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

Abstract

Reactive oxygen species have been proposed to signal the activation of the transcription factor nuclear factor (NF)-κB in response to tumor necrosis factor (TNF)-α challenge. In the present study, we investigated the effects of H2O2 and TNF-α in mediating activation of NF-κB and transcription of the intercellular adhesion molecule (ICAM)-1 gene. Northern blot analysis showed that TNF-α 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-κB-driven reporter gene expression in the transduced HMEC-1 cells, whereas TNF-α increased the NF-κB-driven gene expression ∼10-fold. Gel supershift assay revealed the presence of p65 (Rel A), p50, and c-Rel in both H2O2- and TNF-α-induced NF-κB 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-α exposure induced marked phosphorylation of NF-κB 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-κB to nuclear DNA, whereas TNF-α generates additional signals that induce phosphorylation of the bound NF-κB p65 and confer transcriptional competency to NF-κB.