Compartmentation of Nrf-2 Redox Control: Regulation of Cytoplasmic Activation by Glutathione and DNA Binding by Thioredoxin-1

Jason M. Hansen*, Walter H. Watson{dagger} and Dean P. Jones*,1

* Department of Medicine and Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322, and {dagger} Department of Environmental Health Sciences, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland 21205

Received June 30, 2004; accepted July 19, 2004


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Nrf-2 is a redox-sensitive transcription factor that is activated by an oxidative signal in the cytoplasm but has a critical cysteine that must be reduced to bind to DNA in the nucleus. The glutathione (GSH) and thioredoxin (TRX) systems have overlapping functions in thiol/disulfide redox control in both the cytoplasm and the nucleus, and it is unclear whether these are redundant or have unique functions in control of Nrf-2-dependent signaling. To test whether GSH and Trx-1 have distinct functions in Nrf-2 signaling, we selectively modified GSH by metabolic manipulation and selectively modified Trx-1 expression by transient transfection. Cytoplasmic activation of Nrf-2 was measured by its nuclear translocation and nuclear activity of Nrf-2 was measured by expression of a luciferase reporter construct containing an ARE4 from glutamate cysteine ligase. Results showed that tert-butylhydroquinone (TBHQ), a transcriptional activator that functions through Nrf-2/ARE, promoted Nrf-2 nuclear translocation by a type I (thiylation) redox switch which was regulated by GSH not by Trx-1. In contrast, the ARE reporter was principally controlled by nuclear-targeted Trx-1 and not by GSH. The data show that the GSH and TRX systems have unique, compartmented functions in the control of transcriptional regulation by Nrf-2/ARE.

Key Words: thioredoxin; glutathione; redox; NE-F2 related factor; Nrf-2.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Gene expression responses to oxidative stress are necessary to ensure cell survival and are largely attributed to specific redox-sensitive transcription factors. Activator protein-1 (AP-1) is a redox-sensitive transcription factor that is responsive to low levels of oxidants resulting in AP-1/DNA binding and an increase in gene expression. AP-1 activation is due to the induction of Jun kinase (JNK) activity by oxidants resulting in the phosphorylation of serine63 and serine73 in the c-Jun transactivation domain (Karin, 1995Go; Smeal et al., 1992). With high concentrations of oxidants, AP-1 is inhibited and gene expression is impeded. Inhibition of AP-1/DNA interactions is attributed to the oxidation of specific cysteine residues in c-Jun's DNA binding region, namely cysteine252 (Abate et al., 1990Go). Similarly, nuclear factor kappa B (NF-{kappa}B), contains a critical cysteine residue (cysteine62) in the p50 subunit that is involved in DNA binding (Matthews et al., 1993Go). NF-{kappa}B is normally sequestered in the cytoplasm by I{kappa}B but under oxidative conditions, I{kappa}B is phosphorylated by I-{kappa}B kinase (IKK), ubiquitinated and subsequently degraded. Excessive oxidative stress results in the oxidation of cysteine62 which does not affect its translocation to the nucleus but rather interferes with DNA binding and decreases gene expression (Galter et al., 1994Go).

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, 1999Go; Wild et al., 1998), thioredoxin-1 (Kim et al., 2003Go), heme oxygenase-1 (Inamdar et al., 1996Go), glutathione peroxidase, glutathione disulfide reductase (Kwak et al., 2003b), glutathione S-transferases (Rushmore and Pickett, 1993Go), 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., 2003Go). Like AP-1 and NF-{kappa}B, a cysteine residue (cysteine506) must be in a reduced state for efficient DNA binding to occur (Bloom et al., 2002Go). 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., 2002Go; Nguyen et al., 2003Go).

