Activation of NF-kappaB induced by H(2)O(2) and TNF-alpha and its effects on ICAM-1 expression in endothelial cells.
Reactive oxygen species have been proposed to signal the activation of the transcription factor nuclear factor (NF)-kappaB in response to tumor necrosis factor (TNF)-alpha challenge. In the present study, we investigated the effects of H(2)O(2) and TNF-alpha in mediating activation of NF-kappaB and transcription of the intercellular adhesion molecule (ICAM)-1 gene. Northern blot analysis showed that TNF-alpha exposure of human dermal microvascular endothelial cells (HMEC-1) induced marked increases in ICAM-1 mRNA and cell surface protein expression. In contrast, H(2)O(2) added at subcytolytic concentrations failed to activate ICAM-1 expression. Challenge with H(2)O(2) also failed to induce NF-kappaB-driven reporter gene expression in the transduced HMEC-1 cells, whereas TNF-alpha increased the NF-kappaB-driven gene expression approximately 10-fold. Gel supershift assay revealed the presence of p65 (Rel A), p50, and c-Rel in both H(2)O(2)- and TNF-alpha-induced NF-kappaB complexes bound to the ICAM-1 promoter, with the binding of the p65 subunit being the most prominent. In vivo phosphorylation studies, however, showed that TNF-alpha exposure induced marked phosphorylation of NF-kappaB p65 in HMEC-1 cells, whereas H(2)O(2) had no effect. These results suggest that reactive oxygen species generation in endothelial cells mediates the binding of NF-kappaB to nuclear DNA, whereas TNF-alpha generates additional signals that induce phosphorylation of the bound NF-kappaB p65 and confer transcriptional competency to NF-kappaB.
Department of Pharmacology, University of Illinois College of Medicine, Chicago, Illinois 60612, USA.
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Rahman
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Malik
D001667:Binding, Competitive / Q000235:genetics; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D018799:Intercellular Adhesion Molecule-1 / Q000096:biosynthesis* / Q000235:genetics; D007424:Intracellular Fluid / Q000187:drug effects / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011401:Promoter Regions, Genetic / Q000187:drug effects; D012333:RNA, Messenger / Q000096:biosynthesis; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D051996:Transcription Factor RelA; D015533:Transcriptional Activation / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D016328:NF-kappa B; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha; D018799:Intercellular Adhesion Molecule-1; D004247:DNA; D006861:Hydrogen Peroxide