Publications by authors named "Naiara Beraza"

46 Publications

Multi-Omics Integration Highlights the Role of Ubiquitination in CCl-Induced Liver Fibrosis.

Int J Mol Sci 2020 Nov 27;21(23). Epub 2020 Nov 27.

Liver Disease Lab, CIC bioGUNE, Basque Research and Technology Alliance (BRTA), 48160 Derio, Bizkaia, Spain.

Liver fibrosis is the excessive accumulation of extracellular matrix proteins that occurs in chronic liver disease. Ubiquitination is a post-translational modification that is crucial for a plethora of physiological processes. Even though the ubiquitin system has been implicated in several human diseases, the role of ubiquitination in liver fibrosis remains poorly understood. Here, multi-omics approaches were used to address this. Untargeted metabolomics showed that carbon tetrachloride (CCl)-induced liver fibrosis promotes changes in the hepatic metabolome, specifically in glycerophospholipids and sphingolipids. Gene ontology analysis of public deposited gene array-based data and validation in our mouse model showed that the biological process "protein polyubiquitination" is enriched after CCl-induced liver fibrosis. Finally, by using transgenic mice expressing biotinylated ubiquitin (Ub mice), the ubiquitinated proteome was isolated and characterized by mass spectrometry in order to unravel the hepatic ubiquitinated proteome fingerprint in CCl-induced liver fibrosis. Under these conditions, ubiquitination appears to be involved in the regulation of cell death and survival, cell function, lipid metabolism, and DNA repair. Finally, ubiquitination of proliferating cell nuclear antigen (PCNA) is induced during CCl-induced liver fibrosis and associated with the DNA damage response (DDR). Overall, hepatic ubiquitome profiling can highlight new therapeutic targets for the clinical management of liver fibrosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms21239043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7729774PMC
November 2020

Intestinal Microbiome-Macrophage Crosstalk Contributes to Cholestatic Liver Disease by Promoting Intestinal Permeability in Mice.

Hepatology 2020 Dec;72(6):2090-2108

Gut Microbes and Health Institute Strategic Programme, Quadram Institute Bioscience, Norwich Research Park, Norwich, United Kingdom.

Background And Aims: Mounting evidence supports an association between cholestatic liver disease and changes in the composition of the microbiome. Still, the role of the microbiome in the pathogenesis of this condition remains largely undefined.

Approach And Results: To address this, we have used two experimental models, administering alpha-naphtylisocyanate or feeding a 0.1% 3,5-diethoxycarbonyl-1,4-dihydrocollidine diet, to induce cholestatic liver disease in germ-free mice and germ-free mice conventionalized with the microbiome from wild-type, specific pathogen-free animals. Next, we have inhibited macrophage activation by depleting these cells using clodronate liposomes and inhibiting the inflammasome with a specific inhibitor of NOD-, LRR-, and pyrin domain-containing protein 3. Our results demonstrate that cholestasis, the accumulation of bile acids in the liver, fails to promote liver injury in the absence of the microbiome in vivo. Additional in vitro studies supported that endotoxin sensitizes hepatocytes to bile-acid-induced cell death. We also demonstrate that during cholestasis, macrophages contribute to promoting intestinal permeability and to altered microbiome composition through activation of the inflammasome, overall leading to increased endotoxin flux into the cholestatic liver.

Conclusions: We demonstrate that the intestinal microbiome contributes to cholestasis-mediated cell death and inflammation through mechanisms involving activation of the inflammasome in macrophages.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/hep.31228DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7839474PMC
December 2020

TGF-β2 silencing to target biliary-derived liver diseases.

Gut 2020 Sep 28;69(9):1677-1690. Epub 2020 Jan 28.

Molecular Hepatology-Alcohol Associated Diseases, Department of Medicine II, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany

Objective: TGF-β2 (TGF-β, transforming growth factor beta), the less-investigated sibling of TGF-β1, is deregulated in rodent and human liver diseases. Former data from bile duct ligated and MDR2 knockout (KO) mouse models for human cholestatic liver disease suggested an involvement of TGF-β2 in biliary-derived liver diseases.

Design: As we also found upregulated in liver tissue of patients with primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC), we now fathomed the positive prospects of targeting TGF-β2 in early stage biliary liver disease using the MDR2-KO mice. Specifically, the influence of silencing on the fibrotic and inflammatory niche was analysed on molecular, cellular and tissue levels.

Results: -induced expression of fibrotic genes in cholangiocytes and hepatic stellate cellswas detected. expression in MDR2-KO mice was blunted using -directed antisense oligonucleotides (AON). Upon AON treatment, reduced collagen deposition, hydroxyproline content and αSMA expression as well as induced expression reflected a significant reduction of fibrogenesis without adverse effects on healthy livers. Expression analyses of fibrotic and inflammatory genes revealed AON-specific regulatory effects on , , , and expression. Further, AON treatment of MDR2-KO mice increased tissue infiltration by F4/80-positive cells including eosinophils, whereas the number of CD45-positive inflammatory cells decreased. In line, and CD45 expression correlated positively in PSC/PBC patients and localised in similar areas of the diseased liver tissue.

Conclusions: Taken together, our data suggest a new mechanistic explanation for amelioration of fibrogenesis by TGF-β2 silencing and provide a direct rationale for TGF-β2-directed drug development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1136/gutjnl-2019-319091DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7456737PMC
September 2020

ROS-mediated PI3K activation drives mitochondrial transfer from stromal cells to hematopoietic stem cells in response to infection.

Proc Natl Acad Sci U S A 2019 12 14;116(49):24610-24619. Epub 2019 Nov 14.

Norwich Medical School, University of East Anglia, NR4 7UQ Norwich, United Kingdom;

Hematopoietic stem cells (HSCs) undergo rapid expansion in response to stress stimuli. Here we investigate the bioenergetic processes which facilitate the HSC expansion in response to infection. We find that infection by Gram-negative bacteria drives an increase in mitochondrial mass in mammalian HSCs, which results in a metabolic transition from glycolysis toward oxidative phosphorylation. The initial increase in mitochondrial mass occurs as a result of mitochondrial transfer from the bone marrow stromal cells (BMSCs) to HSCs through a reactive oxygen species (ROS)-dependent mechanism. Mechanistically, ROS-induced oxidative stress regulates the opening of connexin channels in a system mediated by phosphoinositide 3-kinase (PI3K) activation, which allows the mitochondria to transfer from BMSCs into HSCs. Moreover, mitochondria transfer from BMSCs into HSCs, in the response to bacterial infection, occurs before the HSCs activate their own transcriptional program for mitochondrial biogenesis. Our discovery demonstrates that mitochondrial transfer from the bone marrow microenvironment to HSCs is an early physiologic event in the mammalian response to acute bacterial infection and results in bioenergetic changes which underpin emergency granulopoiesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1913278116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6900710PMC
December 2019

Fibrosis and the intestinal microbiome; a focus on chronic liver disease.