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., 2000Go; Matsui et al., 1996Go). 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, 2001Go). 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., 2002Go). 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., 2002Go). 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture. HeLa cells were purchased from American Type Culture Collection (ATCC, Manassas, VA). Cells were grown in Eagle's Minimum Essential Medium (EMEM) supplemented with 10% Fetal Bovine Serum and antibiotics and maintained in a humidified atmosphere of 5% CO2 at 37°C. Eh calculations for cells were done using the Nernst equation with Eo values for pH 7.4 and assuming a 5-µl cell volume per mg of cell protein (Jones, 2002Go). Cells were pretreated with 0.5–2 mM N-acetylcysteine (NAC) or 1–100 µM buthionine sulfoximine (BSO) for 24 h. Similarly, for Nrf-2 activation and reporter experiments, cells were treated with 2 mM NAC or 100 µM BSO for 24 h prior to the addition of 50 µM tert-butylhydroquinone (TBHQ), a known activator of Nrf-2, for 6 h.

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 {gamma}-glutamylglutamate as an internal standard as described by Jones (2002)Go.

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)Go. 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., 2004Go). 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Altered GSH Concentrations and GSH/GSSG Redox State Do Not Affect Trx-1 Redox Status
To evaluate redox equilibration between GSH and Trx-1 in HeLa cells, cultures were treated with GSH modulating agents, NAC, to increase intracellular GSH, and BSO, to decrease GSH. NAC treatments (0.5–2 mM) for 24 h increased GSH concentrations at the highest treatment (2 mM) where concentrations were increased by approximately 15% from control GSH concentrations (Fig. 1). No significant changes in GSSG concentrations were detected. Similarly, no significant changes were seen in GSH/GSSG redox potential, ranging from –256 to –260 mV. Using BSO (1–100 µM) for 24 h, GSH was decreased in a dose-dependent manner, where GSH changes ranged from decreases of 15% (1 µM) to 85% (100 µM) of control GSH concentrations. A 15% increase in GSSG concentrations was noted with the lowest BSO concentration and subsequently higher BSO doses decreased GSSG concentrations. Together these changes in GSH and GSSG concentrations produced a dose-dependent increase in redox potential, increasing maximally by +30 mV at 100 µM BSO.



View larger version (23K):
[in this window]
[in a new window]
 
FIG. 1. (A) Changes in GSH and GSSG concentrations, (B) changes in GSH/GSSG redox potential (Eh), and (C) changes in GSH:GSSG ratios as compared to control levels following treatment with either 0.5–2.0 mM NAC or 1–100 µM BSO for 24 h. Control GSH concentrations measured 4.6 (±0.4) mM. Control GSSG measured 0.04 (±0.004) mM. Data is shown as percent changes from control and is representative of four independently performed experiments. Asterisks (*) denote a significant difference (p < 0.05) from control.

 
Redox Western blot analysis of reduced (Trx-1SH2) and oxidized (Trx-1SS) showed no changes in Trx-1 redox state with either NAC or BSO (Figs. 2A and 2B), where values ranged from –287 (±4) to –282 (±4) mV. These results show that the GSH redox state is independent of Trx-1 levels.



View larger version (29K):
[in this window]
[in a new window]
 
FIG. 2. (A) Redox Western analysis of Trx-1 following treatment with 0.5–2.0 mM NAC or 1–100 µM BSO for 24 h and with 0–50 µM TBHQ for 6 h. The upper band represents the oxidized Trx-1 pool while the reduced Trx-1 pool is represented by the lower band. No redox changes were noted in any of the treatments. (B) Redox potential (Eh) of reduced/oxidized Trx-1 as determined by band densitometry and the Nernst equation (see Methods) as determined by three separate experiments. (C) Representative immunoblots of Trx-1 protein following treatment with 0.5–2.0 mM NAC or 1–100 µM BSO for 24 h. (D) NAC treatments showed no changes in total Trx-1 but treatment with BSO (100 µM) increased total Trx-1 by 112% from control. Densitometric quantitation is an average of three separate experiments. Asterisks (*) denote a significant difference (p < 0.05) from control.

