Nrf2-regulated glutathione recycling independent of biosynthesis is critical for cell survival during oxidative stress

Abstract

Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) is the primary transcription factor protecting cells from oxidative stress by regulating cytoprotective genes, including the antioxidant glutathione (GSH) pathway. GSH maintains cellular redox status and affects redox signaling, cell proliferation, and death. GSH homeostasis is regulated by de novo synthesis as well as GSH redox state; previous studies have demonstrated that Nrf2 regulates GSH homeostasis by affecting de novo synthesis. We report that Nrf2 modulates the GSH redox state by regulating glutathione reductase (GSR). In response to oxidants, lungs and embryonic fibroblasts (MEFs) from Nrf2-deficient (Nrf2−/−) mice showed lower levels of GSR mRNA, protein, and enzyme activity relative to wild type (Nrf2+/+). Nrf2−/− MEFs exhibited greater accumulation of glutathione disulfide and cytotoxicity compared to Nrf2+/+ MEFs in response to t-butylhydroquinone, which was rescued by restoring GSR. Microinjection of glutathione disulfide induced greater apoptosis in Nrf2−/− MEFs compared to Nrf2+/+ MEFs. In silico promoter analysis of the GSR gene revealed three putative antioxidant-response elements (ARE1, −44; ARE2, −813; ARE3, −1041). Reporter analysis, site-directed mutagenesis, and chromatin immunoprecipitation assays demonstrated binding of Nrf2 to two AREs distal to the transcription start site. Overall, Nrf2 is critical for maintaining the GSH redox state via transcriptional regulation of GSR and protecting cells against oxidative stress.

Copper induces Cu-ATPase ATP7A mRNA in a fish cell line, SAF1

“…One of the most sensitive and consistent responses to oxidative stress is an increase in glutathione reductase activity both in mammals where it is regulated by the oxidative stressactivated transcription factor Nrf2 (Harvey et al, 2009) and in fish, where it is used as a biomarker (Sturve et al, 2008). In the present study, in SAF-1 cells GR mRNA levels were induced both by Cd and Cu indicating that these exposures elicited oxidative stress which is in accord with previous in vivo results (Minghetti et al, 2008;Hansen et al, 2006).…”

“…In SAF1 exposure to 100 µM Zn induced a 2.6 fold induction of GR mRNA after 4 hours, but returning to control levels after 24 h exposure suggesting that SAF1 cells have efficient homeostatic mechanisms which regulate Zn intracellular concentration. Murine GR is regulated by the oxidative stress-activated transcription factor Nrf2 (Harvey et al, 2009), and although similar oxidative mechanisms may also play a role in MT induction (Ohtsuji et al, 2008), the lack of change in ATP7A mRNA after Cd treatment suggests that Cu is not affecting ATP7A mRNA expression via a mechanism dependent on oxidative stress. Recently Singleton et al (2010) have provided evidence that the reversible glutaredoxin1-catalysed glutathionylation of the Cu-binding Cys residues of ATP7A is required for normal copper transport.…”

S-glutathionylation: relevance in diabetes and potential role as a biomarker

“…Its inactivation may result in a 400-fold increased activity of NADPH oxidase (NOX), generating superoxide (Gallwitz et al, 1999). As it is the case with many antioxidant enzymes, Nrf2 is a key regulator of GR expression (Harvey et al, 2009).…”

Basic Principles and Emerging Concepts in the Redox Control of Transcription Factors

“…They comprise enzymes that directly or indirectly exert antioxidant functions (5,21,111,202,264,373,491), molecular chaperons (265), and proteins that enhance GSH synthesis and regeneration (164,174,210,276,330,336), phase 2 detoxication, drug metabolism (169,210,326,392,424,479), recognition, repair, and removal of damaged proteins (191,264,265,393), and nucleotide excision repair (8), further proteins that regulate the expression of other transcription factors, growth factors, and receptors (113,163), and inhibit cytokine-mediated inflammation (6,209,245,385,503) and autophagy (394). With its broad range of target genes, Nrf2 is certainly one of the most important transcription factors that protect the organism against exogenous stressors, be they poisonous food ingredients, physical damage, or infection.…”