Defective HNF4alpha-dependent gene expression as a driver of hepatocellular failure in alcoholic hepatitis

RNA sequencing in livers; TGFβ1; Epigenetic driversSeqüenciació d’ARN en el fetge; TGFβ1; Controladors epigenèticsSecuenciación de ARN en el hígado; TGFβ1; Controladores epigenéticosAlcoholic hepatitis (AH) is a life-threatening condition characterized by profound hepatocellular dysfunction for which targeted treatments are urgently needed. Identification of molecular drivers is hampered by the lack of suitable animal models. By performing RNA sequencing in livers from patients with different phenotypes of alcohol-related liver disease (ALD), we show that development of AH is characterized by defective activity of liver-enriched transcription factors (LETFs). TGFβ1 is a key upstream transcriptome regulator in AH and induces the use of HNF4α P2 promoter in hepatocytes, which results in defective metabolic and synthetic functions. Gene polymorphisms in LETFs including HNF4α are not associated with the development of AH. In contrast, epigenetic studies show that AH livers have profound changes in DNA methylation state and chromatin remodeling, affecting HNF4α-dependent gene expression. We conclude that targeting TGFβ1 and epigenetic drivers that modulate HNF4α-dependent gene expression could be beneficial to improve hepatocellular function in patients with AH.This work was mainly supported by NIH/NIAAA funded Consortia "Integrated approaches for identifying molecular targets in alcoholic hepatitis" InTEAM (U01AA021908) (R.B., P.M., P.S-B., I.R., J.Cbl). This work was supported in part by: NIH/NIAAA (R01AA023781), USA (C.W.); Hepacare Project, Fundacion La Caixa, Spain (M.A.A., C.B. and M.U.L); Fond national de la recherche scientifique (FNRS J.0146.17) and Fond de la recherche scientifique medicale (FRSM T.0217.18), Belgium (P.S.); NIH/NCATS (UH3TR000503) and EPA (STAR 83573601), USA (D.L.V. and L.A.T.); MRC, UK (MK/K001949/1) and NIH/NIAAA, USA (UO1AA018663) (J.M.); NIH/NIAAA (1U01AA021908-01-33490), Instituto de Salud Carlos III (PI17/00673) and Miguel Servet (CPII16/00041) and "Una manera de hacer Europa" program, European Regional Development Fund (ERDF), EU (P.S-B.); National Institute for Health Research Imperial Biomedical Research Centre and NIHR Health Technology Assessment Grant 08-14-44 (M.R.T.); NIH T32, DK007052, USA (L.R.E.); NIH/NIAAA (1U01AA021908) and AFEF (P.M., L.D., A. L.)

Restoring cellular magnesium balance through Cyclin M4 protects against acetaminophen-induced liver damage

Acetaminophen overdose is one of the leading causes of acute liver failure and liver transplantation in the Western world. Magnesium is essential in several cellular processess. The Cyclin M family is involved in magnesium transport across cell membranes. Herein, we identify that among all magnesium transporters, only Cyclin M4 expression is upregulated in the liver of patients with acetaminophen overdose, with disturbances in magnesium serum levels. In the liver, acetaminophen interferes with the mitochondrial magnesium reservoir via Cyclin M4, affecting ATP production and reactive oxygen species generation, further boosting endoplasmic reticulum stress. Importantly, Cyclin M4 mutant T495I, which impairs magnesium flux, shows no effect. Finally, an accumulation of Cyclin M4 in endoplasmic reticulum is shown under hepatoxicity. Based on our studies in mice, silencing hepatic Cyclin M4 within the window of 6 to 24 h following acetaminophen overdose ingestion may represent a therapeutic target for acetaminophen overdose induced liver injury.Acknowledgements: This work was supported by Ministerio de Ciencia, Innovación y Universidades MICINN: PID2020-117116RB-I00 integrado en el Plan Estatal de Investigación Cientifica y Técnica y Innovación, cofinanciado con Fondos FEDER (to MLM-C), Ministerio de Ciencia e Innovación CONSOLIDER-INGENIO 2010 Program Grant CSD2008-00005 (to LAMC); Spanish Ministry of Economy and Competitiveness Grant BFU2013-47531-R, BFU2016-77408-R, PID2019-109055RB-100 (to L.A.M.-C.) (MINECO/FEDER, UE); Asociación Española contra el Cáncer (MLM-C, TC-D), Fundación Científica de la Asociación Española Contra el Cáncer (AECC Scientific Foundation) Rare Tumor Calls 2017 (to M.L.M.-C.), La Caixa Foundation Program (to M.L.M.-C.), Fundacion BBVA UMBRELLA project (to M.L.M.-C.), Ayuda RYC2020-029316-I financiada por MICIN/AEI/10.13039/501100011033 (to TC-D), Plataforma de Investigación Clínica-SCReN (PT17 0017 0020) (to M.I.-L.), programa retos RTC2019-007125-1 (to M.L.M.-C, J.S.), Proyectos Investigacion en Salud DTS20/00138 (to M.L.M.-C., J.S), ERA-Net E-Rare EJP RD Joint Translational Call for Rare Diseases FIGHT-CNNM2 (EJPRD19-040) and from Instituto Carlos III, Spain (REF G95229142) (to L.A.M.-C.), US National Institutes of Health under grant CA217817 (to D.B.), Ciberehd_ISCIII_MINECO is funded by the Instituto de Salud Carlos III. We thankMINECO for the Severo Ochoa Excellence Accreditation to CIC bioGUNE (SEV-2016-0644) and PhD fellowship fromMINECO (REF BES-2017-080435) awarded to I.G.-R. The collection and storage of patients tissues was supported by the Newcastle Biomedicine Biobank and the European Community’s Seventh Framework Programme (FP7/2001–2013) and Cancer Research UK awards Cancer Research UK grants C18342/A23390; C9380/A18084 and C9380/A26813. Finally, we would like to acknowledge Begoña Rodríguez Iruretagoyena for the technical support provided

Splicing Factor SLU7 Prevents Oxidative Stress-Mediated Hepatocyte Nuclear Factor 4α Degradation, Preserving Hepatic Differentiation and Protecting From Liver Damage

Background and Aims: Hepatocellular dedifferentiation is emerging as an important determinant in liver disease progression. Preservation of mature hepatocyte identity relies on a set of key genes, predominantly the transcription factor hepatocyte nuclear factor 4α (HNF4α) but also splicing factors like SLU7. How these factors interact and become dysregulated and the impact of their impairment in driving liver disease are not fully understood. Approach and Results: Expression of SLU7 and that of the adult and oncofetal isoforms of HNF4α, driven by its promoter 1 (P1) and P2, respectively, was studied in diseased human and mouse livers. Hepatic function and damage response were analyzed in wild-type and Slu7-haploinsufficient/heterozygous (Slu7) mice undergoing chronic (CCl) and acute (acetaminophen) injury. SLU7 expression was restored in CCl-injured mice using SLU7-expressing adeno-associated viruses (AAV-SLU7). The hepatocellular SLU7 interactome was characterized by mass spectrometry. Reduced SLU7 expression in human and mouse diseased livers correlated with a switch in HNF4α P1 to P2 usage. This response was reproduced in Slu7 mice, which displayed increased sensitivity to chronic and acute liver injury, enhanced oxidative stress, and marked impairment of hepatic functions. AAV-SLU7 infection prevented liver injury and hepatocellular dedifferentiation. Mechanistically we demonstrate a unique role for SLU7 in the preservation of HNF4α1 protein stability through its capacity to protect the liver against oxidative stress. SLU7 is herein identified as a key component of the stress granule proteome, an essential part of the cell’s antioxidant machinery. Conclusions: Our results place SLU7 at the highest level of hepatocellular identity control, identifying SLU7 as a link between stress-protective mechanisms and liver differentiation. These findings emphasize the importance of the preservation of hepatic functions in the protection from liver injury.Supported by MINECO/AEI/FEDER (UE SAF2016-75972-R, PID2019-104265RB-I00/AEI/10.13039/501100011033, and PID2019-104878RB-100/AEI/10.13039/501100011033), CIBERehd, Fundación La Caixa (HEPACARE), an AECC postdoctoral fellowship (POSTD18014AREC, to M.A.), a Ministerio de Educación FPU fellowship (FPU18/01461, to M.G.R.), a Ministerio de Educación FPI fellowship (BES-2017-079883, to M.R.); a Ramón y Cajal Program contract (RYC2018-024475-1, to M.G.F.B.), the Fundación Eugenio Rodríguez Pascual, the Fundación Mario Losantos, the Fundación M. Torres, and a generous donation from Mr. Eduardo Avila

Restoring cellular magnesium balance through Cyclin M4 protects against acetaminophen-induced liver damage

Acetaminophen overdose is one of the leading causes of acute liver failure and liver transplantation in the Western world. Magnesium is essential in several cellular processess. The Cyclin M family is involved in magnesium transport across cell membranes. Herein, we identify that among all magnesium transporters, only Cyclin M4 expression is upregulated in the liver of patients with acetaminophen overdose, with disturbances in magnesium serum levels. In the liver, acetaminophen interferes with the mitochondrial magnesium reservoir via Cyclin M4, affecting ATP production and reactive oxygen species generation, further boosting endoplasmic reticulum stress. Importantly, Cyclin M4 mutant T495I, which impairs magnesium flux, shows no effect. Finally, an accumulation of Cyclin M4 in endoplasmic reticulum is shown under hepatoxicity. Based on our studies in mice, silencing hepatic Cyclin M4 within the window of 6 to 24 h following acetaminophen overdose ingestion may represent a therapeutic target for acetaminophen overdose induced liver injury