 
Although the ratio of reduced:oxidized Trx-1 did not correlate with changes in GSH and GSSG concentrations or redox potential, BSO treatment, but not NAC treatments, caused an increase in the total amount of Trx-1 over a 24 h period (Figs. 2C and 2D). Immunoblot analysis confirmed an overall increase in Trx-1 protein, up to 212% of control, following GSH depletion via BSO. These changes in total Trx-1 protein are consistent with an activation of the antioxidant response element as the Trx-1 gene is controlled by this element (Kim et al., 2003Go).

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.



View larger version (18K):
[in this window]
[in a new window]
 
FIG. 3. Changes in GSH, GSSG, and Eh (GSH/GSSG) redox potential following transfection with wild-type Trx-1, C35S mutant Trx-1, nuclear-targeted Trx-1 (NLS-Trx-1), and C35S mutant NLS-Trx-1. Data is representative of four independently performed experiments. Asterisks (*) denote a significant difference (p < 0.05) from control.

 
TBHQ Alters GSH but Not Trx-1
Tert-butylhydroquinone (TBHQ), a potent activator of the Nrf-2 pathway, is believed to generate reactive oxygen species through a redox cycling mechanism which in turn initiates Nrf-2 dissociation (Nguyen et al., 2003Go). To evaluate the effects of TBHQ on GSH, cultures were pretreated with either 2 mM NAC or 100 µM BSO for 24 h and then treated with 50 µM TBHQ for 6 h. TBHQ in cultures not receiving any pretreatment caused an increase in GSH and GSSG concentrations (Figs. 4A and 4B). In NAC and BSO pretreated cells, TBHQ similarly increased both GSH and GSSG concentrations. With TBHQ co-treatment, NAC or BSO pretreatments produced no significant differences in redox potential as compared to cultures treated with NAC or BSO only (Fig. 4C). However, notable changes in GSH:GSSG ratios were evident with TBHQ treatment. NAC co-treated with TBHQ caused a significant decrease in the GSH:GSSG ratio, while BSO co-treated with TBHQ had the opposite effect resulting in an increase (Fig. 4D). Thus, the data are consistent with an upregulation in GSH synthesis that is coupled with oxidation of GSH to GSSG. In contrast to changes in GSH redox state, TBHQ (0–50 µM) did not alter the Trx-1 redox state (Figs. 5A and 5B). The failure of TBHQ to alter Trx-1 redox state further supports that the regulation of TBHQ-induced Nrf-2 dissociation involves GSH and not Trx-1.



View larger version (14K):
[in this window]
[in a new window]
 
FIG. 4. Changes in (A) GSH, (B) GSSG, (C) Eh (GSH/GSSG) redox potential, and (D) GSH:GSSG ratios following treatment with either 2 mM NAC or 100 µM BSO for 24 h with and without 50 µM TBHQ for 6 h. Data is representative of four independently performed experiments. Asterisks (*) denote a significant difference (p < 0.05) from control. Crosses ({dagger}) denote a significant difference (p < 0.05) between cells treated with 50 µM TBHQ only and cells receiving either 2 mM NAC or 100 µM BSO and 50 µM TBHQ.

 


View larger version (34K):
[in this window]
[in a new window]
 
FIG. 5. TBHQ caused no significant oxidation of Trx-1 redox state as determined by Redox Western Blot analysis (A) and Nernst equation (B). Upper panel: Representative Western blot. Lower panel: Densitometric analysis of three separate experiments.

 
GSH/GSSG Ratio Controls Cytoplasmic Nrf-2 Dissociation/Nuclear Translocation
The ability to independently control the GSH and TRX systems allowed us to test whether cytoplasmic dissociation/nuclear translocation of Nrf-2 was controlled by one or both of these systems. Results showed that depletion of GSH with BSO increased cytoplasmic dissociation/nuclear translocation of Nrf-2 while pretreatment with NAC decreased this process (Fig. 6A). In contrast, increased expression of Trx-1 by transfection had no effect on Nrf-2 cytoplasmic dissociation/nuclear translocation (Fig. 6B).



