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International audience; During liver repair after injury, bile secretion has to be tightly modulated in order to preserve liver parenchyma from bile acid (BA)-induced injury. The mechanisms allowing the liver to maintain biliary homeostasis during repair after injury are not completely understood. Besides their historical role in lipid digestion, bile acids (BA) and their receptors constitute a signalling network with multiple impacts on liver repair, both stimulating regeneration and protecting the liver from BA overload. BA signal through nuclear (mainly Farnesoid X Receptor, FXR) and membrane (mainly G Protein-coupled BA Receptor 1, GPBAR-1 or TGR5) receptors to elicit a wide array of biological responses. While a great number of studies have been dedicated to the hepato-protective impact of FXR signalling, TGR5 is by far less explored in this context. Because the liver has to face massive and potentially harmful BA overload after partial ablation or destruction, BA-induced protective responses crucially contribute to spare liver repair capacities. Based on the available literature, the TGR5 BA receptor protects the remnant liver and maintains biliary homeostasis, mainly through the control of inflammation, biliary epithelial barrier permeability, BA pool hydrophobicity and sinusoidal blood flow. Mouse experimental models of liver injury reveal that in the lack of TGR5, excessive inflammation, leaky biliary epithelium and hydrophobic BA overload result in parenchymal insult and compromise optimal restoration of a functional liver mass. Translational perspectives are thus opened to target TGR5 with the aim of protecting the liver in the context of injury and BA overload.

Backgrounds & AimsAs the bile acid (BA) pool composition is of major impact on liver pathophysiology, we studied its regulation by the BA receptor TGR5, promoting hepatoprotection against BA overload.MethodsWT, total and hepato-specific TGR5-KO, and TGR5-overexpressing mice were used in: partial and 90% extended hepatectomies (EH) upon normal, ursodeoxycholic acid (UDCA)- or cholestyramine (CT)-enriched diet, bile duct ligation (BDL), cholic acid (1%)-enriched diet, and TGR5 agonist (RO) treatments. We thereby studied TGR5 impact on: BA pool composition, liver injury, regeneration and survival. Particular focus was made on gut microbiota (GM) and gallbladder (GB) function analysis. BA pool composition was analyzed in patients undergoing major hepatectomy.ResultsThe TGR5-KO hyperhydrophobic BA pool was not related to BA synthesis alteration, nor to the TGR5-KO GM dysbiosis, as supported by hepatocyte-specific KO mice and cohousing experiments. The TGR5-dependent control of GB dilatation was crucial for BA pool composition, as determined by experiments including RO treatment +/− cholecystectomy. The poor TGR5-KO post-EH survival rate, related with exacerbated peribiliary necrosis and BA overload, was improved by shifting the BA pool towards a more hydrophilic composition (CT and UDCA treatments). After either BDL or CA-enriched diet +/− cholecystectomy, we found that GB dilatation had strong TGR5-dependent hepatoprotective properties. In patients, a more hydrophobic BA pool was correlated with an unfavorable outcome after hepatectomy.ConclusionBA pool composition is crucial for hepatoprotection in mice and humans. We point TGR5 as a key regulator of BA profile and thereby as a potential hepatoprotective target under BA overload conditions.Lay summaryThrough multiple in vivo experimental approaches in mice, together with a patients study, this work brings some new light on the relationships between biliary homeostasis, gallbladder function and liver protection. We showed that the bile acid pool composition is crucial for optimal liver repair, not only in mice but also in human patients undergoing major hepatectomy.

