Ezrin-Radixin-Moesin-Binding Phosphoprotein (EBP50), an Estrogen-Inducible Scaffold Protein, Contributes to Biliary Epithelial Cell Proliferation
2009; Elsevier BV; Volume: 174; Issue: 3 Linguagem: Inglês
10.2353/ajpath.2009.080079
ISSN1525-2191
AutoresLaura Fouassier, Peter Rosenberg, Martine Mergey, Bruno Saubaméa, Audrey Clapéron, Nils Kinnman, Nicolas Chignard, Gunilla Jacobsson‐Ekman, Birgitta Strandvik, Colette Rey, Véronique Barbu, Rolf Hultcrantz, Chantal Housset,
Tópico(s)Cancer Mechanisms and Therapy
ResumoEzrin-radixin-moesin-binding phosphoprotein 50 (EBP50) anchors and regulates apical membrane proteins in epithelia. EBP50 is inducible by estrogen and may affect cell proliferation, although this latter function remains unclear. The goal of this study was to determine whether EBP50 was implicated in the ductular reaction that occurs in liver disease. EBP50 expression was examined in normal human liver, in human cholangiopathies (ie, cystic fibrosis, primary biliary cirrhosis, and primary sclerosing cholangitis), and in rats subjected to bile-duct ligation. The regulation of EBP50 by estrogens and its impact on proliferation were assessed in both bile duct-ligated rats and Mz-Cha-1 human biliary epithelial cells. Analyses of cell isolates and immunohistochemical studies showed that in normal human liver, EBP50 is expressed in the canalicular membranes of hepatocytes and, together with ezrin and cystic fibrosis transmembrane conductance regulator, in the apical domains of cholangiocytes. In both human cholangiopathies and bile duct-ligated rats, EBP50 was redistributed to the cytoplasmic and nuclear compartments. EBP50 underwent a transient increase in rat cholangiocytes after bile-duct ligation, whereas such expression was down-regulated in ovariectomized rats. In addition, in Mz-Cha-1 cells, EBP50 underwent up-regulation and intracellular redistribution in response to 17β-estradiol, whereas its proliferation was inhibited by siRNA-mediated EBP50 knockdown. These results indicate that both the expression and distribution of EBP50 are regulated by estrogens and contribute to the proliferative response in biliary epithelial cells. Ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) anchors and regulates apical membrane proteins in epithelia. EBP50 is inducible by estrogen and may affect cell proliferation, although this latter function remains unclear. The goal of this study was to determine whether EBP50 was implicated in the ductular reaction that occurs in liver disease. EBP50 expression was examined in normal human liver, in human cholangiopathies (ie, cystic fibrosis, primary biliary cirrhosis, and primary sclerosing cholangitis), and in rats subjected to bile-duct ligation. The regulation of EBP50 by estrogens and its impact on proliferation were assessed in both bile duct-ligated rats and Mz-Cha-1 human biliary epithelial cells. Analyses of cell isolates and immunohistochemical studies showed that in normal human liver, EBP50 is expressed in the canalicular membranes of hepatocytes and, together with ezrin and cystic fibrosis transmembrane conductance regulator, in the apical domains of cholangiocytes. In both human cholangiopathies and bile duct-ligated rats, EBP50 was redistributed to the cytoplasmic and nuclear compartments. EBP50 underwent a transient increase in rat cholangiocytes after bile-duct ligation, whereas such expression was down-regulated in ovariectomized rats. In addition, in Mz-Cha-1 cells, EBP50 underwent up-regulation and intracellular redistribution in response to 17β-estradiol, whereas its proliferation was inhibited by siRNA-mediated EBP50 knockdown. These results indicate that both the expression and distribution of EBP50 are regulated by estrogens and contribute to the proliferative response in biliary epithelial cells. Ezrin-radixin-moesin (ERM) binding phosphoprotein 50 (EBP50) is an adapter protein, normally localized in the apical region of epithelial cells.1Weinman EJ Steplock D Shenolikar S cAMP-mediated inhibition of the renal brush border membrane Na+-H+ exchanger requires a dissociable phosphoprotein cofactor.J Clin Invest. 