title: Role of oxidants in NF-kappa B activation and TNF-alpha gene transcription induced by hypoxia and endotoxin.
abstract: The transcription factor NF-kappa B stimulates the transcription of proinflammatory cytokines including TNF-alpha. LPS (endotoxin) and hypoxia both induce NF-kappa B activation and TNF-alpha gene transcription. Furthermore, hypoxia augments LPS induction of TNF-alpha mRNA. Previous reports have indicated that antioxidants abolish NF-kappa B activation in response to LPS or hypoxia, which suggests that reactive oxygen species (ROS) are involved in NF-kappa B activation. This study tested whether mitochondrial ROS are required for both NF-kappaB activation and the increase in TNF-alpha mRNA levels during hypoxia and LPS. Our results indicate that hypoxia (1.5% O2) stimulates NF-kappa B and TNF-alpha gene transcription and increases ROS generation as measured by the oxidant sensitive dye 2',7'-dichlorofluorescein diacetate in murine macrophage J774.1 cells. The antioxidants N-acetylcysteine and pyrrolidinedithiocarbamic acid abolished the hypoxic activation of NF-kappa B, TNF-alpha gene transcription, and increases in ROS levels. Rotenone, an inhibitor of mitochondrial complex I, abolished the increase in ROS signal, the activation of NF-kappa B, and TNF-alpha gene transcription during hypoxia. LPS stimulated NF-kappa B and TNF-alpha gene transcription but not ROS generation in J774.1 cells. Rotenone, pyrrolidinedithiocarbamic acid, and N-acetylcysteine had no effect on the LPS stimulation of NF-kappa B and TNF-alpha gene transcription, indicating that LPS activates NF-kappa B and TNF-alpha gene transcription through a ROS-independent mechanism. These results indicate that mitochondrial ROS are required for the hypoxic activation of NF-kappa B and TNF-alpha gene transcription, but not for the LPS activation of NF-kappa B.
authors.affiliation: Section of Pulmonary and Critical Care Medicine, Department of Medicine, University of Chicago, Chicago, IL 60637, USA. nav@nwu.edu
authors.forename: N S
authors.initials: NS
authors.lastname: Chandel
authors.forename: W C
authors.initials: WC
authors.lastname: Trzyna
authors.forename: D S
authors.initials: DS
authors.lastname: McClintock
authors.forename: P T
authors.initials: PT
authors.lastname: Schumacker
mesh_terms: D000818:Animals; D015687:Cell Hypoxia / Q000235:genetics / Q000276:immunology; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008264:Macrophages / Q000276:immunology / Q000378:metabolism; D051379:Mice; D008928:Mitochondria / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism*; D016877:Oxidants / Q000502:physiology*; D011485:Protein Binding / Q000276:immunology; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000096:biosynthesis; D017382:Reactive Oxygen Species / Q000378:metabolism; D014158:Transcription, Genetic / Q000276:immunology*; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism
chemical_list: D008070:Lipopolysaccharides; D016328:NF-kappa B; D016877:Oxidants; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D014409:Tumor Necrosis Factor-alpha; D004247:DNA