Oncogene (2002) 21, 2191 ± 2200 ã 2002 Nature Publishing Group All rights reserved 0950 ± 9232/02 $25.00 www.nature.com/onc Site-directed mutagenesis of cysteine to serine in the DNA binding region of Nrf2 decreases its capacity to upregulate antioxidant response element-mediated expression and antioxidant induction of NAD(P)H:quinone oxidoreductase1 gene David Bloom1, Saravanakumar Dhakshinamoorthy1 and Anil K Jaiswal*,1 1Department of Pharmacology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas, TX 77030, USA NF-E2 related factor 2 (Nrf2) is a CNC/b-zip protein mation (Thelen et al., 1993), ionizing radiation (Breen that regulates antioxidant response element (ARE)- and Murphy, 1995), and cellular respiration (Grisham mediated expression, and antioxidant induction, of and McCord, 1986). These stimuli result in the detoxifying enzyme genes, including NAD(P)H:quinone production of free radicals, including reactive oxygen oxidoreductase1 (NQO1). A comparison of Nrf2 from species (ROS). ROS, and xenobiotic and antioxidant dierent species, and with other b-zip proteins, revealed generated electrophiles, attack DNA and other cellular the presence of a highly conserved cysteine residue at macromolecules, causing aging, neurodegenerative dis- position 506 in the DNA binding domain of Nrf2. Site- eases, arthritis, arteriosclerosis, and tumor induction directed mutagenesis was used to mutate this cysteine to and promotion (Breen and Murphy, 1995; Grisham serine. Transfection/over expression experiments in hu- and McCord, 1986; Ward, 1994; Rosen et al., 1995). man hepatoblastoma (Hep-G2) cells demonstrated that Cells have developed a defense mechanism to detoxify mutant Nrf2 (mNrf2), containing the C506S mutation, and remove electrophiles and ROS. During times of was signi®cantly less ecient in activating ARE-mediated electrophilic and oxidative stress, a battery of detoxify- gene expression, and induction in response to tert-butyl ing genes is coordinately induced through the anti- hydroquinone (t-BHQ), as copmpared with wild-type oxidant response element (ARE) (Rushmore and Nrf2. N-ethyl malemide (NEM), a sulfhydryl cross- Pickett, 1993; Jaiswal, 1994). These genes include linker, inhibited Nrf2 but not mNrf2C506S-mediated NAD(P)H:quinone oxidoreductases (NQOs), which expression of NQO1. This further implicated the cysteine compete with cytochromes P450 and P450 reductase at position 506 in Nrf2 regulation of ARE-mediated gene and catalyze two-electron reductive metabolism and expression. Nuclear localization experiments revealed detoxi®cation of quinones (Joseph et al., 1994; that C506S mutation did not aect the retention of Radjendirane et al., 1997; Talalay et al., 1995; Riley Nrf2 by INrf2/Keap1 in the cytosol, or its release in and Workman, 1992; Ernster, 1987); glutathione response to antioxidants. However, band and supershift S-transferases (GSTs), which conjugate hydrophobic assays showed a signi®cant reduction in the binding of electrophiles and ROS with glutathione (Pickett and mNrf2C506S to the NQO1 gene ARE as compared with Lu, 1989; Tsuchida and Sato, 1992); UDP-glucurono- wild-type Nrf2. Therefore, the C506S mutation in Nrf2 syl transferases (UDP-GT), which catalyze the con- lowered its anity for the ARE, leading to decreased jugation of glucuronic acid with xenobiotics and drugs, expression, and antioxidant induction, of NQO1. leading to their excretion (Tephly and Burchell, 1990); Oncogene (2002) 21, 2191 ± 2200. DOI: 10.1038/sj/ epoxide hydrolase (EH), which inactivates epoxides onc/1205288 (Oesch et al., 1991); g-glutamylcysteine synthetase (g-GCS), which plays a role in glutathione metabolism Keywords: NQO1; ARE; detoxifying genes; Nrf2; (Mulkahy et al., 1997); ferritin-L gene, which plays an cysteine; redox regulation important role in iron storage (Wasserman and Fahl, 1997); and heme oxygenase-1 (HO-1), which catalyses the ®rst and rate-limiting step in heme catabolism Introduction (Choi and Alam, 1996). In response to xenobiotic and antioxidant stress, Cellular oxidative stress is caused by a variety of Nrf2, a basic leucine zipper transcription factor (bZIP), external and internal stimuli, including xenobiotics and binds to the ARE and positively regulates ARE- antioxidants (Dhakshinamoorthy et al., 2000), in¯am- mediated induction of detoxifying genes, including NQO1 (Venugopal and Jaiswal, 1996). Nrf2 is also known to regulate ARE-mediated expression and induction of GST Ya, g-GCS and HO-1 genes *Correspondence: AK Jaiswal; E-mail: [email protected] Received 5 October 2001; revised 3 January 2002; accepted 7 (Venugopal and Jaiswal, 1996; Nguyen et al., 2000; January 2002 Jayepaul and Jaiswal, 2000; Wild et al., 1999; Alam et Redox regulation of Nrf2 D Bloom et al 2192 al., 1999). Under normal conditions, Nrf2 is held in the amount of pcDNA-mNrf2 in the same experiment cytoplasm by a cytosolic inhibitor INrf2/KEAP1 (Itoh resulted in 25% repression of ARE-mediated CAT et al., 