Reciprocal REGγ-Nrf2 Regulation Promotes ROS Scavenging In Oxidative Stress Induced Cell Aging

Increased accumulation of reactive oxygen species（ROS）and decline of adaptive response of antioxidants to oxidative stimuli has been implicated in the aging process.Nuclear factor erythroid 2-related factor 2（Nrf2）activation is a core event in attenuating oxidative stress associated aging.The activity is modulated by a more complex regulatory network.The proteasome activator REGγ has been shown to bind and activate the 20S proteasome in a ubiquitin-and ATP-independent manner,promoting proteasome-dependent degradation of several important cellular regulatory proteins such as SRC-3 and cyclin-dependent kinase inhibitors p21,GSK-3β and so on.Furthermore,REGγ is also involved in the regulation of a wide range of physiological and pathological processes,including premature aging,oxidative stress,cancer progression and others.However,roles of REGγ in adaptive response during recovering from oxidative stress,which is associated with cell aging,remain to be explored.In this study,we used primary MEF cell senescence model from REGγ KO mice and their control counter parts,which were induced with H₂O₂ to investigate how REGγ affects cell senescence.We demonstrated that the proteasome activator REGγ function as a new regulator of Nrf2 activity upon oxidative stress in cell aging model induced by hydrogen peroxide（H₂O₂）.Assessment of β-galactosidase staining results indicates REGγ deficiency promotes cell senescence in primary MEF cells after H₂O₂ treatment.Since cells exposed to oxidative stress accumulate large amount of ROS,we found that ROS level in WT cells significantly higher that than KO cells after brief exposure of H₂O₂.However,continuous detection of ROS levels at various time points after stimulation revealed that ROS levels in WT cells decreased faster than in KO cells.This result suggests that REGγ may have long lasting effect on scavenging ROS.Molecular mechanism studies showed that ROS accumulation compelled cells to respond oxidative stress by Nrf2 translocation to the nucleus and increased the expression of downstream target genes.Indeed,our qPCR analysis showed increased mRNA expression of Ho1 and Nqo1 genes in REGγ WT cells compared to the REGγ KO cells.However,western blotting results could not confirm the stability of Nrf2 at protein levels in WT cells.Moreover,immunofluorescence assays results indicates that under normal condition the Nrf2 cytoplasm location is more in both REGγ and REGγ deficient primary MEF cells,whereas under brief stimulation with oxidative stress,Nrf2 translocated to the nucleus in almost all REGγ WT and REGγ KO cells.However,after allowing the cells to recover for a while,REGγ WT cells had considerably more Nrf2 in the nucleus than in REGγ KO cells.This indicate that REGγ promotes the nuclear translocation and activity of Nrf2.Inhibition of GSK-3β was reported to lead to Nrf2 nuclear accumulation and activation and our previous study discovered that REGγ boosts GSK-3β decay in an ATP-and ubiquitin-independent manner.Concurrently,our degradation stability result showed that REGγ promotes the nuclear retention and activation of Nrf2 through degrading GSK-3β .The finding further incorporated by GSK-3β inhibition.The results of western blotting,real-time and gel-based PCR showed that oxidative stress alone resulted in a significant increase in Ho1 and Nqo1expression in WT cells compared to KO cells.Interestingly,REGγ expression increase significantly during cell adaption to oxidative stress at both mRNA and protein levels.These results point out that REGγ may be upregulated and maintained to provide a longer-lasting protection for oxidative stress challenged cells.Simultaneously,REGγ was either up-or down-regulated depending on whether Nrf2 was expressed excessively or its activity was silenced.Consistently,transient transfection of Nrf2 and H₂O₂ dramatically increased the activity of wild-typed luciferase reporter gene while blunted by mutated luciferase gene and Nrf2 inhibitor.In conclusion,our data indicates that REGγ ,the 11s family of proteasome activator,prolongs the nuclear retention of Nrf2 and the expression of its downstream antioxidant enzymes Ho1 and Nqo1,to reduce the ROS accumulation and eventually protect against oxidative stress mediated cell senescence.Thus,our results establish a positive feedback loop between REGγ and Nrf2 and a new layer of adaptive response after oxidative stimulation that is the REGγ -GSK-3β -Nrf2 pathway.These results make REGγ a promising drug target since pharmacological manipulation of REGγ could have potential benefits on oxidative damage related disorders and aging.