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-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.
Highlights
The costs of publication of this article were defrayed in part by the payment of page charges
TNF receptor-associated factor 2 (TRAF2) functions as a ubiquitin ligase (E3) for receptor-interacting protein 1 (RIP1) generating polyubiquitin chain linked through lysine 63 of ubiquitin (Lys-63polyUb) that is insensitive to the 26S proteasome [12,13,14], whereas A20 acts sequentially through its two domains to disassemble Lys-63-polyUb and assemble Lys-48-polyUb bound to RIP1 and thereby designates RIP1 for degradation [14]
GSH Depletion by DEM Down-regulates TNF-induced NF-B Activity, but Perturbation of Nuclear Accumulation of NF-B Is Not a Requisite—Previous work in our laboratory demonstrated that GSH depletion by DEM (0.25 and 0.5 mM) inhibits TNF-induced increase of mRNA of endogenous NF-B-responsive genes, such as inhibitor proteins (IBs)␣, iNOS, and cIAP1, sensitizing PMH to TNF-induced apoptosis [20, 21]
Summary
The costs of publication of this article were defrayed in part by the payment of page charges. Profound GSH depletion by 0.5 mM DEM resulted in a marked reduction in IKK activity as early as 5 min post-TNF␣ stimulation followed by decreases in IB␣ degradation and corresponding nuclear translocation of both RelA(p65) and p50 (Fig. 4, A and B, right panels).
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