ObjectiveWe explored the hypothesis that TGR5, the bile acid (BA) G-protein-coupled receptor highly expressed in biliary epithelial cells, protects the liver against BA overload through the regulation of biliary epithelium permeability.DesignExperiments were performed under basal and TGR5 agonist treatment. In vitro transepithelial electric resistance (TER) and FITC-dextran diffusion were measured in different cell lines. In vivo FITC-dextran was injected in the gallbladder (GB) lumen and traced in plasma. Tight junction proteins and TGR5-induced signalling were investigated in vitro and in vivo (wild-type [WT] and TGR5-KO livers and GB). WT and TGR5-KO mice were submitted to bile duct ligation or alpha-naphtylisothiocyanate intoxication under vehicle or TGR5 agonist treatment, and liver injury was studied.ResultsIn vitro TGR5 stimulation increased TER and reduced paracellular permeability for dextran. In vivo dextran diffusion after GB injection was increased in TGR5-knock-out (KO) as compared with WT mice and decreased on TGR5 stimulation. In TGR5-KO bile ducts and GB, junctional adhesion molecule A (JAM-A) was hypophosphorylated and selectively downregulated among TJP analysed. TGR5 stimulation induced JAM-A phosphorylation and stabilisation both in vitro and in vivo, associated with protein kinase C-ζ activation. TGR5 agonist-induced TER increase as well as JAM-A protein stabilisation was dependent on JAM-A Ser285 phosphorylation. TGR5 agonist-treated mice were protected from cholestasis-induced liver injury, and this protection was significantly impaired in JAM-A-KO mice.ConclusionThe BA receptor TGR5 regulates biliary epithelial barrier function in vitro and in vivo through an impact on JAM-A expression and phosphorylation, thereby protecting liver parenchyma against bile leakage.

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. Fil: Lorenzetti, Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; Argentina Fil: Vera, Marina Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; Argentina Fil: Ceballos Mancini, María Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; Argentina Fil: Ronco, Maria Teresa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; Argentina Fil: Pisani, Gerardo Bruno. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Ciencias Fisiológicas. Área Morfología; Argentina Fil: Monti, Juan Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; Argentina Fil: Lucci, Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Ciencias Fisiológicas. Área Morfología; Argentina Fil: Comanzo, Carla Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; Argentina Fil: Tordjmann, Thierry. Université Paris-Saclay; Francia. Institut National de la Santé et de la Recherche Médicale; Francia Fil: Carrillo, María Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Ciencias Fisiológicas. Área Morfología; Argentina Fil: Quiroga, Ariel Dario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Ciencias Fisiológicas. Área Morfología; Argentina. Universidad Abierta Interamericana. Facultad de Medicina. Centro de Altos Estudios en Ciencias de la Salud; Argentina Fil: Alvarez, María de Luján. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Ciencias Fisiológicas. Área Morfología; Argentina. Universidad Abierta Interamericana. Facultad de Medicina. Centro de Altos Estudios en Ciencias de la Salud; Argentina

International audience; 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 characterised 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.

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)

International audience; We investigated the implication of Takeda G protein-coupled receptor 5 (TGR5) in fat preference and fat sensing in taste bud cells (TBC) in C57BL/6 wild-type (WT) and TGR5 knock out (TGR5-/-) male mice maintained for 20 weeks on a high-fat diet (HFD). We also assessed the implication of TGR5 single nucleotide polymorphism (SNP) in young obese humans. The high-fat diet (HFD)-fed TGR5-/- mice were more obese, marked with higher liver weight, lipidemia and steatosis than WT obese mice. The TGR5-/- obese mice exhibited high daily food/energy intake, fat mass and inflammatory status. WT obese mice lost the preference for dietary fat, but the TGR5-/- obese mice exhibited no loss towards the attraction for lipids. In lingual TBC, the fatty acid-triggered Ca2+ signaling was decreased in WT obese mice; however, it was increased in TBC from TGR5-/- obese mice. Fatty acid-induced in vitro release of GLP-1 was higher, but PYY concentrations were lower, in TBC from TGR5-/- obese mice than those in WT obese mice. We noticed an association between obesity and variations in TGR5 rs11554825 SNP. Finally, we can state that TGR5 modulates fat eating behavior and obesity.