View larger version (21K):
[in this window]
[in a new window]
 
FIG. 6. (A) Immunoblot analysis of nuclear Nrf-2 from HeLa cell nuclear fractions following treatment with either 2 mM NAC or 100 µM BSO for 24 h, with or without 50 µM TBHQ for 6 h. Asterisks (*) denote a significant difference between designated treatments. Upper panel: Representative western blot. Lower panel: Densitometric analysis of three separate experiments. (B) Immunoblot analysis of nuclear Nrf-2 from HeLa cell nuclear fractions following transient transfection with wild-type Trx-1 with and without TBHQ. Asterisks (*) denote a significant difference (p < 0.05) from control. Upper panel: Representative Western blot. Lower panel: Densitometric analysis of three separate experiments.

 
Using TBHQ as an Nrf-2 stimulator, results showed that TBHQ-induced cytoplasmic dissociation/nuclear translocation is a process that was blocked by NAC (Fig. 6A) but not by Trx-1 overexpression (Fig. 6B). Under these conditions, NAC prevented TBHQ-induced changes in GSH/GSSG ratios but did not change Eh GSH/GSSG (Fig. 4). Consequently, the data show cytoplasmic dissociation/nuclear translocation of Nrf-2 is controlled by GSH and not Trx-1. Furthermore, because type I thiol switches respond to changes in GSH/GSSG ratios while type II thiol switches respond to changes in Eh (Gilbert, 1990; Schafer and Beuttner, 2001Go), the data are consistent with regulation by a type I redox switch and not consistent with a type II switch.

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.



View larger version (13K):
[in this window]
[in a new window]
 
FIG. 7. Effects of glutathione modulators on ARE4-luciferase induction. (A) Treatment with 50 mM TBHQ for 6 h induces luciferase expression. Data are represented as luciferase activity/ß-galactosidase activity as described in the methods section. (B) Treatment with 2 mM NAC or 100 µM BSO for 24 h without TBHQ stimulation. (C) Treatment with 2 mM NAC or 100 µM BSO for 24 h followed by treatment with 50 µM TBHQ for 6 h. Data in B and C are represented as percent changes from control (depicted in A). Data is representative of at least three independently performed experiments. Asterisks (*) denote a significant difference (p < 0.05) from control.

 
Nuclear Trx-1 Mediates Nrf-2/DNA Interactions
In vitro studies show that Trx-1 functions in the reduction of cysteine residues critical for transcription factor binding to DNA (Hirota et al., 1997Go; Makino et al., 1999Go; Matthews et al., 1993Go). Because overexpression of Trx-1 had no effect on cytoplasmic dissociation of Nrf-2, co-transfection of Trx-1 along with the ARE4 reporter provided an opportunity to determine whether Trx-1 functions in the Nrf-2 activation of the ARE. Trx-1 overexpression caused a significant increase in ARE activation as determined by luciferase activity but Trx-1 active site mutant, C35S Trx-1, inhibited ARE activation (Fig. 8). TBHQ induction of Nrf-2 activity only occurred with wild-type Trx-1 overexpession but Nrf-2 activity was inhibited by C35S Trx-1. Consequently, the data show that Trx-1 functions in transcriptional activation but not in nuclear translocation, as expected from the previous characterized reduction of the cysteine in the DNA binding site (Bloom et al., 2002Go).



View larger version (19K):
[in this window]
[in a new window]
 
FIG. 8. ARE4-Luciferase reporter assay following transient co-transfection with wild-type Trx-1, C35S mutant Trx-1, NLS-Trx-1 and C35S mutant NLS-Trx-1 for 24 h with or without 50 µM TBHQ for 6 h. Data is representative of three independently performed experiments. Asterisks (*) denote a significant difference (p < 0.05) between designated groups.

 
To further test whether this occurred in the nuclei, we used a nuclear targeted fusion protein created with a SV-40 nuclear translocation sequence complexed to Trx-1 (NLS-Trx-1). Transfection with NLS-Trx-1 showed an even greater increase in Nrf-2/ARE activity than with wild-type Trx-1 (Fig. 8). Overexpression of NLS-C35S Trx-1 mutant decreased Nrf-2/ARE activity and appeared to have similar effects as the C35S Trx-1 mutant (Fig. 8). TBHQ treatment increased Nrf-2/ARE activity overexpressing NLS-Trx-1 but decreased in cells transfected with the NLS-C35S Trx-1 mutant. Because Trx-1 overexpression had no effect on Nrf-2 dissociation/nuclear translocation (Fig. 6) it is clear that nuclear Trx-1 plays a distinct role downstream of the cytoplasmic Nrf-2 dissociation/nuclear translocation events, namely Trx-1 regulates Nrf-2 at the DNA interaction level.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Accumulating evidence has shown nonequilibrium between the GSH and TRX systems but, as a result, has not demonstrated independent regulation of specific redox-sensitive signal transduction pathways. In the present study, we establish the independence of these two redox systems in Nrf-2 signaling. Two redox-sensitive steps comprise Nrf-2 signaling: the dissociation of the Nrf-2/Keap-1 complex in the cytoplasm and the binding of Nrf-2 to the ARE in the nucleus. Nrf-2 can be activated by oxidants, and consequently, antioxidants, such as ascorbic acid, decrease Nrf-2 activity (Tarumoto et al., 2004Go). GSH is an abundant antioxidant in cells (Cotgreave and Gerdes, 1998Go) and is responsible for the regulating of oxidative signals that initiate Nrf-2 dissociation. Decreased GSH concentrations and/or a shift in GSH/GSSG ratios may permit Nrf-2/Keap-1 dissociation. GSH depletion via BSO resulted in Nrf-2 nuclear translocation even without TBHQ stimulation but NAC supplementation, which increased GSH concentrations, inhibited translocation even with TBHQ stimulation. Overexpression of Trx-1 did not alter the movement of Nrf-2 to the nucleus during TBHQ stimulation. These results demonstrate that GSH concentration and/or GSH/GSSG ratios, but not necessarily GSH redox potential, are key determinants in Nrf-2/Keap-1 dissociation and Nrf-2 translocation to the nucleus. GSH inhibition of dissociation and translocation may result from the ability of GSH to actively detoxify Nrf-2/Keap-1 dissociation signals (i.e., ROS). Moreover, these results suggest that regulation of this process may rely more on a type I redox switch and are dependent upon thiylation rather than disulfide formation (Gilbert, 1995Go; Schafer and Buettner, 2001Go). On the other hand, Trx-1 has little effect on the early events of Nrf-2 dissociation but Trx-1 overexpression increases activation of the ARE. This finding is consistent with the reduction of the critical cysteine in the DNA binding site (Bloom et al., 2002Go). Mutant Trx-1 does not show the same increase but rather a substantial decrease compared to control levels of ARE activation. Because overexpression of Trx-1 did not cause a decrease in Nrf-2 dissociation/nuclear translocation the data further show that Trx-1 function is limited to the Nrf-2/DNA binding aspect of Nrf-2 signaling. Oxidative stress may activate Nrf-2 but an oxidative environment would promote a decline in Nrf-2/DNA binding in the nucleus. Utilizing nuclear-targeted Trx-1, ARE activation increased significantly from the overexpression of non-targeted, wild-type Trx-1, while the nuclear-targeted mutant Trx-1 inhibited ARE activation. Consequently, the data show that the nuclear activity of Nrf-2 depends upon nuclear Trx-1. This observation may be of fundamental importance because it suggests that the Nrf-2 cysteine in the DNA binding domain is not fully reduced under these conditions. This implies that the cysteines of DNA binding sites of transcription factors may function as redox switches, and are not merely "critical" cysteines but rather may provide some regulatory functions as well. Many transcription factors have such cysteines (e.g., NF-{kappa}B, p53, Fos, Jun, glucocorticoid receptor, etc.). Indeed, transfection with a wild-type Trx-1 produced an increase in NF-{kappa}B activity determined by various reporter constructs, but transfection with a plasmid for nuclear-targeted Trx-1 potentiated NF-{kappa}B activity as compared to transfection with wild-type Trx-1 plasmids (Hirota et al., 1999Go; Kontou et al., 2003Go). With Nrf-2, activation, dissociation, nuclear transport, and DNA binding is a multi-step process. From our findings, there is a redox component which includes both the GSH and TRX systems. While incomplete, involvement of these systems furthers our understanding of Nrf-2 signaling.

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., 1992Go; Voehringer et al., 1998Go), while others arrive at opposing conclusions using GSH antibodies (Cotgreave, 2003Go). 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, 2003Go). Evidence supports differences in nuclear and cytoplasmic Trx-1 pools (Watson and Jones, 2003Go) and a mechanism for nuclear sequestration of Trx-1 during oxidative stress (Hirota et al., 1999Go).

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., 1997Go; Kirlin et al., 1999Go; Nkabyo et al., 2002Go). 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 (40–60 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., 2001Go), 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., 2004Go). 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.


    ACKNOWLEDGMENTS
 
The authors gratefully acknowledge Dr. Young-Mi Go for fruitful discussions concerning experimental design. This research was supported by grants ES011195 and ES013015-01 from the National Institute of Environmental Health Sciences (NIEHS), NIH. Its contents are solely the responsibility of the authors and do not necessarily represent the official view of the NIEHS, NIH.


    NOTES
 

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.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Abate, C., Patel, L., Rauscher, F. J., and Curran, T. (1990). Redox regulation of fos and jun DNA-binding activity in vitro. Science 249, 1157–1161.[ISI][Medline]

Bellomo, G., Vairetti, M., Stivala, L., Mirabelli, F., Richelmi, P., and Orrenius, S. (1992). Demonstration of nuclear compartmentalization of glutathione in hepatocytes. Proc. Natl. Acad. Sci. U.S.A. 89, 4412–4416.[Abstract]

Bloom, D., Dhakshinamoorthy, S., and Jaiswal, A. K. (2002). 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. Oncogene 21, 2191–2200.[CrossRef][ISI][Medline]

Cotgreave, I. A. (2003). Analytical developments in the assay of intra- and extracellular GSH homeostasis: specific protein S-glutathionylation, cellular GSH and mixed disulphide compartmentalisation and interstitial GSH redox balance. Biofactors 17, 269–277.[ISI][Medline]

Cotgreave, I. A., and Gerdes, R. G. (1998). Recent trends in glutathione biochemistry–glutathione-protein interactions: A molecular link between oxidative stress and cell proliferation? Biochem. Biophys. Res. Commun. 242, 1–9.[CrossRef][ISI][Medline]

Dalton, T. P., Dieter, M. Z., Yang, Y., Shertzer, H. G., and Nebert, D. W. (2000) Knockout of the mouse glutamate cysteine ligase catalytic subunit (Gclc) gene: Embryonic lethal when homozygous, and proposed model for moderate glutathione deficiency when heterozygous. Biochem. Biophys. Res. Commun. 279, 324–329.[CrossRef][ISI][Medline]

Dinkova-Kostova, A. T., Holtzclaw, W. D., Cole, R. N., Itoh, K., Wakabayashi, N., Katoh, Y., Yamamoto, M., and Talalay, P. (2002). Direct evidence that sulfhydryl groups of Keap1 are the sensors regulating induction of phase 2 enzymes that protect against carcinogens and oxidants. Proc. Natl. Acad. Sci. U.S.A. 99, 11908–11913.[Abstract/Free Full Text]

Galter, D., Mihm, S., and Dröge, W. (1994). Distinct effects of glutathione disulphide on the nuclear transcription factor kappa B and the activator protein-1. Eur. J. Biochem. 221, 639–648.[Abstract]

Gilbert, H. F. (1995) Thiol/disulfide exchange equilibria and disulfide bond stability. Methods Enzymol. 251, 8–28.[ISI][Medline]

Go, Y. M., Gipp, J. J., Mulcahy, R. T., and Jones, D. P. (2004). H2O2-dependent activation of GCLC-ARE4 reporter occurs by mitogen-activated protein kinase pathways without oxidation of cellular glutathione or thioredoxin-1. J. Biol. Chem. 279, 5837–5845.[Abstract/Free Full Text]

Guo, Y., Einhorn, L., Kelley, M., Hirota, K., Yodoi, J., Reinbold, R., Scholer, H., Ramsey, H., and Hromas, R. (2004). Redox regulation of the embryonic stem cell transcription factor Oct-4 by thioredoxin. Stem Cells 22, 259–264.[Abstract/Free Full Text]

Hansen, J. M., Choe, H. S., Carney, E. W., and Harris, C. (2001). Differential antioxidant enzyme activities and glutathione content between rat and rabbit conceptuses. Free Radic. Biol. Med. 30, 1078–1088.[CrossRef][ISI][Medline]

Hutter, D. E., Till, B. G., and Greene, J. J. (1997). Redox state changes in density-dependent regulation of proliferation. Exp. Cell Res. 232, 435–438.[CrossRef][ISI][Medline]

Hirota, K., Matsui, M., Iwata, S., Nishiyama, A., Mori, K., and Yodoi. J. (1997) AP-1 transcriptional activity is regulated by a direct association between thioredoxin and Ref-1. Proc. Natl. Acad. Sci. U.S.A. 94, 3633–3638.[Abstract/Free Full Text]

Hirota, K., Murata, M., Sachi, Y., Nakamura, H., Takeuchi, J., Mori, K., and Yodoi, J. (1999). Distinct roles of thioredoxin in the cytoplasm and in the nucleus. A two-step mechanism of redox regulation of transcription factor NF-kappaB. J. Biol. Chem. 274, 27891–27897.[Abstract/Free Full Text]

Inamdar, N. M., Ahn, Y. I., and Alam, J. (1996). The heme-responsive element of the mouse heme oxygenase-1 gene is an extended AP-1 binding site that resembles the recognition sequences for MAF and NF-E2 transcription factors. Biochem. Biophys. Res. Commun. 221, 570–576.[CrossRef][ISI][Medline]

Jones, D. P. (2002). Redox potential of GSH/GSSG couple: Assay and biological significance. Methods Enzymol. 348, 93–112.[ISI][Medline]

Karin, M. (1995). The regulation of AP-1 activity by mitogen-activated protein kinases. J. Biol. Chem. 270, 16483–16486.[Free Full Text]

Kim, Y. C., Yamaguchi, Y., Kondo, N., Masutani, H., and Yodoi, J. (2003). Thioredoxin-dependent redox regulation of the antioxidant responsive element (ARE) in electrophile response. Oncogene 22, 1860–1865.[CrossRef][ISI][Medline]

Kirlin, W. G., Cai, J., Thompson, S. A., Diaz, D., Kavanagh, T. J., and Jones, D. P. (1999). Glutathione redox potential in response to differentiation and enzyme inducers. Free Radic. Biol. Med. 27, 1208–1218.[CrossRef][ISI][Medline]

Kontou, M., Adelfalk, C., Hirsch-Kauffmann, M., Schweiger, M. (2003). Suboptimal action of NF-kappaB in Fanconi anemia cells results from low levels of thioredoxin. Biol. Chem. 384, 1501–1507.[CrossRef][ISI][Medline]

Kwak, M. K., Kensler, T. W., and Casero, R. A. (2003a). Induction of phase 2 enzymes by serum oxidized polyamines through activation of Nrf2: Effect of the polyamine metabolite acrolein. Biochem. Biophys. Res. Commun. 305, 662–670.[CrossRef][ISI][Medline]

Kwak, M. K., Wakabayashi, N., Itoh, K., Motohashi, H., Yamamoto, M., and Kensler, T. W. (2003b). Modulation of gene expression by cancer chemopreventive dithiolethiones through the Keap1-Nrf2 pathway. Identification of novel gene clusters for cell survival. J. Biol. Chem. 278, 8135–8145.[Abstract/Free Full Text]

Makino, Y., Yoshikawa, N., Okamoto, K., Hirota, K., Yodoi, J., Makino, I., and Tanaka, H. (1999). Direct association with thioredoxin allows redox regulation of glucocorticoid receptor function. J. Biol. Chem. 274, 3182–3188.[Abstract/Free Full Text]

