Download TCF11/Nrf1 overexpression increases the intracellular

Biochimica et Biophysica Acta 1517 (2001) 212^219
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TCF11/Nrf1 overexpression increases the intracellular glutathione level
and can transactivate the Q-glutamylcysteine synthetase (GCS) heavy
subunit promoter
Mari C.W. Myhrstad a , Cathrine Husberg b , Paula Murphy b , Olov Nordstro«m a ,
Rune Blomho¡ a , Jan Òyvind Moskaug a , Anne-Brit KolstÖ b; *
a
b
Institute for Nutrition Research, University of Oslo, Oslo, Norway
Biotechnology Centre of Oslo, PB: 1125 Blindern, 0316 Oslo, Norway
Received 5 April 2000 ; received in revised form 27 October 2000; accepted 7 November 2000
Abstract
Q-Glutamylcysteinylglycine or glutathione (GSH) performs important protective functions in the cell through maintenance of the
intracellular redox balance and elimination of xenobiotics and free radicals. The production of GSH involves a number of enzymes and
enzyme subunits offering multiple opportunities for regulation. Two members of the CNC subfamily of bZIP transcription factors (TCF11/
Nrf1 and Nrf2) have been implicated in the regulation of detoxification enzymes and the oxidative stress response. Here we investigate the
potential role of one of these factors, TCF11/Nrf1, in the regulation of GSH levels in the cell and particularly its influence on the expression
of one of the enzymatic components necessary for the synthesis of GSH, the heavy subunit of Q-glutamylcysteine synthetase (GCSh ). Using
overexpression of the transcription factor in COS-1 cells we show that TCF11/Nrf1 stimulates GSH accumulation. Using co-transfection
with reporter constructs where reporter expression is driven through the GCSh promoter we show that this increase may be mediated in part
by induced expression of the GCSh gene by TCF11/Nrf1. We further show that a distal portion of the promoter including two antioxidantresponse elements (AREs) predominantly mediates the TCF11/Nrf1 transactivation and an electromobility shift assay showed that just one
of these AREs specifically binds TCF11/Nrf1 as heterodimers with small Maf proteins. We suggest that TCF11/Nrf1 can operate through a
subset of AREs to modulate the expression of GCSh together with other components of the pathway and in this way play a role in
regulating cellular glutathione levels. ß 2001 Elsevier Science B.V. All rights reserved.
Keywords : TCF11/Nrf1 ; Glutathione; Antioxidant-response element; Q-Glutamylcysteine synthetase
1. Introduction
The tripeptide Q-glutamylcysteinylglycine or glutathione
(GSH) is a ubiquitous cellular non-protein sulphydryl.
GSH has two important roles in the cell: maintaining
the intracellular redox balance and eliminating xenobiotics
and free radicals [1]. GSH is synthesised from amino acids
in two enzymatic reactions catalysed by Q-glutamylcysteine
synthetase (GCS) and glutathione synthetase [2]. The reaction catalysed by GCS is the rate-limiting step in the de
novo synthesis of GSH, so that regulation of GCS levels is
likely to be an important aspect in the regulation of GSH
synthesis [3]. GCS consists of two subunits, the heavy subunit (GCSh ) with catalytic activity and the light subunit
* Corresponding author. Fax: +47-22-840-501;
E-mail : [email protected]
(GCSl ) with regulatory activity [4^6]. Regulation of enzyme activity occurs through multiple mechanisms a¡ecting one or both subunits and the heavy subunit in particular is regulated by both transcriptional and posttranscriptional mechanisms [7].
Transcriptional control of GCSh is mediated by a region
spanning approx. 5 kb of the 5P £anking sequence of the
GCSh gene [8]. Analysis of this region by Mulcahy et al.
revealed the presence of response elements including several AP-1 sites, antioxidant-response elements (AREs; also
known as the electrophile-response element EpRE) and
one NF-UB site. Similar ARE motifs are also found in
promoters of other genes that participate in the defence
against free radicals and toxic insults, and this has led to
the de¢nition of a consensus core ARE motif (A/GTGAC/
GnnnGCA/G). Among such genes are NADPH:quinone
oxidoreductase [9], glutathione S-transferase [10] and
UDP-glucuronosyltransferase [11]. Many of these genes
0167-4781 / 01 / $ ^ see front matter ß 2001 Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 7 - 4 7 8 1 ( 0 0 ) 0 0 2 7 6 - 1
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are induced through AREs in response to chemical or oxidative stress [12].
