Bile Acids, Cholesterol, and Cholangiocyte Biology


Rab1 1 facilitates cyclic AMP- and tauroursodeoxycholate-induced MRP2 translocation

Se Won Park1, Christopher M. Schonhoff1, Cynthia Webster2, Mohammed S. Anwer1;

1Biomedical Sciences, Tufts Univ Cum-mings School of Veterinary Medicine, North Grafton, MA; 2Clinical Sciences, Tufts Univ Cummings School of Veterinary Medicine, North Grafton, MA

Rab proteins (Ras homologous for brain) play an important role in vesicle trafficking, including endocytosis, vesicle motility and protein trafficking. Rab1 1 regulates vesicle recycling to the plasma membrane and is expressed in apical vesicular populations in discrete epithelial cell populations, including liver. Bile salt exporting pump (Bsep) cycles between canalicular membrane and Rab1 1-positive endosomes, and Rab1 1 is required for bile canalicular formation in WIF-B9 cells (Proc Natl Acad Sci 102: 15087, 2005; Mol Biol Cell 15: 3485, 2004). Both cAMP and tauroursodeoxycholate (TUDC) have been shown to increase plasma membrane Mrp2 in rat hepa-tocytes. However, the role of Rab1 1 in cAMP- and TUDC-induced MRP2 translocation has not been studied. The goal of the present study was to provide further insight into the role of Rab1 1 in the trafficking of MRP2. More specifically, we tested the hypothesis that Rab1 1 facilitates cAMP- and TUDC-induced MRP2 translocation. Studies were conducted in HuH-NTCP cells (HuH7 cells stably transfected with human NTCP), which con-stitutively express MRP2. HuH-NTCP cells were transfected with GFP-tagged wild type Rab1 1 (Rab1 1-WT) or GDP-locked inactive Rab1 1 (Rab1 1-GDP) to study the role of Rab1 1. Overex-pression of mutant constructs of Rab1 1 was confirmed by GFP immunoblots. Cells were treated with either 100 μM cAMP or 25 μM TUDC; these concentrations were shown to increase plasma membrane Mrp2 in hepatocytes. A biotinylation method and a GTP overlay assay were used to determine the plasma membrane MRP2 and the activation of Rab1 1, respectively. Cyclic AMP and TUDC significantly stimulated Rab1 1 activity by 57 +/- 1 1% and 20 +/- 2.3%, respectively (mean +/- SE, n=3), suggesting that Rab1 1 may be involved in MRP2 translocation by cAMP and TUDC. If Rab1 1 is involved, one would expect overexpression of Rab1 1-WT and Rab1 1-GDP to augment and inhibit, respectively, TUDC- and cAMP-induced increases in plasma membrane MRP2. Overexpression of either Rab1 1-WT or Rab1 1-GDP did not significantly affect the basal level of plasma membrane MRP2. In cells transfected with the empty vector, both cAMP and TUDC increased plasma membrane MRP2 by 13 +/- 3% (n=6) and 12 +/- 2% (n=4), respectively. The effect of cAMP and TUDC on plasma membrane MRP2 was further increased to 36 +/- 13% (n=6) and 76 +/-23% (n=4), respectively, in cells transfected with Rab1 1-WT. In contrast, cAMP and TUDC failed to increase plasma membrane MRP2 in cells transfected with Rab1 1-GDP. Taken together, these results suggest that TUDC- and cAMP-induced MRP2 translocation is facilitated by Rab 11.