1999; Dhakshinamoorthy and Jaiswal, 2001). gene expression. The other two concentrations of the When cells are challenged by oxidative stress, Nrf2 is expression plasmids also showed signi®cant dierences released from INrf2/Keap1 and translocates to the between the eciency of Nrf2 and mNrf2 to upregulate nucleus, where it activates transcription. The signal, ARE-mediated CAT gene expression. This dierence in which causes this release and activates Nrf2, is not activity was not due to a dierence in the amount of clearly understood. Nrf2 has been shown to hetero- expression. HepG2 cells transfected with V5 tagged dimerize with c-Jun and small Maf proteins (MafG and Nrf2, or mNrf2, were lysed with RIPA buer, 100 mg MafK). These heterodimeric complexes bind to the of the whole cell extracts were resolved on an SDS ± ARE, and alter ARE-mediated gene expression PAGE. Western blots were performed with the V5 (Venugopal and Jaiswal, 1998; Dhakshinamoorthy antibody and demonstrated equal expression of the two and Jaiswal, 2000; Itoh et al., 1997; Nguyen et al., proteins in transfected Hep-G2 cells (Figure 2b). 2000; Wild et al., 1999). To determine if mNrf2 would respond to antiox- bZIP proteins, including c-Jun and c-Fos, contain a idants, both wild-type Nrf2 and mNrf2 were transiently highly conserved cysteine in their DNA binding transfected into HepG2 cells. The cells were then domains (Abate et al., 1990). This cysteine residue treated with 100 mM or 200 mM t-BHQ for 16 h. mNrf2 has been shown to be a site of redox regulation in activity was induced in response to 100 mM t-BHQ c-Jun, aecting its binding to the AP1 site (Abate et treatment, but again the response was attenuated as al., 1990). Interestingly, all species of Nrf2 also compared to wild-type Nrf2 (Figure 3). 200 mM t-BHQ contains this cysteine residue in their DNA binding treatment caused an inhibition of Nrf2 activity. mNrf2, region (Venugopal and Jaiswal, 1998). In addition, it however, was still induced by 200 mM t-BHQ treat- has been reported that ARE-mediated expression and ment. induction of the NQO1 gene is sensitive to sulfhydryl N-ethyl malemide (NEM) was used to determine if modifying agents (Li and Jaiswal, 1994). The present C506, in Nrf2, is a target for oxidation-reduction studies were performed to determine the role of this (redox) regulation. Hep-G2 cells cotransfected with the cysteine residue in Nrf2 regulation of ARE-mediated reporter plasmid, NQO1 ARE-tk-CAT, expression gene expression, and induction in response to anti- plasmid pcDNA, or pcDNA-Nrf2 were treated with oxidants and xenobiotics. 0.25 mM or 0.5 mM NEM. These cells showed a In this report, we show that site-directed mutagenesis signi®cant repression of ARE-mediated CAT expres- of cysteine to serine in the DNA binding domain of sion (Figure 4). However, in a similar experiment, the Nrf2 leads to a signi®cant decrease in the capacity of mNrf2 regulation of ARE-mediated CAT expression Nrf2 to upregulate ARE-mediated NQO1 gene expres- was more or less unaected after treatment with NEM. sion, and induction in response to t-BHQ. The C506S GFP tagged proteins were used to show the mutation did not aect Nrf2 interaction with INrf2/ interaction of Nrf2/mNrf2 with INrf2 (Figure 5). The Keap1. This mutation also had no eect on the transfection of QT6 cells, with EGFP-Nrf2 or EGFP- heterodimerization of Nrf2 with MafG, or the binding mNrf2 led to the expression of the respective proteins of the Nrf2/MafG complex to the NQO1 gene ARE. in both the cytosolic and nuclear compartments However, binding of the mNrf2/c-Jun complex to the (Figure 5, left panels). The treatment of transfected ARE was signi®cantly reduced as compared to QT6 cells with DMSO had no eect on the localization Nrf2-c-Jun. Therefore, the cysteine at position 506 of of Nrf2 or mNrf2 (data not shown). The coexpression Nrf2 is required for interaction with speci®c hetero- of INrf2 with EGFP-Nrf2 or EGFP-mNrf2 retained dimeric partner proteins and/or binding of these the respective proteins in the cytosol (Figure 5, middle heterodimeric complexes to the ARE. panels). The treatment of these cells with t-BHQ caused the release of Nrf2, or mNrf2, which then translocated to the nucleus (Figure 5, right panels). Results Nrf2, mNrf2, MafG-V5 and c-Jun were in vitro translated; the translation was con®rmed by SDS ± The cysteine residue at position 506 of Nrf2 was PAGE (Figure 6b) and Western analysis (data not successfully converted to a serine residue using site shown). These proteins were used in band and super directed mutagenesis. The molecule created, shift assays. Nrf2-c-Jun heterodimers, MafG-V5 homo- Nrf2C506S, is referred to as mNrf2 (Figure 1). Over dimers and Nrf2/mNrf2-MafG-V5 heterodimers all expression of either wild-type Nrf2 or mNrf2 in HepG2 bound to the NQO1 gene ARE (Figures 6 and 7). cells lead to a dose dependent increase in ARE activity Nrf2-c-Jun binding to the ARE required pretreatment (Figure 2a).