The transcription factor TCF11/Nrf1 (also known as
LCR-F1) is a member of the CNC subfamily of bZIP
transcription factors, most closely related to Nrf2, Nrf3
and p45-NF-E2 [10,13^16]. Like the other members of
this group, TCF11/Nrf1 can heterodimerise with small
Maf transcription factors to more e¤ciently bind DNA
target sequences [17]. We originally showed that TCF11/
Nrf1 can bind to the same target sequence as p45 NF-E2
in vitro (the NF-E2 site), both alone and in the presence of
small Maf proteins [17]. We have further demonstrated
transactivation of a reporter gene through this site when
TCF11/Nrf1 is transfected alone while the transactivation
is inhibited when MafG is co-transfected in COS-1 cells
[18]. In order to investigate di¡erences in speci¢city between the closely related CNC DNA binding factors we
used a selection approach to identify the optimal binding
sites for TCF11/Nrf1. We found a clear consensus binding
site in the presence of small Maf, which matches both the
NF-E2 site and the ARE motif. We therefore postulated
that genes stimulated in response to oxidative and other
forms of stress through AREs might be targets for TCF11/
Nrf1 transcriptional regulation [18]. In fact, more recent
evidence suggests that both TCF11/Nrf1 and the closely
related CNC factor Nrf2 are involved in the regulation of
expression of a number of phase II detoxifying enzymes.
Both genes were found to positively regulate the expression of the NAD(P)H :quinone oxidoreductase gene [19].
Interaction between Nrf1 or Nrf2 and Jun proteins has
also been described and appears to coordinately induce
at least two genes involved in detoxi¢cation of xenobiotics,
with Nrf2 having a signi¢cantly stronger e¡ect [20]. Recently it has also been shown that Nrf2 was a positive
regulator of ARE dependent GCSh and GCSl subunit
gene expression in cells exposed to L-naphtho£avone and
pyrrolidinedithiocarbamate [21]. More direct evidence of a
role for these two factors in regulating stress response
comes from the genetic manipulation of the tcf11/nrf1
and nrf2 genes in mice: null mutants for nrf2 respond
poorly in phenolic antioxidant induced expression of
NAD(P)H:quinone oxidoreductase [22] and tcf11/nrf1 mutants show reduced protection against the e¡ects of oxidants, reduced levels of glutathione and reduced induction
of the GCSl gene following exposure to paraquat [23].
To investigate whether TCF11/Nrf1 may regulate the
cellular glutathione level and in particular expression of
the GCSh gene, we performed a series of transfection experiments transiently expressing TCF11/Nrf1 in COS-1
cells. We found that such expression led to a signi¢cant
increase in cellular glutathione. When expressed in the
presence of reporter constructs, we showed that TCF11/
Nrf1 transactivates the GCSh promoter and that this
transactivation is predominantly mediated through the distal part of the GCSh promoter. Our data further implicate
TCF11/Nrf1 in the regulation of antioxidant and detoxi¢-
213
cation enzymes and show for the ¢rst time that its in£uence may be exerted in part through GCSh gene regulation.
2. Materials and methods
2.1. Cell culture
COS-1 cells, purchased from the American Type Culture collection, were grown in Dulbecco's modi¢ed Eagle's
medium (DMEM) supplemented with 2 mM L-glutamine,
5 units/ml penicillin, 50 Wg/ml streptomycin and 10% foetal calf serum (COS-1 medium). The cell cultures were
maintained in a humidi¢ed atmosphere with 5% CO2 at
37³C.
2.2. Plasmid constructs
The complete GCSh -luc reporter construct (33802/
GCSh -luc) was kindly provided by Mulcahy et al. [8].
Brie£y, the construct was obtained by cloning a 4.2 kb
fragment from the 5P £anking region of the human
GCSh gene into HindIII-NcoI sites of the pGL3 basic vector (Promega).