1993; 92: 1781-1786Crossref PubMed Scopus (118) Google Scholar This protein contains two postsynaptic density 95/disc-large/zona occludens (PDZ) domains that can bind integral membrane proteins such as transporters, and an ERM-binding domain. EBP50 is required for the maintenance of active ERM proteins at the cortical brush border membranes of polarized epithelia.2Morales FC Takahashi Y Kreimann EL Georgescu MM Ezrin-radixin-moesin (ERM)-binding phosphoprotein 50 organizes ERM proteins at the apical membrane of polarized epithelia.Proc Natl Acad Sci USA. 2004; 101: 17705-17710Crossref PubMed Scopus (177) Google Scholar In addition, EBP50 assembles multiprotein complexes that anchor transporters to the apical actin cytoskeleton and facilitate their regulation. The liver is the tissue that displays the highest level of EBP50 expression.3Yun CH Oh S Zizak M Steplock D Tsao S Tse CM Weinman EJ Donowitz M cAMP-mediated inhibition of the epithelial brush border Na+/H+ exchanger, NHE3, requires an associated regulatory protein.Proc Natl Acad Sci USA. 1997; 94: 3010-3015Crossref PubMed Scopus (404) Google Scholar, 4Ediger TR Kraus WL Weinman EJ Katzenellenbogen BS Estrogen receptor regulation of the Na+/H+ exchange regulatory factor.Endocrinology. 1999; 140: 2976-2982Crossref PubMed Scopus (105) Google Scholar We previously showed that in rat liver, EBP50 is expressed both in hepatocytes and in cholangiocytes, and that, in the latter, the interaction of cystic fibrosis transmembrane conductance regulator (CFTR) with the PDZ1 domain of EBP50 is required for cAMP-dependent chloride secretion,5Fouassier L Duan CY Feranchak AP Yun CH Sutherland E Simon F Fitz JG Doctor RB Ezrin-radixin-moesin-binding phosphoprotein 50 is expressed at the apical membrane of rat liver epithelia.Hepatology. 2001; 33: 166-176Crossref PubMed Scopus (94) Google Scholar suggesting an important role of EBP50 in bile secretory functions. Different lines of evidence suggest that EBP50 has other regulatory functions that potentially include the modulation of cell proliferation.6Voltz JW Weinman EJ Shenolikar S Expanding the role of NHERF, a PDZ-domain containing protein adapter, to growth regulation.Oncogene. 2001; 20: 6309-6314Crossref PubMed Scopus (141) Google Scholar In tumors, ie, in hepatocellular or breast carcinomas, and in non tumor proliferative tissue such as the endometrium, EBP50 can be overexpressed and redistributed to the cytoplasm and/or nucleus of epithelial cells.7Stemmer-Rachamimov AO Wiederhold T Nielsen GP James M Pinney-Michalowski D Roy JE Cohen WA Ramesh V Louis DN NHE-RF, a merlin-interacting protein, is primarily expressed in luminal epithelia, proliferative endometrium, and estrogen receptor-positive breast carcinomas.Am J Pathol. 2001; 158: 57-62Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 8Shibata T Chuma M Kokubu A Sakamoto M Hirohashi S EBP50, a beta-catenin-associating protein, enhances Wnt signaling and is over-expressed in hepatocellular carcinoma.Hepatology. 2003; 38: 178-186Crossref PubMed Scopus (129) Google Scholar, 9Cardone RA Bellizzi A Busco G Weinman EJ Dell'aquila ME Casavola V Azzariti A Mangia A Paradiso A Reshkin SJ The NHERF1 PDZ2 domain regulates PKA-RhoA-p38-mediated NHE1 activation and invasion in breast tumor cells.Mol Biol Cell. 2007; 18: 1768-1780Crossref PubMed Scopus (118) Google Scholar, 10Song J Bai J Yang W Gabrielson EW Chan DW Zhang Z Expression and clinicopathological significance of oestrogen-responsive ezrin-radixin-moesin-binding phosphoprotein 50 in breast cancer.Histopathology. 