Hepatitis C and Alcohol Exacerbate Liver Injury by Suppression of FOXO3
2013; Elsevier BV; Volume: 183; Issue: 6 Linguagem: Inglês
10.1016/j.ajpath.2013.08.013
ISSN1525-2191
AutoresBatbayar Tumurbaatar, Irina Tikhanovich, Zhuan Li, Jinyu Ren, Robert Ralston, Sudhakiranmayi Kuravi, Roosevelt V. Campbell, Gaurav Chaturvedi, Ting‐Ting Huang, Jie Zhao, Junfang Hao, Maura O’Neil, Steven A. Weinman,
Tópico(s)Liver Disease Diagnosis and Treatment
ResumoHepatitis C virus (HCV) infection exacerbates alcoholic liver injury by mechanisms that include enhanced oxidative stress. The forkhead box transcription factor FOXO3 is an important component of the antioxidant stress response that can be altered by HCV. To test whether FOXO3 is protective for alcoholic liver injury, we fed alcohol to FOXO3−/− mice. After 3 weeks, one third of these mice developed severe hepatic steatosis, neutrophilic infiltration, and >10-fold alanine aminotransferase (ALT) elevations. In cell culture, either alcohol or HCV infection alone increased FOXO3 transcriptional activity and expression of target genes, but the combination of HCV and alcohol together caused loss of nuclear FOXO3 and decreased its transcriptional activity. This was accompanied by increased phosphorylation of FOXO3. Mice expressing HCV structural proteins on a background of reduced expression of superoxide dismutase 2 (SOD2; Sod2+/−) also had increased liver sensitivity to alcohol, with elevated ALT, steatosis, and lobular inflammation. Elevated ALT was associated with an alcohol-induced decrease in SOD2 and redistribution of FOXO3 to the cytosol. These results demonstrate that FOXO3 functions as a protective factor preventing alcoholic liver injury. The combination of HCV and alcohol, but not either condition alone, inactivates FOXO3, causing a decrease in expression of its target genes and an increase in liver injury. Modulation of the FOXO3 pathway is a potential therapeutic approach for HCV-alcohol–induced liver injury. Hepatitis C virus (HCV) infection exacerbates alcoholic liver injury by mechanisms that include enhanced oxidative stress. The forkhead box transcription factor FOXO3 is an important component of the antioxidant stress response that can be altered by HCV. To test whether FOXO3 is protective for alcoholic liver injury, we fed alcohol to FOXO3−/− mice. After 3 weeks, one third of these mice developed severe hepatic steatosis, neutrophilic infiltration, and >10-fold alanine aminotransferase (ALT) elevations. In cell culture, either alcohol or HCV infection alone increased FOXO3 transcriptional activity and expression of target genes, but the combination of HCV and alcohol together caused loss of nuclear FOXO3 and decreased its transcriptional activity. This was accompanied by increased phosphorylation of FOXO3. Mice expressing HCV structural proteins on a background of reduced expression of superoxide dismutase 2 (SOD2; Sod2+/−) also had increased liver sensitivity to alcohol, with elevated ALT, steatosis, and lobular inflammation. Elevated ALT was associated with an alcohol-induced decrease in SOD2 and redistribution of FOXO3 to the cytosol. These results demonstrate that FOXO3 functions as a protective factor preventing alcoholic liver injury. The combination of HCV and alcohol, but not either condition alone, inactivates FOXO3, causing a decrease in expression of its target genes and an increase in liver injury. Modulation of the FOXO3 pathway is a potential therapeutic approach for HCV-alcohol–induced liver injury. Alcohol consumption has long been recognized as an important risk factor for more rapidly progressive liver disease in patients with chronic hepatitis C infection.1Yilmaz Y. Dolar E. Ulukaya E. Akgoz S. Keskin M. Kiyici M. Yerci O. Oral A.Y. Gul C.B. Gurel S. Nak S.G. Gulten M. Elevated serum levels of caspase-cleaved cytokeratin 18 (CK18-Asp396) in patients with nonalcoholic steatohepatitis and chronic hepatitis C.Med Sci Monit. 