Background: Most of the literature on the bile acid (BA) membrane receptor TGR5 is dedicated to its potential role in the metabolic syndrome, through its regulatory impact on energy expenditure, insulin and GLP-1 secretion, and inflammatory processes. While the receptor was cloned in 2002, very little data are available on TGR5 functions in the normal and diseased liver. However, TGR5 is highly expressed in Kupffer cells and liver endothelial cells, and is particularly enriched in the biliary tract [cholangiocytes and gallbladder (GB) smooth muscle cells]. We recently demonstrated that TGR5 has a crucial protective impact on the liver in case of BA overload, including after partial hepatectomy. Key Messages: TGR5-KO mice after PH exhibited periportal bile infarcts, excessive hepatic inflammation and defective adaptation of biliary composition (bicarbonate and chloride). Most importantly, TGR5-KO mice had a more hydrophobic BA pool, with more secondary BA than WT animals, suggesting that TGR5-KO bile may be harmful for the liver, mainly in situations of BA overload. As GB is both the tissue displaying the highest level of TGR5 expression and a crucial physiological site for the regulation of BA pool hydrophobicity by reducing secondary BA, we investigated whether TGR5 may control BA pool composition through an impact on GB. Preliminary data suggest that in the absence of TGR5, reduced GB filling dampens the cholecystohepatic shunt, resulting in more secondary BA, more hydrophobic BA pool and extensive liver injury in case of BA overload. Conclusions: In the setting of BA overload, TGR5 is protective of the liver through the regulation of not only secretory and inflammatory processes, but also through the control of BA pool composition, at least in part by targeting the GB. Thereby, TGR5 appears to be crucial for protecting the regenerating liver from BA overload.

Background & Aims Liver fibrosis is characterized by the accumulation of extracellular matrix produced by hepatic myofibroblasts (hMF), the activation of which is critical to the fibrogenic process. Extracellular ATP, released by dying or stressed cells, and its purinergic receptors, constitute a powerful signaling network after injury. Although the purinergic receptor P2X4 (P2RX4) is highly expressed in the liver, its functions in hMF had never been investigated during liver fibrogenesis. Methods In vivo, bile duct ligation was performed and methionine- and choline-deficient diet administered in wild-type and P2x4 knock-out (P2x4-KO) mice. In vitro, hMF were isolated from mouse (wild-type and P2x4-KO) and human liver. P2X4 pharmacological inhibition (in vitro and in vivo) and P2X4 siRNAs (in vitro) were used. Histological, biochemical and cell culture analysis allowed us to study P2X4 expression and its involvement in the regulation of fibrogenic and fibrolytic factors, as well as of hMF activation markers and properties. Results P2X4 genetic invalidation or pharmacological inhibition protected mice from liver fibrosis and hMF accumulation after bile duct ligation or methionine- and choline-deficient diet. Human and mouse hMFs expressed P2X4, mainly in lysosomes. Invalidation of P2X4 in human and mouse hMFs blunted their activation marker expression and their fibrogenic properties. Finally, we showed that P2X4 regulates calcium entry and lysosomal exocytosis in hMF, impacting on ATP release, profibrogenic secretory profile, and transcription factor activation. Conclusion P2X4 expression and activation is critical for hMF to sustain their activated and fibrogenic phenotype. Therefore, the inactivation of P2X4 may be of therapeutic interest during liver fibrotic diseases. Lay summary During chronic injury, the liver often repairs with fibrotic tissue, which impairs liver function, and for which there is currently no treatment. We found that a previously unexplored pathway involving the purinergic receptor P2X4, can modulate fibrotic liver repair. Therefore, this receptor could be of interest in the development of novel therapies for fibrotic liver diseases.

