalities. On the other hand, the role of altered ion transport systems in the
pathogenesis of cholangiopathies is demonstrated in CF, a common genetic disease in which mutations in CFTR lead to pulmonary, pancreatic, and liver disease.7 Roughly 5%-10% of CF patients develop rapidly progressive biliary fibrosis resembling PSC.7 Thus, loss of CFTR ion channel function leads to onset of liver disease in humans. Likewise, reduced gene expression of AE-2 in the bile duct is also related to PBC.8 These transporters are exclusively expressed in the apical membrane of bile duct in the liver, and their being coupled ion transport is essential for bile flow regulation. These results suggest that dysfunction of their coupled ion transport in the bile duct is etiologically associated with cholangiopathies.
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Tietz et al.4 reported that CFTR, AE-2, and the AQP1 water channel are coordinately trafficked to the apical membrane in response to hormonal stimulation by secretin. They also suggested that the interaction between vesicles containing these proteins and cytoskeletal and motor proteins is essential for their apical membrane targeting.5 Disturbed apical membrane localizations of CFTR, AE-2, and AQP1 were found in Vil2kd/kd mice and in mouse cholangiocytes expressing dominant negative ezrin. Thus, ezrin may mediate the interaction between cytoskeletal or motor proteins and vesicles containing these proteins to ensure their appropriate membrane targeting or stabilization. Recently, Ramel et al.18 reported that in Drosophila, moesin, which is another member of the ERM family of proteins, interacts with the small GTPase, Rab11, which plays important roles in vesicle trafficking. Thus, ezrin may play a pivotal role in formation of the complex necessary for trafficking of transportercontaining vesicles. Further experiments will be required to explore this possibility. Ezrin also serves as a scaffold for PKA to regulate the activities of apical membrane proteins, such as CFTR and sodium-hydrogen exchanger 3 9,10,19 2 (NHE3). The Cl transport activity of CFTR is activated by the PKA-dependent phosphorylation of this protein’s intracellular R-domain, whereas the ion transport activity of NHE3 is suppressed by the PKAdependent phosphorylation of serine residues at the NHE3 C-terminus.9,10,19 Recently, Hayashi et al.20 reported that loss of ezrin expression reduced responsiveness to the PKA-dependent inactivation of NHE3 without affecting its apical membrane expression in the small intestine of Vil2kd/kd mice. Thus, loss of ezrin could be related to the defect in PKA-dependent membrane protein regulation. In cholangiocytes, ezrin colocalizes with CFTR at the apical membrane. Possibly, the interaction between ezrin and CFTR is mediated by NHERF1. Loss of ezrin may thus lead to reduced activation of CFTR by secretin-dependent PKA phosphorylation. In our in vitro experiments, the levels of phosphorylated CFTR were significantly reduced in cholangiocytes expressing dominant negative ezrin, even when those cells were treated with dbcAMP, suggesting that ezrin plays an important role in the regulation of CFTR activity in cholangiocytes. In liver of Vil2kd/kd mice, levels of mRNA expression of inflammatory and profibrotic markers were significantly increased, although there were no apparent invasions of inflammatory cells in the periductural area. This absence of inflammatory cell infiltration suggests the possibility that the observed enhanced
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mRNA signals may be derived from cholangiocytes themselves or from other hepatic cells, such as HSCs, although the origin of these inflammatory cytokines is not fully revealed in the present study. In humans, most cholangiopathies are associated with significant quantities of inflammatory infiltrate in the portal spaces, which is believed to be a trigger for periductular fibrosis and hepatocyte injuries, ultimately leading to liver cirrhosis.6,21 However, few studies have addressed the pattern of cytokines produced in the liver in the course of immune-mediated cholangiopathies. Thus, our results suggest that defective bile duct function may itself cause epithelial damage through accumulation of toxic bile components, leading to the release of inflammatory mediators from intrahepatic cells without apparent contributions from inflammatory cell infiltration. Furthermore, increased production of these inflammatory cytokines might secondarily cause the biliary proliferation that is observed in Vil2kd/kd mice. mRNA of these inflammatory cytokines was also expressed in our cultured cholangiocytes, but functional inhibition of ezrin by DN-ezrin itself did not induce the up-regulation of these gene expressions in NMCs (data not shown). We still need further investigation for the source of these inflammatory cytokines by consideration of the secondary effects by intrahepatic bile acid accumulation to these cells. In summary, our results suggest that the defective function of coupled ion transport at the apical membrane of bile duct epithelial cells is the primary cause of intrahepatic cholestasis in Vil2kd/kd mice. Loss of ezrin in cholangiocytes leads to further progression of the characteristic symptoms of cholangiopathies, including bile duct proliferation and periductural fibrosis. These phenotypes in Vil2kd/kd mice resemble some parts of the liver diseases observed in human conditions, including CF, PBC, and PSC. Ezrin and its interacting proteins, such as NHERF1 and CFTR, possibly play a critical role in the regulation of bile duct physiological functions. These mice will be a new and useful animal model to investigate the pathogenesis of these liver diseases and may be utilized to develop and test new therapeutic agents in the future.
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Author names in bold designate shared co-first authorship.
Supporting Information Additional Supporting Information may be found at onlinelibrary.wiley.com/doi/10.1002/hep.27565/suppinfo.
