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.

Fig. 1

Nrf2-dependent induction of glutathione reductase in the lungs of mice in response to cigarette smoke-induced oxidative stress. (A) Quantification of mRNA expression of GSR in Nrf2^+/+ and Nrf2^−/− lungs after CS exposure. Lungs were isolated from mice immediately after 5 h of CS exposure and mRNA expression of GSR was analyzed by real-time RT-PCR. Data are presented as the means±SD; n=3. (B) Protein expression of GSR in Nrf2^+/+ and Nrf2^−/− lungs after CS exposure. Lungs were isolated from mice immediately after 5 h of CS, and GSR expression was analyzed by Western blot. Immunoblot is a representative of two mouse lungs from three independent analyses. (C) Quantification of GSR protein expression in the lungs of air- and CS-exposed Nrf2^+/+and Nrf2^−/− mice; n=6 per group. *Significant compared to air controls; p < 0.05. (D) GSR enzyme activity in the lungs of Nrf2^+/+ and Nrf2⁻ mice 24 h after CS exposure. Enzyme activity was measured in lung lysates and expressed as nmol of NADPH oxidized/min/mg protein; n=4 per group. (E) Immunohistochemical analysis of GSR protein in the lungs of Nrf2^+/+ and Nrf2^−/− mice after CS exposure. At 24 h post-CS exposure, lungs were fixed and processed for immunohistochemical analysis using anti-GSR antibody. Intense staining was observed in the epithelial cells lining the small and large airways of CS-exposed Nrf2^+/+ mice (E-II), whereas weak or no staining was observed in the lung sections of the CS-exposed Nrf2^−/− mice (E-IV) and the air-exposed Nrf2^+/+ (E-I) and Nrf2^−/− mice (E-III). Original magnification: 20×. Abbreviations are as follows: BV, blood vessel; AW, airway; and AE, airway epithelium. For (A) and (C), relative fold change is abbreviated RFC.

Fig. 2

Activation of Nrf2 by tBHQ treatment and disruption of Keap1 induces GSR expression. (A) Expression of GSR mRNA in Nrf2^+/+ and Nrf2^−/− MEFs treated with tBHQ. MEFs were challenged with either vehicle (DMSO) or tBHQ (50 μM) for 16 h. After challenge, mRNA expression was analyzed by RT-PCR. The mRNA levels were normalized to GAPDH endogenous control; n=3. (B) Protein expression of GSR in Nrf2^+/+ and Nrf2^−/− MEFs treated with tBHQ. MEFs were challenged with tBHQ (50 μM), and 16 h later, GSR protein expression was analyzed by immunoblot. Immunoblot is representative of two biological duplicates of three independent analyses. (C) Quantification of GSR protein expression in the MEFs after tBHQ treatment; n=6. (D) Basal expression of GSR mRNA in Nrf2^+/+, Nrf2^−/−, and Keap^−/− MEFs as analyzed by real-time RT-PCR. Data are represented as fold change relative (RFC) to Nrf2^+/+ cells; n=3. *Significant compared to Nrf2^+/+ or vehicle; p < 0.05.

Fig. 3

Oxidized glutathione induces apoptosis in Nrf2^+/+ and Nrf2^−/− MEFs. Approximately 100 MEFs per genotype were microinjected with GSSG (50 μM), GSH (50 μM), or PBS solution. Apoptosis was analyzed by annexin V binding 90 min after microinjection. (A) Photomicrographs illustrating annexin V staining in cells microinjected with GSSG compared to no staining seen in cells microinjected with GSH and PBS. (B) Percentage of apoptotic cells in Nrf2^+/+ and Nrf2^−/− MEFs 90 min after GSSG microinjection. Values presented are percentage annexin V-positive cells of the total number of injected cells. (C) Transfection of GSR overexpression vector increased GSR mRNA expression in Nrf2^−/− MEFs 48 h posttransfection. *Significant relative to Nrf2^+/+ vehicle, and †significant relative to Nrf2^−/− MEF cells transfected with empty vector (p < 0.05). (D) Overexpression of GSR rescues Nrf2^−/− MEFs from tBHQ-induced cytotoxicity. Results are presented as percentage cell death relative to untreated Nrf2^+/+ MEFs; n=3. *Significant relative to control (p < 0.05).

Fig. 4

Glutathione reductase maintains GSH redox state and is critical for cell survival. (A) GSR mRNA expression in Nrf2^+/+ MEFs 48 h after GSR siRNA, SS siRNA, and mock transfection. Data are expressed as the relative fold change (RFC)±SD; n=3. (B) Basal GSR enzyme activity in MEFs 48 h after transfection with SS siRNA and GSR siRNA. Enzyme activity was measured in cell lysates and is expressed as nmol of NADPH oxidized/min/mg protein; n=3. (C) tBHQ-induced cell death in MEFs transfected with GSR siRNA. After 48 h of GSR siRNA transfection, MEFs were treated with tBHQ (150 μM) for 4 h, and cell death was analyzed by MTT assay; n=3. (D) Levels of total GSH in MEFs 16 h after BCNU, a potent inhibitor of GSR treatment; n=3. (E) Levels of reduced GSH and GSSG are expressed as a percentage of total GSH. Significant compared to vehicle control at p<0.05. (F) Levels of total, oxidized, and reduced glutathione in Nrf2^+/+ and Nrf2^−/− MEFs 24 h after treatment with tBHQ (100 μM). For each treatment and genotype, reduced GSH and GSSG levels are presented as relative percentages of total glutathione in control Nrf2^+/+ vehicle-treated cells (RTC).

Fig. 5

Nrf2 mediates transcriptional regulation of the GSR gene via the ARE. Functional analysis of three AREs in the GSR promoter by transient transfection in Nrf2^−/−, Nrf2^+/+, and Keap^−/− MEFs. (A) A 2-kb portion 5′ upstream of the translation start site of the GSR gene was analyzed for the presence of AREs. The three AREs identified are highlighted in red with orientation indicated by the black arrows. The transcription start site is indicated in yellow. (B) The sequence alignment of the individual AREs and the NQO1 ARE is shown along with the consensus sequence. (C) Fragments of the GSR promoter containing all three AREs and deletion constructs containing ARE1 and ARE2 or only ARE1 were cloned into the pGL3 Basic luciferase reporter vector. These constructs were transiently transfected into Nrf2^−/− and Keap^−/− MEFs and luciferase activity was recorded after 48 h. (D) The full-length promoter region and individual AREs were cloned into the pTAL luciferase reporter vector with minimal promoter (ARE3, −1703 to −997; ARE2, −857 to −725; and ARE1, ⁻143 to +14). The putative AREs in individual constructs were subjected to site-directed mutagenesis using the primers described under Materials and methods. These constructs (individual and mu-AREs) were transiently transfected into Nrf2^−/− MEFs (no Nrf2 activity) and Keap^−/− MEFs (high Nrf2 activity) and luciferase activity was measured after 48 h. Luciferase activity was normalized by measuring the Renilla luciferase activity from a cotransfected reporter vector. Values are means±SE from three different experiments (p<0.05). *Significant compared to Nrf2^−/−. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

Nrf2 mediates transcriptional regulation of the GSR gene via the ARE. Functional analysis of three AREs in the GSR promoter by transient transfection in Nrf2^−/−, Nrf2^+/+, and Keap^−/− MEFs. (A) A 2-kb portion 5′ upstream of the translation start site of the GSR gene was analyzed for the presence of AREs. The three AREs identified are highlighted in red with orientation indicated by the black arrows. The transcription start site is indicated in yellow. (B) The sequence alignment of the individual AREs and the NQO1 ARE is shown along with the consensus sequence. (C) Fragments of the GSR promoter containing all three AREs and deletion constructs containing ARE1 and ARE2 or only ARE1 were cloned into the pGL3 Basic luciferase reporter vector. These constructs were transiently transfected into Nrf2^−/− and Keap^−/− MEFs and luciferase activity was recorded after 48 h. (D) The full-length promoter region and individual AREs were cloned into the pTAL luciferase reporter vector with minimal promoter (ARE3, −1703 to −997; ARE2, −857 to −725; and ARE1, ⁻143 to +14). The putative AREs in individual constructs were subjected to site-directed mutagenesis using the primers described under Materials and methods. These constructs (individual and mu-AREs) were transiently transfected into Nrf2^−/− MEFs (no Nrf2 activity) and Keap^−/− MEFs (high Nrf2 activity) and luciferase activity was measured after 48 h. Luciferase activity was normalized by measuring the Renilla luciferase activity from a cotransfected reporter vector. Values are means±SE from three different experiments (p<0.05). *Significant compared to Nrf2^−/−. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

CHIP analysis demonstrating Nrf2 binding to GSR AREs. (A) Nuclear extracts from Nrf2^−/−, Nrf2^+/+, and Keap^−/− MEFs were used to assess the Nrf2 binding activity to ARE3 and ARE2. (A) CHIP assay was performed with Nrf2^−/−, Nrf2^+/+, and Keap^−/− MEFs using anti-Nrf2 antibody and rabbit IgG1. (B) Quantification of CHIP assay results ((GSR promoter binding-antibody)/input ratio). Quantification was performed on representative CHIP data from n=3 independent experiments.