International audience; Transforming growth factor β1 (TGF-β1) is a master cytokine in many biological processes, including tissue homeostasis, epithelial-to-mesenchymal transition, and wound repair. Here, we report that four and a half LIM-only protein 2 (FHL2) is a critical regulator of TGF-β1 expression. Devoid of a DNA-binding domain, FHL2 is a transcriptional cofactor that plays the role of coactivator or corepressor, depending on the cell and promoter contexts. We detected association of FHL2 with the TGF-β1 promoter, which showed higher activity in Fhl2-/- cells than in wild-type (WT) cells in a reporter assay. Overexpression of FHL2 abrogates the activation of the TGF-β1 promoter, whereas the upregulation of TGF-β1 gene transcription correlates with reduced occupancy of FHL2 on the promoter. Moreover, ablation of FHL2 facilitates recruitment of RNA polymerase II on the TGF-β1 promoter, suggesting that FHL2 may be involved in chromatin remodeling in the control of TGF-β1 gene transcription. Enhanced expression of TGF-β1 mRNA and cytokine was evidenced in the livers of Fhl2-/- mice. We tested the in vivo impact of Fhl2 loss on hepatic fibrogenesis that involves TGF-β1 activation. Fhl2-/- mice developed more severe fibrosis than their WT counterparts. These results demonstrate the repressive function of FHL2 on TGF-β1 expression and contribute to the understanding of the TGF-β-mediated fibrogenic response.

Zizyphin, isolated from Zizyphus sps. leaf extracts, has been shown to modulate sugar taste perception, and the palatability of a sweet solution is increased by the addition of fatty acids. We, therefore, studied whether zizyphin also modulates fat taste perception. Zizyphin was purified from edible fruit of Zizyphus lotus L. Zizyphin induced increases in [Ca2+]i in human taste bud cells (hTBC). Zizyphin shared the endoplasmic reticulum Ca2+ pool and also recruited, in part, Ca2+ from extracellular environment via the opening of store-operated Ca2+ (SOC) channels. Zizyphin exerted additive actions on linoleic acid-induced increases in [Ca2+]i in these cells, indicating that zizyphin does not exert its action via fatty acid receptors. However, zizyphin seemed to exert, at least in part, its action via bile acid receptor Takeda-G-protein-receptor-5 (TGR5) in hTBC. In behavioural tests, mice exhibited preference for both linoleic acid or zizyphin. Interestingly, zizyphin increased the preference for a solution containing-linoleic acid. The present study is the first evidence of the modulation of fat taste perception by zizyphin at the cellular level in hTBC. Our study might be helpful for considering the synthesis of zizyphin analogues as “taste modifiers” with a potential in the management of obesity and lipid-mediated disorders. This article is protected by copyright. All rights reserved.

Sphingosine kinases (SphKs) and their product sphingosine-1-phosphate (S1P) have been reported to regulate apoptosis and survival of liver cells. Cholestatic liver diseases are characterized by cytotoxic levels of bile salts inducing liver injury. It is unknown whether SphKs and/or S1P play a role in this pathogenic process. Here, we investigated the putative involvement of SphK1 and S1P in bile salt-induced cell death in hepatocytes. Primary rat hepatocytes were exposed to glycochenodeoxycholic acid (GCDCA) to induce apoptosis. GCDCA-exposed hepatocytes were co-treated with S1P, the SphK1 inhibitor Ski-II and/or specific antagonists of S1P receptors (S1PR1 and S1PR2). Apoptosis and necrosis were quantified. Ski-II significantly reduced GCDCA-induced apoptosis in hepatocytes (−70%, P

Many regulatory pathways are involved in liver regeneration after partial hepatectomy (PH) to initiate growth, protect liver cells, and sustain functions of the remnant liver. Bile acids (BAs), whose levels rise in the blood early after PH, stimulate both hepatocyte proliferation and protection, in part through their binding to the nuclear farnesoid X receptor (FXR). However, the effect of the BA receptor, TGR5 (G-protein-coupled BA receptor 1) after PH remains to be studied. Liver histology, hepatocyte proliferation, BA concentrations (plasma, bile, liver, urine, and feces), bile flow and composition, and cytokine production were studied in wild-type (WT) and TGR5 KO (knockout) mice before and after PH. BA composition (plasma, bile, liver, urine, and feces) was more hydrophobic in TGR5 KO than in WT mice. After PH, severe hepatocyte necrosis, prolonged cholestasis, exacerbated inflammatory response, and delayed regeneration were observed in TGR5 KO mice. Although hepatocyte adaptive response to post-PH BA overload was similar in WT and TGR5 KO mice, kidney and biliary adaptive responses were strongly impaired in TGR5 KO mice. Cholestyramine treatment, as well as Kupffer cell depletion, significantly improved the post-PH TGR5 KO mice phenotype. After bile duct ligation or upon a cholic acid-enriched diet, TGR5 KO mice exhibited more severe liver injury than WT as well as impaired BA elimination in urine. Conclusion: TGR5 is crucial for liver protection against BA overload after PH, primarily through the control of bile hydrophobicity and cytokine secretion. In the absence of TGR5, intrahepatic stasis of abnormally hydrophobic bile and excessive inflammation, in association with impaired bile flow adaptation and deficient urinary BA efflux, lead to BA overload-induced liver injury and delayed regeneration. (Hepatology 2013;58:1451-1460)