Authors:
Naiara Beraza

Curr Opin Pharmacol 2019 12;49:76-81

Gut Microbes and Health Institute Strategic Programme, Quadram Institute Bioscience, Norwich Research Park, Norwich, Norfolk, NR4 7UQ, UK. Electronic address:

The role of the microbiome in progression of liver disease is an exciting area of research that is advancing rapidly supported by the development of next-generation sequencing and bioinformatics tools that simultaneously identify the composition and function of the microbiome. Changes in the microbiome are associated with pathogenesis of chronic liver disease; specifically, changes in microbiome composition predict disease severity and specific microbial signatures can be used to distinguish between mild disease, advanced fibrosis and cirrhosis. Future work combining functional metagenomic analysis with preclinical mechanistic studies will be key to advancing our understanding of how the microbiome affects the pathogenesis of different chronic liver disease aetiologies and to identify personalised therapeutics based on modulation of the microbiome and its function.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.coph.2019.09.012DOI Listing
December 2019

Cholestasis induced by bile duct ligation promotes changes in the intestinal microbiome in mice.

Sci Rep 2019 08 23;9(1):12324. Epub 2019 Aug 23.

Gut Microbes and Health Institute Strategic Programme, Quadram Institute, Norwich Research Park, Norwich, UK.

Increasing evidence point to the relevance of intestinal disfunction and changes in the microbiome composition during chronic liver disease. More specifically, recent studies have highlighted that cholestatic diseases associate with a reduction in the microbiome diversity in patients. Still, the dynamics of the changes in the microbiome composition observed, as well as their implication in contributing to the pathogenesis of this disease remain largely undefined. Hence, experimental mouse models resembling the human pathogenesis are crucial to move forward our understanding on the mechanisms underpinning cholestatic disease and to enable the development of effective therapeutics. Our results show that the bile duct ligation (BDL) experimental model of cholestasis leads to rapid and significant changes in the microbiome diversity, with more than 100 OTUs being significantly different in faecal samples obtained from WT mice at 3 days and 7 days after BDL when compared to control animals. Changes in the microbial composition in mice after BDL included the enrichment of Akkermansia, Prevotella, Bacteroides and unclassified Ruminococcaceae in parallel with a drastic reduction of the presence of Faecalibacterium prausnitzii. In conclusion, our results support that bile duct ligation induces changes in the microbiome that partly resemble the gut microbial changes observed during human cholestatic disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-019-48784-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6707139PMC
August 2019

Hepatocellular carcinomas: evolution to sorafenib resistance through hepatic leukaemia factor.

Gut 2019 10 3;68(10):1728-1730. Epub 2019 Jul 3.

Gut Microbes and Health Research Programme, Quadram Institute, Norwich, UK.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1136/gutjnl-2019-318999DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6839724PMC
October 2019

SUMOylation regulates LKB1 localization and its oncogenic activity in liver cancer.

EBioMedicine 2019 Feb 26;40:406-421. Epub 2018 Dec 26.

Liver Disease and Liver Metabolism Lab, CIC bioGUNE, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), 48160 Derio, Bizkaia, Spain. Electronic address:

Background: Even though liver kinase B1 (LKB1) is usually described as a tumor suppressor in a wide variety of tissues, it has been shown that LKB1 aberrant expression is associated with bad prognosis in Hepatocellular Carcinoma (HCC).

Methods: Herein we have overexpressed LKB1 in human hepatoma cells and by using histidine pull-down assay we have investigated the role of the hypoxia-related post-translational modification of Small Ubiquitin-related Modifier (SUMO)ylation in the regulation of LKB1 oncogenic role. Molecular modelling between LKB1 and its interactors, involved in regulation of LKB1 nucleocytoplasmic shuttling and LKB1 activity, was performed. Finally, high affinity SUMO binding entities-based technology were used to validate our findings in a pre-clinical mouse model and in clinical HCC.

Findings: We found that in human hepatoma cells under hypoxic stress, LKB1 overexpression increases cell viability and aggressiveness in association with changes in LKB1 cellular localization. Moreover, by using site-directed mutagenesis, we have shown that LKB1 is SUMOylated by SUMO-2 at Lys178 hampering LKB1 nucleocytoplasmic shuttling and fueling hepatoma cell growth. Molecular modelling of SUMO modified LKB1 further confirmed steric impedance between SUMOylated LKB1 and the STe20-Related ADaptor cofactor (STRADα), involved in LKB1 export from the nucleus. Finally, we provide evidence that endogenous LKB1 is modified by SUMO in pre-clinical mouse models of HCC and clinical HCC, where LKB1 SUMOylation is higher in fast growing tumors.

Interpretation: Overall, SUMO-2 modification of LKB1 at Lys178 mediates LKB1 cellular localization and its oncogenic role in liver cancer. FUND: This work was supported by grants from NIH (US Department of Health and Human services)-R01AR001576-11A1 (J.M.M and M.L.M-C.), Gobierno Vasco-Departamento de Salud 2013111114 (to M.L.M.-C), ELKARTEK 2016, Departamento de Industria del Gobierno Vasco (to M.L.M.-C), MINECO: SAF2017-87301-R and SAF2014-52097-R integrado en el Plan Estatal de Investigación Cientifica y Técnica y Innovación 2013-2016 cofinanciado con Fondos FEDER (to M.L.M.-C and J.M.M., respectively), BFU2015-71017/BMC MINECO/FEDER, EU (to A.D.Q. and I.D.M.), BIOEF (Basque Foundation for Innovation and Health Research): EITB Maratoia BIO15/CA/014; Instituto de Salud Carlos III:PIE14/00031, integrado en el Plan Estatal de Investigación Cientifica y Técnica y Innovacion 2013-2016 cofinanciado con Fondos FEDER (to M.L.M.-C and J.M.M), Asociación Española contra el Cáncer (T.C.D, P·F-T and M.L.M-C), Daniel Alagille award from EASL (to T.C.D), Fundación Científica de la Asociación Española Contra el Cancer (AECC Scientific Foundation) Rare Tumor Calls 2017 (to M.L.M and M.A), La Caixa Foundation Program (to M.L.M), Programma di Ricerca Regione-Università 2007-2009 and 2011-2012, Regione Emilia-Romagna (to E.V.), Ramón Areces Foundation and the Andalusian Government (BIO-198) (A.D.Q. and I.D.M.), ayudas para apoyar grupos de investigación del sistema Universitario Vasco IT971-16 (P.A.), MINECO:SAF2015-64352-R (P.A.), Institut National du Cancer, FRANCE, INCa grant PLBIO16-251 (M.S.R.), MINECO - BFU2016-76872-R to (E.B.). Work produced with the support of a 2017 Leonardo Grant for Researchers and Cultural Creators, BBVA Foundation (M.V-R). Finally, Ciberehd_ISCIII_MINECO is funded by the Instituto de Salud Carlos III. We thank MINECO for the Severo Ochoa Excellence Accreditation to CIC bioGUNE (SEV-2016-0644). Funding sources had no involvement in study design; in the collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ebiom.2018.12.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6412020PMC
February 2019

The ATG5-binding and coiled coil domains of ATG16L1 maintain autophagy and tissue homeostasis in mice independently of the WD domain required for LC3-associated phagocytosis.

