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

• Published in: Free radical biology & medicine Vol. 46; no. 4; pp. 443 - 453
• Main Authors: Harvey, C.J., Thimmulappa, R.K., Singh, A., Blake, D.J., Ling, G., Wakabayashi, N., Fujii, J., Myers, A., Biswal, S.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15-02-2009
• Subjects: Animals; Cell Line; Cell Survival - drug effects; Cell Survival - physiology; Cigarette smoke; COPD; Emphysema; Fibroblasts - pathology; Fibroblasts - physiology; Free radicals; Glutathione; Glutathione - analogs & derivatives; Glutathione - biosynthesis; Glutathione - genetics; Glutathione - metabolism; Glutathione reductase; Glutathione Reductase - genetics; Glutathione Reductase - metabolism; Hydroquinones - pharmacology; Lung - enzymology; Lung - pathology; Mice; Mice, Knockout; Mutagenesis, Site-Directed; NF-E2-Related Factor 2 - genetics; NF-E2-Related Factor 2 - metabolism; Nrf2; Oxidation-Reduction; Oxidative stress; Oxidative Stress - physiology; Promoter Regions, Genetic - genetics; Response Elements - genetics; RNA Interference; RNA, Small Interfering; Smoking; Transcriptional Activation - physiology; Oxidative stress; Free radicals; Nrf2; Glutathione reductase; Emphysema; Cigarette smoke; Glutathione; COPD
• ISSN: 0891-5849; 1873-4596
• DOI: 10.1016/j.freeradbiomed.2008.10.040

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.