Tissue repair is orchestrated by a finely tuned interplay between processes of regeneration, inflammation and cell protection, allowing organisms to restore their integrity after partial loss of cells or organs. An important, although largely unexplored feature is that after injury and during liver repair, liver functions have to be maintained to fulfill the peripheral demand. This is particularly critical for bile secretion, which has to be finely modulated in order to preserve liver parenchyma from bile-induced injury. However, mechanisms allowing the liver to maintain biliary homeostasis during repair after injury are not completely understood. Besides cytokines and growth factors, bile acids (BA) and their receptors constitute an insufficiently explored signaling network during liver regeneration and repair. BA signal through both nuclear (mainly Farnesoid X Receptor, FXR) and membrane (mainly G Protein-coupled BA Receptor 1, GPBAR-1 or TGR5) receptors which distributions are large in the organism, and which activation elicits a wide array of biological responses. While a number of studies have been dedicated to FXR signaling in liver repair processes, TGR5 remains poorly explored in this context. Because of the massive and potentially harmful BA overload that faces the remnant liver after partial ablation or destruction, both BA-induced adaptive and proliferative responses may stand in a central position to contribute to the regenerative response. Based on the available literature, both BA receptors may act in synergy during the regeneration process, in order to protect the remnant liver and maintain biliary homeostasis, otherwise potentially toxic BA overload would result in parenchymal insult and compromise optimal restoration of a functional liver mass.

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/β-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.

Background/Aims During liver regeneration, a network of cytokines and growth factors interact with hepatocytes, helping to restore the liver mass and functions after partial tissue loss. Agonists that trigger Ca 2+ signals in the liver contribute to this process, although little is known about calcium signalling during liver regeneration. Results We observed two phases in which the hepatocyte response to calcium-mobilising agonists was greatly reduced versus control cells at 24h and five days after partial hepatectomy. We found that both phases of hepatocyte desensitisation involved the down-regulation of cell surface receptors and the type II InsP 3 receptor. Single cell studies with flash photolysis of caged InsP 3 revealed that InsP 3 -mediated Ca 2+ release was slower in regenerating hepatocytes at 24, 48h and 5 days than in control cells. Also, the temporal pattern of vasopressin-elicited intracellular calcium oscillations studied on fura2-loaded cells was altered, with the duration of each Ca 2+ peak being longer. Finally, we showed an association between hepatocyte desensitisation and progression through the cell cycle towards the S phase at 24h after hepatectomy. Conclusions Our study supports the remodelling of hepatocyte calcium signalling during liver regeneration, and that this change is partly linked with cell cycle progression.

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.