The distal GCSh -luc construct (33258:32946/GCSh luc) was obtained by ampli¢cation of a 312 bp fragment
containing ARE 3 and ARE 4 of GCSh from human genomic DNA by PCR using primers 1 and 2 (1: 5P-ACT
GCG GCA ATC CTA GCA GC-3P and 2: 5P-AAG CTT
CTG GAC CGT GGA GAT CC-3P). The PCR product
was cloned into the pCR 2.1-TOPO vector (Invitrogen,
USA). This plasmid was digested with HindIII and the
resulting fragment was ligated into the HindIII site of a
TATA luc vector [24].
The proximal GCSh -luc construct (32752/GCSh -luc)
was obtained by digestion of the recombinant 33802/
GCSh -luc plasmid with SacI. The resulting fragment was
isolated and religated.
The expression construct was obtained by cloning a fulllength TCF11/Nrf1 cDNA (clone pZcEA20, up to the
EcoRV site in the 3P non-coding sequence at bp 3550)
into the expression vector pcDNA3 (Invitrogen).
MBP-TCF11/Nrf1-A, a fusion protein between Escherichia coli maltose binding protein (MBP) and the 300 Cterminal amino acids of TCF11/Nrf1, and the two MBPMaf fusions, in which MBP was fused to chicken MafG or
MafK, were prepared as previously described [17].
pRSVCAT is also described previously [17].
2.3. Transient transfection of COS-1 cells
The cells were plated in 35 mm tissue culture wells the
day before transfection at a density of approx. 60% con£uence and transfected using a standard dextran-chloroquine method as described previously [25] with DNA
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concentrations as indicated in the results. Two hours after
transfection the cells were subjected to a DMSO shock
(Sigma, St. Louis, MO, USA) (10% in PBS), after which
they were incubated overnight in COS-1 medium. COS-1
cells were also transfected using a standard calcium phosphate method and a total amount of 10 Wg DNA as previously described [18], including CAT as a control plasmid
for transfection e¤ciency.
2.4. Luciferase activity measurements
Luciferase activity was measured in the cell lysates according to the manufacturer's protocol (Promega). Brie£y,
the cells were washed in PBS without Ca2‡ or Mg2‡ prior
to the addition of 300 Wl lysis bu¡er (Promega). The cells
were incubated at room temperature for 20 min before
removal by scraping and subsequent vigorous vortexing.
The lysate was then centrifuged to remove cell debris. Luciferase activity was measured by adding 100 Wl luciferase
assay solution (Promega) to 20 Wl of the lysate and the
luminescence was detected in a TD 20/20 luminometer
(Turner Design, Sunnyvale, CA, USA). Protein concentration was measured in the cell lysate using Coomassie brilliant blue reagent from Bio-Rad (CA, USA).
2.5. Electrophoretic mobility shift assay
Bacterially expressed MBP-TCF11/Nrf1-A (25 ng) alone
or together with either MBP-MafG (2 ng) or MBP-MafK
(2 ng) were used in an electrophoretic mobility shift assay
(EMSA) essentially as described previously [18], with the
exception that 32 P-labelled DNA probes were puri¢ed using a spin column (Bio-Rad).
2.6. GSH measurements
Cells were harvested in a phosphate bu¡er (10 mM, pH
6) containing 10 WM EDTA and thereafter lysed by several
freeze-thaw cycles. Proteins were precipitated with approx.
2% TFA and the resulting supernatant was neutralised to
pH 2 with 1 M KOH. GSH was analysed on a HPLC
system (Hewlett Packard 1100 system) equipped with an
electrochemical detector (ESA Coulochem II model 5500
with an ESA detector cell model 5010 and a 5020 guard
cell, both with inline ¢lters). The guard cell was set to
900 mM and the analytical cell to 865 mV. A damper
(Scienti¢c System, model LP-21) was placed between the
pump and the injector. Chromatography was performed
on a Hypersil ODS column (150U4.6 mm I.D. with 3 Wm
particle size) with an ODS precolumn (2 cm, Supelco,
Bellefonte, USA) placed before the separation column.
