Decreased oxidant buffering impairs NF-kappa B activation and ICAM-1 transcription in endothelial cells

The DNA binding activity of the transcription factor, NF-kappaB, is regulat ed by the phosphorylation and degradation of its inhibitory protein, I kapp aB, and post-translational modification involving redox reaction of a cyste ine residue (Cys62) of NF-kappaB. We addressed the role of the redox state of endothelial cells in modulating TNF alpha -induced NF-kappaB activity. T he effects of TNF alpha on DNA-binding activity of NF-kappaB and expression of mRNA encoding ICAM-1 (an NF-kappaB-activated gene) were studied in huma n pulmonary artery endothelial (HPAE) cells under basal conditions and afte r decreasing the intracellular glutathione (GSH) concentration. HPAE cells were treated with buthionine sulfoximine (BSO) (16 h), an inhibitor of GSH synthesis, which caused concentration-dependent decreases in GSH concentrat ion. Stimulation of control cells with TNF alpha resulted in reactive oxyge n species (ROS) generation and activation of NF-kappaB binding to the ICAM- 1 promoter and ICAM-1 transcription. However, stimulation of GSH-depleted c ells with TNF alpha resulted in ROS accumulation secondary to the decreased ROS buffering capacity, and marked impairment of NF-kappaB-binding activit y and ICAM-1 mRNA expression. Exposure of BSO-treated cells to the reducing agent dithiothreitol (DTT) before TNF alpha treatment or supplementation o f nuclear extract (isolated after TNF alpha challenge of BSO-treated cells) with DTT significantly augmented the effect of TNF alpha on NF-kappaB-bind ing activity and ICAM-1 mRNA expression. Thus the oxidative modification of NF-kappaB secondary to the loss of ROS buffering capacity may regulate NF- kappaB binding to ICAM-1 promoter, and thereby ICAM-1 transcription in endo thelial cells.