311 Regulation of hepatobiliary transporters in response to hypoxia

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hepatocytes, reaching 94• (p < 0.001), and 93• 11% (p < 0.001) peak immunofluorescence compared to glutamine synthetase-negative pericentral hepatocytes of controls, respectively. Likewise, Oatplb2 was induced in periportal hepatocytes, reaching 92• (p < 0.01) peak immunofluorescence compared to pericentral hepatocytes of controls. Down-regulation of Oatplal occurred homogenously in periportal and pericentral hepatocytes. Conclusion: Down-regulation of Ntcp and Bsep in obstructive cholestasis is largely confined to periportal hepatocytes. Increasing intracellular Bsepand Ntcp-positive vesicles point to a predominant endocytic transporter retrieval in the periportal area. Induction of periportal Oatp 1a4 und Oatp lb2 suggests a potential role in compensatory basolateral export of bile acids and organic anions in hepatocytes with profound down-regulation of Bsep, Ntcp and Mrp2.

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HUMAN C H O L A N G I O C A R C I N O M A CELLS EXPRESS LEPTIN RECEPTORS AND RESPOND TO LEPTIN WITH INCREASE OF PROLIFERATION

G. Fava 1, G. Alpini 2'3, C. Rychlicki 1, G. Svegliati-Baroni 1, A. di Sario 1, S. De Morrow 2, H. Francis 2, J. Venter2, S. Glaser 2, R. Reichenbach 3, A. Benedetti 1. 1Dept. of Gastroenterology, Polytechnic University of

Marche, Ancona, Italy," 2Scott & White Hospital, The Texas A&M University System, 3Central Texas' Veterans HCS, Temple, TX, USA B a c k g r o u n d : Leptin, a hormone primarily produced by adipocytes, regulates food intake and energy expenditure. Serum leptin is elevated in obesity, a risk factor for tumors of the biliary tract. Leptin is a mitogenic factor for many cancer types but its role on cholangiocarcinoma cell growth is unknown. Aims: We aimed to evaluate if: (i) malignant cholangiocytes express long and short leptin receptor isoforms (ObR1 and ObRs) and leptin protein; (ii) leptin regulates cholangiocarcinoma proliferation; and (iii) the effect of leptin is associated with changes in PI3K/AKT, STAT-3 and ERKI/2 pathways. M a t e r i a l and Methods: The expression of ObR was evaluated in normal (H69) and malignant (Mz-ChA-1, HUH-28 and TFK-1) cholangiocytes by immunofluorescence, RT-PCR and immunoblots. The expression of leptin protein was assessed by immunofluorescence and immunoblots. The dose and time-dependent effect of leptin on cholangiocarcinoma growth was evaluated by BrDU incorporation by stimulating HUH-28 cells with human recombinant leptin. The effect of leptin on intracellular signaling pathways was assessed by immunoblots. Results: Normal and malignant cholangiocytes express ObR1, ObRs and Leptin protein. Leptin is more expressed in malignant than in normal cholangiocytes. Leptin (10 100ng/ml) significantly stimulated HUH-28 cell growth, and the stimulatory effect of leptin persisted up to 48 hours. Leptin increase of cholangiocarcinoma cell growth was associated with phosphorylation of STAT-3 and ERKI/2, without change in AKT protein phosphorylation. Pretreatment with AG490, a JAK/STAT specific inhibitor (AG490, 20 mM), blocked the stimulatory effect of leptin and inhibited leptin-induced ERKI/2 phosphorylation. Preincubation with PD98059, a MEK inhibitor (PD98059, 20 mM), did not significantly change leptinmediated STAT-3 phosphorylation levels. Conclusions: Leptin, interacting with ObR1, stimulates cholangiocarcinoma cell proliferation through STAT-3 dependent ERKI/2 activation. High circulating levels of leptin could explain the increased incidence of biliary tumors in particular clinic conditions such as obesity. Thus, modulation of leptin signal may be useful to modulate growth of tumors of the biliary tract.

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HEPATIC EXPRESSION OF PPAR-y COACTIVATOR 1 (PGC-1) IS REDUCED IN HUMAN CHOLELITHIASIS

M. Bertolotti 1, C. Gabbi 1, C. Anzivino 1, N. Mitro 2, C. Godio 2, E. De Fabiani 2, M. Crestani 2, L. Carulli 1, M. Del Puppo 3, A. Rossi4, R Loria 1, N. Carulli 1. 1Dipartimento di Medicine e Specialitf Mediche, Universitf

di Modena e Reggio Emilia, Modena, Italy," 2Dipartimento di Scienze Farmacologiche, Universit& di Milano, Milano, Italy," 3DIMESAB, Universit& di Milano Bicocca, Monza, Italy," 4Dipartimento di Scienze Chirurgiche, Universit& di Modena e Reggio Emilia, Modena, Italy The prevalence of cholesterol cholelithiasis in the Western world is high. Even if laparoscopic cholecystectomy represents a satisfactory treatment in most instances, some drawbacks need to be considered such as stone recurrence, pancreatitis due to biliary sludge and postoperative complications. Further insight in the pathophysiology and molecular regulation of biliary lipid secretion is therefore highly desirable. Aim of the present study was to analyze the hepatic expression of a number of nuclear receptors involved in bile acid metabolism in human cholelithiasis. Methods: Surgical liver biopsies were obtained in 11 patients with untreated cholesterol cholelithiasis and 9 gallstone-free subjects; mRNA levels of CYP7A1 and related nuclear receptors and coactivators were assayed by real-time quantitative RT-PCR. Results: No differences between the two groups were detected in gene expression of CYP7A1 and other nuclear receptors, with the exception of PPAR-7 coactivator 1 (PGC-1), a transcriptional coactivator of CYP7A1 also involved in insulin sensitivity and energy balance, which was significantly (p <0.01 on a log scale) less expressed in gallstone subjects. Expression of PGC-1 was linearly correlated with FXR both in the whole population (r 0.55 on a log scale, p <0.05) and in gallstone patients (r 0.87, p <0.01); in gallstone patients PGC-1 expression was also correlated with HNF-4 (r 0.78, p <0.01). Conclusions: PGC-1 appears to play a role in the prevention of cholesterol gallstone disease in humans; the finding might suggest a link with insulin resistance conditions. This effect is likely to take place via interaction with the bile acid receptor FXR, whose protective role in cholelithiasis has been suggested by recent evidence in animal models. Involvement of target genes coding for biliary lipid transporters is likely. PGC-1 and related genes might therefore represent molecular targets for the prevention and/or treatment of gallstone disease. Grant support: Supported by 5th Framework Program grant QLGI-CT2001~01513, by COFIN-PRIN grant 2002062991 and by COFIN-PRIN grant 2004 067491.

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REGULATION OF HEPATOBILIARY T R A N S P O R T E R S IN RESPONSE TO HYPOXIA

L. Fouassier 1, M. Beaussier 1'2, E. Schiffer3, C. Rey 1, E. Lasnier4, J.Y. Scoazec 5, A. Lienhart 2, V. Barbu 1,4, C. Housset 1. lInserm U680,

UPMC, Paris', France," 2Anesthesiology Department, H@ital SaintAntoine, Paris', France," 3Anesthesiology Department, University Hospital Geneva, Geneva, Switzerland," 4Biochemistry Department, H@ital saintAntoine, Paris', France," 5Pathology Department, Hdpital Edourd Herriot, Lyon, France Ischemia/hypoxia causes cholestatic disorders, in particular in the setting of liver transplantation, by mechanisms that are yet poorly defined. Because hypoxia may induce transcriptional regulations of various genes, we examined potential changes in the expression of transporter genes expressed in hepatocytes or in cholangiocytes, in response to hypoxia. Methods: In vivo, ischemia was induced by complete arterial deprivation of the rat liver. Dynamic tests included the injection of fluorescent ursodeoxycholic acid aimed to visualize potential leakage of bile ducts. In vitro, hepatocytes and cholangiocytes were isolated from normal rat