2009; 15: CR189-CR193PubMed Google Scholar Chronic hepatitis C virus (HCV) infection also predisposes to more severe outcomes of drug-induced liver disease2Nguyen G.C. Sam J. Thuluvath P.J. Hepatitis C is a predictor of acute liver injury among hospitalizations for acetaminophen overdose in the United States: a nationwide analysis.Hepatology. 2008; 48: 1336-1341Crossref PubMed Scopus (89) Google Scholar and alcoholic hepatitis.3Singal A.K. Sagi S. Kuo Y.F. Weinman S. Impact of hepatitis C virus infection on the course and outcome of patients with acute alcoholic hepatitis.Eur J Gastroenterol Hepatol. 2011; 23: 204-209Crossref PubMed Scopus (25) Google Scholar Although no single mechanism has been shown to be responsible for this synergy, several potential mechanisms have been identified. These include effects on mitochondrial reactive oxygen species production and cell death pathways, endoplasmic reticulum stress, activation of mitogen-activated protein kinases, innate immune responses, dendritic cell function, and proteasome inhibition, and direct effects of alcohol on viral replication pathways.4Seronello S. Ito C. Wakita T. Choi J. 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Hepatitis C virus core protein, cytochrome P450 2E1, and alcohol produce combined mitochondrial injury and cytotoxicity in hepatoma cells.Gastroenterology. 2005; 128: 96-107Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar Several mechanisms normally protect the liver from alcoholic injury, and these include antioxidant enzymes, such as superoxide dismutase8Wheeler M.D. Nakagami M. Bradford B.U. Uesugi T. Mason R.P. Connor H.D. Dikalova A. Kadiiska M. Thurman R.G. Overexpression of manganese superoxide dismutase prevents alcohol-induced liver injury in the rat.J Biol Chem. 2001; 276: 36664-36672Crossref PubMed Scopus (186) Google Scholar and thioredoxin.9Cohen J.I. Roychowdhury S. DiBello P.M. Jacobsen D.W. Nagy L.E. Exogenous thioredoxin prevents ethanol-induced oxidative damage and apoptosis in mouse liver.Hepatology. 2009; 49: 1709-1717Crossref PubMed Scopus (70) Google Scholar HCV can impair antioxidant defenses by decreasing activation of nuclear factor erythroid 2-related factor 2 (Nrf-2)10Carvajal-Yepes M. Himmelsbach K. Schaedler S. Ploen D. Krause J. Ludwig L. Weiss T. Klingel K. Hildt E. Hepatitis C virus impairs the induction of cytoprotective Nrf2 target genes by delocalization of small Maf proteins.J Biol Chem. 2011; 286: 8941-8951Crossref PubMed Scopus (88) Google Scholar and heme oxygenase-1,11Wen F. Brown K.E. Britigan B.E. Schmidt W.N. Hepatitis C core protein inhibits induction of heme oxygenase-1 and sensitizes hepatocytes to cytotoxicity.Cell Biol Toxicol. 2008; 24: 175-188Crossref PubMed Scopus (30) Google Scholar two important components of the antioxidant response, and these have been postulated to play a role in ethanol-HCV synergy.11Wen F. Brown K.E. Britigan B.E. Schmidt W.N. Hepatitis C core protein inhibits induction of heme oxygenase-1 and sensitizes hepatocytes to cytotoxicity.Cell Biol Toxicol. 2008; 24: 175-188Crossref PubMed Scopus (30) Google Scholar Understanding the mechanisms by which these occur could, therefore, lead to novel prophylactic and therapeutic approaches to alcoholic liver disease. There is emerging evidence that the FOXO transcription factor family plays a critical role in hepatic metabolic, antioxidant, and cell death responses.12Tikhanovich I. Cox J. Weinman S. FOXO transcription factors in liver function and disease.J Gastroenterol Hepatol. 