2007; 51: 40-53Crossref PubMed Scopus (56) Google Scholar In addition, some EBP50 binding partners, eg, platelet-derived growth factor or epidermal growth factor receptors, PTEN, β-catenin, and Pin1 signaling molecules, are directly involved in cell proliferation.8Shibata T Chuma M Kokubu A Sakamoto M Hirohashi S EBP50, a beta-catenin-associating protein, enhances Wnt signaling and is over-expressed in hepatocellular carcinoma.Hepatology. 2003; 38: 178-186Crossref PubMed Scopus (129) Google Scholar, 11Maudsley S Zamah AM Rahman N Blitzer JT Luttrell LM Lefkowitz RJ Hall RA Platelet-derived growth factor receptor association with Na(+)/H(+) exchanger regulatory factor potentiates receptor activity.Mol Cell Biol. 2000; 20: 8352-8363Crossref PubMed Scopus (180) Google Scholar, 12He J Lau AG Yaffe MB Hall RA Phosphorylation and cell cycle-dependent regulation of Na+/H+ exchanger regulatory factor-1 by Cdc2 kinase.J Biol Chem. 2001; 276: 41559-41565Crossref PubMed Scopus (61) Google Scholar, 13Lazar CS Cresson CM Lauffenburger DA Gill GN The Na+/H+ exchanger regulatory factor stabilizes epidermal growth factor receptors at the cell surface.Mol Biol Cell. 2004; 15: 5470-5480Crossref PubMed Scopus (89) Google Scholar, 14Takahashi Y Morales FC Kreimann EL Georgescu MM PTEN tumor suppressor associates with NHERF proteins to attenuate PDGF receptor signaling.EMBO J. 2006; 25: 910-920Crossref PubMed Scopus (168) Google Scholar, 15Kreimann EL Morales FC de Orbeta-Cruz J Takahashi Y Adams H Liu TJ McCrea PD Georgescu MM Cortical stabilization of beta-catenin contributes to NHERF1/EBP50 tumor suppressor function.Oncogene. 2007; 26: 5290-5299Crossref PubMed Scopus (84) Google Scholar However, the exact impact of EBP50 on cell proliferation remains unclear. Whereas in vitro experiments have suggested anti-proliferative functions of EBP50,14Takahashi Y Morales FC Kreimann EL Georgescu MM PTEN tumor suppressor associates with NHERF proteins to attenuate PDGF receptor signaling.EMBO J. 2006; 25: 910-920Crossref PubMed Scopus (168) Google Scholar, 15Kreimann EL Morales FC de Orbeta-Cruz J Takahashi Y Adams H Liu TJ McCrea PD Georgescu MM Cortical stabilization of beta-catenin contributes to NHERF1/EBP50 tumor suppressor function.Oncogene. 2007; 26: 5290-5299Crossref PubMed Scopus (84) Google Scholar, 16Pan Y Wang L Dai JL Suppression of breast cancer cell growth by Na+/H+ exchanger regulatory factor 1 (NHERF1).Breast Cancer Res. 2006; 8: R63Crossref PubMed Scopus (53) Google Scholar a positive correlation between EBP50 expression, estrogen receptor status, and tumor progression has been found in human breast cancer.9Cardone RA Bellizzi A Busco G Weinman EJ Dell'aquila ME Casavola V Azzariti A Mangia A Paradiso A Reshkin SJ The NHERF1 PDZ2 domain regulates PKA-RhoA-p38-mediated NHE1 activation and invasion in breast tumor cells.Mol Biol Cell. 2007; 18: 1768-1780Crossref PubMed Scopus (118) Google Scholar, 10Song J Bai J Yang W Gabrielson EW Chan DW Zhang Z Expression and clinicopathological significance of oestrogen-responsive ezrin-radixin-moesin-binding phosphoprotein 50 in breast cancer.Histopathology. 2007; 51: 40-53Crossref PubMed Scopus (56) Google Scholar Of particular interest with respect to biliary pathophysiology, the major regulators of EBP50 expression are estrogens,4Ediger TR Kraus WL Weinman EJ Katzenellenbogen BS Estrogen receptor regulation of the Na+/H+ exchange regulatory factor.Endocrinology. 1999; 140: 2976-2982Crossref PubMed Scopus (105) Google Scholar, 17Ediger TR Park SE Katzenellenbogen BS Estrogen receptor inducibility of the human Na+/H+ exchanger regulatory factor/ezrin-radixin-moesin binding protein 50 (NHE-RF/EBP50) gene involving multiple half-estrogen response elements.Mol Endocrinol. 