04B. Molecular and cellular biology liver, allowed to adhere in culture and submitted to a catalytic system, which reduces oxygen concentration to less than 1% within 30 minutes. Gene expressions were assessed by quantitative real-time RT-PCR. Results: In vivo, 24 hours after the onset of arterial liver ischemia in rats, cholestatic changes in serum were detected together with a decrease in bile flow and in bile acid and bicarbonate output in bile. No regurgitation of fluorescent ursodeoxycholic acid was detected, eliminating bile duct leakage as a major mechanism of cholestasis in this model. VEGF, a hypoxia-regulated gene, was induced in both hepatocytes and cholangiocytes, as ascertained by immunohistochemistry. Concomitantly, hepatic mRNA levels of ntcp, bsep and mrp2 were significantly reduced (by 70%, 50% and 70%, respectively), whereas Cftr mRNA levels were increased (by more than 4 fold), even though ductular reaction was not yet present at this time. In vitro, hypoxia caused a decrease in ntcp, bsep and mrp2 transcripts in hepatocytes (by 70%, 80% and 70%, respectively), and an increase in cftr transcripts (by 3 fold), associated with a rise in cAMP (by more than 10 fold) in cholangiocytes. Conclusion: These results demonstrate that hypoxia induces differential regulations in the expression of hepatobiliary transporters. The downregulation of transporters involved in bile salt-dependent and -independent secretion in hepatocytes may contribute to cholestasis. By contrast the upregulation of CFTR in cholangiocytes may, together with increased cAME a major regulator of CFTR expression and activity, and with subsequent ductular proliferation, provide a defense mechanism.

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EBP50, A SCAFFOLD PROTEIN PARTICIPATING IN THE PROLIFERATION OF CHOLANGIOCYTES, IS DELOCALIZED IN THE DUCTULAR REACTION ASSOCIATED WITH CYSTIC FIBROSIS LIVER DISEASE

L. Fouassier1, R Rosenberg2, M. Mergey 1, N. Kinnman 3, B. Strandvik4, R. Hultcrantz 2, C. Housset1. lInserm U680, UPMC, Paris', France,"

2Karolinska institute, Stockholm, Sweden," 3Serono International SA, Geneva, Switzerland," 4Gothenburg University, Gothenburg, Sweden Background and Alms: While bile duct epithelium is a primary target, bile duct proliferation is a typical feature in cystic fibrosis (CF) liver disease. The expression of ezrin-moesin-radixin binding phosphoprotein 50 (EBP50), a scaffold protein interacting with CFTR, has been previously detected in rat cholangiocytes. Different lines of evidence suggest that EBP50 may be involved in cell proliferation. The aim of this study was to examine the expression of EBP50 in bile duct alteration associated with CF liver disease and its potential implication in cholangiocyte proliferation. Methods: The expression of EBP50 and of its partner ezrin, was first examined by RT-PCR in fresh isolates of the different epithelial cell types lining the human biliary tree. EBP50, ezrin and CFTR localizations were determined by immunohistochemistry in normal human liver (n 3) and in the liver from CF patients carrying the ?F508 mutation (n 9). To examine the contribution of EBP50 to cell proliferation, a knock-down of endogenous EBP50 was achieved in a human biliary epithelial cell line, using siRNA. Results: RT-PCR analyses showed that EBP50 is expressed in the different epithelial cell types lining the human biliary tree, i.e. hepatocytes, bile duct and gallbladder epithelial cells (cholangiocytes), while ezrin is expressed in cholangiocytes but not in hepatocytes. EBP50 mRNA expression was not changed in CF liver. Immunohistochemical analyses showed that in native interlobular bile ducts from normal and CF liver, EBP50 and ezrin were localized beneath the apical membrane of cholangiocytes, while mutant ?F508 CFTR was delocalized in the cytoplasm. By contrast, in proliferating bile ducts of the liver from CF patients, while ezrin remained localized in the apical region of cholangiocytes, EBP50 showed a heterogeneous distribution in the cytoplasm. The knock-down of endogenous EBP50 by siRNA in the MzChA-1 biliary epithelial cell line caused a marked inhibition in cell proliferation, as demonstrated by a 70% reduction in BrDU incorporation and a 90% reduction in PCNA expression.