Autophagy 2019 04 7;15(4):599-612. Epub 2018 Nov 7.

a Norwich Medical School , University of East Anglia , Norwich , Norfolk , UK.

Macroautophagy/autophagy delivers damaged proteins and organelles to lysosomes for degradation, and plays important roles in maintaining tissue homeostasis by reducing tissue damage. The translocation of LC3 to the limiting membrane of the phagophore, the precursor to the autophagosome, during autophagy provides a binding site for autophagy cargoes, and facilitates fusion with lysosomes. An autophagy-related pathway called LC3-associated phagocytosis (LAP) targets LC3 to phagosome and endosome membranes during uptake of bacterial and fungal pathogens, and targets LC3 to swollen endosomes containing particulate material or apoptotic cells. We have investigated the roles played by autophagy and LAP in vivo by exploiting the observation that the WD domain of ATG16L1 is required for LAP, but not autophagy. Mice lacking the linker and WD domains, activate autophagy, but are deficient in LAP. The LAP mice survive postnatal starvation, grow at the same rate as littermate controls, and are fertile. The liver, kidney, brain and muscle of these mice maintain levels of autophagy cargoes such as LC3 and SQSTM1/p62 similar to littermate controls, and prevent accumulation of SQSTM1 inclusions and tissue damage associated with loss of autophagy. The results suggest that autophagy maintains tissue homeostasis in mice independently of LC3-associated phagocytosis. Further deletion of glutamate E230 in the coiled-coil domain required for WIPI2 binding produced mice with defective autophagy that survived neonatal starvation. Analysis of brain lysates suggested that interactions between WIPI2 and ATG16L1 were less critical for autophagy in the brain, which may allow a low level of autophagy to overcome neonatal lethality. Abbreviations: CCD: coiled-coil domain; CYBB/NOX2: cytochrome b-245: beta polypeptide; GPT/ALT: glutamic pyruvic transaminase: soluble; LAP: LC3-associated phagocytosis; LC3: microtubule-associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; NOD: nucleotide-binding oligomerization domain; NADPH: nicotinamide adenine dinucleotide phosphate; RUBCN/Rubicon: RUN domain and cysteine-rich domain containing Beclin 1-interacting protein; SLE: systemic lupus erythematosus; SQSTM1/p62: sequestosome 1; TLR: toll-like receptor; TMEM: transmembrane protein; TRIM: tripartite motif-containing protein; UVRAG: UV radiation resistance associated gene; WD: tryptophan-aspartic acid; WIPI: WD 40 repeat domain: phosphoinositide interacting.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/15548627.2018.1534507DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6526875PMC
April 2019

MiR-873-5p acts as an epigenetic regulator in early stages of liver fibrosis and cirrhosis.

Cell Death Dis 2018 09 20;9(10):958. Epub 2018 Sep 20.

CIC bioGUNE, Centro de Investigación Cooperativa en Biociencias, Derio, Bizkaia, Spain.

Glycine N-methyltransferase (GNMT) is the most abundant methyltransferase in the liver and a master regulator of the transmethylation flux. GNMT downregulation leads to loss of liver function progressing to fibrosis, cirrhosis, and hepatocellular carcinoma. Moreover, GNMT deficiency aggravates cholestasis-induced fibrogenesis. To date, little is known about the mechanisms underlying downregulation of GNMT levels in hepatic fibrosis and cirrhosis. On this basis, microRNAs are epigenetic regulatory elements that play important roles in liver pathology. In this work, we aim to study the regulation of GNMT by microRNAs during liver fibrosis and cirrhosis. Luciferase assay on the 3'UTR-Gnmt was used to confirm in silico analysis showing that GNMT is potentially targeted by the microRNA miR-873-5p. Correlation between GNMT and miR-873-5p in human cholestasis and cirrhosis together with miR-873-5p inhibition in vivo in different mouse models of liver cholestasis and fibrosis [bile duct ligation and Mdr2 (Abcb4) mouse] were then assessed. The analysis of liver tissue from cirrhotic and cholestatic patients, as well as from the animal models, showed that miR-873-5p inversely correlated with the expression of GNMT. Importantly, high circulating miR-873-5p was also detected in cholestastic and cirrhotic patients. Preclinical studies with anti-miR-873-5p treatment in bile duct ligation and Mdr2 mice recovered GNMT levels in association with ameliorated inflammation and fibrosis mainly by counteracting hepatocyte apoptosis and cholangiocyte proliferation. In conclusion, miR-873-5p emerges as a novel marker for liver fibrosis, cholestasis, and cirrhosis and therapeutic approaches based on anti-miR-873-5p may be effective treatments for liver fibrosis and cholestatic liver disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41419-018-1014-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6148053PMC
September 2018

Fine-Tuning of Sirtuin 1 Expression Is Essential to Protect the Liver From Cholestatic Liver Disease.

Hepatology 2019 02;69(2):699-716

Gut Microbes and Health Institute Strategic Programme, Quadram Institute, Norwich, United Kingdom.

Cholestasis comprises aetiologically heterogeneous conditions characterized by accumulation of bile acids in the liver that actively contribute to liver damage. Sirtuin 1 (SIRT1) regulates liver regeneration and bile acid metabolism by modulating farnesoid X receptor (FXR); we here investigate its role in cholestatic liver disease. We determined SIRT1 expression in livers from patients with cholestatic disease, in two experimental models of cholestasis, as well as in human and murine liver cells in response to bile acid loading. SIRT1-overexpressing (SIRT ) and hepatocyte-specific SIRT1-KO (knockout) mice (SIRT ) were subjected to bile duct ligation (BDL) and were fed with a 0.1% DDC (3,5-diethoxycarbonyl-1,4-dihydrocollidine) diet to determine the biological relevance of SIRT1 during cholestasis. The effect of NorUDCA (24-norursodeoxycholic acid) was tested in BDL/SIRT mice. We found that SIRT1 was highly expressed in livers from cholestatic patients, mice after BDL, and Mdr2 knockout mice (Mdr2 ) animals. The detrimental effects of SIRT1 during cholestasis were validated in vivo and in vitro. SIRT mice showed exacerbated parenchymal injury whereas SIRT mice evidenced a moderate improvement after BDL and 0.1% DDC feeding. Likewise, hepatocytes isolated from SIRT mice showed increased apoptosis in response to bile acids, whereas a significant reduction was observed in SIRT hepatocytes. Importantly, the decrease, but not complete inhibition, of SIRT1 exerted by norUDCA treatment correlated with pronounced improvement in liver parenchyma in BDL/SIRT mice. Interestingly, both SIRT1 overexpression and hepatocyte-specific SIRT1 depletion correlated with inhibition of FXR, whereas modulation of SIRT1 by NorUDCA associated with restored FXR signaling. Conclusion: SIRT1 expression is increased during human and murine cholestasis. Fine-tuning expression of SIRT1 is essential to protect the liver from cholestatic liver damage.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/hep.30275DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6492079PMC
February 2019

mTORC1-dependent AMD1 regulation sustains polyamine metabolism in prostate cancer.