Matsui, M., Oshima, M., Oshima, H., Takaku, K., Maruyama, T., Yodoi, J., and Taketo, M. M. (1996) Early embryonic lethality caused by targeted disruption of the mouse thioredoxin gene. Dev. Biol. 178, 179–185.[CrossRef][ISI][Medline]

Matthews, J. R., Kaszubska, W., Turcatti, G., Wells, T. N., and Hay, R. T. (1993). Role of cysteine62 in DNA recognition by the P50 subunit of NF-kappa B. Nucleic Acids Res. 21, 1727–1734.[Abstract]

Moinova, H. R., and Mulcahy, R. T. (1999). Up-regulation of the human gamma-glutamylcysteine synthetase regulatory subunit gene involves binding of Nrf-2 to an electrophile responsive element. Biochem. Biophys. Res. Commun. 261, 661–668.[CrossRef][ISI][Medline]

Nguyen, T., Sherratt, P. J., and Pickett, C. B. (2003). Regulatory mechanisms controlling gene expression mediated by the antioxidant response element. Annu. Rev. Pharmacol. Toxicol. 43, 233–260.[CrossRef][ISI][Medline]

Nkabyo, Y. S., Ziegler, T. R., Gu, L. H., Watson, W. H., and Jones, D. P. (2002). Glutathione and thioredoxin redox during differentiation in human colon epithelial (Caco-2) cells. Am. J. Physiol. Gastrointest. Liver Physiol. 283, G1352–G1259.[Abstract/Free Full Text]

Rushmore, T. H., and Pickett, C. B. (1993). Transcriptional regulation of the rat NAD(P)H:quinone reductase gene. Characterization of a DNA-protein interaction at the antioxidant responsive element and induction by 12-O-tetradecanoylphorbol 13-acetate. J. Biol. Chem. 268, 11475–11478.[Free Full Text]

Schafer, F. Q., and Buettner, G. R. (2001). Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple. Free Radic. Biol. Med. 30, 1191–1212.[CrossRef][ISI][Medline]

Smeal, T., Binetruy, B., Mercola, D., Grover-Bardwick, A., Heidecker, G., Rapp, U. R., and Karin, M. (1992). Oncoprotein-mediated signalling cascade stimulates c-Jun activity by phosphorylation of serines 63 and 73. Mol. Cell Biol. 12, 3507–3513.[Abstract]

Tarumoto, T., Nagai, T., Ohmine, K., Miyoshi, T., Nakamura, M., Kondo, T., Mitsugi, K., Nakano, S., Muroi, K., Komatsu, N., and Ozawa, K. (2004). Ascorbic acid restores sensitivity to imatinib via suppression of Nrf2-dependent gene expression in the imatinib-resistant cell line. Exp. Hematol. 32, 375–381.[CrossRef][ISI][Medline]

Voehringer, D. W., McConkey, D. J., McDonnell, T. J., Brisbay, S., and Meyn, R. E. (1998). Bcl-2 expression causes redistribution of glutathione to the nucleus. Proc. Natl. Acad. Sci. U.S.A. 95, 2956–2960.[Abstract/Free Full Text]

Watson, W. H., Pohl, J., Montfort, W. R., Stuchlik, O., Reed, M. S., Powis, G., and Jones, D. P. (2003). Redox potential of human thioredoxin 1 and identification of a second dithiol/disulfide motif. J. Biol. Chem. 278, 33408–33415.[Abstract/Free Full Text]

Watson, W. H., and Jones, D. P. (2003). Oxidation of nuclear thioredoxin during oxidative stress. FEBS Lett. 543, 144–147.[CrossRef][ISI][Medline]

Wild, A. C., Gipp, J. J., and Mulcahy, T. (1988). Overlapping antioxidant response element and PMA response element sequences mediate basal and beta-napthoflavone-induced expession of the human gamma-glutamaylcysteine synthetase catalytic subunit gene. Biochem J. 332, 373–381.