Background/Aims : In the liver, InsP 3 -dependent agonists such as vasopressin and noradrenaline induce tightly coordinated sequences of intracellular Ca 2+ increases, leading to apparent unidirectional Ca 2+ waves. In previous works, we have postulated that cell-to-cell differences in hormone receptor density create a cellular sensitivity gradient that determines which cell initiates the intercellular Ca 2+ wave and the direction of propagation of the Ca 2+ signal. The aim of this study was to test directly this hypothesis. Methods : Lobular distribution of V1a vasopressin receptors and α1 adrenergic receptors were observed by autoradiography in rat liver sections. Cell-to-cell differences in the number of these receptors were evaluated on hepatocyte multiplets using specific fluorescent probes. Results : The relative amount of fluorescence associated with the V1a receptor differed significantly between cells within multiplets. The ‘cell-after-cell' Ca 2+ increase induced by vasopressin was correlated with the number of V1a receptors. These observations may be more general, as autoradiography revealed similar lobular distributions of V1a receptors and α1 adrenergic receptors; the amounts of both were greatest in hepatocytes surrounding central veins. Conclusions : These data confirm that a fine gradient along liver cell plates contributes to the molecular basis of the unidirectional hormone-induced Ca 2+ signalling observed in the liver lobule.

After its discovery in Drosophila, the Hippo signalling pathway has been shown to regulate organ size in mammals as well. Based on recent studies, this kinase cascade appears in particular crucial for liver tissue homeostasis, by regulating both cellular proliferation and apoptosis. Thereby, Hippo signalling may appear as a key pathway in liver carcinogenesis.

Because it fosters our dreams of immortality, regeneration has been for centuries a matter of fascination for countless biologists and clinicians. While in mammals regeneration is obviously less spectacular than in hydra, newt or zebrafish, it allows, in any case, repairing intestine, skin or liver. The latter organ is seen as a paradigmatic regeneration model because, as for an amputated fin's zebrafish or newt's forelimb, it is indeed possible to follow the complete restoration of a rodent liver mass after a 2/3 partial hepatectomy (PH) within a few days. The caveat is that most of our knowledge about liver regeneration is based on this specific surgical model. The amazing conclusion is that liver regeneration relies mainly on highly differentiated hepatocytes, which, while actively dividing to restore the missing part, maintain their vital functions.1 Hepatocytes are in a quiescent state (G0) in a normal adult liver. Following 2/3 PH, >95% of these parenchymal cells present in the remnant lobes divide in a rather synchronous manner, for one or two rounds of cell division. Other liver cell types, such as macrophages, cholangiocytes or endothelial cells, will divide afterwards. Schematically, one can distinguish two successive main steps allowing proper regeneration. The first phase requires the secretion of cytokines such as tumour necrosis factor-α and interleukin-6 in the very first minutes after PH, which poise hepatocytes to enter G1 phase and to become receptive to growth factors. The second step involves concomitantly metabolic changes, consisting in particular in a transient accumulation of lipid droplets, and in the activation of two growth factor pathways, epidermal growth factor receptor and c-Met. Both pathways then recruit scaffolding proteins and activate multiple intracellular intertwined networks, such as mitogen-activated protein kinases, signal transducer and activator of transcription 3 (STAT3), …

Glycogenolytic agonists induce coordinated Ca(2+) oscillations in multicellular rat hepatocyte systems as well as in the intact liver. The coordination of intercellular Ca(2+) signals requires functional gap-junction coupling. The mechanisms ensuring this coordination are not precisely known. We investigated possible roles of Ca(2+) or inositol 1,4,5-trisphosphate (InsP(3)) as a coordinating messengers for Ca(2+) spiking among connected hepatocytes. Application of ionomycin or of supra-maximal concentrations of agonists show that Ca(2+) does not significantly diffuse between connected hepatocytes, although gap junctions ensure the passage of small signaling molecules, as demonstrated by FRAP experiments. By contrast, coordination of Ca(2+) spiking among connected hepatocytes can be favored by a rise in the level of InsP(3), via the increase of agonist concentrations, or by a shift in the affinity of InsP(3) receptor for InsP(3). In the same line, coordination cannot be achieved if the InsP(3) is rapidly metabolized by InsP(3)-phosphatase in one cell of the multiplet. These results demonstrate that even if small amounts of Ca(2+) diffuse across gap junctions, they most probably do not play a significant role in inducing a coordinated Ca(2+) signal among connected hepatocytes. By contrast, coordination of Ca(2+) oscillations is fully dependent on the diffusion of InsP(3) between neighboring cells.