The elution was isocratic with a mobile phase consisting
of 40 mM NaH2 PO4 (AnalaR, BDH, Poole, UK), 50 WM
sodium octanesulphonic acid (Sigma) which was adjusted
to pH 2.7 with 85% phosphoric acid and acetonitrile
(Rathburn, Walkerburn, UK) in a ratio of 49:1. GSH
(Sigma) stock solution (1 mg/ml) was made weekly in mobile phase containing 1 mM EDTA (Sigma). Standard
curves were made daily in the range of 30^650 WM in
the same bu¡er used for analyses of cell lysates. The injection volume was 10 Wl for all analyses. Data were collected and analysed with HP Chem Station software.
3. Results
3.1. TCF11/Nrf1 increases the glutathione level in
transfected cells
To investigate the potential role of TCF11/Nrf1 in regulating GSH production, we measured the level of GSH in
COS-1 cells following transfection of the TCF11/Nrf1 expression construct. Thirty-six hours post transfection the
cells were harvested and the reduced glutathione level was
measured by HPLC. We found that when 1 Wg of Nrf1
expression plasmid was transfected per 35 mm culture well
the glutathione level was signi¢cantly increased approx.
24% compared to empty expression vector pcDNA3 (Table 1). This shows that TCF11/Nrf1 can activate the GSH
synthesis pathway. The e¡ect, however, could be mediated
by in£uencing the expression of one or more of several
components of the pathway including GCSl , GCSh and
glutathione S-transferase. In fact both GCSl and glutathione S-transferase were shown to be downregulated in
Nrf13/3 ¢broblasts [23]. We wished to further explore the
possibility that TCF11/Nrf1 can also regulate the GCSh
gene.
3.2. TCF11/Nrf1 transactivates a GCSh driven reporter
TCF11/Nrf1 has been shown to bind to sequences often
found in the promoter region of genes involved in cellular
stress response [17,19,20,26]. To test the possibility that
expression of GCSh is regulated by TCF11/Nrf1, COS-1
cells were co-transfected with a luciferase reporter construct including 4.2 kb of the 5P £anking region of the
Table 1
Increased glutathione level in COS-1 cells expressing TCF11/Nrf1
Amount DNA (Wg)
pcDNA3 vector (control)
TCF11/Nrf1 expression vector
0.5
65.0 ( þ 3.3)
76.5 ( þ 8.2)
1
60.8 ( þ 1.7)
75.3 ( þ 4.8)*
GSH is expressed as Wmol/g protein. Data are presented as mean þ S.D. of three experiments each performed in duplicate. * indicates that there is a statistically signi¢cant di¡erence between empty control vector and Nrf1 expression vector (P 6 0.05), as determined using a two-tailed Student's t-test.
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215
Fig. 1. A schematic illustration of the reporter constructs with cis-acting elements depicted by shaded boxes.
GCSh gene (Fig. 1) and a TCF11/Nrf1 expression plasmid.
The cells were transfected with a constant amount of the
complete GCSh -luc reporter (0.5 Wg) together with 0.1 Wg
or 0.5 Wg TCF11/Nrf1 expression plasmid or the same
amount of the empty expression vector pcDNA3 per
35 mm culture well. Thirty-six to 40 h post transfection
the luciferase activity in cell lysates was measured. A 2.7fold increase in luciferase activity was observed at the
highest concentration of the TCF11/Nrf1 expression plasmid used (Fig. 2). The same results were obtained when
cells were transfected using calcium phosphate and including CAT as internal control for transfection e¤ciency.
TCF11/Nrf1 was also able to activate transcription
through the complete GCSh promoter in HepG2 cells
although the e¡ect was lower in HepG2 cells compared
to COS-1 cells (data not shown).