The following people have nothing to disclose: Se Won Park, Christopher M. Schonhoff, Cynthia Webster, Mohammed S. Anwer


Role of MKK/p38 MAPK pathway in cAMP-induced translocation and TLC-induced retrieval of Mrp2 in mouse hepatocytes

Christopher M. Schonhoff1, Cynthia R. Webster2, Mohammed S. Anwer1;

1Biomedical Sciences, Tufts Veterinary School, North Grafton, MA; 2Clinical Sciences, Tufts Veterinary School, North Grafton, MA

There are four known isoforms (α, β, γ and δ) of p38 mitogen activated protein kinase (MAPK) with only α and β isoforms expressed in human livers. Three upstream MAPK kinases (MKKs), namely MKK3, MKK6 and MKK4, are known to activate p38 MAPK. Studies in non-hepatic cell suggest that MKK6 activates p38α and p38β MAPK, while MKK3 and MKK4 activate p38α MAPK. Cyclic AMP has been shown to increase plasma membrane MRP2 (PM-MRP2) by activating a MKK3/p38α in HuH-7 cells. In contrast, taurolithocholate (TLC), which also activates p38 MAPK in hepatocytes, decreases PM-Mrp2. Since cAMP and TLC both activate p38MAPK but have opposing effects on PM-MRP2 levels, we speculated that the effects of cAMP and TLC may be mediated via activation of MKK3/p38α and MKK6/p38β, respectively. Thus, the aim of the present study was to test the hypothesis that cAMP-induced increases in PM-Mrp2, but not TLC-induced decreases in PM-Mrp2, is mediated through activation of MKK3/p38α in primary hepatocytes. In order to define the importance of MKKs/p38 MAPK in PM-Mrp2 regulation, studies were conducted in 24 hours cultured hepatocytes isolated from C57BL/6 WT and MKK3 knockout (KO) mice. A biotinylation method was used to determine PM-Mrp2. Activations of MKK3, MKK4, p38 MAPK and p38α MAPK were determined using immunoblot analysis of phosphorylated (active) forms. As in rat hepatocytes, PM-Mrp2 was increased and decreased by cAMP and TLC, respectively, in WT mouse hepatocytes. Protein expression of MKK6, but not of MKK4, was increased by 3 fold in MKK3 KO hepatocytes. Despite this increase in MKK6 expression, cAMP failed to increase PM-Mrp2 in MKK3 KO hepatocytes, indicating that effect of cAMP on PM-Mrp2 requires MKK3 and not MKK6. In contrast, the ability of TLC to decrease PM-Mrp2 was not altered in MKK3 KO hepatocytes, indicating that MKK3 is not required for the TLC-mediated decreases in PM-Mrp2. In addition, cAMP but not TLC activated MKK3 in WT mouse hepatocytes. While cAMP as well as TLC activated p38 MAPK in WT hepatocytes, the activation of p38 MAPK by cAMP, but not by TLC, was decreased in MKK3 KO hepatocytes. In addition, cAMP and not TLC activated p38α MAPK in WT mouse hepatocytes. In MKK3 KO hepatocytes, basal level of activated p38α MAPK decreased by 70% and cAMP was unable to activate p38α MAPK. Neither cAMP nor TLC activated MKK4. Taken together, these results suggest that: a) cAMP increases PM-Mrp2 by activating MKK3/p38α MAPK, b) MKK4 is not involved in the activation of p38 MAPK by cAMP and TLC, c) TLC-induced activation of p38 MAPK and decreases in PM-Mrp2 are not mediated via MKK3, and d) MKK3 is the major activator of p38α MAPK in unstimulated hepatocytes.


The following people have nothing to disclose: Christopher M. Schonhoff, Cynthia R. Webster, Mohammed S. Anwer


Reversal of NAFLD Through Selective Increased Intracellular Hepatic Cholesterol Catabolism

Genta Kakiyama, Dalila M. Marques, Shunlin Ren, Daniel Rodriguez-Agudo, Patricia Cooper, Phillip Hylemon, Huiping Zhou, William M. Pandak;

McGuire VA Medical Center/Virginia Commonwealth University, Richmond, VA

Nonalcoholic fatty liver disease (NAFLD) is a leading cause of cirrhosis in the U.S.; and predicted as a leading indication for liver transplantation. Just as important, NAFLD is associated with an insulin resistant state and is an independent risk factor for CVD. Current therapies have not been effective. We embarked upon providing evidence that increased liver cholesterol catabolism would not only lower intrahepatic cholesterol, but triglycerides & free fatty acids; i.e. attenuating liver injury and promoting glucose homeostasis. Hypothesis: increased cholesterol degradation with subsequent increase in regulatory oxysterols combined with an increase in FXR activating bile acids would reverse fatty liver in vivo under conditions which closely simulate an American dietary lifestyle. A 12-14 wk old NAFLD mouse model (1 29S/SV) was fed a Western diet for 2 weeks. On day 7, mice were injected with a CMV-βGal (control), -StarD1, or -CYP7A1 recombinant adenovirus to overex-press genes. On day 14, serum & hepatic lipid and oxysterol levels were determined. Histology was performed, and hepatic gene levels determined. S1P2-/- mice, a 2nd NAFLD animal model, underwent similar studies. Liver free (fxol), total cholesterol (txol), triglycerides (tg), and free fatty acids (ffa) were dramatically decreased by increasing StarD1 or CYP7A1 expression, with a more greater effect seen with StarD1 over-expression (↓45% fxol, ↓45% txol, ↓77% tg, & ↓30% ffa). His-tologic changes reflected biochemical changes. Both StarD1 and CYP7A1 overexpression led to a ~50% decrease in serum glucose. Serum tg were unchanged. HDL levels were reduced >50% while the non-HDL fraction was increased, c/w rapid movement of lipids from liver. SHP mRNA levels were increased with decreases observed in endogenous CYP7A1, CYP8B1, LDLR, HMGR, SREBP2, and in regulators of fatty acid synthesis, LXR & SREBP1c. G6Pase and PEPCK1 levels responded in a manner consistent with increased glucose utilization and serum lowering. A marked reduction (>80%) was found in CYP7B1 mRNA with an associated increase in the regulatory oxysterol, 25HC3S (StarD1: ↑4.9x; CYP7A1: ↑2.6x). Similar changes in liver chemistries and histology were found in S1 P2-/- mice following StarD1 and CYP7A1 overexpression. Conclusion: Increased cholesterol catabolism via either StarD1 or CYP7A1 overexpression led to a rapid reduction in liver lipids in 2 NAFLD models. A coupled increase in regulatory oxysterols and FXR activation appears responsible for the lipid homeosta-tic response with greater 25HC3S increases implicated in the StarD1 vs. CYP7A1 responses. The full therapeutic effects of higher levels of 25HC3S are in study.


Shunlin Ren - Grant/Research Support: Durect Corporation

William M. Pandak - Patent Held/Filed: Virginia Commonwealth University, Veterans Affairs Medical Center

The following people have nothing to disclose: Genta Kakiyama, Dalila M. Marques, Daniel Rodriguez-Agudo, Patricia Cooper, Phillip Hylemon, Huiping Zhou


Cholesterol and non-cholesterol sterols in serum and gallstones interfere with pathogenesis of pediatric gallstone disease

Markku J. Nissinen1, Mikko P. Pakarinen2, Helena Gylling3, Antti Koivusalo2;

1 Department of Medicine, Division of Gastroenterol-ogy, Peijas Hospital, Helsinki University Central Hospital, Helsinki, Finland; 2Pediatric Surgery and Pediatric Transplantation Surgery, Children's Hospital, Helsinki University Central Hospital, Helsinki, Finland; 3Department of Medicine, Division of Internal Medicine, Helsinki University Central Hospital, Helsinki, Finland

Study purposes. We performed a controlled study in children 1) to evaluate characteristics of sterol composition in pediatric cholesterol (C) stones (CS), brown pigment stones (brown PS) and black pigment stones (black PS), 2) to reveal any specific etiopathogenetic factor affecting stone sterol profile, and 3) to elucidate characteristics of C metabolism in children with CS and PS. Patients and methods. C and non-C sterols (lathosterol, campesterol, sitosterol and stigmasterol, of which three latter ones are phytosterols) were examined by gas-liquid chro-matography in gallstones and preoperatively in serum (subgroup) of consecutively cholecystectomised children in pediatric tertiary referral center between years 2004 - 201 2. Gallstone C was determined as mg/1 00mg of stone and non-C sterols as proportions (μg/mg of C). In serum, C precursor sterol (lathosterol) as ratio to C served as surrogate marker of C synthesis, whereas campesterol- and sitosterol-ratios served as relative markers of C absorption. Gallstones from adult CS (n=178), adult PS (n=16), and serum from age- and BMI-matched children (n=82) were controls. Results. Altogether 42 children were cholecystectomized. 23 children had black PS (median age 12.1 yrs, 57% girls), 3 had brown PS (median age 6.4 yrs, 1 girl) and 16 had CS (median age 15.0 yrs, 88% girls) (p=0.027 for age between PS and CS). C content in black PS was 20% of respective adult value, and lathosterol proportion was ~50% below CS-value (p<0.05 for both). Proportions of campesterol, sitosterol and stigmasterol in black PS were 3.5-13 -fold higher than respectively in CS and adult PS (p<0.05 for all). Brown PS had especially high proportions of phytosterols, and all these children were on parenteral nutrition (PN). Overall, four children with stones of very high stigmasterol proportion had history of or ongoing PN. In serum, C concentrations were comparable, but relative synthesis of C was increased in PS-and CS-groups by ~80% (lathosterol)(p<0.01). Relative absorption of C was decreased in CS-group by ~21% (campesterol and sitosterol)(p<0.05). Serum stigmasterol ratios were ~38% below control levels equally in both stone subclasses (p<0.05 for both). Conclusions. Children with CS were associated with higher age and female gender compared to those with PS. Both black PS and especially brown PS were characterized by high proportions of phytosterols. Brown PS and high stone phytosterols, especially stigmasterol proportions were associated with PN. Stigmasterol accumulated in PS despite its low serum levels. In both stone subclasses, metabolism of C was shifted to increased synthesis, and, in CS-group, to decreased absorption.


The following people have nothing to disclose: Markku J. Nissinen, Mikko P. Pakarinen, Helena Gylling, Antti Koivusalo


Analysis of the serum bile acid composition in patients with liver disease determined according to the LC-MS/MS method

Tomonori Sugita1, Katsushi Amano1, Masanori Nakano1, Noriko Masubuchi2, Masahiro Sugihara3, Tomokazu Matsuura4, Hisao Tajiri1;

1Division of Gastroenterology and Hepatology, Department of Internal Medicine, Jikei University School of Medicine, Tokyo, Japan; 2Drug Metabolism & Pharmacokinetics Research Laboratories, R&D Division, Daiichi Sankyo Co., Ltd., Tokyo, Japan; 3Clini-cal Data & Biostatistics Department, R&D Division, Daiichi Sankyo Co., Ltd., Tokyo, Japan; 4Department of Laboratory Medicine, Jikei University School of Medicine, Tokyo, Japan

Background: Bile acids (BA) are synthesized from cholesterol in the liver and are crucial for lipid absorption. Liver diseases can affect BA synthesis and disposition; therefore, the serum BA concentration has been utilized as a prognostic and diagnostic marker. However, the detailed metabolism of BA in patients with liver disease remains to be clarified. In this study, we determined the serum BA composition in patients with liver diseases and healthy volunteers in order to investigate the relationship between etiology and BA metabolism. Methods: Sera from 150 patients with liver disease attending Jikei University Hospital and 40 healthy volunteers were obtained. The serum concentrations of the following 17 different BA were determined according to the LC-MS/MS method: cholic acid, chenodeoxy-cholic acid, deoxycholic acid (DCA), glycocholic acid, gly-cochenodeoxycholic acid, glycodeoxycholic acid, glycolithocholic acid, glycoursodeoxycholic acid (GUDCA), lithocholic acid, sulfated BA, taurocholic acid, taurochen-odeoxycholic acid, taurodeoxycholic acid, taurolithocholic acids, tauroursodeoxycholic acid, ursodeoxycholic acid (UDCA) and 12- ketolithocholic acid. The concentrations of the BA were log-transformed to approximately normalize the distributions and then were compared between the patients with different liver disease etiologies using multiple linear regression models adjusted for sex, age, body mass index, drinking status, the use of liver-protective agents, hyperlipidemia, diabetes and type of hepatic disease. Results: A total of 150 subjects, including patients with chronic hepatitis C (n=44), hepatitis B (n=23), alcoholic liver disease (ALD) (n=21), biliary tract disease (n=20), nonalcoholic fatty liver disease (n=13), autoimmune hepatitis (n=6), primary biliary cirrhosis (n=8) and other liver diseases (n=15), were recruited. The least square geometric mean concentrations of UDCA and GUDCA were higher in the patients with ALD than in those with viral hepatitis (UDCA: 1.153 μMvs 0.237 μM, (p<0.01); GUDCA: 3.337 μM vs 0.900 μM, (p=0.02)). On the other hand, the least square geometric mean concentrations of DCA and UDCA were significantly lower in the patients with biliary tract disease than in those with viral hepatitis (DCA: 0.032 μM vs 0.129 μM, (p<0.01); UDCA: 0.088 μM vs 0.237 μM, (p=0.03)). Conclusions: ALD and biliary tract disease often present with a similar abnormal liver function. Therefore, making a diagnosis is sometimes difficult. In this study, the BA metabolism differed between the liver diseases. Analyses of the BA composition are useful for revealing pathophysiological processes in liver disease.


Noriko Masubuchi - Employment: Daiichi Sankyo Co., Ltd.

Masahiro Sugihara - Employment: Daiichi-Sankyo Co., LTD.

The following people have nothing to disclose: Tomonori Sugita, Katsushi Amano, Masanori Nakano, Tomokazu Matsuura, Hisao Tajiri


Leukemia Inhibitory Factor Protects Cholangiocarcinoma Cells from Cytotoxicity via Mcl-1

Massimiliano Cadamuro1, Stuart D. Morton1, Tommaso Stecca2, Marco Massani2, Nicolò Bassi2, Annarosa Floreani1, Mario Straz-zabosco3,4, Luca Fabris1,4;

1Dep. of Surgery, Oncology and Gastroenterology, University of Padova, Padova, Italy; 24th Surgery Division, Treviso Regional Hospital, Treviso, Italy; 3Department of Surgery and Interdisciplinary Medicine, University of Milan-Bic-occa, Milan, Italy; 4Section of Digestive Diseases, Yale University, New Haven, CT

BACKGROUND AND AIM Cytokines released within the tumor reactive stroma of epithelial cancers are pivotal for cell proliferation and survival; their modulation may represent a potential therapeutic target. IL-6 is a known potent growth factor for normal and neoplastic cholangiocytes. Leukemia inhibitory factor (LIF), an IL-6 family member mainly secreted by inflammatory cells, induces proliferation in breast and colon cancers, but very little is known about its effects in cholangiocarcinoma (CCA). The aim of the study was to elucidate the role of LIF and its cognate receptor (LIFR) in CCA, a cancer with strong chemo-resistance. METHODS We investigated established human CCA cell lines (HuCCT-1, TFK-1, EGI-1) and primary cholangiocytes isolated from resected CCA (n=8) and alcoholic cirrhosis (serving as controls, n=2) livers. Tissue sections (immunohistochemistry, IHC) and cultured cholangiocytes (Western blotting, WB) were tested for LIFR expression; LIF secretion (ELISA) was assessed in cultured cells. Expression of gp130, the co-receptor for LIFR, was also studied (IHC). Chosen for their LIFR expression, TFK-1 and HuCCT-1 were evaluated for proliferation and cell viability (MTS) in response to recom-binant human (rh) LIF (0.1-1 00ng/mL) and following cytotoxic stimuli (paclitaxel 500nM, camptothecin 15μM, platinum 5μg/ml, gemcitabine 30μM, platinum 5μg/ml + gemcitabine 30μM) with/without rhLIF pre-treatment. In TFK-1 and HuCCT-1, expression of Bax (pro-apoptotic) and Mcl-1 (anti-apoptotic) were analyzed by WB after treatment with rhLIF (10, 1 00ng/mL). RESULTS In tissue sections, LIFR expression was restricted to neoplastic cholangiocytes along with positive expression of gp130. Confirmed by WB, LIFR was expressed >7 times in cultured CCA cholangiocytes with respect to controls. CCA and control cholangiocytes secreted variable amounts of LIF (0-95.7pg/mL). Following administration of rhLIF, TFK-1 and HuCCT-1 cells proliferated mildly (up to 5% and 21%, respectively). Administration of cytotoxic agents decreased the number of TFK-1 by up to 21-68% and HuCCT-1 by up to 39-89%. This effect was counteracted by pre-treatment with rhLIF, which induced a significant increase in cell viability (up to 25% in both cell lines). In LIF-stimulated CCA cells, expression of Mcl-1 increased significantly whilst Bax levels remained unchanged as respect to untreated cells. CONCLUSIONS CCA cells express LIFR and secrete variable amounts of LIF. As LIF is also commonly secreted by infiltrating macrophages, autocrine and paracrine LIF-dependent mechanisms are responsible for chemo-resistance in CCA by enhancing tumoral cholangiocyte survival through up-regulation of Mcl-1.


