Activation of NF-kappa B induced by H2O2 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)-kappa B in response to tumor necro sis 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 tr anscription 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 concen trations failed to activate ICAM-1 expression. Challenge with H2O2 also fai led 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 expres sion similar to 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 comp lexes bound to the ICAM-1 promoter, with the binding of the p65 subunit bei ng the most prominent. In vivo phosphorylation studies, however, showed tha t TNF-alpha exposure induced marked phosphorylation of NF-kappa B p65 in HM EC-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 transcriptio nal competency to NF-kappa B.