Modelling the impact of liver regeneration on hepatoblastoma patient-derived-xenograft tumor growth.

BACKGROUND: Twenty percent of children with hepatoblastoma (HB) have lung metastasis at diagnosis. Treatment protocols recommend surgical removal of chemotherapy-refractory lung nodules, however no chronological order is established. As hepatectomy is followed by release of growth factors, it has been proposed that partial hepatectomy (PH) could boost local or distant residual tumor growth. METHODS: To evaluate the impact of PH on distant tumor growth, PH was performed in mice subcutaneously implanted with a HB patient-derived xenograft (PDX). The influence of PH on tumor growth at primary site was assessed by performing PH concomitantly to HB PDXs orthotopic implantation. RESULTS: Subcutaneously implanted HB PDX failed to show any influence of hepatectomy on tumor growth. Instead, intrahepatic tumor growth of one of the 4 HB PDXs implanted orthotopically was clearly enhanced. Cells derived from the hepatectomy-sensitive HB PDX exposed to hepatic growth factor (HGF) showed increased proliferation rate compared to cells derived from a hepatectomy-insensitive model, suggesting that the HGF/MET pathway could be one of the effectors of the crosstalk between liver regeneration and HB growth. CONCLUSION: These results suggest that hepatectomy can contribute to HB growth in some patients, further studies will be necessary to identify biomarkers predictive of patient risk of PH-induced HB recurrence. IMPACT: Key message: Cytokines and growth factors secreted following partial hepatectomy can contribute to intrahepatic tumor growth in some hepatoblastoma models. What does it add to the existing literature: It is the first article about the impact of liver regeneration induced by partial hepatectomy on hepatoblastoma local or distant tumoral growth in nude mice. What is the impact: It is important to identify the secreted factors that enhance tumor growth and to define biomarkers predictive of patient risk of partial hepatectomy-induced hepatoblastoma recurrence.

Tight junction proteins and biliary diseases.

PURPOSE OF REVIEW: In the pathophysiological context of cholangiopathies and more broadly of hepatopathies, while it is conceptually clear that the maintenance of inter-cholangiocyte and inter-hepatocyte tight junction integrity would be crucial for liver protection, only scarce studies have been devoted to this topic. Indeed, in the liver, alteration of tight junctions, the intercellular adhesion complexes that control paracellular permeability would result in leaky bile ducts and bile canaliculi, allowing bile reflux towards hepatic parenchyma, contributing to injury during the disease process. RECENT FINDINGS: Last decades have provided a great deal of information regarding both tight junction structural organization and signaling pathways related to tight junctions, providing clues about potential intervention to modulate paracellular permeability during cholangiopathies pathogenesis. Interestingly, several liver diseases have been reported to be associated with abnormal expression of one or several tight junction proteins. However, the question remains unanswered if these alterations would be primarily involved in the disease pathogenesis or if they would occur secondarily in the pathological course. SUMMARY: In this review, we provide an overview of tight junction disruptions described in various biliary diseases that should pave the way for defining new therapeutic targets in this field.

Fibroblast growth factor 19 stimulates water intake.

Fibroblast growth factor 19 (FGF19) is a hormone with pleiotropic metabolic functions, leading to ongoing development of analogues for treatment of metabolic disorders. On the other hand, FGF19 is overexpressed in a sub-group of hepatocellular carcinoma (HCC) patients and has oncogenic properties. It is therefore crucial to precisely define FGF19 effects, notably in the context of chronic exposure to elevated concentrations of the hormone. Here, we used hydrodynamic gene transfer to generate a transgenic mouse model with long-term FGF19 hepatic overexpression. We describe a novel effect of FGF19, namely the stimulation of water intake. This phenotype, lasting at least over a 6-month period, depends on signaling in the central nervous system and is independent of FGF21, although it mimics some of its features. We further show that HCC patients with high levels of circulating FGF19 have a reduced natremia, indicating dipsogenic features. The present study provides evidence of a new activity of FGF19, which could be clinically relevant in the context of FGF19 overexpressing cancers and in the course of treatment of metabolic disorders by FGF19 analogues.

Bile acid-receptor TGR5 deficiency worsens liver injury in alcohol-fed mice by inducing intestinal microbiota dysbiosis.