Intercellular calcium signals are propagated in multicellular hepatocyte systems as well as in the intact liver. The stimulation of connected hepatocytes by glycogenolytic agonists induces reproducible sequences of intracellular calcium concentration increases, resulting in unidirectional intercellular calcium waves. Hepatocytes are characterized by a gradient of vasopressin binding sites from the periportal to perivenous areas of the cell plate in hepatic lobules. Also, coordination of calcium signals between neighboring cells requires the presence of the agonist at each cell surface as well as gap junction permeability. We present a model based on the junctional coupling of several hepatocytes differing in sensitivity to the agonist and thus in the intrinsic period of calcium oscillations. In this model, each hepatocyte displays repetitive calcium spikes with a slight phase shift with respect to neighboring cells, giving rise to a phase wave. The orientation of the apparent calcium wave is imposed by the direction of the gradient of hormonal sensitivity. Calcium spikes are coordinated by the diffusion across junctions of small amounts of inositol 1,4, 5-trisphosphate (InsP(3)). Theoretical predictions from this model are confirmed experimentally. Thus, major physiological insights may be gained from this model for coordination and spatial orientation of intercellular signals.-Dupont, G., Tordjmann, T., Clair, C., Swillens, S., Claret, M., Combettes, L. Mechanism of receptor-oriented intercellular calcium wave propagation in hepatocytes.

In freshly isolated rat hepatocyte multiplets, Ca2+ signals in response to vasopressin are highly organized. In this study we used specific probes to visualize, by fluorescence and confocal microscopy, the main signaling molecules involved in vasopressin-mediated Ca2+ responses. V1a receptors were detected with a novel fluorescent antagonist, Rhm8 -PVA. The Gαq/Gα11, PLCβ3, PIP2, and InsP3 receptors were detected with specific antibodies. V1a vasopressin receptors and PIP2 were associated with the basolateral membrane and were not detected in the bile canalicular domain. Gαq/Gα11, PLCβ3, and InsP3 receptors were associated with the basolateral membrane and also with other intracellular structures. We used double labeling, Western blotting, and drugs (cytochalasin D, colchicine) known to disorganize the cytoskeleton to demonstrate the partial co-localization of Gαq/Gα11 with F-actin.

Morphological and functional heterogeneity of hepatocytes according to their position in the liver lobule has been known for many years. The digitonin-collagenase perfusion technique is widely used to study hepatocyte heterogeneity and has yielded reliable data. However, with this procedure, periportal (PP) or perivenous (PV) hepatocytes are isolated from different livers, allowing only comparison between cell populations issued from two separate animals. To overcome this drawback, we have modified this technique by perfusing the two main rat liver lobes of a single animal in succession. The procedure involved alternate clamping of the median and the left lateral lobes, restricting digitonin infusion to one lobe via the portal vein, and to the other via the caudal vena cava. Lobe exclusion during digitonin perfusion, and zonal restriction of digitonin-induced damage, were monitored using macroscopic and histological controls. We compared our results with previous data on PP and PV hepatocytes issued from two different livers using the conventional digitonin-collagenase perfusion technique. First, we found that the cellular sensitivity to angiotensin II, a calcium-mobilizing agonist, was 60% to 80% higher in PV than in PP hepatocytes, whereas, previously, no difference had been recorded. Second, we found that albumin messenger RNAs (mRNAs) were 35% more abundant in PP than in PV hepatocytes, whereas, previously, larger differences had been reported. Our results show that PP and PV hepatocytes may be isolated from a single liver using an improved digitonin-collagenase perfusion technique. Furthermore, we suggest that zonal differences can be artificially masked or amplified when comparing PP and PV cell populations from two different livers, indicating that it is preferable to use a single liver for accurate zonal comparisons between hepatocytes.

International audience; 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.

BACKGROUND & AIMS: In multicellular systems of rat hepatocytes and in the intact liver, inositol 1,4,5-trisphosphate (IP3)-dependent agonists induce sequentially ordered calcium ion signals. The mechanisms by which sequential waves are oriented from one hepatocyte to another are unknown. The aim of this study was to investigate the relationship between hepatocyte location in the acinus and cellular sensitivity to noradrenaline, vasopressin, adenosine triphosphate, and angiotensin II. METHODS: Periportal (PP) and pericentral (PC) rat hepatocyte suspensions, isolated by the digitonin-collagenase technique, were loaded with quin2-acetoxymethyl ester, and hormonal responses were studied in a spectrofluorimeter. The function of the IP3 receptor was studied by measuring the IP3-mediated 45Ca2+ release from permeabilized PP and PC hepatocytes. RESULTS: Increases in noradrenaline and vasopressin-induced intracellular Ca2+ concentration were greater in PC than in PP hepatocytes. In contrast, PP cells were more responsive than PC cells to adenosine triphosphate, and angiotensin II induced similar intracellular Ca2+ concentration increases in both hepatocyte populations. In permeabilized PP and PC hepatocytes, internal Ca2+ stores showed the same loading kinetics, the responses to IP3 were similar, and the sizes of the IP3 sensitive compartment were not different. CONCLUSIONS: Hepatocyte location in the acinus determines cellular sensitivity to Ca(2+)-mobilizing agonists. Intercellular Ca2+ waves in the liver could be driven by sensitivity gradients along the hepatocyte plate. (Gastroenterology 1996 Nov;111(5):1343-52)

After partial hepatectomy (PH) the initial mass of the organ is restored through a complex network of cellular interactions that orchestrate both proliferative and hepatoprotective signalling cascades. Among agonists involved in this network many of them drive Ca2+movements. During liver regeneration in the rat, hepatocyte cytosolic Ca2+signalling has been shown on the one hand to be deeply remodelled and on the other hand to enhance progression of hepatocytes through the cell cycle. Mechanisms through which cytosolic Ca2+signals impact on hepatocyte cell cycle early after PH are not completely understood, but at least they include regulation of immediate early gene transcription and ERK and CREB phosphorylation. In addition to cytosolic Ca2+, there is also evidence that mitochondrial Ca2+and also nuclear Ca2+may be critical for the regulation of liver regeneration. Finally, Ca2+movements in hepatocytes, and possibly in other liver cells, not only impact hepatocyte progression in the cell cycle but more generally may regulate cellular homeostasis after PH.

International audience; 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.

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. (HEPATOLOGY 2010;)

International audience; BACKGROUND & AIMS: Early neuroendocrine pathways contribute to liver regeneration after partial hepatectomy (PH). We investigated one of these pathways involving acute cholestasis, immediate portal hyperpressure, and arginine vasopressin (AVP) secretion. METHODS: Surgical procedure (PH, Portal vein stenosis (PVS), bile duct ligation (BDL), spinal cord lesion (SCL)) and treatments (capsaicin, bile acids (BA), oleanolic acid (OA)) were performed on rats and/or wild type or TGR5 (GPBAR1) knock-out mice. In these models, the activation of AVP-secreting supraoptic nuclei (SON) was analyzed, as well as plasma BA, AVP, and portal vein pressure (PVP). Plasma BA, AVP, and PVP were also determined in human living donors for liver transplantation. RESULTS: Acute cholestasis (mimicked by BDL or BA injection) as well as portal hyperpressure (mimicked by PVS) independently activated SON and AVP secretion. BA accumulated in the brain after PH or BDL, and TGR5 was expressed in SON. SON activation was mimicked by the TGR5 agonist OA and inhibited in TGR5 KO mice after BDL. An afferent nerve pathway also contributed to post-PH AVP secretion, as capsaicin treatment or SCL resulted in a weaker SON activation after PH. CONCLUSIONS: After PH in rodents, acute cholestasis and portal hypertension, via the nervous and endocrine routes, stimulate the secretion of AVP that may protect the liver against shear stress and bile acids overload. Data in living donors suggest that this pathway may also operate in humans.