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

C. J. Harvey; R. K. Thimmulappa; A. Singh; D. J. Blake; G. Ling; N. Wakabayashi; J. Fujii; A. Myers; S. Biswal

doi:10.1016/j.freeradbiomed.2008.10.040

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

C. J. Harvey, R. K. Thimmulappa, A. Singh, D. J. Blake, G. Ling, N. Wakabayashi, J. Fujii, A. Myers, S. Biswal

Bloomberg School of Public Health

Research output: Contribution to journal › Article › peer-review

268 Scopus citations

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.

Original language	English (US)
Pages (from-to)	443-453
Number of pages	11
Journal	Free Radical Biology and Medicine
Volume	46
Issue number	4
DOIs	https://doi.org/10.1016/j.freeradbiomed.2008.10.040
State	Published - Feb 15 2009

Keywords

COPD
Cigarette smoke
Emphysema
Free radicals
Glutathione
Glutathione reductase
Nrf2
Oxidative stress

ASJC Scopus subject areas

Biochemistry
Physiology (medical)

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10.1016/j.freeradbiomed.2008.10.040

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title = "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.",

keywords = "COPD, Cigarette smoke, Emphysema, Free radicals, Glutathione, Glutathione reductase, Nrf2, Oxidative stress",

author = "Harvey, {C. J.} and Thimmulappa, {R. K.} and A. Singh and Blake, {D. J.} and G. Ling and N. Wakabayashi and J. Fujii and A. Myers and S. Biswal",

note = "Funding Information: 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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doi = "10.1016/j.freeradbiomed.2008.10.040",

language = "English (US)",

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T1 - Nrf2-regulated glutathione recycling independent of biosynthesis is critical for cell survival during oxidative stress

AU - Harvey, C. J.

AU - Thimmulappa, R. K.

AU - Singh, A.

AU - Blake, D. J.

AU - Ling, G.

AU - Wakabayashi, N.

AU - Fujii, J.

AU - Myers, A.

AU - Biswal, S.

N1 - Funding Information: 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.

PY - 2009/2/15

Y1 - 2009/2/15

N2 - 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.

AB - 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.

KW - COPD

KW - Cigarette smoke

KW - Emphysema

KW - Free radicals

KW - Glutathione

KW - Glutathione reductase

KW - Nrf2

KW - Oxidative stress

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ER -

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

Abstract

Keywords

ASJC Scopus subject areas

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