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Am J Physiol Lung Cell Mol Physiol 279: L302-L311, 2000;
1040-0605/00 $5.00
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Vol. 279, Issue 2, L302-L311, August 2000

Activation of NF-kappa B induced by H2O2 and TNF-alpha and its effects on ICAM-1 expression in endothelial cells

Andrea L. True, Arshad Rahman, and Asrar B. Malik

Department of Pharmacology, University of Illinois College of Medicine, Chicago, Illinois 60612

Reactive oxygen species have been proposed to signal the activation of the transcription factor nuclear factor (NF)-kappa B in response to tumor necrosis factor (TNF)-alpha challenge. In the present study, we investigated the effects of H2O2 and TNF-alpha in mediating activation of NF-kappa B 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, H2O2 added at subcytolytic concentrations failed to activate ICAM-1 expression. Challenge with H2O2 also failed to induce NF-kappa B-driven reporter gene expression in the transduced HMEC-1 cells, whereas TNF-alpha increased the NF-kappa B-driven gene expression ~10-fold. Gel supershift assay revealed the presence of p65 (Rel A), p50, and c-Rel in both H2O2- and TNF-alpha -induced NF-kappa B 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-kappa B p65 in HMEC-1 cells, whereas H2O2 had no effect. These results suggest that reactive oxygen species generation in endothelial cells mediates the binding of NF-kappa B to nuclear DNA, whereas TNF-alpha generates additional signals that induce phosphorylation of the bound NF-kappa B p65 and confer transcriptional competency to NF-kappa B.

redox state; intercellular adhesion molecule-1 promoter; tumor necrosis factor-alpha ; oxidants; nuclear factor-kappa B; signaling; hydrogen peroxide


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