Nature 2017 07 28;547(7661):109-113. Epub 2017 Jun 28.

CIC bioGUNE, Bizkaia Technology Park, 801 building, 48160, Derio, Spain.

Activation of the PTEN-PI3K-mTORC1 pathway consolidates metabolic programs that sustain cancer cell growth and proliferation. Here we show that mechanistic target of rapamycin complex 1 (mTORC1) regulates polyamine dynamics, a metabolic route that is essential for oncogenicity. By using integrative metabolomics in a mouse model and human biopsies of prostate cancer, we identify alterations in tumours affecting the production of decarboxylated S-adenosylmethionine (dcSAM) and polyamine synthesis. Mechanistically, this metabolic rewiring stems from mTORC1-dependent regulation of S-adenosylmethionine decarboxylase 1 (AMD1) stability. This novel molecular regulation is validated in mouse and human cancer specimens. AMD1 is upregulated in human prostate cancer with activated mTORC1. Conversely, samples from a clinical trial with the mTORC1 inhibitor everolimus exhibit a predominant decrease in AMD1 immunoreactivity that is associated with a decrease in proliferation, in line with the requirement of dcSAM production for oncogenicity. These findings provide fundamental information about the complex regulatory landscape controlled by mTORC1 to integrate and translate growth signals into an oncogenic metabolic program.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nature22964DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5505479PMC
July 2017

Deregulated neddylation in liver fibrosis.

Hepatology 2017 02 30;65(2):694-709. Epub 2016 Dec 30.

Center for Cooperative Research in Bioscience (CIC bioGUNE), CIBERehd, Derio, Bizkaia, Spain.

Hepatic fibrosis is a global health problem currently without effective therapeutic approaches. Even though the ubiquitin-like posttranslational modification of neddylation, that conjugates Nedd8 (neural precursor cell expressed developmentally downregulated) to specific targets, is aberrant in many pathologies, its relevance in liver fibrosis (LF) remained unexplored. Our results show deregulated neddylation in clinical fibrosis and both in mouse bileductligation- and CCl -induced fibrosis. Importantly, neddylation inhibition, by using the pharmacological inhibitor, MLN4924, reduced liver injury, apoptosis, inflammation, and fibrosis by targeting different hepatic cell types. On one hand, increased neddylation was associated with augmented caspase 3 activity in bile-acid-induced apoptosis in mouse hepatocytes whereas neddylation inhibition ameliorated apoptosis through reduction of expression of the Cxcl1 and Ccl2 chemokines. On the other hand, chemokine receptors and cytokines, usually induced in activated macrophages, were reduced after neddylation inhibition in mouse Kupffer cells. Under these circumstances, decreased hepatocyte cell death and inflammation after neddylation inhibition could partly account for reduction of hepatic stellate cell (HSC) activation. We provide evidence that augmented neddylation characterizes activated HSCs, suggesting that neddylation inhibition could be important for resolving LF by directly targeting these fibrogenic cells. Indeed, neddylation inhibition in activated HSCs induces apoptosis in a process partly mediated by accumulation of c-Jun, whose cullin-mediated degradation is impaired under these circumstances.

Conclusion: Neddylation inhibition reduces fibrosis, suggesting neddylation as a potential and attractive therapeutic target in liver fibrosis. (Hepatology 2017;65:694-709).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/hep.28933DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5258821PMC
February 2017

Methionine and S-adenosylmethionine levels are critical regulators of PP2A activity modulating lipophagy during steatosis.

J Hepatol 2016 Feb 21;64(2):409-418. Epub 2015 Sep 21.

Metabolomic Unit, CIC bioGUNE, CIBERehd, Parque Tecnológico de Bizkaia, Derio 48160, Spain. Electronic address:

Background & Aims: Glycine N-methyltransferase (GNMT) expression is decreased in some patients with severe non-alcoholic fatty liver disease. Gnmt deficiency in mice (Gnmt-KO) results in abnormally elevated serum levels of methionine and its metabolite S-adenosylmethionine (SAMe), and this leads to rapid liver steatosis development. Autophagy plays a critical role in lipid catabolism (lipophagy), and defects in autophagy have been related to liver steatosis development. Since methionine and its metabolite SAMe are well known inactivators of autophagy, we aimed to examine whether high levels of both metabolites could block autophagy-mediated lipid catabolism.

Methods: We examined methionine levels in a cohort of 358 serum samples from steatotic patients. We used hepatocytes cultured with methionine and SAMe, and hepatocytes and livers from Gnmt-KO mice.

Results: We detected a significant increase in serum methionine levels in steatotic patients. We observed that autophagy and lipophagy were impaired in hepatocytes cultured with high methionine and SAMe, and that Gnmt-KO livers were characterized by an impairment in autophagy functionality, likely caused by defects at the lysosomal level. Elevated levels of methionine and SAMe activated PP2A by methylation, while blocking PP2A activity restored autophagy flux in Gnmt-KO hepatocytes, and in hepatocytes treated with SAMe and methionine. Finally, normalization of methionine and SAMe levels in Gnmt-KO mice using a methionine deficient diet normalized the methylation capacity, PP2A methylation, autophagy, and ameliorated liver steatosis.

Conclusions: These data suggest that elevated levels of methionine and SAMe can inhibit autophagic catabolism of lipids contributing to liver steatosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jhep.2015.08.037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4718902PMC
February 2016

Histone deacetylase 4 promotes cholestatic liver injury in the absence of prohibitin-1.

Hepatology 2015 Oct 31;62(4):1237-48. Epub 2015 Jul 31.

CIC bioGUNE, Metabolomics Unit, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Bizkaia, Spain.

Unlabelled: Prohibitin-1 (PHB1) is an evolutionarily conserved pleiotropic protein that participates in diverse processes depending on its subcellular localization and interactome. Recent data have indicated a diverse role for PHB1 in the pathogenesis of obesity, cancer, and inflammatory bowel disease, among others. Data presented here suggest that PHB1 is also linked to cholestatic liver disease. Expression of PHB1 is markedly reduced in patients with primary biliary cirrhosis and biliary atresia or with Alagille syndrome, two major pediatric cholestatic conditions. In the experimental model of bile duct ligation, silencing of PHB1 induced liver fibrosis, reduced animal survival, and induced bile duct proliferation. Importantly, the modulatory effect of PHB1 is not dependent on its known mitochondrial function. Also, PHB1 interacts with histone deacetylase 4 (HDAC4) in the presence of bile acids. Hence, PHB1 depletion leads to increased nuclear HDAC4 content and its associated epigenetic changes. Remarkably, HDAC4 silencing and the administration of the HDAC inhibitor parthenolide during obstructive cholestasis in vivo promote genomic reprogramming, leading to regression of the fibrotic phenotype in liver-specific Phb1 knockout mice.

