Suppression of nuclear factor-κB activity by nitric oxide and hyperoxia in oxygen-resistant cells

Abstract

Inhaled nitric oxide (iNO) is used clinically to treat pulmonary hypertension in newborns, often in conjunction with hyperoxia (NO/O2). Prolonged exposure to NO/O2 causes synergistic lung injury and death of lung epithelial cells. To explore the mechanisms involved, oxygen-resistant HeLa-80 cells were exposed to NO ± O2. Exposure to NO and O2 induced a synergistic cytotoxicity, accompanied with apoptotic characteristics, including elevated caspase-3-like activity, Annexin V incorporation, and nuclear condensation. This apoptosis was associated with a synergistic suppression of NF-κB activity. Cells lacking functional NF-κB p65 subunit were more sensitive to NO/O2 than their wild type counterparts. This injury was partially rescued by transfection with a p65 expression construct, suggesting an inverse relationship between NF-κB and susceptibility to the cytotoxicity of NO/O2. Despite the reduced NF-κB activity in cells exposed to NO ± O2, IκBα was degraded, suggesting that pathways regulating the steady-state levels of IκB were not involved. However, exposure to NO/O2 caused a marked reduction in nuclear localization and an increase in protein carbonyl formation of NF-κB p65 subunit. These results suggest that NO/O2-induced apoptosis occurs by suppressing NF-κB activity.