BACKGROUND & AIMS: Bile-acid metabolism and the intestinal microbiota are impaired in alcohol-related liver disease. Activation of the bile-acid receptor TGR5 (or GPBAR1) controls both biliary homeostasis and inflammatory processes. We examined the role of TGR5 in alcohol-induced liver injury in mice. METHODS: We used TGR5-deficient (TGR5-KO) and wild-type (WT) female mice, fed alcohol or not, to study the involvement of liver macrophages, the intestinal microbiota (16S sequencing), and bile-acid profiles (high-performance liquid chromatography coupled to tandem mass spectrometry). Hepatic triglyceride accumulation and inflammatory processes were assessed in parallel. RESULTS: TGR5 deficiency worsened liver injury, as shown by greater steatosis and inflammation than in WT mice. Isolation of liver macrophages from WT and TGR5-KO alcohol-fed mice showed that TGR5 deficiency did not increase the pro-inflammatory phenotype of liver macrophages but increased their recruitment to the liver. TGR5 deficiency induced dysbiosis, independently of alcohol intake, and transplantation of the TGR5-KO intestinal microbiota to WT mice was sufficient to worsen alcohol-induced liver inflammation. Secondary bile-acid levels were markedly lower in alcohol-fed TGR5-KO than normally fed WT and TGR5-KO mice. Consistent with these results, predictive analysis showed the abundance of bacterial genes involved in bile-acid transformation to be lower in alcohol-fed TGR5-KO than WT mice. This altered bile-acid profile may explain, in particular, why bile-acid synthesis was not repressed and inflammatory processes were exacerbated. CONCLUSIONS: A lack of TGR5 was associated with worsening of alcohol-induced liver injury, a phenotype mainly related to intestinal microbiota dysbiosis and an altered bile-acid profile, following the consumption of alcohol. LAY SUMMARY: Excessive chronic alcohol intake can induce liver disease. Bile acids are molecules produced by the liver and can modulate disease severity. We addressed the specific role of TGR5, a bile-acid receptor. We found that TGR5 deficiency worsened alcohol-induced liver injury and induced both intestinal microbiota dysbiosis and bile-acid pool remodelling. Our data suggest that both the intestinal microbiota and TGR5 may be targeted in the context of human alcohol-induced liver injury.

Participation of 5-lipoxygenase and LTB4 in liver regeneration after partial hepatectomy.

Regeneration is the unmatched liver ability for recovering its functional mass after tissue lost. Leukotrienes (LT) are a family of eicosanoids with the capacity of signaling to promote proliferation. We analyzed the impact of blocking LT synthesis during liver regeneration after partial hepatectomy (PH). Male Wistar rats were subjected to two-third PH and treated with zileuton, a specific inhibitor of 5-lipoxygenase (5-LOX). Our first find was a significant increment of intrahepatic LTB4 during the first hour after PH together with an increase in 5-LOX expression. Zileuton reduced hepatic LTB4 levels at the moment of hepatectomy and also inhibited the increase in hepatic LTB4. This inhibition produced a delay in liver proliferation as seen by decreased PCNA and cyclin D1 nuclear expression 24 h post-PH. Results also showed that hepatic LTB4 diminution by zileuton was associated with a decrease in NF-ĸB activity. Additionally, decreased hepatic LTB4 levels by zileuton affected the recruitment of neutrophils and macrophages. Non-parenchymal cells (NPCs) from zileuton-treated PH-rats displayed higher apoptosis than NPCs from PH control rats. In conclusion, the present work provides evidences that 5-LOX activation and its product LTB4 are involved in the initial signaling events for liver regeneration after PH and the pharmacological inhibition of this enzyme can delay the initial time course of the phenomenon.

Human Peripheral Blood Eosinophils Express High Levels of the Purinergic Receptor P2X4.

Extracellular nucleotides are important mediators of cell activation and trigger multiple responses via membrane receptors known as purinergic receptors (P2). P2X receptors are ligand-gated ion channels, activated by extracellular ATP. P2X4 is one of the most sensitive purinergic receptors, that is typically expressed by neurons, microglia, and some epithelial and endothelial cells. P2X4 mediates neuropathic pain via brain-derived neurotrophic factor and is also involved in inflammation in response to high ATP release. It is therefore involved in multiple inflammatory pathologies as well as neurodegenerative diseases. We have produced monoclonal antibodies (mAb) directed against this important human P2X4 receptor. Focusing on two mAbs, we showed that they also recognize mouse and rat P2X4. We demonstrated that these mAbs can be used in flow cytometry, immunoprecipitation, and immunohistochemistry, but not in Western blot assays, indicating that they target conformational epitopes. We also characterized the expression of P2X4 receptor on mouse and human peripheral blood lymphocytes (PBL). We showed that P2X4 is expressed at the surface of several leukocyte cell types, with the highest expression level on eosinophils, making them potentially sensitive to adenosine triphosphate (ATP). P2X4 is expressed by leucocytes, in human and mouse, with a significant gender difference, males having higher surface expression levels than females. Our findings reveal that PBL express significant levels of P2X4 receptor, and suggest an important role of this receptor in leukocyte activation by ATP, particularly in P2X4high expressing eosinophils.

Hepatocellular Carcinomas With Mutational Activation of Beta-Catenin Require Choline and Can Be Detected by Positron Emission Tomography.

BACKGROUND & AIMS: In one-third of hepatocellular carcinomas (HCCs), cancer cells have mutations that activate ß-catenin pathway. These cells have alterations in glutamine, bile, and lipid metabolism. We investigated whether positron emission tomography (PET) imaging allows identification of altered metabolic pathways that might be targeted therapeutically. METHODS: We studied mice with activation of ß-catenin in liver (Apcko-liv mice) and male C57Bl/6 mice given injections of diethylnitrosamine, which each develop HCCs. Mice were fed a conventional or a methionine- and choline-deficient diet or a choline-deficient (CD) diet. Choline uptake and metabolism in HCCs were analyzed by micro-PET imaging of mice; livers were collected and analyzed by histologic, metabolomic, messenger RNA quantification, and RNA-sequencing analyses. Fifty-two patients with HCC underwent PET imaging with 18F-fluorodeoxyglucose, followed by 18F-fluorocholine tracer metabolites. Human HCC specimens were analyzed by immunohistochemistry, quantitative polymerase chain reaction, and DNA sequencing. We used hepatocytes and mouse tumor explants for studies of incorporation of radiolabeled choline into phospholipids and its contribution to DNA methylation. We analyzed HCC progression in mice fed a CD diet. RESULTS: Livers and tumors from Apcko-liv mice had increased uptake of dietary choline, which contributes to phospholipid formation and DNA methylation in hepatocytes. In patients and in mice, HCCs with activated ß-catenin were positive in 18F-fluorocholine PET, but not 18F-fluorodeoxyglucose PET, and they overexpressed the choline transporter organic cation transporter 3. The HCC cells from Apcko-liv mice incorporated radiolabeled methyl groups of choline into phospholipids and DNA. In Apcko-liv mice, the methionine- and choline-deficient diet reduced proliferation and DNA hypermethylation of hepatocytes and HCC cells, and the CD diet reduced long-term progression of tumors. CONCLUSIONS: In mice and humans, HCCs with mutations that activate ß-catenin are characterized by increased uptake of a fluorocholine tracer, but not 18F-fluorodeoxyglucose, revealed by PET. The increased uptake of choline by HCCs promotes phospholipid formation, DNA hypermethylation, and hepatocyte proliferation. In mice, the CD diet reverses these effects and promotes regression of HCCs that overexpress ß-catenin.

The P2X4 purinergic receptor impacts liver regeneration after partial hepatectomy in mice through the regulation of biliary homeostasis.

UNLABELLED: Many regulatory pathways are involved in liver regeneration after partial hepatectomy (PH), to initiate growth, protect liver cells, and sustain remnant liver functions. Extracellular adenosine triphosphate rises in blood and bile after PH and contributes to liver regeneration, although purinergic receptors and mechanisms remain to be precisely explored. In this work we analyzed during regeneration after PH the involvement of P2X4 purinergic receptors, highly expressed in the liver. P2X4 receptor expression in the liver, liver histology, hepatocyte proliferation, plasma bile acid concentration, bile flow and composition, and lysosome distribution in hepatocytes were studied in wild-type and P2X4 knockout (KO) mice, before and after PH. P2X4 receptors were expressed in hepatocytes and Kupffer cells; in hepatocytes, P2X4 was concentrated in subcanalicular areas closely costained with lysosomal markers. After PH, delayed regeneration, hepatocyte necrosis, and cholestasis were observed in P2X4-KO mice. In P2X4-KO mice, post-PH biliary adaptation was impaired with a smaller increase in bile flow and HCO3 (-) biliary output, as well as altered biliary composition with reduced adenosine triphosphate and lysosomal enzyme release. In line with these data, lysosome distribution and biogenesis were altered in P2X4-KO compared with wild-type mice. CONCLUSION: During liver regeneration after PH, P2X4 contributes to the complex control of biliary homeostasis through mechanisms involving pericanalicular lysosomes, with a resulting impact on hepatocyte protection and proliferation. (Hepatology 2016;64:941-953).

LIM-only protein FHL2 activates NF-κB signaling in the control of liver regeneration and hepatocarcinogenesis.

Four-and-a-half LIM-only protein 2 (FHL2) is an important mediator in many signaling pathways. In this study, we analyzed the functions of FHL2 in nuclear factor κB (NF-κB) signaling in the liver. We show that FHL2 enhanced tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) activity in transcriptional activation of NF-κB targets by stabilizing the protein. TRAF6 is a binding partner of FHL2 and an important component of the Toll-like receptor-NF-κB pathway. Knockdown of FHL2 in 293-hTLR4/MD2-CD14 cells impaired lipopolysaccharide (LPS)-induced NF-κB activity, which regulates expression of inflammatory cytokines. Indeed, FHL2(-/-) macrophages showed significantly reduced production of TNF and interleukin 6 (IL-6) following LPS stimulation. TNF and IL-6 are the key cytokines that prime liver regeneration after hepatic injury. Following partial hepatectomy, FHL2(-/-) mice exhibited diminished induction of TNF and IL-6 and delayed hepatocyte regeneration. In the liver, NF-κB signaling orchestrates inflammatory cross talk between hepatocytes and hepatic immune cells that promote chemical hepatocarcinogenesis. We found that deficiency of FHL2 reduced susceptibility to diethylnitrosamine-induced hepatocarcinogenesis, correlating with the activator function of FHL2 in NF-κB signaling. Our findings demonstrate FHL2 as a positive regulator of NF-κB activity in liver regeneration and carcinogenesis and highlight the importance of FHL2 in both hepatocytes and hepatic immune cells.

The four and a half LIM-only protein 2 regulates liver homeostasis and contributes to carcinogenesis.

BACKGROUND & AIMS: The four and a half LIM-only protein 2 (FHL2) is upregulated in diverse pathological conditions. Here, we analyzed the effects of FHL2 overexpression in the liver of FHL2 transgenic mice (Apo-FHL2). METHODS: We first examined cell proliferation and apoptosis in Apo-FHL2 livers and performed partial hepatectomy to investigate high FHL2 expression in liver regeneration. Expression of FHL2 was then analyzed by real time PCR in human hepatocellular carcinoma and adjacent non-tumorous livers. Finally, the role of FHL2 in hepatocarcinogenesis was assessed using Apo-FHL2;Apc(lox/lox) mice. RESULTS: Six-fold increase in cell proliferation in transgenic livers was associated with concomitant apoptosis, resulting in normal liver mass. In Apo-FHL2 livers, both cyclin D1 and p53 were markedly increased. Evidence supporting a p53-dependent cell death mechanism was provided by the findings that FHL2 bound to and activated the p53 promoter, and that a dominant negative p53 mutant compromised FHL2-induced apoptosis in hepatic cells. Following partial hepatectomy in Apo-FHL2 mice, hepatocytes displayed advanced G1 phase entry and DNA synthesis leading to accelerated liver weight restoration. Interestingly, FHL2 upregulation in human liver specimens showed significant association with increasing inflammation score and cirrhosis. Finally, while Apo-FHL2 mice developed no tumors, the FHL2 transgene enhanced hepatocarcinogenesis induced by liver-specific deletion of the adenomatous polyposis coli gene and aberrant Wnt/ß-catenin signaling in Apc(lox/lox) animals. CONCLUSIONS: Our results implicate FHL2 in the regulation of signaling pathways that couple proliferation and cell death machineries, and underscore the important role of FHL2 in liver homeostasis and carcinogenesis.

Cytosolic calcium regulates liver regeneration in the rat.