Conclusion: PHB1 is an important mediator of cholestatic liver injury that regulates the activity of HDAC4, which controls specific epigenetic markers; these results identify potential novel strategies to treat liver injury and fibrosis, particularly as a consequence of chronic cholestasis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/hep.27959DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4589448PMC
October 2015

Stabilization of LKB1 and Akt by neddylation regulates energy metabolism in liver cancer.

Oncotarget 2015 Feb;6(4):2509-23

CIC bioGUNE, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Bizkaia Science and Technology Park, Derio, Bizkaia, Spain.

The current view of cancer progression highlights that cancer cells must undergo through a post-translational regulation and metabolic reprogramming to progress in an unfriendly environment. In here, the importance of neddylation modification in liver cancer was investigated. We found that hepatic neddylation was specifically enriched in liver cancer patients with bad prognosis. In addition, the treatment with the neddylation inhibitor MLN4924 in Phb1-KO mice, an animal model of hepatocellular carcinoma showing elevated neddylation, reverted the malignant phenotype. Tumor cell death in vivo translating into liver tumor regression was associated with augmented phosphatidylcholine synthesis by the PEMT pathway, known as a liver-specific tumor suppressor, and restored mitochondrial function and TCA cycle flux. Otherwise, in protumoral hepatocytes, neddylation inhibition resulted in metabolic reprogramming rendering a decrease in oxidative phosphorylation and concomitant tumor cell apoptosis. Moreover, Akt and LKB1, hallmarks of proliferative metabolism, were altered in liver cancer being new targets of neddylation. Importantly, we show that neddylation-induced metabolic reprogramming and apoptosis were dependent on LKB1 and Akt stabilization. Overall, our results implicate neddylation/signaling/metabolism, partly mediated by LKB1 and Akt, in the development of liver cancer, paving the way for novel therapeutic approaches targeting neddylation in hepatocellular carcinoma.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4385867PMC
http://dx.doi.org/10.18632/oncotarget.3191DOI Listing
February 2015

TRAIL-producing NK cells contribute to liver injury and related fibrogenesis in the context of GNMT deficiency.

Lab Invest 2015 Feb 22;95(2):223-36. Epub 2014 Dec 22.

1] Department of Metabolomics, CIC bioGUNE, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (Ciberehd), Derio, Spain [2] Ikerbasque, Basque Foundation for Science, Bilbao, Spain.

Glycine-N-methyltransferase (GNMT) is essential to preserve liver homeostasis. Cirrhotic patients show low expression of GNMT that is absent in hepatocellular carcinoma (HCC) samples. Accordingly, GNMT deficiency in mice leads to steatohepatitis, fibrosis, cirrhosis, and HCC. Lack of GNMT triggers NK cell activation in GNMT(-/-) mice and depletion of TRAIL significantly attenuates acute liver injury and inflammation in these animals. Chronic inflammation leads to fibrogenesis, further contributing to the progression of chronic liver injury regardless of the etiology. The aim of our study is to elucidate the implication of TRAIL-producing NK cells in the progression of chronic liver injury and fibrogenesis. For this we generated double TRAIL(-/-)/GNMT(-/-) mice in which we found that TRAIL deficiency efficiently protected the liver against chronic liver injury and fibrogenesis in the context of GNMT deficiency. Next, to better delineate the implication of TRAIL-producing NK cells during fibrogenesis we performed bile duct ligation (BDL) to GNMT(-/-) and TRAIL(-/-)/GNMT(-/-) mice. In GNMT(-/-) mice, exacerbated fibrogenic response after BDL concurred with NK1.1(+) cell activation. Importantly, specific inhibition of TRAIL-producing NK cells efficiently protected GNMT(-/-) mice from BDL-induced liver injury and fibrogenesis. Finally, TRAIL(-/-)/GNMT(-/-) mice showed significantly less fibrosis after BDL than GNMT(-/-) mice further underlining the relevance of the TRAIL/DR5 axis in mediating liver injury and fibrogenesis in GNMT(-/-) mice. Finally, in vivo silencing of DR5 efficiently protected GNMT(-/-) mice from BDL-liver injury and fibrogenesis, overall underscoring the key role of the TRAIL/DR5 axis in promoting fibrogenesis in the context of absence of GNMT. Overall, our work demonstrates that TRAIL-producing NK cells actively contribute to liver injury and further fibrogenesis in the pathological context of GNMT deficiency, a molecular scenario characteristic of chronic human liver disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/labinvest.2014.151DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4310762PMC
February 2015

Targeting of Gamma-Glutamyl-Cysteine Ligase by miR-433 Reduces Glutathione Biosynthesis and Promotes TGF-β-Dependent Fibrogenesis.

Antioxid Redox Signal 2015 Nov 9;23(14):1092-105. Epub 2015 Jan 9.

1 Departamento de Biología Celular e Inmunología, Centro de Biología Molecular "Severo Ochoa, " Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid , Madrid, Spain .

Aims: Glutathione (GSH) is the main antioxidant against cell damage. Several pathological states course with reduced nucleophilic tone and perturbation of redox homeostasis due to changes in the 2GSH/GSSG ratio. Here, we investigated the regulation of the rate-limiting GSH biosynthetic heterodimeric enzyme γ-glutamyl-cysteine ligase (GCL) by microRNAs (miRNAs).

Results: "In silico" analysis of the 3'- untranslated regions (UTRs) of both catalytic (GCLc) and regulatory (GCLm) subunits of GCL enabled an identification of miR-433 as a strong candidate for the targeting of GCL. Transitory overexpression of miR-433 in human umbilical vein endothelial cells (HUVEC) showed a downregulation of both GCLc and GCLm in a nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-independent manner. Increases in pro-oxidant stimuli such as exposure to hydrogen peroxide or GSH depletion in endothelial and hepatic cells caused an expected increase in GCLc and GCLm protein expression and abrogation of miR-433 levels, thus supporting a cross-regulation of these pathways. Treatment of HUVEC with miR-433 resulted in reduced antioxidant and redox potentials, increased S-glutathionylation, and reduced endothelial nitric oxide synthase activation. In vivo models of renal and hepatic fibrosis were associated with transforming growth factor β1 (TGF-β1)-related reduction of GCLc and GCLm levels that were miR-433 dependent.

Innovation And Conclusion: We describe for the first time an miRNA, miR-433, capable of directly targeting GCL and promoting functional consequences in endothelial physiology and fibrotic processes by decreasing GSH levels.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/ars.2014.6025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4657521PMC
November 2015

Regulation of oxidative stress by methylation-controlled J protein controls macrophage responses to inflammatory insults.

J Infect Dis 2015 Jan 14;211(1):135-45. Epub 2014 Jul 14.

Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst Proteomics Unit Ikerbasque, Basque Foundation for Science, Bilbao, Spain.

Mitochondria contribute to macrophage immune function through the generation of reactive oxygen species, a byproduct of the mitochondrial respiratory chain. MCJ (also known as DnaJC15) is a mitochondrial inner membrane protein identified as an endogenous inhibitor of respiratory chain complex I. Here we show that MCJ is essential for the production of tumor necrosis factor by macrophages in response to a variety of Toll-like receptor ligands and bacteria, without affecting their phagocytic activity. Loss of MCJ in macrophages results in increased mitochondrial respiration and elevated basal levels of reactive oxygen species that cause activation of the JNK/c-Jun pathway, lead to the upregulation of the TACE (also known as ADAM17) inhibitor TIMP-3, and lead to the inhibition of tumor necrosis factor shedding from the plasma membrane. Consequently, MCJ-deficient mice are resistant to the development of fulminant liver injury upon lipopolysaccharide administration. Thus, attenuation of the mitochondrial respiratory chain by MCJ in macrophages exquisitely regulates the response of macrophages to infectious insults.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/infdis/jiu389DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4326314PMC
January 2015

Ubiquitin profiling in liver using a transgenic mouse with biotinylated ubiquitin.

J Proteome Res 2014 Jun 30;13(6):3016-26. Epub 2014 Apr 30.

CIC bioGUNE, Bizkaia Teknologia Parkea , Building 801-A, 48160 Derio, Basque Country, Spain.

Ubiquitination is behind most cellular processes, with ubiquitin substrates being regulated variously according to the number of covalently conjugated ubiquitin molecules and type of chain formed. Here we report the first mammalian system for ubiquitin proteomics allowing direct validation of the MS-identified proteins. We created a transgenic mouse expressing biotinylated ubiquitin and demonstrate its use for the isolation of ubiquitinated proteins from liver and other tissues. The specificity and strength of the biotin-avidin interaction allow very stringent washes, so only proteins conjugated to ubiquitin are isolated. In contrast with recently available antibody-based approaches, our strategy allows direct validation by immunoblotting, therefore revealing the type of ubiquitin chains (mono or poly) formed in vivo. We also identify the conjugating E2 enzymes that are ubiquitin-loaded in the mouse tissue. Furthermore, our strategy allows the identification of candidate cysteine-ubiquitinated proteins, providing a strategy to identify those on a proteomic scale. The novel in vivo system described here allows broad access to tissue-specific ubiquitomes and can be combined with established mouse disease models to investigate ubiquitin-dependent therapeutical approaches.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/pr5001913DOI Listing
June 2014

SIRT1 controls liver regeneration by regulating bile acid metabolism through farnesoid X receptor and mammalian target of rapamycin signaling.

Hepatology 2014 May 31;59(5):1972-83. Epub 2014 Mar 31.

Department of Metabolomics, CIC bioGUNE, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (Ciberehd), Technology Park of Bizkaia, Bizkaia, Spain.

Unlabelled: Sirtuin1 (SIRT1) regulates central metabolic functions such as lipogenesis, protein synthesis, gluconeogenesis, and bile acid (BA) homeostasis through deacetylation. Here we describe that SIRT1 tightly controls the regenerative response of the liver. We performed partial hepatectomy (PH) to transgenic mice that overexpress SIRT1 (SIRT). SIRT mice showed increased mortality, impaired hepatocyte proliferation, BA accumulation, and profuse liver injury after surgery. The damaging phenotype in SIRT mice correlated with impaired farnesoid X receptor (FXR) activity due to persistent deacetylation and lower protein expression that led to decreased FXR-target gene expression; small heterodimer partner (SHP), bile salt export pump (BSEP), and increased Cyp7A1. Next, we show that 24-norUrsodeoxycholic acid (NorUDCA) attenuates SIRT protein expression, increases the acetylation of FXR and neighboring histones, restores trimethylation of H3K4 and H3K9, and increases miR34a expression, thus reestablishing BA homeostasis. Consequently, NorUDCA restored liver regeneration in SIRT mice, which showed increased survival and hepatocyte proliferation. Furthermore, a leucine-enriched diet restored mammalian target of rapamycin (mTOR) activation, acetylation of FXR and histones, leading to an overall lower BA production through SHP-inhibition of Cyp7A1 and higher transport (BSEP) and detoxification (Sult2a1) leading to an improved liver regeneration. Finally, we found that human hepatocellular carcinoma (HCC) samples have increased presence of SIRT1, which correlated with the absence of FXR, suggesting its oncogenic potential.

Conclusion: We define SIRT1 as a key regulator of the regenerative response in the liver through posttranscriptional modifications that regulate the activity of FXR, histones, and mTOR. Moreover, our data suggest that SIRT1 contributes to liver tumorigenesis through dysregulation of BA homeostasis by persistent FXR deacetylation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/hep.26971DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3999184PMC
May 2014

Excess S-adenosylmethionine reroutes phosphatidylethanolamine towards phosphatidylcholine and triglyceride synthesis.

Hepatology 2013 Oct 14;58(4):1296-305. Epub 2013 Aug 14.

Department of Physiology, University of the Basque Country UPV/EHU, Medical School, Bilbao, Spain.

Unlabelled: Methionine adenosyltransferase 1A (MAT1A) and glycine N-methyltransferase (GNMT) are the primary genes involved in hepatic S-adenosylmethionine (SAMe) synthesis and degradation, respectively. Mat1a ablation in mice induces a decrease in hepatic SAMe, activation of lipogenesis, inhibition of triglyceride (TG) release, and steatosis. Gnmt-deficient mice, despite showing a large increase in hepatic SAMe, also develop steatosis. We hypothesized that as an adaptive response to hepatic SAMe accumulation, phosphatidylcholine (PC) synthesis by way of the phosphatidylethanolamine (PE) N-methyltransferase (PEMT) pathway is stimulated in Gnmt(-/-) mice. We also propose that the excess PC thus generated is catabolized, leading to TG synthesis and steatosis by way of diglyceride (DG) generation. We observed that Gnmt(-/-) mice present with normal hepatic lipogenesis and increased TG release. We also observed that the flux from PE to PC is stimulated in the liver of Gnmt(-/-) mice and that this results in a reduction in PE content and a marked increase in DG and TG. Conversely, reduction of hepatic SAMe following the administration of a methionine-deficient diet reverted the flux from PE to PC of Gnmt(-/-) mice to that of wildtype animals and normalized DG and TG content preventing the development of steatosis. Gnmt(-/-) mice with an additional deletion of perilipin2, the predominant lipid droplet protein, maintain high SAMe levels, with a concurrent increased flux from PE to PC, but do not develop liver steatosis.

Conclusion: These findings indicate that excess SAMe reroutes PE towards PC and TG synthesis and lipid sequestration.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/hep.26399DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3720726PMC
October 2013

NF-κB essential modifier is required for hepatocyte proliferation and the oval cell reaction after partial hepatectomy in mice.

