title: Induced oxidative stress and activated expression of manganese superoxide dismutase during hepatitis C virus replication: role of JNK, p38 MAPK and AP-1.
abstract: Activation of cellular kinases and transcription factors mediates the early phase of the cellular response to chemically or biologically induced stress. In the present study we investigated the oxidant/antioxidant balance in Huh-7 cells expressing the HCV (hepatitis C virus) subgenomic replicon, and observed a 5-fold increase in oxidative stress during HCV replication. We used MnSOD (manganese-superoxide dismutase) as an indicator of the cellular antioxidant response, and found that its activity, protein levels and promoter activity were significantly increased, whereas Cu/ZnSOD was not affected. The oxidative stress-induced protein kinases p38 MAPK (mitogen-activated protein kinase) and JNK (c-Jun N-terminal kinase) were activated in the HCV repliconcontaining cells and in Huh-7 cells transduced with Ad-NS5A [a recombinant adenovirus encoding NS5A (non-structural protein 5A)], coupled with a 4-5-fold increase in AP-1 (activator protein-1) DNA binding. Ava.1 cells, which encode a replication-defective HCV replicon, showed no significant changes in MnSOD, p38 MAPK or JNK activity. The AP-1 inhibitors dithiothreitol and N -acetylcysteine, as well as a dominant negative AP-1 mutant, significantly reduced AP-1 activation, demonstrating that this activation is oxidative stress-related. Exogenous NS5A had no effect on AP-1 activation in vitro, suggesting that NS5A acts at the upstream targets of AP-1 involving p38 MAPK and JNK signalling cascades. AP-1-dependent gene expression was increased in HCV subgenomic replicon-expressing Huh-7 cells. MnSOD activation was blocked by inhibitors of JNK (JNKI1) and p38 MAPK (SB203580), but not by an ERK (extracellular-signal-regulated kinase) inhibitor (U0126), in HCV-replicating and Ad-NS5A-transduced cells. Our results demonstrate that cellular responses to oxidative stress in HCV subgenomic replicon-expressing and Ad-NS5A-transduced cells are regulated by two distinct signalling pathways involving p38 MAPK and JNK via AP-1 that is linked to increased oxidative stress and therefore to an increased antioxidant MnSOD response.
authors.affiliation: Department of Medicine, Division of Gastroenterology and Hepatology, University of Colorado Health Sciences Center, 4200 E. 9th Ave., Denver, CO 80262, USA. ishtiaq.qadri@uchsc.edu
authors.forename: Ishtiaq
authors.initials: I
authors.lastname: Qadri
authors.forename: Mieko
authors.initials: M
authors.lastname: Iwahashi
authors.forename: Juan M
authors.initials: JM
authors.lastname: Capasso
authors.forename: Matthew W
authors.initials: MW
authors.lastname: Hopken
authors.forename: Sonia
authors.initials: S
authors.lastname: Flores
authors.forename: Jerome
authors.initials: J
authors.lastname: Schaack
authors.forename: Francis R
authors.initials: FR
authors.lastname: Simon
mesh_terms: D002460:Cell Line; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D016174:Hepacivirus / Q000235:genetics / Q000472:pathogenicity* / Q000502:physiology; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*; D018384:Oxidative Stress; D011401:Promoter Regions, Genetic; D016755:Proto-Oncogene Proteins c-jun / Q000096:biosynthesis; D012093:Replicon; D013482:Superoxide Dismutase / Q000235:genetics / Q000378:metabolism*; D018808:Transcription Factor AP-1 / Q000502:physiology*; D015533:Transcriptional Activation; D017361:Viral Nonstructural Proteins / Q000378:metabolism; D014779:Virus Replication; D048051:p38 Mitogen-Activated Protein Kinases; D012324:RNA-Dependent RNA Polymerase
chemical_list: D004791:Enzyme Inhibitors; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D016755:Proto-Oncogene Proteins c-jun; D013482:Superoxide Dismutase; D018808:Transcription Factor AP-1; D017361:Viral Nonstructural Proteins; D048051:p38 Mitogen-Activated Protein Kinases; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase