308.full

TOXICOLOGICAL SCIENCES 82, 308–317 (2004)

doi:10.1093/toxsci/kfh231

Advance Access publication July 28, 2004

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

Jason M. Hansen,* Walter H. Watson,† and Dean P. Jones*,1

*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

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 selec-

tivelymodifiedTrx-1 expressionby transient transfection.Cytoplas-

mic 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 switchwhichwas regulated byGSHnot 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.

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 induc-

tion of Jun kinase (JNK) activity by oxidants resulting in the

phosphorylation of serine63 and serine73 in the c-Jun transacti-

vation domain (Karin, 1995; 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.,

1990). Similarly, nuclear factor kappa B (NF-kB), contains a

critical cysteine residue (cysteine62) in the p50 subunit that is

involved in DNA binding (Matthews et al., 1993). NF-kB is

normally sequestered in the cytoplasm by IkB but under oxida-

tive conditions, IkB is phosphorylated by I-kB 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., 1994).

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-kB, a cysteine residue

(cysteine506) must be in a reduced state for efficient DNA bind-

ing 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 thio-

redoxin (TRX) systems, provide control of intracellular thiol/

disulfide redox environments. Both are ubiquitously distributed

among mammalian cell types and function in peroxide elimina-

tion, 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 concentra-

tions while Trx-1 is a 12 kDa protein present in micromolar

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: [email protected].

Toxicological Sciences vol. 82 no. 1 # Society of Toxicology 2004; all rights reserved.

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 prolif-

eration, differentiation, and apoptosis (Schafer and Buettner,

2001). While there is substantial evidence for overlapping func-

tions of the GSH and TRX systems, evidence shows that their

redox potentials are not in equilibrium. For instance, Caco-2

cells undergo a 140 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). Conse-

quently, 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 cyto-

plasm 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 dis-

sociation 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

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-ml cell volume per mg of cell protein (Jones, 2002). Cells

werepretreatedwith 0.5–2 mM N-acetylcysteine (NAC) or 1–100mM buthionine

sulfoximine (BSO) for 24 h. Similarly, for Nrf-2 activation and reporter experi-

ments, cells were treated with 2 mM NAC or 100 mM BSO for 24 h prior to the

addition of 50 mM 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 g-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 mM 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 mem-

brane. 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.

RedoxWestern. 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 iodo-

acetic 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 53 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 5 E0 1 2.3 3 RT/nF3 log ([TRX-SS]/

[TRX-SH2]; E0 5 �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). Densito-

metry 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-antigennuclear localizationsequence(PPKKKRKVEDP).Site-directedmuta-

genesis was performed on the Trx-1-NLS construct to introduce the C35S muta-

tion 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 50-CCACGTGGCTGAGAAGTCAACTATA-

CAAGT-30 and 50-TTGACTTCTCAGCCACTGGGTGTGGGCCTTCA-30,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

b-galactosidase enzyme assays were measured as described previously (Go et al.,

2004). Luciferase measurements were normalized to b-galactosidase activity as

determined with the b-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.

COMPARTMENTATION OF Nrf-2 REDOX CONTROL 309

RESULTS

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 concen-

trations at the highest treatment (2 mM) where concentrations

were increased by approximately 15% from control GSH con-

centrations (Fig. 1). No significant changes in GSSG concentra-

tions were detected. Similarly, no significant changes were seen

in GSH/GSSG redox potential, ranging from�256 to�260 mV.

Using BSO (1–100 mM) for 24 h, GSH was decreased in a dose-

dependent manner, where GSH changes ranged from decreases

of 15% (1mM) to 85% (100mM) 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 by130 mV at

100 mM BSO.

Redox Western blot analysis of reduced (Trx-1SH2) and oxi-

dized (Trx-1SS) showed no changes in Trx-1 redox state with

either NAC or BSO (Figs. 2A and 2B), where values ranged from

�287 (64) to �282 (64) mV. These results show that the GSH

redox state is independent of Trx-1 levels.

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 mM BSO for 24 h. Control GSH concentrations measured 4.6 (60.4) mM. Control

GSSG measured 0.04 (60.004) mM. Data is shown as percent changes from control and is representative of four independently performed experiments. Asterisks

(*) denote a significant difference (p 5 0.05) from control.

310 HANSEN, WATSON, AND JONES

Although the ratio of reduced:oxidized Trx-1 did not correlate

with changes in GSH and GSSG concentrations or redox poten-

tial, 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., 2003).

Increased Trx-1 Expression Does Not Affect GSH

Redox State

Although the redox states of Trx-1 and GSH are not equili-

brated, increased expression of Trx-1 could affect GSH redox

state. To test this, cells were transiently transfected to over-

express Trx-1 and related mutants. Evaluation by immuno-

blotting confirmed overxpression of Trx-1 over endogenous,

wild-type Trx-1 (data not shown). Overexpression of Trx-1

FIG. 2. (A) Redox Western analysis of Trx-1 following treatment with 0.5–2.0 mM NAC or 1–100 mM BSO for 24 h and with 0–50 mM 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 mM BSO for 24 h. (D) NAC

treatments showed no changes in total Trx-1 but treatment with BSO (100 mM) increased total Trx-1 by 112% from control. Densitometric quantitation is an

average of three separate experiments. Asterisks (*) denote a significant difference (p 5 0.05) from control.


and related mutants did not have any significant effect on GSH

redoxstateorGSHconcentration. (Figs.3Aand3C).After48hof

transient transfection with the various Trx-1 expression plas-

mids, GSH/GSSG redox potential ranged from �257 (64) to

�261 (65) mV and were not different (Fig. 3). Together with the

above data, these results show that the GSH andTRX redox states

are independently controlled and that BSO and transient trans-

fection with Trx-1 and Trx-1 mutants provide an approach to

evaluate selective functions of the two systems.

