Glutathione depletion down-regulates tumor necrosis factor alpha-induced NF-kappaB activity via IkappaB kinase-dependent and -independent mechanisms.
Reduced glutathione (GSH) plays a crucial role in hepatocyte function, and GSH depletion by diethyl maleate was shown previously to inhibit expression of NF-kappaB target genes induced by tumor necrosis factor alpha (TNFalpha) 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-kappaB transactivation via two mechanisms, depending on the extent of the depletion. With moderate GSH depletion (approximately 50%), the down-regulation is IkappaB kinase (IKK)-independent and likely acts on NF-kappaB transcriptional activity because TNF-induced IKK activation, IkappaBalpha phosphorylation and degradation, NF-kappaB nuclear translocation, NF-kappaB DNA binding in vitro, and NF-kappaB subunit RelA(p65) recruitment to kappaB sites of target gene promoters all appear unaltered. On the other hand, with profound GSH depletion (approximately 80%), the down-regulation 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, IkappaBalpha phosphorylation and degradation, and NF-kappaB nuclear translocation. Of note, pretreatment with antioxidant trolox protects against the inhibitory effect of profound GSH depletion on IKK activation and NF-kappaB nuclear translocation but fails to restore expression of NF-kappaB 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-kappaB pathway.
Lou
Huan
H
Research Center for Liver Diseases, Keck School of Medicine, University of Southern California, Los Angeles, California 90033, USA.
Kaplowitz
Neil
N
D021581:Active Transport, Cell Nucleus; D000818:Animals; D000975:Antioxidants / Q000378:metabolism; D015536:Down-Regulation; D005978:Glutathione / Q000378:metabolism*; D051550:I-kappa B Kinase / Q000378:metabolism*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D016328:NF-kappa B / Q000378:metabolism*; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010766:Phosphorylation; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
D000975:Antioxidants; D016328:NF-kappa B; D047888:Receptors, Tumor Necrosis Factor, Type I; D014409:Tumor Necrosis Factor-alpha; D051550:I-kappa B Kinase; D005978:Glutathione