(b) Biliary tract pathophysiology

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Conclusion: These results indicate that in CF liver disease, EBP50 displays an abnormal localization in the cytoplasm of cholangiocytes of the ductular reaction independently of CFTR delocalization, and that EBP50 participates in the proliferation of these cells.

~3-] DIFFERENT CLAUDIN-4 EXPRESSION IN BILIARY AND HEPATOCELLULAR CARCINOMAS C. Lddi 1, E. Szab61 , E. Batmunkh 1, A. Holczbauer 1, S. Paku 2, R Kupcsulik3, G. Illy~s 1, A. Kiss 1, Z. Schaff1. 12nd Department of

Pathology, 2First Department of Pathology and Experimental Cancer Researeh, 3First Department of Surgery, Semmelweis University, Budapest, Hungary Background and Aims: Cell adhesion molecules play an important role in carcinogenesis, especially claudins, being part of tight junctions. The importance of certain claudins has been shown, especially of claudin-4, in the development of several tumors, and some of them have even been suggested as a future therapeutic target. For this reason, the aim of our study was to analyse the expression of claudin-4 in biliary and hepatocellular carcinomas. Methods: A total of 103 cases were investigated: 53 biliary carcinomas (BCs), arising from different localisations (gallbladder, common bile duct, intrahepatic bile ducts) and 50 hepatocellular carcinomas (HCCs). Immunohistochemistry was performed on formalin-fixed paraffin-embedded tissue specimens using anti-claudin-4 antibody. Claudin-4 was further investigated by Western blot analysis on 5 5 human surgically resected, snap frozen BC and HCC specimens. Subcellular localisation of claudin-4 was detected by confocal laser scanning microscopy. Results: Intensive membranous immunolabeling was found for claudin-4 in BCs of different origin, however none of the HCCs showed positive immunostaining for claudin-4. A single band of the expected size was detected on Western immunoblots and revealed a strong expression of claudin-4 solely in case of BC. Confocal microscopy confirmed the expression of claudin-4 on the membrane of BC cells. Conclusions: Our data report for the first time a significantly increased claudin-4 expression in BCs. The results represent a novel feature of BCs and claudin-4 protein could well become a useful marker in differentiating biliary carcinomas from hepatocellular carcinomas. The project was supported by grants: NKFP-1/0023/2002, NKFP1A/002/2004, ETT- 228/2001, OTKA-T037838.

~THE ACTIVATION OF IGF1 SYSTEM IS A REQUISITE FOR CHOLANGIOCYTE SURVIVAL DURING THE PROGRESSION OF PRIMARY BILIARY CIRRHOSIS (PBC) D. Alvaro 1, A. Floreani 3, R Onori4, A. Franchitto 4, M.G. Mancinol~ M. Guido 3, G. Carpino4, A. De Santis 1, M. Angelico 2, A.F. Attili 1, E. Gaudio 4. 1Department of Clinical Medicine,

Division of Gastroenterology, University "La Sapienza", Rome, Italy," 2Gastroenterology Unit, University Tor Vergata, Rome, Italy," 3Department of Surgical and Gastroentero-logical Sciences, University of Padua, Padua, Italy," 4Department of Anatomy, University 'Za Sapienza '" Rome, Italy We have recently shown, in experimental studies, that IGF1 (insulinlike growth factor-l) system activation plays a key role in determining cholangiocyte survival and resistance to apoptosis. The aim of this study was to evaluate IGF1 and IGF1-R (receptor) in cholangiocytes of patients with different stages of PBC and correlate their expression with markers of proliferation and apoptosis. Liver biopsies from PBC patients (n 35) and normal subjects (n 5) were investigated by immunohistochemistry (IHC) for IGF1 and IGF1-R as well as for markers of proliferation (PCNA) and