RESUMO
UNLABELLED: Hepatitis C virus (HCV) infection produces chronic liver injury that is significantly exacerbated by alcohol consumption. While multiple mechanisms contribute to this synergy, a viral-induced loss of antioxidant responses has been shown to play an important role. This study examined the effects of HCV infection and alcohol on the regulation of the transcription factor FOXO3, an important regulator of Mn-superoxide dismutase (SOD2) expression, a tumor suppressor, and a component of the hepatic antioxidant response system. FOXO3 was activated by either HCV or alcohol alone but suppressed by the combination. To understand this paradoxical result, we applied a capillary isoelectric focusing (IEF) method to determine the pattern of FOXO3 posttranslational modifications (PTMs) induced by HCV and alcohol. We observed the presence of multiple different nuclear and cytosolic species of FOXO3 and used antiphosphoserine, acetyl-lysine, methylarginine, and ubiquitin antibodies to identify the PTM patterns present in each species. HCV caused multiple changes including phosphorylation of FOXO3 at S-574, a novel c-Jun N-terminal kinase (JNK) site, which promoted nuclear translocation and transcription. Ethanol suppressed arginine-methylation of FOXO3 promoting nuclear export and degradation of the JNK phosphorylated form. Human liver biopsy samples showed the presence of the HCV-specific form of FOXO3 in HCV-infected livers but not in normal liver or nonalcoholic steatohepatitis. CONCLUSION: The development of this novel IEF method for the simultaneous quantification of differently modified FOXO3 species allowed us to demonstrate how HCV and alcohol combine to modify a complex pattern of FOXO3 PTMs that contribute to pathogenesis. This approach will allow further dissection of the role of protein PTMs in viral liver disease.
Assuntos
Consumo de Bebidas Alcoólicas/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Hepatite C/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Processamento de Proteína Pós-Traducional , Substituição de Aminoácidos , Linhagem Celular Tumoral , Etanol/farmacologia , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/efeitos dos fármacos , Humanos , Focalização Isoelétrica , Metilação/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Solventes/farmacologiaRESUMO
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.
Assuntos
Depressores do Sistema Nervoso Central/efeitos adversos , Etanol/efeitos adversos , Fatores de Transcrição Forkhead , Hepacivirus/metabolismo , Hepatite C , Hepatopatias Alcoólicas , Animais , Depressores do Sistema Nervoso Central/farmacologia , Etanol/farmacologia , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Hepacivirus/genética , Hepatite C/genética , Hepatite C/metabolismo , Hepatite C/patologia , Humanos , Hepatopatias Alcoólicas/genética , Hepatopatias Alcoólicas/metabolismo , Hepatopatias Alcoólicas/patologia , Camundongos , Camundongos Knockout , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Fatores de Tempo , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genéticaRESUMO
Viral infections frequently alter mitochondrial function with suppression or induction of apoptosis and enhanced generation of reactive oxygen species. The mechanisms of these effects are varied, and mitochondria are affected by both direct interactions with viral proteins and by secondary effects of viral-activated signaling cascades. This chapter describes methods used in our laboratory to assess the effects of the hepatitis C virus core protein on mitochondrial ROS production, electron transport, and Ca(2+) uptake. These include measurements of the effects of in vitro incubation of liver mitochondria with purified core protein and assessment of the function of mitochondria in cells and tissues expressing core and other viral proteins. These methods are generally applicable to the study of viral-mitochondrial interactions.