UNLABELLED: Liver regeneration is regulated by growth factors, cytokines, and other endocrine and metabolic factors. Calcium is important for cell division, but its role in liver regeneration is not known. The purpose of this study was to understand the effects of cytosolic calcium signals in liver growth after partial hepatectomy (PH). The gene encoding the calcium-binding protein parvalbumin (PV) targeted to the cytosol using a nuclear export sequence (NES), and using a discosoma red fluorescent protein (DsR) marker, was transfected into rat livers by injecting it, in recombinant adenovirus (Ad), into the portal vein. We performed two-thirds PH 4 days after Ad-PV-NES-DsR or Ad-DsR injection, and liver regeneration was analyzed. Calcium signals were analyzed with fura-2-acetoxymethyl ester in hepatocytes isolated from Ad-infected rats and in Ad-infected Hela cells. Also, isolated hepatocytes were infected with Ad-DsR or Ad-PV-NES-DsR and assayed for bromodeoxyuridine incorporation. Ad-PV-NES-DsR injection resulted in PV expression in the hepatocyte cytosol. Agonist-induced cytosolic calcium oscillations were attenuated in both PV-NES-expressing Hela cells and hepatocytes, as compared to DsR-expressing cells. Bromodeoxyuridine incorporation (S phase), phosphorylated histone 3 immunostaining (mitosis), and liver mass restoration after PH were all significantly delayed in PV-NES rats. Reduced cyclin expression and retinoblastoma protein phosphorylation confirmed this observation. PV-NES rats exhibited reduced c-fos induction and delayed extracellular signal-regulated kinase 1/2 phosphorylation after PH. Finally, primary PV-NES-expressing hepatocytes exhibited less proliferation and agonist-induced cyclic adenosine monophosphate responsive element binding and extracellular signal-regulated kinase 1/2 phosphorylation, as compared with control cells. CONCLUSION: Cytosolic calcium signals promote liver regeneration by enhancing progression of hepatocytes through the cell cycle.

Beneficial paracrine effects of cannabinoid receptor 2 on liver injury and regeneration.

UNLABELLED: The cannabinoid receptor 2 (CB2) plays a pleiotropic role in innate immunity and is a crucial mediator of liver disease. In this study, we investigated the impact of CB2 receptors on the regenerative process associated with liver injury. Following acute hepatitis induced by carbon tetrachloride (CCl(4)), CB2 was induced in the nonparenchymal cell fraction and remained undetectable in hepatocytes. Administration of CCl(4) to CB2(-/-) mice accelerated liver injury, as shown by increased alanine/aspartate aminotransferase levels and hepatocyte apoptosis, and delayed liver regeneration, as reflected by a retarded induction of hepatocyte proliferating cell nuclear antigen expression; proliferating cell nuclear antigen induction was also delayed in CB2(-/-) mice undergoing partial hepatectomy. Conversely, following treatment with the CB2 agonist JWH-133, CCl(4)-treated WT mice displayed reduced liver injury and accelerated liver regeneration. The CCl(4)-treated CB2(-/-) mice showed a decrease in inducible nitric oxide synthase and tumor necrosis factor-alpha expression, and administration of the nitric oxide donor moldomine (SIN-1) to these animals reduced hepatocyte apoptosis, without affecting liver regeneration. Impaired liver regeneration was consecutive to an interleukin-6 (IL-6)-mediated decrease in matrix metalloproteinase 2 (MMP-2) activity. Indeed, CCl(4)-treated CB2(-/-) mice displayed lower levels of hepatic IL-6 messenger RNA and increased MMP-2 activity. Administration of IL-6 to these mice decreased MMP-2 activity and improved liver regeneration, without affecting hepatocyte apoptosis. Accordingly, administration of the MMP inhibitor CTTHWGFTLC to CCl(4)-treated CB2(-/-) mice improved liver regeneration. Finally, in vitro studies demonstrated that incubation of hepatic myofibroblasts with JWH-133 increased tumor necrosis factor-alpha and IL-6 and decreased MMP-2 expressions. CONCLUSION: CB2 receptors reduce liver injury and promote liver regeneration following acute insult, via distinct paracrine mechanisms involving hepatic myofibroblasts. These results suggest that CB2 agonists display potent hepatoprotective properties, in addition to their antifibrogenic effects.

ATP release after partial hepatectomy regulates liver regeneration in the rat.