2002; 16: 1828-1839Crossref PubMed Scopus (39) Google Scholar which are known to target the biliary tree, where they modulate the proliferative activities of cholangiocytes.18Alvaro D Alpini G Onori P Perego L Svegliata Baroni G Franchitto A Baiocchi L Glaser SS Le Sage G Folli F Gaudio E Estrogens stimulate proliferation of intrahepatic biliary epithelium in rats.Gastroenterology. 2000; 119: 1681-1691Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 19Alvaro D Alpini G Onori P Franchitto A Glaser S Le Sage G Gigliozzi A Vetuschi A Morini S Attili AF Gaudio E Effect of ovariectomy on the proliferative capacity of intrahepatic rat cholangiocytes.Gastroenterology. 2002; 123: 336-344Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 20Alvaro D Barbaro B Franchitto A Onori P Glaser SS Alpini G Francis H Marucci L Sterpetti P Ginanni-Corradini S Onetti Muda A Dostal DE De Santis A Attili AF Benedetti A Gaudio E Estrogens and insulin-like growth factor 1 modulate neoplastic cell growth in human cholangiocarcinoma.Am J Pathol. 2006; 169: 877-888Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar In the present study, we examined the expression of EBP50 and binding partners in the human liver, under normal conditions and in the setting of biliary disorders, ie, in different types of human cholangiopathies and after bile duct ligation (BDL) in rats. We tested the hypothesis that EBP50 could be regulated by estrogens, control cell proliferation in cholangiocytes, and thereby participate to ductular reactions in biliary disorders. Human tissue samples were used with informed consent of the patients on approval by the Regional Ethical Committees at Karolinska and Göteborg University Hospitals. Liver tissue was obtained by liver biopsy from 10 patients with cystic fibrosis (CF) liver disease (4 were females; mean age, 15.3 years; range, 3.6 to 38 years), 5 with primary biliary cirrhosis (PBC) (all females; mean age, 60.8 years; range, 45 to 68 years), 3 with primary sclerosing cholangitis (PSC) (all females; mean age, 51.3 years; range, 42 to 58 years), and 1 (male) who had bile stone obstruction for 7 months. Most of the patients had early-stage liver disease, with the exception of one CF patient having extensive fibrosis, one PBC, and one PSC patient, having cirrhosis. All CF patients were either homozygous or compound heterozygous for the ΔF508 CFTR mutation. Normal liver and gallbladder tissue specimens (ie, with no histological abnormality) were obtained from patients who underwent cholecystectomy or liver surgery for focal lesion(s). Immediately after tissue collection, part of the sample was snap-frozen in liquid nitrogen and stored at −70°C. BDL was performed by double ligation and section of the common bile duct in male and female Sprague-Dawley rats (Janvier, Le Genest Saint-Isle, France) 10 to 12 weeks of age, as reported.21Beaussier M Wendum D Schiffer E Dumont S Rey C Lienhart A Housset C Prominent contribution of portal mesenchymal cells to liver fibrosis in ischemic and obstructive cholestatic injuries.Lab Invest. 2007; 87: 292-303Crossref PubMed Scopus (129) Google Scholar Sham operation consisted in laparotomy and bile duct exposure without ligation. To assess the impact of estrogens, female rats were ovariectomized 3 to 5 weeks before BDL was performed, as previously described.19Alvaro D Alpini G Onori P Franchitto A Glaser S Le Sage G Gigliozzi A Vetuschi A Morini S Attili AF Gaudio E Effect of ovariectomy on the proliferative capacity of intrahepatic rat cholangiocytes.Gastroenterology. 