GPx-1 Gene Delivery Modulates NF κ B Activation Following Diverse Environmental Injuries Through a Specific Subunit of the IKK Complex

Abstract

Numerous environmental stimuli alter cell functions by the induction of intracellular reactive oxygen species, such as superoxide and hydrogen peroxide (H₂O₂). These redox alterations can change the activity of kinases and phosphatases responsible for controlling intracellular signal transduction cascades important in determining how cells react to their environment. One such well known pathway includes nuclear factor-κB (NFκB); however, the exact redox-sensitive factors important in controlling H₂O₂-mediated activation of NFκB remain unclear. In the present study, we have investigated how intracellular clearance of H₂O₂, using a recombinant adenovirus expressing glutathione peroxidase-1 (GPx-1), modulates NFκB activation following UV irradiation, tumor necrosis factor-α, or H₂O₂ treatment of MCF-7 cells. Findings from these studies demonstrate that GPx-1 overexpression can down-regulate NFκB DNA binding, and transcriptional activation of an NFκB-dependent luciferase reporter, to varying extents following these environmental stimuli. Studies using dominant negative adenoviral vectors expressing IKKα(KM) and IKKβ(KA) suggest that GPx-1-mediated H₂O₂ clearance appears to preferentially inhibit the activity of IKKα, but not IKKβ. These studies demonstrate for the first time that redox regulation of NFκB activation by intracellular H₂O₂ may be specific for a unique subunit in the IKK complex. Such findings suggest that IKK kinases or IKK phosphatases may have unique redox-regulated components. These studies have shed mechanistic insight into the potential application of redox-modulating gene therapies aimed at altering NFκB activation following environmental injury.