Gastroenterology 2012 Dec 23;143(6):1597-1608.e11. Epub 2012 Aug 23.

Department of Internal Medicine, University Hospital (RWTH), Aachen, Germany.

Background & Aims: The transcription factor nuclear factor κB (NF-κB) is activated by the IκB kinase complex. The regulatory subunit of this complex, NF-κB essential modifier (NEMO or IKBKG), is a tumor suppressor. Hepatocyte-specific deletion of NEMO induces chronic liver inflammation that leads to apoptosis, oxidative stress, development of nonalcoholic steatohepatitis, and hepatocarcinogenesis.

Methods: We performed partial hepatectomies in mice with hepatocyte-specific disruption of NEMO (Nemo(Δhepa)). Some mice were fed a diet that contained the antioxidant butylated hydroxyanisole (BHA), and others were given daily intraperitoneal injections of the oxidant phenetyl isothiocyanate (PEITC).

Results: Nemo(Δhepa) mice had impaired liver regeneration after partial hepatectomy and 50% mortality, indicating that NEMO is required for the regenerative response. Liver cells of the mice had a strong oxidative stress response; these cells down-regulated the NF-κB-dependent antioxidant response and reduced levels of proteins that repair DNA double-strand breaks. However, the impairments to hepatocyte proliferation were compensated by a response of oval cells in Nemo(Δhepa) mice. Oval cells expressed low levels of albumin and thereby expressed normal levels of NEMO. Repopulation of the liver with oval cells that expressed NEMO reversed liver damage in Nemo(Δhepa) mice. Interestingly, these mice still developed hepatocellular carcinomas 6 months after partial hepatectomy, whereas Nemo(Δhepa) mice fed the BHA diet were protected from carcinogenesis.

Conclusions: In livers of mice, expression of NEMO and activation of NF-κB are required for hepatocyte proliferation and liver regeneration. These mechanisms require control of oxidative stress and DNA integrity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1053/j.gastro.2012.08.030DOI Listing
December 2012

Hepatoma cells from mice deficient in glycine N-methyltransferase have increased RAS signaling and activation of liver kinase B1.

Gastroenterology 2012 Sep 8;143(3):787-798.e13. Epub 2012 Jun 8.

CIC bioGUNE, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas, Technology Park of Bizkaia, Bizkaia, Spain.

Background & Aims: Patients with cirrhosis are at high risk for developing hepatocellular carcinoma (HCC), and their liver tissues have abnormal levels of S-adenosylmethionine (SAMe). Glycine N-methyltransferase (GNMT) catabolizes SAMe, but its expression is down-regulated in HCC cells. Mice that lack GNMT develop fibrosis and hepatomas and have alterations in signaling pathways involved in carcinogenesis. We investigated the role of GNMT in human HCC cell lines and in liver carcinogenesis in mice.

Methods: We studied hepatoma cells from GNMT knockout mice and analyzed the roles of liver kinase B1 (LKB1, STK11) signaling via 5'-adenosine monophosphate-activated protein kinase (AMPK) and Ras in regulating proliferation and transformation.

Results: Hepatoma cells from GNMT mice had defects in LKB1 signaling to AMPK, making them resistant to induction of apoptosis by adenosine 3',5'-cyclic monophosphate activation of protein kinase A and calcium/calmodulin-dependent protein kinase kinase 2. Ras-mediated hyperactivation of LKB1 promoted proliferation of GNMT-deficient hepatoma cells and required mitogen-activated protein kinase 2 (ERK) and ribosomal protein S6 kinase polypeptide 2 (p90RSK). Ras activation of LKB1 required expression of RAS guanyl releasing protein 3 (RASGRP3). Reduced levels of GNMT and phosphorylation of AMPKα at Thr172 and increased levels of Ras, LKB1, and RASGRP3 in HCC samples from patients were associated with shorter survival times.

Conclusions: Reduced expression of GNMT in mouse hepatoma cells and human HCC cells appears to increase activity of LKB1 and RAS; activation of RAS signaling to LKB1 and RASGRP3, via ERK and p90RSK, might be involved in liver carcinogenesis and be used as a prognostic marker. Reagents that disrupt this pathway might be developed to treat patients with HCC.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1053/j.gastro.2012.05.050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3429651PMC
September 2012

Human antigen R contributes to hepatic stellate cell activation and liver fibrosis.

Hepatology 2012 Nov 9;56(5):1870-82. Epub 2012 Oct 9.

Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas, Technology Park of Bizkaia, Derio, Bizkaia, Spain.

Unlabelled: RNA-binding proteins (RBPs) play a major role in the control of messenger RNA (mRNA) turnover and translation rates. We examined the role of the RBP, human antigen R (HuR), during cholestatic liver injury and hepatic stellate cell (HSC) activation. HuR silencing attenuated fibrosis development in vivo after BDL, reducing liver damage, oxidative stress, inflammation, and collagen and alpha smooth muscle actin (α-SMA) expression. HuR expression increased in activated HSCs from bile duct ligation mice and during HSC activation in vitro, and HuR silencing markedly reduced HSC activation. HuR regulated platelet-derived growth factor (PDGF)-induced proliferation and migration and controlled the expression of several mRNAs involved in these processes (e.g., Actin, matrix metalloproteinase 9, and cyclin D1 and B1). These functions of HuR were linked to its abundance and cytoplasmic localization, controlled by PDGF, by extracellular signal-regulated kinases (ERK) and phosphatidylinositol 3-kinase activation as well as ERK/LKB1 (liver kinase B1) activation, respectively. More important, we identified the tumor suppressor, LKB1, as a novel downstream target of PDGF-induced ERK activation in HSCs. HuR also controlled transforming growth factor beta (TGF-β)-induced profibrogenic actions by regulating the expression of TGF-β, α-SMA, and p21. This was likely the result of an increased cytoplasmic localization of HuR, controlled by TGF-β-induced p38 mitogen-activated protein kinase activation. Finally, we found that HuR and LKB1 (Ser428) levels were highly expressed in activated HSCs in human cirrhotic samples.

Conclusion: Our results show that HuR is important for the pathogenesis of liver fibrosis development in the cholestatic injury model, for HSC activation, and for the response of activated HSC to PDGF and TGF-β.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/hep.25828DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3433583PMC
November 2012

Inhibition of natural killer cells protects the liver against acute injury in the absence of glycine N-methyltransferase.

Hepatology 2012 Aug 6;56(2):747-59. Epub 2012 Jul 6.

Department of Metabolomics, CIC bioGUNE, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (Ciberedh), Technology Park of Bizkaia, 48160-Derio, Bizkaia, Spain.

