Oxidative Stress Induces NF- B Nuclear Translocation Without Degradation of I B

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Abstract
Background— Rel/NF-κB, an oxidative stress–responsive transcription factor, participates transiently in the control of gene expression. The cellular mechanisms that mediate NF-κB activation during ischemia (and during reperfusion in the course of treating ischemia) are not known. Methods and Results— To investigate the NF-κB activation induced during oxidative stress, we examined human cardiac tissue obtained during surgical procedures requiring cardiopulmonary bypass. In vitro, we examined human umbilical vein endothelial cells (HUVECs) exposed to hypoxia, reoxygenation after hypoxia, or a reactive oxygen intermediate (H2O2). Electrophoretic mobility shift assays performed on right atrial tissue revealed prominent NF-κB activation after hearts had been exposed to ischemia and reperfusion. The assays also showed that NF-κB activation was observed in hypoxic HUVECs after reoxygenation and in cultures treated with H2O2 (500 μmol/L). Pervanadate (200 μmol/L) also induced marked NF-κB activation in HUVECs, indicating that H2O2-induced NF-κB activation is potentiated by the inhibition of tyrosine phosphatases. Western blotting of cytoplasmic IκBα demonstrated that NF-κB activation induced by oxidative stress was not associated with IκBα degradation. In contrast, tumor necrosis factor-α–induced NF-κB activation occurred in concert with degradation of IκBα. Inhibition of IκBα degradation with a proteasome inhibitor, MG-115, blocked NF-κB activation induced by tumor necrosis factor-α; however, MG-115 had no effect on NF-κB activation during oxidative stress. Conclusions— This study demonstrated a stimulus-specific mechanism of NF-κB activation in endothelial cells that acts independently of IκBα degradation and may require tyrosine phosphorylation.