Activation of NF-kB induced by H2O2 and TNF-a and its effects on ICAM-1 expression in endothelial cells

True, Andrea L., Arshad Rahman, and Asrar B. Malik. Activation of NF-kB induced by H2O2 and TNF-a and its effects on ICAM-1 expression in endothelial cells. Am J Physiol Lung Cell Mol Physiol 279: L302–L311, 2000.— Reactive oxygen species have been proposed to signal the activation of the transcription factor nuclear factor (NF)-kB in response to tumor necrosis factor (TNF)-a challenge. In the present study, we investigated the effects of H2O2 and TNF-a in mediating activation of NF-kB and transcription of the intercellular adhesion molecule (ICAM)-1 gene. Northern blot analysis showed that TNF-a 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-kB-driven reporter gene expression in the transduced HMEC-1 cells, whereas TNF-a increased the NF-kB-driven gene expression ;10-fold. Gel supershift assay revealed the presence of p65 (Rel A), p50, and c-Rel in both H2O2and TNF-a-induced NF-kB 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-a exposure induced marked phosphorylation of NF-kB 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-kB to nuclear DNA, whereas TNF-a generates additional signals that induce phosphorylation of the bound NF-kB p65 and confer transcriptional competency to NF-kB.