The following people have nothing to disclose: Massimiliano Cadamuro, Stuart D. Morton, Tommaso Stecca, Marco Massani, Nicolò Bassi, Annarosa Floreani, Mario Strazzabosco, Luca Fabris


Src tyrosine kinase mediates the increased TLR4-dependent stimulation of innate immune responses to endotoxins in Cftr-defective cholangiocytes

Romina Fiorotto1, Ambra Villani1,2, Roberto Scirpo2, Carlo Spirli1, Mario Strazzabosco1,2;

1Yale University, Section of Digestive Diseases, New Haven, CT; 2Department of Surgery and Interdisciplinary Medicine, University of Milano-Bicocca, Milan, Italy

BACKGROUND and AIMS: Cystic Fibrosis-related liver diseases (CFLD) have been classically considered a consequence of the impaired biliary secretion caused by the defective CFTR channel function. Instead, we have recently described that exposure of Cftr-KO mice to intestinal endotoxins enhanced the TLR4-dependent innate immune responses of the biliary epithelium causing inflammation and liver damage. Furthermore, in isolated Cftr-defective cholangiocytes a stronger TLR4/NF-κB-mediated inflammatory response to bacterial lipopolysaccha-ride (LPS) was associated with increased phosphorylation of TLR4. Src tyrosine kinase was responsible for increased TLR4 phosphorylation. The hypothesis of this study is that CFTR expression negatively regulates Src activity by destabilizing its membrane anchorage and that Src activity plays a role in the altered responsiveness of CF cholangiocytes to LPS. METHODS: C57BL/6J-Cftrtm1Unc mice (Cftr-KO) and WT littermates were used for in vivo experiments and cholangiocyte isolation. Dex-tran sodium sulfate (DSS)-colitis was used to induce a portal endotoxemia in vivo, while LPS (100ng/ml) was used to challenge cholangiocytes in vitro. Administration of PP2 was used in vivo (1mg/Kg/day) and in vitro (10μM) to inhibit Src activation. Ductular reaction and inflammation were quantified by morphometric analysis of K19 and CD45 immunolabeling, respectively. Secretion of multiple cytokines was quantified by Luminex. NF-κB activation was determined by immunoblot for p65 in nuclear fraction. Confocal imaging was used to visualize the distribution of EBP50, CBP and Csk in polarized cholangiocytes. RESULTS: Treatment with PP2 significantly attenuated DSS-induced biliary damage and inflammation in Cftr-KO mice. In Cftr-defective but not in WT cholangiocytes, treatment with PP2 decreased NF-κB activation both at basal condition and after exposure to LPS. Similarly, PP2 decreased LPS-induced, TLR4/NF-κB regulated cytokines secretion (G-CSF, KC, LIX, MIP-2) in CF-cholangiocytes. Confocal images and X-Z projection analysis showed that Csk, the negative regulator of Src and its membrane and cytoskeleton adaptors CBP and EBP-50 were mislocalized in Cftr-KO cells as compared to the apical membrane localization in normal cells. CONCLUSIONS: In line with the known role of CFTR as a regulator of membrane trafficking, its deficiency alters the membrane location of proteins involved in the negative regulation of Src, thereby resulting in TLR4 phosphorylation and increased TLRs responsiveness. These findings suggest that Src is as a potential therapeutic target in CFLD.