Hepatic matrix metalloproteinase-10 exerts a hepatoprotective role after acute liver injury

After injuries that lead to a loss of liver tissue a regenerative and reparative response is performed in order to restore an adequate hepatic mass. The remodeling of the extracellular matrix, accompanies the liver regeneration and when the reparative reaction goes awry in the setting of chronic liver injury, could be involved in the carcinogenic process (1,2). Following the damage, a provisional matrix is deposed, intended to be successively replaced, which has the function of stabilizing the lesional area and constitutes a support for guiding regenerating cells. Matrix metalloproteinases are increasingly recognized as important modulators of the matrix remodeling process. Matrix metalloproteinase-10 (MMP-10) has been implicated in the reparative process in other organs and has effects on the plasminogen system, which plays a fundamental role in liver repair (3). The hepatic expression of MMP10 in animal models of acute liver injury was tested in order to investigate the role of MMP-10 in liver repair and regeneration. The liver regeneration after two thirds partial hepatectomy (PH) and bile duct ligation (BDL) models were examined. Hepatic MMP-10 expression, analyzed by immunohistochemistry, western blot and qPCR showed a rise early after injury. In the MMP10-deficient mice a diminished and delayed resolution of necrotic lesions, enhanced fibrogenesis and a fibrinogen/fibrin and fibronectin compromised turnover were observed. These findings showed that the MMP10 expression plays a role in the hepatic wound healing response probably through its profibrinolytic activity

Pilot multi-omic analysis of human bile from benign and malignant biliary strictures: a machine-learning approach

Cholangiocarcinoma (CCA) and pancreatic adenocarcinoma (PDAC) may lead to the development of extrahepatic obstructive cholestasis. However, biliary stenoses can also be caused by benign conditions, and the identification of their etiology still remains a clinical challenge. We performed metabolomic and proteomic analyses of bile from patients with benign (n = 36) and malignant conditions, CCA (n = 36) or PDAC (n = 57), undergoing endoscopic retrograde cholangiopancreatography with the aim of characterizing bile composition in biliopancreatic disease and identifying biomarkers for the differential diagnosis of biliary strictures. Comprehensive analyses of lipids, bile acids and small molecules were carried out using mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (1H-NMR) in all patients. MS analysis of bile proteome was performed in five patients per group. We implemented artificial intelligence tools for the selection of biomarkers and algorithms with predictive capacity. Our machine-learning pipeline included the generation of synthetic data with properties of real data, the selection of potential biomarkers (metabolites or proteins) and their analysis with neural networks (NN). Selected biomarkers were then validated with real data. We identified panels of lipids (n = 10) and proteins (n = 5) that when analyzed with NN algorithms discriminated between patients with and without cancer with an unprecedented accuracy.This research was funded by: Instituto de Salud Carlos III (ISCIII) co-financed by Fondo Europeo de Desarrollo Regional (FEDER) Una manera de hacer Europa, grant numbers: PI16/01126 (M.A.A.), PI19/00819 (M.J.M. and J.J.G.M.), PI15/01132, PI18/01075 and Miguel Servet Program CON14/00129 (J.M.B.); Fundación Científica de la Asociación Española Contra el Cáncer (AECC Scientific Foundation), grant name: Rare Cancers 2017 (J.M.U., M.L.M., J.M.B., M.J.M., R.I.R.M., M.G.F.-B., C.B., M.A.A.); Gobierno de Navarra Salud, grant number 58/17 (J.M.U., M.A.A.); La Caixa Foundation, grant name: HEPACARE (C.B., M.A.A.); AMMF The Cholangiocarcinoma Charity, UK, grant number: 2018/117 (F.J.C. and M.A.A.); PSC Partners US, PSC Supports UK, grant number 06119JB (J.M.B.); Horizon 2020 (H2020) ESCALON project, grant number H2020-SC1-BHC-2018–2020 (J.M.B.); BIOEF (Basque Foundation for Innovation and Health Research: EiTB Maratoia, grant numbers BIO15/CA/016/BD (J.M.B.) and BIO15/CA/011 (M.A.A.). Department of Health of the Basque Country, grant number 2017111010 (J.M.B.). La Caixa Foundation, grant number: LCF/PR/HP17/52190004 (M.L.M.), Mineco-Feder, grant number SAF2017-87301-R (M.L.M.), Fundación BBVA grant name: Ayudas a Equipos de Investigación Científica Umbrella 2018 (M.L.M.). MCIU, grant number: Severo Ochoa Excellence Accreditation SEV-2016-0644 (M.L.M.). Part of the equipment used in this work was co-funded by the Generalitat Valenciana and European Regional Development Fund (FEDER) funds (PO FEDER of Comunitat Valenciana 2014–2020). Gobierno de Navarra fellowship to L.C. (Leticia Colyn); AECC post-doctoral fellowship to M.A.; Ramón y Cajal Program contracts RYC-2014-15242 and RYC2018-024475-1 to F.J.C. and M.G.F.-B., respectively. The generous support from: Fundación Eugenio Rodríguez Pascual, Fundación Echébano, Fundación Mario Losantos, Fundación M Torres and Mr. Eduardo Avila are acknowledged. The CNB-CSIC Proteomics Unit belongs to ProteoRed, PRB3-ISCIII, supported by grant PT17/0019/0001 (F.J.C.). Comunidad de Madrid Grant B2017/BMD-3817 (F.J.C.).Peer reviewe

Toll-like receptor-4 expression by hepatic progenitor cells and biliary epithelial cells in HCV-related chronic liver disease

Notwithstanding numerous evidences implicating toll-like receptor-4 (TLR4) in the pathogenesis of chronic hepatitis C virus (HCV) infection, the localization and level of TLR4 expression in the liver of patients with hepatitis C have never been investigated. We aimed to evaluate, by means of immunohistochemistry and real-time PCR (rt-PCR), hepatic TLR4 expression in patients with chronic HCV infection. Fifty patients who had undergone liver biopsy and 11 patients transplanted because of chronic HCV infection, and 12 controls free of liver disease, were included in the study. Each case was analyzed by immunohistochemistry for TLR4, se smooth muscle actin and cytokeratin-7 (CK-7), and a subgroup of patients and all controls by rt-PCR for TLR4. Immunohistochemistry for a-smooth muscle actin was used to derive a score of activation of hepatic stellate cells and portal/septal myofibroblasts, while immunohistochemistry for CK-7 was used to evaluate and count hepatic progenitor cells, interlobular bile ducts and intermediate hepatocytes. In patients, the parenchymal elements responsible for the highest TLR4 level of expression were hepatic progenitor cells and biliary epithelial cells of interlobular bile ducts. Double-labeling experiments between anti-TLR4 and anti-CK7, anti-CD133, anti-CD44, anti-neural cell adhesion molecule, anti-epithelial cell adhesion molecule and anti-sex determining region Y-box 9, confirmed these findings. TLR4-positive hepatic progenitor cells and interlobular bile ducts were significantly correlated with the stage of liver disease (P<0.001), the grade of inflammation (P<0.001), and the activity of portal/septal myofibroblasts (P<0.001). rt-PCR study confirmed an increased TLR4 expression in the 26 patients analyzed with respect to controls (P<0.001). TLR4 expression positively correlated with fibrosis (P<0.05) and inflammation (P<0.05). The present results suggest that TLR4 expression by hepatic progenitor cells and biliary epithelial cells contributes to the progression of liver damage in the course of chronic HCV-related infection. Modern Pathology (2012) 25, 576-589; doi:10.1038/modpathol.2011.197; published online 16 December 201

Lack of Abcc3 expression impairs bile-acid induced liver growth and delays hepatic regeneration after partial hepatectomy in mice

Background & Aims: Bile acids (BA) are increasingly recognized as important modulators of liver regeneration. Increased enterohepatic BA flux has been proposed to generate specific signals that activate hepatocyte proliferation after partial hepatectomy (PH). We have investigated the role of the BA membrane transporter Mrp3 (Abcc3), which is expressed in the liver and gut, in the hepatic growth response elicited by BA and in liver regeneration after PH. Methods: Liver growth and regeneration, and the expression of growth-related genes, were studied in and Mrp3(-/-) mice fed a cholic acid (CA) supplemented diet and after 2/3 PH. Activation of the BA receptor FXR was measured in mice after in vivo transduction of the liver with a FXR-Luciferase reporter plasmid. BA levels were measured in portal serum and liver tissue by high performance liquid chromatography-tandem mass spectrometry. Results: Liver growth elicited by CA feeding was significantly reduced in Mrp3(-/-) mice. These animals showed reduced FXR activation in the liver after CA administration and decreased portal serum levels of BA. Liver regeneration after PH was significantly delayed in Mrp3-deficient mice. Proliferation-related gene expression and peak DNA synthesis in Mrp3(-/-) mice occurred later than in wild types, coinciding with a retarded elevation in intra-hepatic BA levels. Conclusions: Lack of Abcc3 expression markedly impairs liver growth in response to BA and after PH. Our data suggest that Mrp3 plays a non-redundant role in the regulation of BA flux during liver regeneration. (C) 2011 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserve