Decreased oxidant buffering impairs NF-kappaB activation and ICAM-1 transcription in endothelial cells.

The DNA binding activity of the transcription factor, NF-kappaB, is regulated by the phosphorylation and degradation of its inhibitory protein, IkappaB, and post-translational modification involving redox reaction of a cysteine residue (Cys62) of NF-kappaB. We addressed the role of the redox state of endothelial cells in modulating TNFalpha-induced NF-kappaB activity. The effects of TNFalpha on DNA-binding activity of NF-kappaB and expression of mRNA encoding ICAM-1 (an NF-kappaB-activated gene) were studied in human pulmonary artery endothelial (HPAE) cells under basal conditions and after 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 concentration. Stimulation of control cells with TNFalpha resulted in reactive oxygen species (ROS) generation and activation of NF-kappaB binding to the ICAM-1 promoter and ICAM-1 transcription. However, stimulation of GSH-depleted cells with TNFalpha resulted in ROS accumulation secondary to the decreased ROS buffering capacity, and marked impairment of NF-kappaB-binding activity and ICAM-1 mRNA expression. Exposure of BSO-treated cells to the reducing agent dithiothreitol (DTT) before TNFalpha treatment or supplementation of nuclear extract (isolated after TNFalpha challenge of BSO-treated cells) with DTT significantly augmented the effect of TNFalpha on NF-kappaB-binding 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 endothelial cells.