2002; 123: 336-344Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar The time course of cholangiocyte proliferation including after ovariectomy, was previously established.19Alvaro D Alpini G Onori P Franchitto A Glaser S Le Sage G Gigliozzi A Vetuschi A Morini S Attili AF Gaudio E Effect of ovariectomy on the proliferative capacity of intrahepatic rat cholangiocytes.Gastroenterology. 2002; 123: 336-344Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 22Tuchweber B Desmoulière A Brochaton-Piallat ML Rubbia-Brandt L Gabbiani G Proliferation and phenotypic modulation of portal fibroblasts in early stages of cholestatic fibrosis in the rat.Lab Invest. 1996; 74: 265-278PubMed Google Scholar, 23Marucci L Baroni GS Mancini R Benedetti A Jezequel AM Orlandi F Cell proliferation following extrahepatic biliary obstruction. Evaluation by immunohistochemical methods.J Hepatol. 1993; 17: 163-169Abstract Full Text PDF PubMed Scopus (25) Google Scholar Experiments were conducted in compliance with the national ethical guidelines for the care and use of laboratory animals. The animals were anesthetized with a subcutaneous injection of chlorpromazine (2 mg/kg) and ketamine (20 mg/kg). Investigations were performed on postoperative days 1, 2, and 7. For immunofluorescence analyses, the liver was perfused in situ with 4% paraformaldehyde, cut in small pieces, postfixed in 4% paraformaldehyde for 1 hour at 4°C, and stored in 1% paraformaldehyde overnight at 4°C. Human hepatocytes, intrahepatic bile ducts, and gallbladder epithelial cells were isolated from samples of normal human liver and gallbladder, using established methods.24Chignard N Mergey M Barbu V Finzi L Tiret E Paul A Housset C VPAC1 expression is regulated by FXR agonists in the human gallbladder epithelium.Hepatology. 2005; 42: 549-557Crossref PubMed Scopus (40) Google Scholar Isolation of intrahepatic bile ducts from rat liver was performed as described.25Kinnman N Hultcrantz R Barbu V Rey C Wendum D Poupon R Housset C PDGF-mediated chemoattraction of hepatic stellate cells by bile duct segments in cholestatic liver injury.Lab Invest. 2000; 80: 697-707Crossref PubMed Scopus (120) Google Scholar More than 90% of the cells in bile duct preparations were cholangiocytes, as ascertained by cytokeratin 19 and γ-glutamyltransferase staining.25Kinnman N Hultcrantz R Barbu V Rey C Wendum D Poupon R Housset C PDGF-mediated chemoattraction of hepatic stellate cells by bile duct segments in cholestatic liver injury.Lab Invest. 2000; 80: 697-707Crossref PubMed Scopus (120) Google Scholar The human biliary epithelial cell line Mz-Cha-126Knuth A Gabbert H Dippold W Klein O Sachsse W Bitter-Suermann D Prellwitz M Meyer zum Buschenfelde K Biliary adenocarcinoma. Characterisation of three new human tumor cell lines.J Hepatol. 1985; 1: 579-596Abstract Full Text PDF PubMed Scopus (169) Google Scholar was provided by Alexander Knuth, (Zurich University Hospital, Zurich, Switzerland). Mz-Cha-1 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1% Hepes. To test the effect of estrogens, Mz-Cha-1 cells were placed in serum- and phenol red-free Dulbecco's modified Eagle's medium/1% Hepes for 24 hours before treatment with 17β-estradiol (Sigma-Aldrich Chemie S.a.r.l., L'Isle d'Abeau Chesnes, France) at 10−10 or 10−7 mol/L for 6 to 24 hours. Alternatively, to completely deprive Mz-Cha-1 cells of estrogen receptor stimulation, the cells were maintained in phenol red-free Dulbecco's modified Eagle's medium/1% Hepes supplemented with 10% charcoal-stripped serum for 2 to 3 weeks. Total RNA was extracted from liver tissue and cell preparations using RNA plus lysis solution (Quantum, Montreuil-sous-Bois, France). Complementary DNA was synthesized from 1 μg of total RNA using pd(N)6 primers (Amersham, GE Health Care Europe GmbH, Saclay, France) and the Moloney murine leukemia virus reverse transcriptase (Invitrogen, Cergy-Pontoise, France). EBP50, ezrin, CFTR, and β-actin transcripts were detected by conventional PCR, using the following primers: 5′-GATCGCATTGTGGAGGTGAA-3′ (forward) and 5′-GGAGATGTTGAAGTCTAGGA-3′ (reverse) to amplify a 389-bp fragment of EBP50 cDNA; 5′-GCAGGACTATGAGGAGAAGAC-3′ (forward) and 5′-GTGATGCGCTTCTCCTCATTG-3′ (reverse) to amplify a 503-bp fragment of ezrin cDNA; 5′-AACTGCTGAACGAGAGGAGC-3′ (forward) and 5′-TTGACTATTGCCAGGAAGCC-3′ (reverse) to amplify a 367-pb fragment of CFTR cDNA; 5′-CCTCATGAAGATCCTCACCG-3′ (forward) and 5′-CAGTGATCTCCTTCTGCATCC-3′ (reverse) to amplify a 660-pb fragment of β-actin cDNA. PCR products obtained after completion of 28 cycles were separated by electrophoresis through a 2% agarose gel stained with ethidium bromide. Quantitative real-time PCR was performed with the TaqMan system, using the SYBR green master mix (Applied Biosystems, Courtaboeuf, France). The primers were designed according to published human cDNA sequences in GenBank database using the Primer Express software v1.5 (Applied Biosystems). EBP50 (SLC9A3R1, accession no. NM_004252), 5′-CCAGGATCGCATTGTGGAG-3′ (forward) and 5′-CCATTGGTGAAGGGCACAG-3′ (reverse); ezrin (VIL2, accession no. NM_003379), 5′-CTAGAGGCTGACCGTATGGCTG-3′ (forward) and 5′-GAGGGCAATCTTGGCAGTGT-3′ (reverse); CFTR (ABCC7, accession no. NM_000492), 5′-CCATCAGCCCCTCCGAC-3′ (forward) and 5′-AAAGCCTTGTATCTTGCACCTCT-3′ (reverse); 18S rRNA (accession no. NM_002801), 5′-GAGCGAAAGCATTTGCCAAG-3′ (forward) and 5′-GGCATCGTTTATGGTCGGAA-3′ (reverse). 18S rRNA TaqMan assay reagent was used for internal control. One-step RT-PCR was performed for both target gene and endogenous controls. Duplicate CT values were analyzed in Microsoft Excel (Microsoft Corp., Redmond, WA) using the comparative CT (ΔΔCT) method as described by the manufacturer (Applied Biosystems). The amount of target (2−ΔΔCT) was obtained as normalized to 18S. Human liver cryosections (6 μm) were subjected to immunolabeling with anti-EBP50 rabbit polyclonal antibody (catalog no. 324620; Calbiochem, Fontenay sous Bois, France) at a dilution of 1:300; or mouse monoclonal antibodies raised against EBP50 (catalog no. 611160; R&D Systems Europe Ltd., Lille, France) at 1:50; ezrin (catalog no. MS-661-P1; NeoMarker, Montlucon, France) at 1:40; CFTR (catalog no. MAB-25031; R&D Systems Europe Ltd., Abingdon, UK) at 1:100, or Ki-67 (catalog no. dia607; Dianova, Hamburg, Germany) at 1:100. Except for ezrin detection by an alkaline phosphatase anti-alkaline phosphatase/Fast Red protocol (Vector Laboratories, Paris, France), immunolabeling was performed using an avidin-biotin method. For the latter, the sections were fixed in 4% paraformaldehyde and were incubated subsequently with serum and avidin-biotin blocking reagents (Vectastain ABC, Vector Laboratories), with the primary antibody overnight at 4°C, with a biotinylated secondary antibody (Vector Laboratories) for 30 minutes at room temperature and with Cy3-streptavidin (catalog no. S6402, Sigma-Aldrich Chemie S.a.r.l.) for 60 minutes at room temperature. For double immunofluorescence, secondary antibodies were conjugated with tetramethylrhodamine isothiocyanate (Jackson ImmunoResearch Europe Ltd., Monlutcon, France) or Alexa 488 (Invitrogen Molecular Probes, Cergy-Pontoise, Paris); 4′,6-diamidino-2-phenylindole (Sigma-Aldrich Chemie S.a.r.l.) at a dilution of 1:10,000 or SYTO16 (Invitrogen Molecular Probes) at 1:25,000 were added to the second-last wash for nuclear staining. The samples were mounted with an anti-fading medium (Vectashield, Vector Laboratories). The slides were examined with a Nikon Eclipse E800 microscope connected to a Nikon DXM1200 digital camera and images were acquired with ACT-1 software (Nikon, Tokyo, Japan). Laser confocal microscopy was performed with a Leica TCS SP confocal laser-scanning microscope and images were analyzed with Leica confocal software (Leica, Wetzlar, Germany). Triple staining of EBP50, actin, and DNA was achieved on thick sections (50 μm) of rat liver, obtained with a vibrating blade microtome (VT1000E, Leica Microsystems) and permeabilized in saponin 0.1% (Sigma-Aldrich Chemie S.a.r.l.) for 1 hour. All incubations were performed on floating sections at room temperature (unless otherwise stated), under gentle rocking. After quenching of the aldehydes [30 minutes in phosphate-buffered saline (PBS)/NH4Cl 50 mmol/L], the sections were incubated for 2 hours in blocking buffer (PBS with bovine serum albumin 1%, goat serum 10% and saponin 0.1%). All subsequent incubations were performed in PBS, saponin 1%. For EBP50 staining, the sections were incubated successively with anti-EBP50 rabbit polyclonal antibody (Calbiochem) (2 μg/ml, overnight at 4°C) and Alexa Fluor 488-conjugated goat anti-rabbit IgG (Invitrogen Molecular Probes) (10 μg/ml, 2 hours). Filamentous actin was stained with Alexa Fluor 555 phalloidin (5 U/ml for 40 minutes) and eventually cell nuclei were counterstained with TO-PRO-3 (1 μmol/L for 20 minutes). Sections were mounted in glycerol/PBS (90/10:v/v). Images were recorded on a Leica TCS SP2 confocal microscope (Leica Microsystems) equipped with a ×63 oil-immersion objective (NA = 1.32). The three channels were acquired sequentially with the following excitation and emission parameters: 488 nm, 500 to 540 nm, for Alexa 488; 543 nm, 555 to 615 nm, for Alexa 555; and 633 nm, 645 to 750 nm, for TO-PRO-3. Gains were adjusted to avoid saturation in pixels intensity. The same procedure was used to perform triple staining in Mz-Cha-1 cells, except that incubation times were reduced to 15 minutes for fixation, 10 minutes for permeabilization, 1 hour for each antibody, 20 minutes for phalloidin, and 10 minutes for TO-PRO-3. β-Catenin immunostaining was performed with an anti-β-catenin polyclonal antibody (Cell Signaling, Ozyme, Saint Quentin en Yvelines, France). Ki-67 immunolabeling of Mz-Cha-1 cells was analyzed using an anti-Ki-67 polyclonal antibody coupled with fluorescein isothiocyanate (dilution 1:100; Abcam, Paris, France). Nuclei were stained with TO-PRO-3, and the percentage of Ki-67-labeled nuclei was determined. Mz-Cha-1 cells were stably transfected with a plasmid encoding human EBP50 siRNA (provided by Brian R. Doctor, University of Colorado Health Sciences Center, Denver, CO) or scrambled siRNA by incubation in the presence of Lipofectamine 2000 (Invitrogen) for 2 days. Transfected cells were then 10-fold serially diluted into 10-cm Petri dishes and incubated in the absence of selection for 2 additional days. Puromycin was added at a final concentration of 1 μg/ml to select cells that had acquired the plasmids. Culture medium containing puromycin was changed every 2 to 3 days and when colonies appeared, generally 12 to 14 days later, individual puromycin-resistant colonies were selected and cultured in 12-well culture dishes. Mz-Cha-1 cells were cultured in 96-wells plates (10,000 cells/well) in serum-deprived medium for 3 days and then incubated with 10 μmol/L BrdU in the presence of serum for 1 hour. BrdU incorporation was measured in triplicate, using a cell proliferation enzyme-linked immunosorbent assay kit (Roche Applied Science, Meylan, France). Immunoblotting was performed with mouse monoclonal antibodies raised against proliferating cell nuclear antigen (PCNA) (clone PC10, Cell Signaling) at a dilution of 1:2000, EBP50 (clone 6; BD Biosciences, Le Pont-De-Claix, France) at 1:250, α-tubulin (clone DM1A; Abcam, Paris, France) at 1:5000, lamin A/C (clone JOL2; Millipore Chemicon, Paris, France) at 1:500 