The following people have nothing to disclose: Romina Fiorotto, Ambra Villani, Roberto Scirpo, Carlo Spirli, Mario Strazzabosco


Activation of the bile acid receptor TGR5 (Gpbar-1) induces cholangiocyte proliferation through a ROS-Src-EGFR-ERK signalling pathway

Verena Keitel, Maria Reich, Annika Sommerfeld, Stefanie Kluge, Ralf Kubitz, Dieter Häussinger;

Clinic for Gastroenterology, Hepa-tology and Infectious Diseases, Heinrich-Heine-University Düssel-dorf, Düsseldorf, Germany

Background and Aims: TGR5 is a G-protein coupled bile acid receptor, expressed in different liver cells, including cholangiocytes (1). Bile acids can promote cholangiocyte secretion and proliferation (2), however, the function of TGR5 in cholangiocytes is largely unknown. Aim of the present study was to elucidate the role of TGR5 for cholangiocyte proliferation and to identify TGR5-dependent signalling pathways. Methods: TGR5 knockout and wildtype mice were fed a cholic acid diet (0.5%) for 7 days. Ductular proliferation was quantified by cytokeratin-1 9 staining. In isolated cholangiocytes proliferation was measured by BrDU incorporation after stimulation with bile acids/TGR5 agonists. TGR5-dependent pathways were studied with kinase inhibitors (SU6656,PP1 ,PP2,AG1478, U0126). Reactive oxygen species (ROS) and EGF shedding were measured using a fluorescent dye or an ELISA assay, respectively. Western blotting was used to study EGFR and ERK1/2 phosphorylation. Results: While the amount of CK19-positive bile ducts was similar between TGR5 wildtype and knockout mice on chow diet, a significant increase of bile duct proliferation was detected in wildtype mice after 7 days of cholic acid diet as compared to the TGR5 knockout mice on the same diet. Treatment of isolated cholangiocytes from TGR5 wildtype and knockout mice with taurolithocholic acid (TLC 10 and 25 μM) orTGR5 specific agonists resulted in an increased cholangiocyte proliferation exclusively in wildtype-derived cells. Preincubation of the wildtype cholangiocytes with inhibitors of ERK1/2, EGFR and Src formation abolished TLC and TGR5 agonist induced BrDU incorporation. Treatment with inhibitors of ROS-formation such as N-acetylcystein or apocynin also reduced TLC mediated BrDU incorporation. In contrast inhibition of adenylate cyclase (SQ22536 or dideoxyadenosine) had no effect on TLC/TGR5 agonist dependent cholangiocyte proliferation. Stimulation of wildtype cholangiocytes with TLC and a TGR5 agonist significantly elevated ROS formation and induced EGF shedding. Furthermore, increased tyrosine phosphorylation of the EGFR at positions 845 and 1045 was detected following TGR5 activation. TLC and the TGR5 agonist also led to a significant increase in ERK1/2 phosphorylation exclusively in TGR5 wildtype-derived cholangiocytes. Conclusion: TGR5 mediates bile acid induced cholangiocyte proliferation through a ROS-Src-EGFR-ERK signalling pathway, which is independent of adenylate cyclase activation. 1. Keitel, V. and Häussinger, D. (2012) Clin. Res. Hepatol. Gastroenterol. 36, 412-419 2. Xia, X., Francis, H., Glaser, S., Alpini, G., and LeSage, G. (2006) World J. Gastroenterol. 12, 3553-3563


The following people have nothing to disclose: Verena Keitel, Maria Reich, Annika Sommerfeld, Stefanie Kluge, Ralf Kubitz, Dieter Häussinger


Cholangiocyte proliferation and Osteopontin secretion is induced by Schistosoma mansoni egg antigens and correlates with fibrosis and portal hypertension in human and murine schistosomiasis

Thiago A. Pereira1,2, Wing-Kin Syn4, Steve S. Choi1, Izabela Voieta3, Vivian Resende3, Marcia M. Souza2, William E. Secor5, Paula V. Vidigal3, Rafal P. Witek7, Zilton A. Andrade2, Fausto E. Pereira6, José R. Lambertucci3, Anna Mae Diehl1;

1Division ofGas-troenterology, Duke University Medical Center, Durham, NC; 2Lab-oratório de Patologia Experimental, Centro de Pesquisas Gonçalo Moniz/FIOCRUZ, Salvador, Brazil; 3Faculdade de Medicina, Uni-versidade Federal de Minas Gerais, Belo Horizonte, Brazil; 4Liver Regeneration and Repair Research Group, Institute of Hepatology, Foundation for Liver Research, London, United Kingdom; 5Centers for Disease Control and Prevention, Atlanta, GA; 6Núcleo de Doenças Infecciosas, Universidade Federal do Espírito Santo, Vitória, Brazil; 7Life Technologies, Carlsbad, CA

Background: Schistosomiasis is a leading cause of portal fibrosis and portal hypertension worldwide. Osteopontin (OPN) is a profibrogenic protein associated with hepatic stellate cell activation and correlates with fibrosis stage in chronic liver diseases. Our aims were to investigate if Schistosoma mansoni egg antigens could induce cholangiocyte proliferation and secretion of OPN, and if OPN levels correlate with fibrosis stage and/or degree of portal hypertension. Methods: Mouse cholangiocyte line 603B and primary human cholangiocytes (from Life Technologies) were incubated with 1 0ug/mL of Soluble egg antigen (SEA) or 0.0001 ug/mL LPS (control) for 2,4,6,12 and 24 hours. Cells were harvested and analyzed for OPN mRNA by qRTPCR. OPN protein in conditioned media was measured by ELISA, and cell proliferation assessed by BrdU. Mice were infected with S. mansoni for 6 weeks (early; n=5) or 1 6 weeks (advanced; n=8). Uninfected, age and strain matched mice were controls (n=8). At the end of treatment, mice were sacrificed; liver OPN and αSMA expression was assessed by qRTPCR and IHC; serum OPN was measured by ELISA; liver fibrosis evaluated by Sirius Red staining and mor-phometry. Livers from patients with schistosomiasis mansoni (early fibrosis n=6; advanced fibrosis n= 58) or healthy adults (donors, n=8) were stained for OPN and αSMA, and serum collected for OPN measurements. Splenic vein pressures were studied in a subgroup of patients with advanced fibrosis at the time of splenectomy. This study was approved by the Ethics Committee of UFMG (204-06). Results: SEA stimulated cholangiocyte proliferation and upregulated cholangiocyte-associated OPN secretion (p<0.001). In both mice and humans, liver and serum OPN levels correlated with fibrosis stage (mice: r=0.848; human r=0.61, p=0.000) and αSMA expression (mice: r=0.772, p=0.005; human: r=0.74, p=0.001). A positive correlation was also observed between the number of OPN+ bile ducts and the level of splenic vein pressure(r=0.79; r2=0.624; p=0.01). Conclusions: S. mansoni egg antigens stimulate cholangiocyte proliferation and OPN secretion. OPN levels in the liver and blood correlate with fibrosis stage and severity of portal hypertension.


Rafal P. Witek- Stock Shareholder: Life Technologies

Anna Mae Diehl - Consulting: Bristol Myers Squibb, Synergy, GlaxoSmithKline, Norgine; Grant/Research Support: GlaxoSmithKline

The following people have nothing to disclose: Thiago A. Pereira, Wing-Kin Syn, Steve S. Choi, Izabela Voieta, Vivian Resende, Marcia M. Souza, William E. Secor, Paula V. Vidigal, Zilton A. Andrade, Fausto E. Pereira, José R. Lambertucci