* Department of Medicine and Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322, and Department of Environmental Health Sciences, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland 21205
Received June 30, 2004; accepted July 19, 2004
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ABSTRACT |
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Key Words: thioredoxin; glutathione; redox; NE-F2 related factor; Nrf-2.
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INTRODUCTION |
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NE-F2 related factor (Nrf-2) is a redox-sensitive transcription factor that has been implicated in cellular responses to oxidative stress. It regulates numerous genes through the antioxidant response element (ARE), such as glutathione synthesis enzymes (Moinova and Mulchahy, 1999; Wild et al., 1998), thioredoxin-1 (Kim et al., 2003
), heme oxygenase-1 (Inamdar et al., 1996
), glutathione peroxidase, glutathione disulfide reductase (Kwak et al., 2003b), glutathione S-transferases (Rushmore and Pickett, 1993
), and others. While Nrf-2 is normally sequestered in the cytoplasm by an inhibitor molecule, Keap-1, oxidative stress can stimulate its release and translocation to the nucleus (Nguyen et al., 2003
). Like AP-1 and NF-
B, a cysteine residue (cysteine506) must be in a reduced state for efficient DNA binding to occur (Bloom et al., 2002
). Regulation of Nrf-2 function is controlled by numerous factors, but the dissociation of the Nrf-2/Keap-1 complex is largely a result of the modification of cysteine residues in Keap-1 (cysteine257, 273, 288, 297) in the cytoplasm through either direct conjugation or oxidation (Dinkova-Kostova et al., 2002
; Nguyen et al., 2003
).
Two major redox systems, the glutathione (GSH) and thioredoxin (TRX) systems, provide control of intracellular thiol/disulfide redox environments. Both are ubiquitously distributed among mammalian cell types and function in peroxide elimination, protein thiol/disulfide regulation and as coenzymes. In addition, both are absolutely essential for mammalian life as evidenced by embryonic lethality in GCL and Trx-1 knockout mice (Dalton et al., 2000; Matsui et al., 1996
). Major differences are that GSH is a small peptide present in millimolar concentrations while Trx-1 is a 12 kDa protein present in micromolar concentrations. GSH is a monothiol ideally suited for regulation of protein function involving S-thiylation while Trx-1 contains a conserved dithiol reactive center ideally suited for 2-electron reductions.
However, both GSH and TRX can regulate type I (thiylation) and II (dithiol/disulfide) redox switches, which provide the mechanistic bases for control of the transitions between proliferation, differentiation, and apoptosis (Schafer and Buettner, 2001). While there is substantial evidence for overlapping functions of the GSH and TRX systems, evidence shows that their redox potentials are not in equilibrium. For instance, Caco-2 cells undergo a +40 mV oxidation in the GSH/GSSG redox couple during differentiation but the TRX/TRX-SS redox couple is unaffected (Nkabyo et al., 2002
). Due to the lack of rapid redox equilibration between GSH and TRX in cells, each system may regulate independent processes within signal transduction pathways.
Nrf-2 activity is triggered by the dissociation of the Nrf-2/Kelch-like ECH-associating protein 1 (Keap-1) complex and is controlled by a separate set of cysteine residues (on Keap-1 [cysteine257, 273, 288, 297]) to those residues that regulate DNA binding (cysteine506) (Dinkova-Kostova et al., 2002). Consequently, compartmentation of Nrf-2 signaling suggests that the GSH and the TRX systems may dictate different steps in the Nrf-2 pathway. GSH is present at high concentrations in the cytoplasm and acts as the major detoxification system during ROS generation, while Trx-1 is better suited for reducing oxidized proteins. In the present study, we used selective modification of the GSH and TRX systems to test whether the cytoplasmic dissociation of Nrf-2 is primarily regulated by cytoplasmic GSH concentrations and the nuclear reduction of Nrf-2 cysteine506 for DNA binding is primarily regulated by Trx-1. The results show that these two redox control systems function at different sites in the Nrf-2 signaling pathway. Nonequilibrium of the GSH and TRX systems and compartmentation of Nrf-2 signaling thereby provide a mechanism for maintaining the integrity of Nrf-2 signaling, which involved both oxidative and reductive steps, during periods of oxidative stress.
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MATERIALS AND METHODS |
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Glutathione and glutathione disulfide measurement by high performance liquid chromatography (HPLC). GSH and GSSG were assayed by HPLC as S-carboxymethyl, N-dansyl derivatives using -glutamylglutamate as an internal standard as described by Jones (2002)
.
Immunoblot Analysis of Trx-1. HeLa cells were incubated with 2 mM NAC or 100 µM BSO for 24 h after which cells were collected in a cell lysis buffer (350 mM NaCl, 1 mM MgCl2, 0.5 mM EDTA, 0.1 mM EGTA, 1% Igepal in 20 mM Hepes [pH 7.5]) containing protein inhibitors (Complete mini-tab, Roche, Indianapolis, IN). Samples were separated by SDS-PAGE on a 12% polyacylamide gel after which they were transferred to a nitrocellulose membrane. Trx-1 was detected with a goat primary antibody raised against human Trx-1 (American Diagnostica, Greenwich, CT) and with an AlexaFluor 680 nm anti-goat IgG secondary antibody (Molecular Probes, Eugene, OR). Membranes were scanned with an Odyssey fluorescence scanner (Li-Cor, Lincoln, NE). Densitometric analysis was performed with the Odyssey scanning software.
Redox Western. Redox Western analysis of Trx-1 redox state was slightly modified from the original protocol as described by Watson et al. (2003). Trx-1 was carboxymethylated in guanidine-Tris solution (6 M guanidine-HCl, 50 mM Tris, pH 8.3, 3 mM EDTA, 0.5% (v/v) Triton X-100) containing 50 mM iodoacetic acid (IAA) and incubated for 30 min at 37°C. Excess IAA was removed by Sephadex chromatography (MicroSpin G-25 columns, Amersham Biosciences) after which samples were diluted in 5x nonreducing sample buffer (0.1 M Tris-HCl, pH 6.8, 50% (v/v) glycerol, 0.05% (w/v) bromophenol blue) and separated on a discontinuous native polyacrylamide gel (5% stacking gel, 15% resolving gel). Gels were electroblotted to polyvinylidene difluoride membrane and probed for Trx1 using anti-Trx1 primary antibody (American Diagnostica, Greenwich, CT) and AlexaFluor 680 nm anti-goat IgG secondary antibody (Molecular Probes, Eugene, OR). Membranes were scanned with an Odyssey fluorescence scanner (Li-Cor, Lincoln, NE). Densitometric analysis was performed with the Odyssey scanning software. Redox potentials were determined using band intensities and the Nernst equation (Eh = E0 + 2.3 x RT/nF x log ([TRX-SS]/[TRX-SH2]; E0 = 254 mV at pH 7.4)
Nrf-2 localization. Nuclear and cytoplasmic isolates were collected with the TransFactor Extraction kit (Clontech, Palo Alto, CA). Nrf-2 localization in the nucleus was determined by SDS-PAGE (12% polyacrylamide) analysis using a goat Nrf-2 antibody for primary detection (Santa Cruz Biotechnology, Santa Cruz, CA). A donkey anti-goat AlexaFluor 680 IgG antibody (Molecular Probes, Eugene, OR) was used for secondary detection. Membranes were subsequently scanned by an Odyssey fluorescence scanner (Li-Cor, Lincoln, NE). Densitometry was quantified by the Odyssey scanning software.
Expression vector transfections. Cells were grown until they reached 70% confluence. All transfections were performed using Fugene6 transfection reagent (Roche, Indianapolis, IN) per the manufacturer's instructions. Plasmid pcDNA3.1 encoding human Trx-1 was a kind gift from Dr. Jiyang Cai of Vanderbilt University. The nuclear-targeted Trx-1 was sublconed into pcDNA3.1 encoding C35S Trx-1 and wild-type Trx-1 containing the SV40 T-antigen nuclear localization sequence (PPKKKRKVEDP). Site-directed mutagenesis was performed on the Trx-1-NLS construct to introduce the C35S mutation into the active site by Gene Tailor site-directed mutagenesis kit (Invitrogen). The C35S Trx-1-NLS mutant was generated by hybridization of the plasmid with oligonucleotides of the sequence 5'-CCACGTGGCTGAGAAGTCAACTATACAAGT-3' and 5'-TTGACTTCTCAGCCACTGGGTGTGGGCCTTCA-3', respectively. Clones containing the desired mutations were selected and verified by DNA sequencing. The ARE4-luciferase reporter construct was a kind gift from Dr. Jerry J. Gipp (University of Wisconsin, Madison, WI). Luciferase and ß-galactosidase enzyme assays were measured as described previously (Go et al., 2004
). Luciferase measurements were normalized to ß-galactosidase activity as determined with the ß-galactosidase enzyme assay kit under conditions outlined by the manufacturer (Promega, Madison, WI).
Statistical analysis. Each measurement is the result of three independently performed experiments. The one-way analysis of variance (ANOVA) was employed to determine whether the means of different groups were significantly different. The Tukey's post-hoc test was used to determine the significance for all pairwise comparisons of interest.
