Nuclear factor-kappaB-dependent induction of interleukin-8 gene expression by tumor necrosis factor alpha: evidence for an antioxidant sensitive activating pathway distinct from nuclear translocation.
Tumor necrosis factor alpha (TNFalpha) is a pluripotent activator of inflammation by inducing a proinflammatory cytokine cascade. This phenomenon is mediated, in part, through inducible expression of the CXC chemokine, interleukin-8 (IL-8). In this study, we investigate the role of TNFalpha-inducible reactive oxygen species (ROS) in IL-8 expression by "monocyte-like" U937 histiocytic lymphoma cells. TNFalpha is a rapid activator of IL-8 gene expression by U937, producing a 50-fold induction of mRNA within 1 hour of treatment. In gene transfection assays, the effect of TNFalpha requires the presence of an inducible nuclear factor-kappaB (NF-kappaB) (Rel A) binding site in the IL-8 promoter. TNFalpha treatment induces a rapid translocation of the 65 kD transcriptional activator NF-kappaB subunit, Rel A, whose binding in the nucleus occurs before changes in intracellular ROS. Pretreatment (or up to 15 minutes posttreatment) relative to TNFalpha with the antioxidant dimethyl sulfoxide (DMSO) (2% [vol/vol]) blocks 80% of NF-kappaB-dependent transcription. Surprisingly, however, DMSO has no effect on inducible Rel A binding. Similar selective effects on NF-kappaB transcription are seen with the unrelated antioxidants, N-acetylcysteine (NAC) and vitamin C. These data indicate that TNFalpha induces a delayed ROS-dependent signalling pathway that is required for NF-kappaB transcriptional activation and is separable from that required for its nuclear translocation. Further definition of this pathway will yield new insights into inflammation initiated by TNFalpha signalling.
Vlahopoulos
S
S
Department of Internal Medicine, Sealy Center for Molecular Sciences, Galveston, TX, USA.
Boldogh
I
I
Casola
A
A
Brasier
A R
AR
D000975:Antioxidants / Q000494:pharmacology*; D001483:Base Sequence; D001665:Binding Sites; D002467:Cell Nucleus / Q000502:physiology*; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D016209:Interleukin-8 / Q000235:genetics*; D007700:Kinetics; D009000:Monocytes; D016328:NF-kappa B / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000502:physiology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000494:pharmacology; D051996:Transcription Factor RelA; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D020298:U937 Cells
D000975:Antioxidants; D017931:DNA Primers; D004268:DNA-Binding Proteins; D016209:Interleukin-8; D016328:NF-kappa B; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha