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

doi:10.1016/j.freeradbiomed.2008.10.040

Free Radical Biology and Medicine

Volume 46, Issue 4, 15 February 2009, Pages 443-453

https://doi.org/10.1016/j.freeradbiomed.2008.10.040 Get rights and content

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.

Section snippets

Animals and care

Nrf2^−/− CD-1 (ICR) mice were generated as described [12]. All experimental procedures conducted on the mice were performed in accordance with the standards established by the U.S. Animal Welfare Acts, set forth in the National Institutes of Health guidelines and the Policy and Procedures Manual of the Johns Hopkins University Animal Care and Use Committee.

Mouse exposure to cigarette smoke

Eight-week-old mice were divided into four groups (n = 3 per group): I, air control Nrf2^+/+ mice; II, experimental Nrf2^+/+ mice; III, air

Nrf2-dependent expression of glutathione reductase in mouse lung after acute exposure to CS

The basal levels of mRNA expression of GSR in the lungs were similar in Nrf2^+/+ and Nrf2^−/− mice as determined by real-time RT-PCR analysis. In response to CS exposure, the mRNA expression of GSR showed an approximately fourfold induction in the lungs of Nrf2^+/+ mice compared to air, whereas the lungs of Nrf2^−/− mice showed no induction of GSR (Fig. 1A). In corroboration, immunoblot analysis showed no differences in the basal levels of GSR protein in Nrf2^+/+ and Nrf2^−/− lungs; however, CS

Discussion

Through the use of Nrf2-disrupted models (cells and mice), our laboratory and others have previously reported that Nrf2 protects from cell death induced by multiple oxidants (H₂O₂, tBHQ, cigarette smoke, hyperoxia, and anticancer drugs) mainly by alleviating cellular ROS levels [17]. Nrf2 positively regulates antioxidant and electrophile detoxification enzymes (NQO1, GSTs, and GPX2) as well as enzymes that directly regulate levels of glutathione (GCLM, GCLC). Because electrophilic

Acknowledgments

The authors thank Dr. Thomas W. Kensler for critical review of the manuscript, Dr. Masayuki Yamamoto for sharing the Nrf2^−/− mice, and Drs. Michael A. Trush and James P. Kehrer for discussion of chemical inhibitors of GSR. This work was supported by NIH Grant HL081205, NHLBI SCCOR Grant P50HL084945, FAMRI, a Maryland Cigarette Restitution Fund research grant, GM079239, and NIEHS Center Grant ES03819. C.J.H. was supported by NIEHS Training Grant ES07141.

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Original ContributionNrf2-regulated glutathione recycling independent of biosynthesis is critical for cell survival during oxidative stress

Abstract

Section snippets

Animals and care

Mouse exposure to cigarette smoke

Nrf2-dependent expression of glutathione reductase in mouse lung after acute exposure to CS

Discussion

Acknowledgments

Free Radic. Biol. Med.

Biochem. Pharmacol.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Methods Enzymol.

Toxicology

J. Biol. Chem.

J. Biol. Chem.

Toxicol. Lett.

Toxicol. Lett.

Clin. Chim. Acta

Fundam. Appl. Toxicol.

Glutathione, stress responses, and redox signaling in lung inflammation

Antioxid. Redox Signaling

Deficiency in Nrf2–GSH signaling impairs type II cell growth and enhances sensitivity to oxidants

Am. J. Respir. Cell Mol. Biol.

Role of glutathione depletion and reactive oxygen species generation in apoptotic signaling in a human B lymphoma cell line

Cell Death Differ.

Rescue of cells from apoptosis by inhibition of active GSH extrusion

FASEB J.

Glutathione disulfide induces apoptosis in U937 cells by a redox-mediated p38 MAP kinase pathway

FASEB J.

Decreased GSSG reductase activity enhances cellular zinc toxicity in three human lung cell lines

Arch. Toxicol.

Glutathione reductase in the south-western province of Saudi Arabia—genetic variation vs. acquired deficiency

Haematologia (Budapest)

Riboflavin status in Saudi Arabia—a comparative study in different regions

Trop. Geogr. Med.

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