Glutathione depletion down-regulates tumor necrosis factor α-induced NF-κB activity via IκB kinase-dependent and -independent mechanisms

Abstract

Reduced glutathione (GSH) plays a crucial role in hepatocyte function, and GSH depletion by diethyl maleate was shown previously to inhibit expression of NF-κB target genes induced by tumor necrosis factor α(TNFα) and sensitize primary cultured mouse hepatocytes to TNF-mediated apoptotic killing. Here we demonstrate in the same system that GSH depletion down-regulates TNF-induced NF-κB transactivation via two mechanisms, depending on the extent of the depletion. With moderate GSH depletion (∼50%), the down-regulation is IκB kinase (IKK)-independent and likely acts on NF-κB transcriptional activity because TNF-induced IKK activation, IκBα phosphorylation and degradation, NF-κB nuclear translocation, NF-κB DNA binding in vitro, and NF-κB subunit RelA(p65) recruitment to κB sites of target gene promoters all appear unaltered. On the other hand, with profound GSH depletion (∼80%), the downregulation also is IKK-dependent, and a timeline is established linking the inhibition of polyubiquitination of receptor-interacting protein 1 in TNF receptor 1 complex to partial blockage of IKK activation, IκBα phosphorylation and degradation, and NF-κB nuclear translocation. Of note, pretreatment with antioxidant trolox protects against the inhibitory effect of profound GSH depletion on IKK activation and NF-κB nuclear translocation but fails to restore expression of NF-κB target genes, revealing both IKK-dependent and -independent inhibition. These findings provide new insights into the complex effects of oxidative stress and redox perturbations on the NF-κB pathway. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.