Unlabelled: Glycine N-methyltransferase (GNMT) catabolizes S-adenosylmethionine (SAMe), the main methyl donor of the body. Patients with cirrhosis show attenuated GNMT expression, which is absent in hepatocellular carcinoma (HCC) samples. GNMT(-/-) mice develop spontaneous steatosis that progresses to steatohepatitis, cirrhosis, and HCC. The liver is highly enriched with innate immune cells and plays a key role in the body's host defense and in the regulation of inflammation. Chronic inflammation is the major hallmark of nonalcoholic steatohepatitis (NASH) progression. The aim of our study was to uncover the molecular mechanisms leading to liver chronic inflammation in the absence of GNMT, focusing on the implication of natural killer (NK) / natural killer T (NKT) cells. We found increased expression of T helper (Th)1- over Th2-related cytokines, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-R2/DR5, and several ligands of NK cells in GNMT(-/-) livers. Interestingly, NK cells from GNMT(-/-) mice were spontaneously activated, expressed more TRAIL, and had strong cytotoxic activity, suggesting their contribution to the proinflammatory environment in the liver. Accordingly, NK cells mediated hypersensitivity to concanavalin A (ConA)-mediated hepatitis in GNMT(-/-) mice. Moreover, GNMT(-/-) mice were hypersensitive to endotoxin-mediated liver injury. NK cell depletion and adoptive transfer of TRAIL(-/-) liver-NK cells protected the liver against lipopolysaccharide (LPS) liver damage.

Conclusion: Our data allow us to conclude that TRAIL-producing NK cells actively contribute to promote a proinflammatory environment at early stages of fatty liver disease, suggesting that this cell compartment may contribute to the progression of NASH.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/hep.25694DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3378767PMC
August 2012

Murine double minute 2 regulates Hu antigen R stability in human liver and colon cancer through NEDDylation.

Hepatology 2012 Apr 1;55(4):1237-48. Epub 2012 Mar 1.

CIC bioGUNE, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (Ciberehd), Derio, Spain.

Unlabelled: Hu antigen R (HuR) is a central RNA-binding protein regulating cell dedifferentiation, proliferation, and survival, which are well-established hallmarks of cancer. HuR is frequently overexpressed in tumors correlating with tumor malignancy, which is in line with a role for HuR in tumorigenesis. However, the precise mechanism leading to changes in HuR expression remains unclear. In the liver, HuR plays a crucial role in hepatocyte proliferation, differentiation, and transformation. Here, we unraveled a novel mean of regulation of HuR expression in hepatocellular carcinoma (HCC) and colon cancer. HuR levels correlate with the abundance of the oncogene, murine double minute 2 (Mdm2), in human HCC and colon cancer metastases. HuR is stabilized by Mdm2-mediated NEDDylation in at least three lysine residues, ensuring its nuclear localization and protection from degradation.

Conclusion: This novel Mdm2/NEDD8/HuR regulatory framework is essential for the malignant transformation of tumor cells, which, in turn, unveils a novel signaling paradigm that is pharmacologically amenable for cancer therapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/hep.24795DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3298572PMC
April 2012

Loss of caspase-8 protects mice against inflammation-related hepatocarcinogenesis but induces non-apoptotic liver injury.

Gastroenterology 2011 Dec 28;141(6):2176-87. Epub 2011 Aug 28.

Department of Medicine III, University Hospital, Aachen, Germany.

Background & Aims: Disruption of the nuclear factor-κB (NF-κB) essential modulator (NEMO) in hepatocytes of mice (NEMO(Δhepa) mice) results in spontaneous liver apoptosis and chronic liver disease involving inflammation, steatosis, fibrosis, and development of hepatocellular carcinoma. Activation of caspase-8 (Casp8) initiates death receptor-mediated apoptosis. We investigated the pathogenic role of this protease in NEMO(Δhepa) mice or after induction of acute liver injury.

Methods: We created mice with conditional deletion of Casp8 in hepatocytes (Casp8(Δhepa)) and Casp8(Δhepa)NEMO(Δhepa) double knockout mice. Acute liver injury was induced by Fas-activating antibodies, lipopolysaccharides, or concanavalin A. Spontaneous hepatocarcinogenesis was monitored by magnetic resonance imaging.

Results: Hepatocyte-specific deletion of Casp8 protected mice from induction of apoptosis and liver injury by Fas or lipopolysaccharides but increased necrotic damage and reduced survival times of mice given concanavalin A. Casp8(Δhepa)NEMO(Δhepa) mice were protected against steatosis and hepatocarcinogenesis but had a separate, spontaneous phenotype that included massive liver necrosis, cholestasis, and biliary lesions. The common mechanism by which inactivation of Casp8 induces liver necrosis in both injury models involves the formation of protein complexes that included the adaptor protein Fas-associated protein with death domain and the kinases receptor-interacting protein (RIP) 1 and RIP3-these have been shown to be required for programmed necrosis. We demonstrated that hepatic RIP1 was proteolytically cleaved by Casp8, whereas Casp8 inhibition resulted in accumulation of RIP complexes and subsequent liver necrosis.

Conclusions: Inhibition of Casp8 protects mice from hepatocarcinogenesis following chronic liver injury mediated by apoptosis of hepatocytes but can activate RIP-mediated necrosis in an inflammatory environment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1053/j.gastro.2011.08.037DOI Listing
December 2011

Role of AMP-activated protein kinase in the control of hepatocyte priming and proliferation during liver regeneration.

Exp Biol Med (Maywood) 2011 Apr 22;236(4):402-8. Epub 2011 Mar 22.

CIC bioGUNE, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (Ciberehd), Technology Park of Bizkaia, 48160-Derio, Bizkaia, Spain.

The enzyme AMP-activated protein kinase (AMPK) is the main energy sensor in cells and is responsible for controlling the balance of anabolic/catabolic processes under metabolic stress conditions. This metabolic control exerted by AMPK is critical for energy-demanding situations, such as liver regeneration. Immediately after partial hepatectomy (PH), the liver undergoes the priming phase, mediated by the proinflammatory cytokines tumor necrosis factor (TNF) and interleukin-6, which promote responsiveness of hepatocytes to growth factors, such as hepatocyte growth factor (HGF) and epidermal growth factor, which lead to proliferation. In addition to its metabolic function, AMPK is likely to be a key mediator in both hepatocyte priming and the proliferative phases, induced by TNF-α and HGF, respectively. TNF-α-induced AMPK activation has been shown to be necessary for nuclear factor κappa B (NF-κB)-induced inducible nitric oxide synthase expression and for blocking TNF-α-induced apoptosis. On the other hand, HGF-induced LKB1/AMPK activation has been found to play a critical role in controlling Hu antigen R cytosolic localization and endothelial nitric oxide synthase activation, and consequently Cyclin D1 and Cyclin A expressions, and nitric oxide generation, respectively. During PH, levels of S-adenosylmethionine (SAMe), the principal methyl donor in the liver, have to decrease to allow liver proliferation. Our studies also show that SAMe inhibits hepatocyte proliferation by controlling the hepatocyte's responsiveness to mitogenic signals such as HGF through the inhibition of AMPK activity. In summary, these data highlight the essential role of AMPK in controlling the balance between hepatocyte metabolic adaptations, cell cycle progression and apoptosis during liver regeneration.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1258/ebm.2011.010352DOI Listing
April 2011