Regulation of FOXO3 by phosphorylation and methylation in hepatitis C virus infection and alcohol exposure

Abstract

Hepatitis C 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 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 post-translational modifications (PTMs) induced by HCV and alcohol. We observed the presence of multiple different nuclear and cytosolic species of FOXO3 and used anti phosphoserine, 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 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.

Serine 574 phosphorylation alters transcriptional programming of FOXO3 by selectively enhancing apoptotic gene expression

“…The discovery that p-574-FOXO3 is a specifically pro-apoptotic form of the protein is thus an important advance in our understanding of FOXO3. S-574 was first reported to be a site of FOXO3 phosphorylation by Brunet et al 17 We previously observed this phosphorylated form after HCV infection 23 and now have determined that it occurs after EtOH treatment of hepatocytes or LPS treatment of macrophages as well. We were able to demonstrate a strong connection between JNK activation and the appearance of p-574-FOXO3 as it was induced by activated JNK1 expression, its formation was blocked by JNK inhibitor and the time course of its appearance closely followed that of JNK activation for either LPS-treated macrophages or alcohol-treated hepatoma cells (Supplementary Figure S2).…”

“…32 They were chosen for this study as previous isoelectric focusing studies showed that HCV infection and alcohol generated specific and different FOXO3 PTMs and these appeared to determine antioxidant responses. 23 In this earlier study we observed that HCV caused a specific species of FOXO3 that contained S-574 phosphorylation but this particular species was not identified after EtOH treatment. This implies that the HCVinduced and EtOH-induced p-574 phosphorylated forms of FOXO3 may differ at other sites as they have different pIs.…”

“…We previously observed that hepatitis C virus (HCV) infection induced JNK-dependent phosphorylation of FOXO3 at S-574. 23 The specificity and functional consequences of this phosphorylation are unknown. We generated a specific antibody to phospho-S-574-FOXO3 (p-574-FOXO3) and used it to examine the effects of EtOH on this FOXO3 modification.…”

“…23 In the present study, we sought to determine whether these modified forms of FOXO3 cause preferential target gene transcription. We observed that ethanol (EtOH) induces a c-Jun N-terminal kinase (JNK)-dependent phosphorylation of FOXO3 at serine-574 (S-574) and this altered the transcriptional specificity of FOXO3, converting it from an antioxidant to an apoptosis-inducing transcription factor.…”

Hepatitis C and Alcohol Exacerbate Liver Injury by Suppression of FOXO3

“…It is likely that an ethanol-induced FOXO3 post-translational modification change makes it a better substrate for Akt phosphorylation. Recent studies from our laboratory have shown that the combination of HCV and alcohol specifically suppresses arginine methylation of FOXO3, 47 and this is expected to make it a better substrate for Akt phosphorylation, causing nuclear export and degradation. 48 Because the effects can be seen in our transgenic mice and infected cells, the HCV structural proteins, particularly core, may play a critical role in FOXO3 modulation.…”