or β-actin (Sigma-Aldrich Chemie S.a.r.l.) at 1:10,000. Proteins were extracted from Mz-Cha-1 cells using a lysis buffer composed of 150 mmol/L NaCl, 2 mmol/L phenylmethyl sulfonyl fluoride, 1% Nonidet P-40, 0.5% deoxycholate, 1 mg/L aprotinin, 0.1% sodium dodecyl sulfate, and 50 mmol/L Tris-HCl, pH 7.5. Lysates were precleared by centrifugation at 13,000 × g for 30 minutes at 4°C. Subcellular fractions were prepared with the NE-PER nuclear and cytoplasmic extraction reagents kit (Pierce, Perbio Science France SAS, Brebières, France). Protein concentration was determined by the bicinchoninic acid-based BCA protein assay kit (Pierce, Perbio Science France SAS). Proteins (5 to 20 μg) were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Membranes were incubated with the primary antibodies overnight and with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling) at 1:2000, for 1 hour. Immunoreactive bands were detected by enhanced chemiluminescence using an enhanced chemiluminescence kit (Amersham, GE Health Care Europe GmbH). After exposure to X-OMAT film, the autoradiographic bands were scanned and quantified with ChemiImager 4400 (Astec Co. Ltd., Osaka, Japan). Comparisons between pairs were made using the Mann-Whitney U-test. Comparisons between multiple groups were made using two-way analysis of variance with repeated measures (Statview, Abacus Concept, CA) followed by pairwise comparison. Differences of P < 0.05 were considered statistically significant. The expressions of EBP50 and its binding partners, ezrin and CFTR, were examined in isolated human hepatocytes, bile duct epithelial cells (cholangiocytes), and gallbladder epithelial cells. EBP50 transcripts were detected by RT-PCR in all epithelial cell types, whereas CFTR and ezrin transcripts were detected only in biliary epithelial cells (Figure 1A). qRT-PCR analyses showed that EBP50 is expressed at similar levels in bile ducts and in hepatocytes, whereas in gallbladder epithelial cells, the amount of EBP50 transcripts is ∼40-fold higher, along with a higher expression of ezrin (by twofold) and of CFTR (by eightfold) in the gallbladder compared with bile duct epithelial cells (Figure 1B). Immunohistochemical analyses showed that in normal conditions, EBP50 protein is localized in the canalicular/apical domains of human hepatocytes, bile duct, and gallbladder epithelial cells (Figure 1C, middle and right). In hepatocytes, EBP50 immunostaining decorated juxtacanalicular vesicles in addition to canalicular membranes (Figure 1C, left), suggesting a possible link between this protein and canalicular transporter(s). In the liver, ezrin immunostaining was confined to the apical domain of bile ducts (Figure 1D), and double staining showed an overlapping of EBP50 with CFTR in this region (Figure 1E), consistent with the view that EBP50 acts as a linker between ezrin and CFTR in cholangiocytes. Next, the pattern of EBP50 expression was examined by immunohistochemistry in the liver of patients with cholangiopathies, ie, with CF liver disease, PBC, PSC, or bile stone-induced obstruction. The CF patients were either homozygous or heterozygous for ΔF508, a mutation that impairs the trafficking of CFTR to the cell surface. In their liver specimens, EBP50 was detected not only in hepatocytes and in native bile ducts, but also in cells of the ductular reaction. A basolateral and intracellular distribution of EBP50 was detected selectively in these cells (Figure 2A), as opposed to native bile duct cells (not shown). Ezrin was also detected but remained strictly apical in the ductular reactive cells (Figure 2A). In addition, the immunostaining of EBP50 only partly overlapped with that of CFTR mutant protein in these cells (
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