Glutathione Depletion Down-regulates Tumor Necrosis Factor α-induced NF-κB Activity via IκB Kinase-dependent and -independent Mechanisms

Huan Lou, Neil Kaplowitz
2007 Journal of Biological Chemistry  
Reduced glutathione (GSH) plays a crucial role in hepatocyte function, and GSH depletion by diethyl maleate was shown previously to inhibit expression of NF-B target genes induced by tumor necrosis factor ␣ (TNF␣) 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-B transactivation via two mechanisms, depending on the extent of the depletion. With moderate GSH depletion
more » ... GSH depletion (ϳ50%), the down-regulation is IB kinase (IKK)-independent and likely acts on NF-B transcriptional activity because TNF-induced IKK activation, IB␣ phosphorylation and degradation, NF-B nuclear translocation, NF-B DNA binding in vitro, and NF-B subunit RelA(p65) recruitment to B sites of target gene promoters all appear unaltered. On the other hand, with profound GSH depletion (ϳ80%), 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, IB␣ phosphorylation and degradation, and NF-B nuclear translocation. Of note, pretreatment with antioxidant trolox protects against the inhibitory effect of profound GSH depletion on IKK activation and NF-B nuclear translocation but fails to restore expression of NF-B 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-B pathway. TNF␣ 2 plays an important role in liver injury in many animal models and has been implicated in the progression of human alcoholic liver disease and hepatic viral infections (1-4). TNF␣ exerts diverse biological effects by activation of multiple pathways promoting inflammation and both cell survival and death (5). Like many cell types, normal hepatocytes do not undergo apoptosis in response to TNF␣ because successful activation of TNF-induced NF-B pathway blocks the TNF-induced apoptotic pathway (5, 6). NF-B, a family of dimeric transcriptional factors, regulates the expression of a spectrum of genes involved in cell survival and anti-apoptotic functions and inflammation (7) . It exists mainly as the RelA(p65) and p50 heterodimer and is retained in cytoplasm by inhibitor proteins (IBs) in unstimulated cells (7) . TNF␣ activates various pathways through two receptors, TNFR1 and TNFR2 (5). The signaling from TNF␣ to NF-B activation, mediated mainly through TNFR1, has been most extensively studied (5, 8, 9) . Upon TNF␣ binding, TNFR1 recruits TNF receptor-associated death domain protein (TRADD), receptor-interacting protein 1 (RIP1), and TNF receptor-associated factor 2 (TRAF2) (5, 10). RIP1 is polyubiquitinated in TNFR1 complex (10, 11) . This polyubiquitination process is promoted by TRAF2 and reversed by A20 (8). TRAF2 functions as a ubiquitin ligase (E3) for RIP1 generating polyubiquitin chain linked through lysine 63 of ubiquitin (Lys-63-polyUb) that is insensitive to the 26S proteasome (12-14), 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). RIP1 recruits IKK complexes (15) through the interaction of the polyUb chains of polyUb-RIP1 with IKK␥/Nemo in the IKK complex (16, 17) and leads to eventual activation of IKK␤ by phosphorylation of serines 177 and 181 in the activation loop (8, 9) . Activated IKK complex phosphorylates IB␣ at two serine sites (32 and 36), which trigger its ubiquitination and degradation by 26S proteasome (7). Released NF-B then enters the nucleus where with further modifications (phosphorylation, acetylation, etc.) it binds to specific promoter sequences termed B sites and turns on the transcription of target genes (9, 18). It is reasonable to speculate that in the context of specific liver injury models or disease conditions, hepatocytes become unable to respond normally to the diverse effects of TNF␣ and thereby become sensitized to TNF-induced death. GSH, a cysteine containing tripeptide, as the single most abundant antioxidant for detoxifying enzymes and a determinant of the thioldisulfide state, is crucial to hepatocyte function (19). Previous
doi:10.1074/jbc.m706145200 pmid:17690092 fatcat:okrymdyg75bara2bs6q536bk7y