2013; 28: 125-131Crossref PubMed Scopus (122) Google Scholar The O branch of the forkhead box superfamily consists of four mammalian proteins, FOXO1, FOXO3, FOXO4, and FOXO6, and controls expression of genes regulating antioxidant response, cell cycle arrest, gluconeogenesis, apoptosis, and autophagy.13Calnan D.R. Brunet A. The FoxO code.Oncogene. 2008; 27: 2276-2288Crossref PubMed Scopus (917) Google Scholar, 14Burgering B.M. A brief introduction to FOXOlogy.Oncogene. 2008; 27: 2258-2262Crossref PubMed Scopus (166) Google Scholar FOXO3, in particular, is activated by oxidative stress and plays a role in longevity, cell cycle arrest, protection from oxidative stress, induction of apoptosis, and tumor suppression.13Calnan D.R. Brunet A. The FoxO code.Oncogene. 2008; 27: 2276-2288Crossref PubMed Scopus (917) Google Scholar, 15van der Horst A. Burgering B.M. Stressing the role of FoxO proteins in lifespan and disease.Nat Rev Mol Cell Biol. 2007; 8: 440-450Crossref PubMed Scopus (587) Google Scholar, 16Huang H. Tindall D.J. Dynamic FoxO transcription factors.J Cell Sci. 2007; 120: 2479-2487Crossref PubMed Scopus (878) Google Scholar It is a direct transcriptional activator of the antioxidant proteins superoxide dismutase 2 (SOD2), catalase, and peroxiredoxin III.17Miao L. St Clair D.K. Regulation of superoxide dismutase genes: implications in disease.Free Radic Biol Med. 2009; 47: 344-356Crossref PubMed Scopus (569) Google Scholar FOXO3 transcriptional activity is regulated by a complex pattern of post-translational modifications that regulate both the nuclear-cytosolic distribution and affinity for specific transcriptional sites. The best understood of these is phosphorylation by Akt, considered to be an inactivating modification, which causes nuclear export of the protein, but other modifications, such as alternative phosphorylation sites, acetylation, ubiquitination, and methylation, also regulate protein function.18Zhao Y. Wang Y. Zhu W.G. Applications of post-translational modifications of FoxO family proteins in biological functions.J Mol Cell Biol. 2011; 3: 276-282Crossref PubMed Scopus (144) Google Scholar The role of FOXO in injury processes is complex because FOXO transcriptional programs can be either cytoprotective or cytotoxic, and well-documented examples of both phenomena are numerous.19Hedrick S.M. The cunning little vixen: foxo and the cycle of life and death.Nat Immunol. 2009; 10: 1057-1063Crossref PubMed Scopus (127) Google Scholar There have been a limited number of reports of the effect of HCV infection on FOXO activity, and these have shown that activation of FOXO1 or FOXO3 by HCV may contribute to insulin resistance20Deng L. Shoji I. Ogawa W. Kaneda S. Soga T. Jiang D.P. Ide Y.H. Hotta H. Hepatitis C virus infection promotes hepatic gluconeogenesis through an NS5A-mediated FoxO1-dependent pathway.J Virol. 2011; 85: 8556-8568Crossref PubMed Scopus (76) Google Scholar and autophagy induction.21Honda M. Takehana K. Sakai A. Tagata Y. Shirasaki T. Nishitani S. Muramatsu T. Yamashita T. Nakamoto Y. Mizukoshi E. Sakai Y. Yamashita T. Nakamura M. Shimakami T. Yi M. Lemon S.M. Suzuki T. Wakita T. Kaneko S. Hokuriku Liver Study GroupMalnutrition impairs interferon signaling through mTOR and FoxO pathways in patients with chronic hepatitis C.Gastroenterology. 2011; 141 (140.e1–2): 128-140Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar FOXO3 has also recently been shown to be part of a proapoptotic pathway in human peripheral blood cells.22Kopycinska J. Kempinska-Podhorodecka A. Haas T. Elias E. Depinho R.A. Paik J. Milkiewicz P. Milkiewicz M. Activation of FoxO3a/Bim axis in patients with primary biliary cirrhosis.Liver Int. 2013; 33: 231-238Crossref PubMed Scopus (20) Google Scholar Whether FOXOs play a role in protection against alcoholic liver injury and whether HCV effects on FOXO3 are part of the liver synergy have not been examined. In this study, we used FOXO3 knockout mice to determine whether this transcription factor is required for hepatic protection from alcohol and further examined the effects of HCV and alcohol separately and in combination on FOXO3 activity. The results indicate that FOXO3 is a novel alcohol protection factor that is disrupted by the HCV-alcohol combination. Huh7.5 cells [obtained from Dr. Charles Rice (Rockefeller University, New York, NY)] were maintained in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum, 50 U/mL penicillin, and 50 mg/mL streptomycin. DNA coding for the Japanese fulminant hepatitis 1 (JFH1) sequence was obtained from Dr. Takaji Wakita (National Institute of Infectious Diseases, Tokyo, Japan). Plasmid was propagated and reverse transcribed, and the resulting RNA was used to transfect (by electroporation) Huh7.5 cells for the production of intact viral particles, as described.23Kato T. Date T. Murayama A. Morikawa K. Akazawa D. Wakita T. Cell culture and infection system for hepatitis C virus.Nat Protoc. 2006; 1: 2334-2339Crossref PubMed Scopus (155) Google Scholar 3 × 105 Huh7.5 cells were seeded onto T-25 flasks and infected the following day with HCV at a multiplicity of infection of 0.5-1.0. At 48 hours after infection, control and infected cells were seeded onto 96-well plates or 8-well chamber slides and treated as described. pECE-HA-FOXO3 expression plasmid was provided by Michael Greenberg (Harvard Medical School, Boston, MA) via Addgene (Cambridge, MA). pMirTarget reporter construct with a 3′-untranslated region (UTR) of SOD2 gene (transcript variant 1) downstream of firefly luciferase was from OriGene (Rockville, MD). Cells grown on coverslips were washed in PBS three times and fixed for 10 minutes in 4% paraformaldehyde in PBS at room temperature. For indirect immunofluorescence, fixed cells were permeabilized in 1% Triton X-100 in PBS for 5 minutes at room temperature. The coverslips were inverted and touched to 40-μL droplets of blocking buffer [4% goat serum and 1% bovine serum albumin in PBS-Tween (0.05%) on a clean parafilm sheet for 45 minutes at room temperature]. Cells were then incubated in PBS with mouse anti-HCV core antibody (MA1-080; ThermoScientific, Pittsburgh, PA) and rabbit anti-FOXO3 antibody (75D8; Cell Signaling, Danvers, MA) for 1 hour at room temperature. After washing with PBS, coverslips were incubated with Alexa Fluor 568–conjugated goat anti-mouse IgG (1:1000) and Alexa Fluor 488–conjugated goat anti-rabbit IgG (1:1000; Molecular Probes, Carlsbad, CA) for 1 hour in the dark at room temperature. Coverslips were additionally incubated with DAPI for 20 minutes at room temperature to stain nuclear DNA. Images were acquired using a 40× oil objective on an Eclipse Ti microscope (Nikon Instruments Inc., Melville, NY) equipped with a CoolSNAP HQ2Nguyen G.C. Sam J. Thuluvath P.J. Hepatitis C is a predictor of acute liver injury among hospitalizations for acetaminophen overdose in the United States: a nationwide analysis.Hepatology. 