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RESULTS |
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Increased Trx-1 Expression Does Not Affect GSH Redox State
Although the redox states of Trx-1 and GSH are not equilibrated, increased expression of Trx-1 could affect GSH redox state. To test this, cells were transiently transfected to overexpress Trx-1 and related mutants. Evaluation by immunoblotting confirmed overxpression of Trx-1 over endogenous, wild-type Trx-1 (data not shown). Overexpression of Trx-1 and related mutants did not have any significant effect on GSH redox state or GSH concentration. (Figs. 3A and 3C). After 48 h of transient transfection with the various Trx-1 expression plasmids, GSH/GSSG redox potential ranged from 257 (±4) to 261 (±5) mV and were not different (Fig. 3). Together with the above data, these results show that the GSH and TRX redox states are independently controlled and that BSO and transient transfection with Trx-1 and Trx-1 mutants provide an approach to evaluate selective functions of the two systems.
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Oxidation of GSH/GSSG Is Associated with Nuclear Activation of the ARE
To measure nuclear activity of Nrf-2, HeLa cells were transiently transfected with the ARE4-L reporter construct. Modulation of total GSH concentrations was achieved via 24 h incubation with either 2 mM NAC or 100 µM BSO and then treated with or without TBHQ for 6 h as described above. The expression of the reporter increased with TBHQ treatment only (Fig. 7). Expression of the reporter was increased by BSO and decreased by NAC pretreatment. As expected, TBHQ increased expression of the reporter. Furthermore, BSO increased and NAC decreased TBHQ-induced expression. Because these changes in transcriptional activity so completely paralleled the nuclear translocation of Nrf-2, the results provide evidence for GSH/GSSG control solely in the cytoplasmic compartment and provide no evidence for function of GSH in the nuclear regulation of Nrf-2 binding to the ARE.
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DISCUSSION |
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Compartmentation of specific Nrf-2 events appears to be dependent upon the concentration of GSH in the cytoplasm and Trx-1 in the nucleus. Much study has focused on compartmentation of GSH, especially between the cytoplasm and nucleus, but due to pores in the nuclear envelope, it is difficult to measure nuclear GSH concentrations by traditional chromatography methods. Still, some studies have concluded that there are higher concentrations of GSH in the nucleus vs. the cytoplasm using fluorescent dyes (Bellomo et al., 1992; Voehringer et al., 1998
), while others arrive at opposing conclusions using GSH antibodies (Cotgreave, 2003
). Trx-1 is much larger and is not freely permeable across the nuclear envelope, allowing measurement in the nucleus (i.e., via Redox Western analysis) (Watson and Jones, 2003
). Evidence supports differences in nuclear and cytoplasmic Trx-1 pools (Watson and Jones, 2003
) and a mechanism for nuclear sequestration of Trx-1 during oxidative stress (Hirota et al., 1999
).
Nrf-2 is a critical component in the response to oxidative stress and upregulates multiple genes to ensure cell survival (Kwak et al., 2003a). Cell models have illustrated changes in GSH redox potential with proliferation and differentiation (Hutter et al., 1997; Kirlin et al., 1999
; Nkabyo et al., 2002
). In combination with the results in the present study, one may speculate that proliferating cells would be less likely to activate Nrf-2 due to higher concentrations of GSH and a lower redox potential (4060 mV more reduced). Conversely, differentiated cells that contain less GSH would be prone to activate the Nrf-2 pathway even with a milder oxidative signal. Embryonic development is composed of multiple subpopulations of proliferating and differentiating cells, which have different GSH redox potentials and regulate different redox-sensitive transcription factors. Measurements of GSH, cysteine, and Trx-1 concentrations in early organogenesis stage rat and rabbit embryo limbs, trunks, and head regions showed marked differences in GSH concentrations, GSSG concentrations, and GSH/GSSG redox potentials (Hansen et al., 2001
), and recent studies have shown stem cell totipotentiality and blastocyst differentiation are regulated by the activity of the redox-sensitive transcription factor Oct-4 (Guo et al., 2004
). Because redox mechanisms control both differentiation (Oct-4) and detoxification (Nrf-2) cellular protection may vary in proliferating and differentiating cells, where proliferating cells (with higher GSH concentrations and GSH/GSSG ratios) may not activate Nrf-2 are readily as differentiating cells. This has important implications for developmental toxicity where disruption of redox control can substantially alter gene expression patterns and result in teratogenesis.
In summary, the present data show that oxidant-induced Nrf-2 dissociation in the cytoplasm is controlled by GSH while DNA interactions are controlled by nuclear Trx-1. Thus, even though the GSH and TRX systems have overlapping activities, compartmentation and specificity of interactions allow distinct roles in Nrf-2 signaling. Activation in response to an oxidant signal can occur in the cytoplasm and is dependent upon the GSH system, while nuclear Trx-1 maintains the Nrf-2 signal by reducing critical cysteines in the DNA binding domain. With tandem, complementary functions, GSH and Trx-1 regulate Nrf-2 activity for proper responses to oxidative insult.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at 1510 Clifton Rd NE, Rollins Research Center Room 4131, Department of Medicine, Emory University School of Medicine, Atlanta, GA 30322. Fax: (404) 727-3452. E-mail: dpjones{at}emory.edu.
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