BACKGROUND & AIMS: Paracrine interactions are critical to liver physiology, particularly during regeneration, although physiological involvement of extracellular ATP, a crucial intercellular messenger, remains unclear. The physiological release of ATP into extracellular milieu and its impact on regeneration after partial hepatectomy were investigated in this study. METHODS: Hepatic ATP release after hepatectomy was examined in the rat and in human living donors for liver transplantation. Quinacrine was used for in vivo staining of ATP-enriched compartments in rat liver sections and isolated hepatocytes. Rats were treated with an antagonist for purinergic receptors (Phosphate-6-azo(benzene-2,4-disulfonic acid), PPADS), and liver regeneration after hepatectomy was analyzed. RESULTS: A robust and transient ATP release due to acute portal hyperpressure was observed immediately after hepatectomy in rats and humans. Clodronate liposomal pre-treatment partly inhibited ATP release in rats. Quinacrine-stained vesicles, co-labeled with a lysosomal marker in liver sections and isolated hepatocytes, were predominantly detected in periportal areas. These vesicles significantly disappeared after hepatectomy, in parallel with a decrease in liver ATP content. PPADS treatment inhibited hepatocyte cell cycle progression after hepatectomy, as revealed by a reduction in bromodeoxyuridine incorporation, phosphorylated histone 3 immunostaining, cyclin D1 and A expression and immediate early gene induction. CONCLUSION: Extracellular ATP is released immediately after hepatectomy from hepatocytes and Kupffer cells under mechanical stress and promotes liver regeneration in the rat. We suggest that in hepatocytes, ATP is released from a lysosomal compartment. Finally, observations made in living donors suggest that purinergic signalling could be critical for human liver regeneration.

Substitution of hexon hypervariable region 5 of adenovirus serotype 5 abrogates blood factor binding and limits gene transfer to liver.

Liver tropism potentially leading to massive hepatocyte transduction and hepatotoxicity still represents a major drawback to adenovirus (Ad)-based gene therapy. We previously demonstrated that substitution of the hexon hypervariable region 5 (HVR5), the most abundant capsid protein, constituted a valuable platform for efficient Ad retargeting. The use of different mouse strains revealed that HVR5 substitution also led to dramatically less adenovirus liver transduction and associated toxicity, whereas HVR5-modified Ad were still able to transduce different cell lines efficiently, including primary hepatocytes. We showed that HVR5 modification did not significantly change Ad blood clearance or liver uptake at early times. However, we were able to link the lower liver transduction to enhanced HVR5-modified Ad liver clearance and impaired use of blood factors. Most importantly, HVR5-modified vectors continued to transduce tumors in vivo as efficiently as their wild-type counterparts. Taken together, our data provide a rationale for future design of retargeted vectors with a safer profile.

Activation of CREB by tauroursodeoxycholic acid protects cholangiocytes from apoptosis induced by mTOR inhibition.

Tauroursodeoxycholic acid (TUDCA) is a cytoprotective bile acid frequently prescribed to patients with cholestatic diseases. Several mechanisms of action have been investigated, but the possibility that cyclic adenosine monophosphate responsive element binding protein (CREB), a transcription factor promoting cell survival, mediates TUDCA's protective effects has not been considered. We examined whether TUDCA activates CREB and whether this activation can protect biliary epithelial cells. Cholangiocytes were stressed by exposure to CCI-779, which inhibits signaling though the kinase mTOR (mammalian target of rapamycin), resulting in cell cycle arrest and apoptosis. Incubation of normal rat cholangiocytes (NRC) cells, with TUDCA resulted in phosphorylation of CREB (Western blotting analysis) and activation of CREB transcription activity (luciferase reporter assay). Inhibition of calcium signals and inhibition of protein kinase C prevented the TUDCA-induced activation of CREB. CCI-779 decreased the viability of rat cholangiocytes in a dose-dependent manner (MTT [3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay). TUDCA protected against CCI-779 cytotoxicity. A dominant negative form of CREB was stably transduced in NRC cells (NRC-M1). TUDCA protection was decreased in NRC-M1. While CCI-779 induced apoptosis in NRC cells as determined by caspase 3 activity, TUDCA attenuated CCI-779-induced apoptosis, an effect absent in NRC-M1. Finally, CCI-779 blocked proliferation of both NRC and NRC-M1 (thymidine incorporation) and this was unaffected by TUDCA. In conclusion, TUDCA activates CREB in cholangiocytes, reducing the apoptotic effect of CCI-779. These findings suggest a novel cytoprotective mechanism for this bile acid.