2008; 48: 1336-1341Crossref PubMed Scopus (89) Google Scholar charge-coupled device camera and MetaMorph version 7.1.1.0 software (Molecular Devices, Sunnyvale, CA). Image analysis was performed using CellProfiler version 2.0 release 11052 (Broad Institute of MIT and Harvard, Cambridge, MA, http://www.cellprofiler.org) run on Linux kernel 3.0.29 in Linuxmint version 13 operating system, using a quad core Intel i5 processor (Intel Corp., Santa Clara, CA) with 8 GB ram. Post-processing and statistical analysis were done with R version 3.01 (http://www.r-project.org) using packages Cairo, coin, ggplot2, grid, plyr, and psych. The CellProfiler pipeline and R scripts used for the analysis are available from author R.R. Briefly, DAPI channel images were used to segment nucleus objects and fluorescein isothiocyanate channel images were used to measure FOXO3 staining intensity for each pixel in each nucleus object. Fluorescein isothiocyanate channel images also were used to segment cell objects and cytoplasm objects. Texas Red channel objects were used to measure HCV core staining intensity in cytoplasm objects. Mean per-pixel FOXO3 staining per nucleus was calculated for each nucleus object to control for variation in nuclear area. The distributions of mean FOXO3 nuclear intensities were compared across treatment groups. For treatment groups HCV and HCV-ethanol, nuclear objects from HCV core-negative cells were excluded from the analysis. RNA was extracted from cultured hepatoma cells using the RNeasy Mini Kit (Qiagen, Venlo, the Netherlands). cDNA was generated with the random primer method using the RNA reverse transcription kit (catalogue no. 4368814; Applied Biosystems, Grand Island, NY). Quantitative real-time RT-PCR was performed in a CFX96 real-time system (Bio-Rad, Hercules, CA) using specific sense and antisense primers in a 25-μL reaction volume containing 12.5 μL of IQ SYBR Green supermix (Bio-Rad), 0.5 μL of 10 μmol/L primer stock, 8 μL of cDNA (1:10 diluted), and 0.5 μL double-distilled H2O. The amplification consisted of an initial incubation of 95°C for 5 minutes (hot start), followed by 40 amplification cycles (15 seconds at 95°C, 30 seconds at 60°C, and 30 seconds at 72°C), and a final melting curve from 55°C to 95°C. Oligonucleotide primers were the following: glyceraldehyde-3-phosphate dehydrogenase, 5′-CAGCCTCAAGATCATCAGCA-3′ (sense) and 5′-GTCTTCTGGGTGGCAGTGAT-3′ (antisense); FOXO3, 5′-TCTACGAG TGGATGGTGCGTT-3′ (sense) and 5′-CGACTATGCAGTGACAGGTTGTG-3′ (antisense); Bcl-2-like protein 11 (Bim), 5′- CACATGAGCACATTTCCCTCT-3′ (sense) and 5′-AAGGCACAAAACCTGCAGTAA-3′ (antisense); SOD2, 5′-TTCAATAAGGAACGGGGACA-3′ (sense) and 5′-GTAAGCGTGCTCCCACACA-3′ (antisense); growth arrest and DNA damage-inducible protein GADD45 alpha (GADD45a), 5′-CGCCTGTGAGTGAGTGC-3′ (sense) and 5′-CTTATCCATCCTTTCGGTCTT-3′ (antisense); and SOD1, 5′-AAGATGACTTGGGCAAAGGT-3′ (sense) and 5′-AATCCCAATCACACCACAAG-3′ (antisense). Huh7.5 cells were cultured either without infection or for 4 days after JFH1 infection. Cells were then seeded onto 24-well plates. After 24 hours, cells were serum starved for 16 hours and then cotransfected using Lipofectamine-LTX (Invitrogen, Carlsbad, CA) with FHRE-luciferase reporter vector24Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor.Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5412) Google Scholar and pRL-tk vector (renilla luciferase reporter) for normalization of transfection efficiency. The pFHRE-luciferase plasmid was provided by M. Greenberg via Addgene. Cells were subsequently incubated for 24 hours before lysis and luciferase determination. For ethanol treatment, experimental cells were incubated with 50 mmol/L ethanol for 48 hours before lysis. Lysates were used to measure luciferase activity using the Dual luciferase assay kit (Promega, Madison, WI) on a single-tube Glomax 20/20 luminometer (Promega). Results were expressed as firefly luciferase/renilla luciferase activity. Proteins were extracted from liver tissue by homogenization using a glass tissue grinder (Kontes Duall 20; Kontes Glass Co, Vineland, NJ) in tissue lysis buffer containing 50 mmol/L Tris, pH 7.4, 50 mmol/L NaCl, 0.5% sodium deoxycholate, 1% SDS, 1% NP-40, and protease and phosphatase inhibitor mixture (Sigma-Aldrich, St. Louis, MO) on ice. Whole cell lysates were prepared from cells that had been washed and harvested by centrifugation in PBS, pH 7.5. Cell pellets were resuspended in radioimmunoprecipitation assay buffer that contained 50 mmol/L Tris, pH 7.5, 150 mmol/L NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1 mmol/L EDTA, and 1% protease and phosphatase inhibitors (Sigma-Aldrich). Lysates were centrifuged at 15,000 × g for 15 minutes; supernatants were collected, and protein concentration was measured using the Bio-Rad protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA). Cytosolic and nuclear proteins were prepared using NE-PER nuclear and cytoplasmic extraction reagents (Thermo Fisher Scientific, Rockford, IL), according to the manufacturer’s instructions. Protein extracts (15 μg) were subjected to 10% SDS-PAGE, electrophoretically transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA), and blocked in 3% bovine serum albumin and Tris-buffered saline (TBS)/0.1% Tween-20 (TBS-T) at room temperature for 1 hour. Primary antibodies used were anti-FOXO3 (75D8), anti–phospho-FOXO3 (Ser253), anti-pan Akt (11E7), anti–phospho-Akt (Ser473), anti-Bim, and rabbit anti-caspase 3 (D175) from Cell Signaling, anti-SOD2 (sc-30080), goat anti–intercellular adhesion molecule (ICAM) 1, and β-actin from Santa Cruz Biotechnology (Santa Cruz, CA), anti-HCV core from Affinity Bioreagents (Golden, CO), and rabbit anti-SOD2 and mouse anti–β-actin from Sigma-Aldrich. Secondary antibodies were from Southern Biotechnology Associates (Birmingham, AL). Immunoblots were detected using the Electrochemiluminescence Plus Western Blotting Detection System (Amersham Biosciences, Piscataway, NJ). Expression levels were evaluated by quantification of relative density of each band normalized to that of the corresponding β-actin band density using a digital imager, AlphaEase FC (α Innotech, San Leandro, CA), with NIH ImageJ software version 1.48c (NIH, Bethesda, MD). For protein half-life measurement, cells were treated for indicated times with 100 μg/mL cycloheximide before harvesting. FOXO3−/− mice were provided by Dr. Kana Miyamoto (Keio University, Tokyo, Japan) and were generated as described.25Miyamoto K. Araki K.Y. Naka K. Arai F. Takubo K. Yamazaki S. Matsuoka S. Miyamoto T. Ito K. Ohmura M. Chen C. Hosokawa K. Nakauchi H. Nakayama K. Nakayama K.I. Harada M. Motoyama N. Suda T. Hirao A. Foxo3a is essential for maintenance of the hematopoietic stem cell pool.Cell Stem Cell. 2007; 1: 101-112Abstract Full Text Full Text PDF PubMed Scopus (683) Google Scholar Heterozygotes were bred together, obtaining both knockout and wild-type (WT) littermates. They were used for alcohol feeding experiments at 3 to 6 months of age. HCV transgenic mice (SL-139 line) were generated on a C57BL/6J background, as previously described.26Korenaga M. Wang T. Li Y. Showalter L.A. Chan T. Sun J. Weinman S.A. Hepatitis C virus core protein inhibits mitochondrial electron transport and increases reactive oxygen species (ROS) production.J Biol Chem. 2005; 280: 37481-37488Crossref PubMed Scopus (347) Google Scholar These mice are a hemizygous model and express HCV structural proteins (core, E1, E2, and p7) in hepatocytes under control of the murine albumin enhancer/promoter. Sod2+/− mice on a C57BL/6J background have been previously described.27Van Remmen H. Salvador C. Yang H. Huang T.T. Epstein C.J. Richardson A. Characterization of the antioxidant status of the heterozygous manganese superoxide dismutase knockout mouse.Arch Biochem Biophys. 1999; 363: 91-97Crossref PubMed Scopus (141) Google Scholar They were crossed with the HCV transgenic mice to produce the HCV/Sod2+/− genotype. All mice were housed in a temperature-controlled, specific, pathogen-free environment with 12-hour light-dark cycles and fed regular mouse chow and water ad libitum. HCV/Sod2+/− mice were used for alcohol feeding at 6 to 9 months old. All animal handling procedures were approved by the Institutional Animal Care and Use Committees at the University of Texas Medical Branch (Galveston, TX) and the University of Kansas Medical Center (Kansas City, KS). Mice were fed either a high-fat liquid diet with 35% of the calories supplied by ethanol (product F1258; Bio-Serv Inc., Frenchtown, NJ) or a control diet with maltodextrin isocalorically substituted for ethanol (product F1259; Bio-Serv Inc.). No other food was provided, but they had free access to water. Mice were initially acclimated to the control liquid diet for 3 days, and then ethanol concentration was increased stepwise to 1.6%, 3.2%, 4.8%, and, finally, 6.4% (v/v) at 3-day intervals. This ethanol concentration was maintained to the end of a 3-week period. The duration of alcohol exposure was calculated from the first day of the 1.6% alcohol feeding solution. Control mice were pair fed the amount consumed the previous day by the alcohol-fed mice to prevent differences in caloric intake. At the end of the feeding period, mice were sacrificed and livers and venous blood were obtained. Serum was stored at −80°C. Liver samples were fixed in formalin for histological examination or snap frozen in liquid nitrogen and stored at −80°C. Liver tissue sections (5 μm thick) were prepared from paraffin-embedded samples. For analysis of histological features, H&E sections were evaluated by an experienced liver pathologist (M.O.) in a blinded manner and scored for percentage steatosis, severity of liver inflammation (0, none; 1, mild; 2, moderate; 3, severe), and percentage necrosis. Immunostaining on formalin-fixed sections was performed by deparaffinization and rehydration, followed by antigen retrieval by heating in a pressure cooker (121°C) for 5 minutes in 10 mmol/L sodium citrate, pH 6.0. Peroxidase activity was blocked by incubation in 3% hydrogen peroxide for 10 minutes. Sections were rinsed three times in TBS/TBS-T and incubated in 5% normal goat serum in TBS-T at room temperature for 1 hour. After removal of blocking solution, slides were placed into a humidified chamber and incubated overnight with rabbit anti-FOXO3 (EP1949Y) purchased from Millipore, diluted 1:500 in TBS-T containing 1% normal goat serum at 4°C. Antigen was detected using the SignalStain Boost IHC detection reagent (no. 8114; Cell Signaling Technology, Beverly, MA), developed with diaminobenzidene (Dako, Carpinteria, CA), counterstained with hematoxylin (Sigma-Aldrich), and mounted. Cell death was detected in situ in mouse liver paraffin-embedded sections by enzymatic labeling of DNA strand breaks with a TUNEL assay (In Situ Cell Death Detection Kit, catalogue no. 11684809910; Roche Diagnostics, Indianapolis, IN), according to the manufacturer’s instructions, and followed by counterstaining with N-ethyl-N-[4-([4-(ethyl[(3-sulfophenyl)methyl]amino)phenyl](4-hydroxy-2-sulfophenyl)methylene)-2,5-cyclohexadien-1-ylidene]-3-sulfo-benzenemethanaminium, disodium salt (fast green FCF). Intensity of TUNEL staining was quantitated on a scale of 1 to 5 by determining the percentage of positive nuclei per high-power field. Results are expressed as means ± SD. A Student’s t-test, rank-sum test, χ2 test, or one-way analysis of variance with Tukey’s post hoc test was performed as indicated. P < 0.05 was considered significant. To determine whether FOXO3 is a modifier of alcoholic liver injury, we assessed the effects of ethanol on FOXO3−/− mice. We placed constitutive FOXO3−/− mice on a Lieber-DeCarli alcohol diet for 3 weeks and compared them with WT littermates. Figure 1A demonstrates that ethanol feeding of WT mice caused a modest alanine aminotransferase (ALT) elevation (103 ± 91, n = 17) compared with control diet (59 ± 51, n = 10) (P = 0.17), and histological characteristics showed only moderate to mild zone 2 steatosis with minimal inflammation or necrosis (Figure 1B). These results are consistent with the widely reported observation that Lieber-DeCarli alcohol feeding produces only mild liver injury in mice.28Dolganiuc A. Petrasek J. Kodys K. Catalano D. Mandrekar P. Velayudham A. Szabo G. MicroRNA expression profile in Lieber-DeCarli diet-induced alcoholic and methionine choline deficient diet-induced nonalcoholic steatohepatitis models in mice.Alcohol Clin Exp Res. 2009; 33: 1704-1710Crossref PubMed Scopus (163) Google Scholar, 29Gyamfi M.A. Damjanov I. French S. Wan Y.J. The pathogenesis of ethanol versus methionine and choline deficient diet-induced liver injury.Biochem Pharmacol. 2008; 75: 981-995Crossref PubMed Scopus (43) Google Scholar, 30de la M Hall P. Lieber C.S. DeCarli L.M. French S.W. Lindros K.O. Jarvelainen H. Bode C. Parlesak A. Bode J.C. Models of alcoholic liver disease in rodents: a critical evaluation.Alcohol Clin Exp Res. 2001; 25: 254S-261SCrossref PubMed Scopus (87) Google Scholar In contrast, after 3 weeks, alcohol-fed FOXO3−/− mice had a wide variation in ALT, with a mean of 302 ± 396 (n = 23). Seven of 23 FOXO3−/− mice, but no WT mice, had ALT elevations >10-fold (P = 0.014), and each of these livers showed severe macrovesicular steatosis with pan-lobular inflammation, prominent presence of neutrophils, and frank necrosis (Figure 1B). Examples of TUNEL staining of WT and FOXO3−/− mice on ethanol are shown in Figure 1C, and higher-magnification examples of necrosis and neutrophilic infiltration are shown in Figure 1D. Steatosis, inflammation, and TUNEL positivity were significantly greater in the ethanol-fed FOXO3−/− mice compared with ethanol-fed WT mice (Figure 1E). As shown in Figure 1A, not all FOXO−/− mice developed severe liver injury. The lower half of the ALT distribution was identical for ethanol-treated WT and FOXO−/− mice [45 ± 16 (n = 8) versus 49 ± 15 (n = 11)], but the half of the mice with the greatest ALT elevations were dramatically different [155 ± 99 (n = 9) for WT versus 534 ± 438 (n = 12) for FOXO3−/− (P = 0.03)]. The previously described results suggested that FOXO3 could be an alcohol hepatoprotection factor. We then used Huh7.5 cells as a model system to directly examine the effects of HCV and alcohol on FOXO3. These cells were chosen because they support the full life cycle of HCV infection31Blight K.J. McKeating J.A. Rice C.M. Highly permissive cell lines for subgenomic and genomic hepatitis C virus RNA replication.J Virol. 2002; 76: 13001-13014Crossref PubMed Scopus (969) Google Scholar and metabolize alcohol via alcohol dehydrogenase (ADH). Figure 2A demonstrates that Huh7.5 cells express comparable levels of ADH protein, as found in human or mouse liver. Infection with the JFH1 strain of HCV increased endogenous FOXO3 transcriptional activity, as evidenced by increased FHRE-luciferase reporter activi
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