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., 2003). To evaluate

the effects of TBHQ on GSH, cultures were pretreated with

either 2 mM NAC or 100 mM BSO for 24 h and then treated

with 50 mM TBHQ for 6 h. TBHQ in cultures not receiving any

pretreatment caused an increase in GSH and GSSG concentra-

tions (Figs.4A and4B). InNAC andBSOpretreated 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 treat-

ment. 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

coupledwithoxidationofGSHtoGSSG.Incontrast tochanges in

GSH redox state, TBHQ (0–50mM) 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.

GSH/GSSG Ratio Controls Cytoplasmic Nrf-2 Dissociation/

Nuclear Translocation

The ability to independently control the GSH and TRX sys-

tems 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 transfec-

tion had no effect on Nrf-2 cytoplasmic dissociation/nuclear

translocation (Fig. 6B).

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 pre-

vented TBHQ-induced changes in GSH/GSSG ratios but did

not change Eh GSH/GSSG (Fig. 4). Consequently, the data

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 5 0.05) from control.


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, 2001), the data are consistent with regula-

tion 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 tran-

siently transfected with the ARE4-L reporter construct. Mod-

ulation of total GSH concentrations was achieved via 24 h

incubation with either 2 mM NAC or 100 mM 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.

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 mM BSO for 24 h with and without 50 mM TBHQ for 6 h. Data is

representative of four independently performed experiments. Asterisks (*)

denote a significant difference (p 5 0.05) from control. Crosses (†) denote a

significant difference (p 5 0.05) between cells treated with 50 mM TBHQ

only and cells receiving either 2 mM NAC or 100 mM BSO and 50 mM TBHQ.

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 ana-

lysis of three separate experiments.


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., 1997; Makino et al., 1999; Matthews et al., 1993).

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 over-

expression 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 overexpes-

sion but Nrf-2 activity was inhibited by C35S Trx-1. Conse-

quently, 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., 2002).

To further test whether this occurred in the nuclei, we used a

nuclear targetedfusionprotein created withaSV-40 nuclear trans-

locationsequencecomplexed toTrx-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 over-

expressing 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

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 trans-

duction pathways. In the present study, we establish the inde-

pendence 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., 2004). GSH is an abun-

dant antioxidant in cells (Cotgreave and Gerdes, 1998) 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 poten-

tial, 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). More-

over, 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, 1995; Schafer

and Buettner, 2001). 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., 2002). Mutant Trx-1 does not show the same

increase but rather a substantial decrease compared to control

FIG. 6. (A) Immunoblot analysis of nuclear Nrf-2 from HeLa cell nuclear

fractions following treatment with either 2 mM NAC or 100 mM BSO for

24 h, with or without 50 mM 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 5 0.05)

from control. Upper panel: Representative Western blot. Lower panel:

Densitometric analysis of three separate experiments.


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 funda-

mental importance because it suggests that the Nrf-2 cysteine in

the DNA binding domain is not fully reduced under these con-

ditions. 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 reg-

ulatory functions as well. Many transcription factors have such

cysteines (e.g., NF-kB, p53, Fos, Jun, glucocorticoid receptor,

etc.). Indeed, transfection with a wild-type Trx-1 produced an

increase in NF-kB activity determined by various reporter

constructs, but transfection with a plasmid for nuclear-targeted

Trx-1 potentiated NF-kB activity as compared to transfection

with wild-type Trx-1 plasmids (Hirota et al., 1999; Kontou et al.,

2003). 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 compart-

mentation 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 chroma-

tography methods. Still, some studies have concluded that there

are higher concentrations of GSH in the nucleus vs. the cyto-

plasm using fluorescent dyes (Bellomo et al., 1992; Voehringer

et al., 1998), while others arrive at opposing conclusions using

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/b-galactosidase activity as described in the methods section. (B) Treatment with 2 mM NAC or 100 mM BSO for 24 h without

TBHQ stimulation. (C) Treatment with 2 mM NAC or 100 mM BSO for 24 h followed by treatment with 50 mM 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 5 0.05) from control.

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 mM TBHQ for 6 h. Data is representative of

three independently performed experiments. Asterisks (*) denote a significant

difference (p5 0.05) between designated groups.


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 (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 prolif-

erating and differentiating cells, which have different GSH

redox potentials and regulate different redox-sensitive transcrip-

tion 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 dif-

ferentiation 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 detoxifica-

tion (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 implica-

tions 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, com-

partmentation 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 redu-

cing critical cysteines in the DNA binding domain. With tandem,

complementary functions, GSH and Trx-1 regulate Nrf-2 activ-

ity 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.

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