As outlined in Fig. 1, the 4.2 kb GCSh promoter construct contains several response elements that could mediate the transactivation observed in Fig. 2. These include
four ARE motifs, two located at the distal end of the
promoter (ARE3 and ARE4), and two at the more proximal end (ARE1 and ARE2). ARE3 and ARE4 completely match the consensus ARE sequence while ARE1
and ARE2 show di¡erences in the ¢rst and the ¢rst and
last positions, respectively. None of the AREs perfectly
match the consensus described for TCF11/Maf [18], but
ARE4 di¡ers only in the £anking base pairs whereas all
the others show di¡erences in the core sequences. To further de¢ne the part of the GCSh promoter through which
TCF11/Nrf1 exerts its e¡ect, we produced two reporter
constructs where luciferase is driven either from the proximal part of the promoter containing ARE1 and ARE2
(proximal GCSh -luc, Fig. 1) or from the distal promoter
containing AREs 3 and 4 (distal GCSh -luc, Fig. 1). As
shown in Fig. 3, we found that the construct containing
the distal AREs was induced 2.3-fold following transfection with 0.1 Wg TCF11/Nrf1 expression construct whereas
the proximal promoter was not clearly induced. Taken
Fig. 2. E¡ect of TCF11/Nrf1 expression on GCSh promoter mediated
luciferase activity. COS-1 cells were transfected with 0.5 Wg of the complete GCSh -luc reporter plasmid together with 0.1 Wg or 0.5 Wg of the
empty expression vector pcDNA3 (control) or pcDNA3-TCF11/Nrf1.
Thirty-six to 40 h after transfection, the cells were washed in PBS, lysed
and scraped from the wells. Cell lysates were collected and luciferase activities measured. Luciferase activity was corrected for variation in protein concentration in each well. Data are given as fold induction related
to transfection with the corresponding amount of the empty expression
vector pcDNA3. The means from three di¡erent experiments each preformed in triplicate þ S.D. are presented. COS-1 cells were also transfected using a standard calcium phosphate method and a total amount
of 10 Wg DNA as previously described [18], including CAT as a control
plasmid for transfection e¤ciency. This gave the same small, but signi¢cant e¡ect.
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Fig. 3. E¡ect of TCF11/Nrf1 expression on the distal end or the proximal part of a GCSh -driven reporter. COS-1 cells were transfected with
0.5 Wg of the reporter plasmid distal GCSh -luc or proximal GCSh -luc
together with either 0.1 Wg of the empty expression vector pcDNA3
(control) or pcDNA3-TCF11/Nrf1. Thirty-six to 40 h after transfection,
the cells were washed in PBS, lysed and scraped from the wells. Cell lysates were collected and luciferase activities measured. Luciferase activity
was corrected for variation in protein concentration in each well. Data
are given as fold induction related to that of the empty expression vector pcDNA3. The means from three di¡erent experiments each preformed in triplicate þ S.D. are presented.
together, our results show that Nrf1 induces the GCSh
promoter and that this induction may be mediated predominantly through the distal part of the promoter.
3.3. TCF11/Nrf1/MafG heterodimers bind speci¢cally to
ARE4
The luciferase induction we observed through the distal
region of the promoter (Fig. 3) together with a previous
report indicating that ARE4 is particularly important in
GCSh regulation, prompted us to measure the binding of
TCF11/Nrf1 to the distal ARE motifs. TCF11/Nrf1 heterodimerises with small Maf bZIP proteins and such dimers
are known to bind to DNA more e¤ciently than TCF11/
Nrf1 alone [18]. EMSA was therefore used to measure the
binding of TCF11/Nrf1 to ARE3 and ARE4 both in the
absence and presence of the small Maf proteins, MafG
and MafK.
Using a 66 bp oligonucleotide (pARE34, Table 2) containing both ARE3 and ARE4 as a probe, neither MBPTCF11/Nrf1 nor MBP-Maf fusions show any binding as
homodimers in EMSA (Fig. 4A, lanes 2^4). Such inability
to bind a probe in vitro has previously been shown with a
Table 2
Oligonucleotides used in EMSA
5P CAA AGC GCT GAG TCA CGG GGA GGC GGT GCG
GGG GAG GCG GGC CCG CGC AGT CAC GTG GCG 3P
pARE3
5P GCG GGG GAG GCG GGC CCG CGC AGT CAC GTG
pARE4
5P CAA AGC GCT GAG TCA CGG GGA GGC GGT GCG
pARE4m 5P CAA AGC GCT GAG TCC CGG GGA GGC GGT GCG
pARE34
CGC GCG
GCG 3P
CGC 3P
CGC 3P
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M.C.W. Myhrstad et al. / Biochimica et Biophysica Acta 1517 (2001) 212^219
217
Fig. 4. Electrophoretic mobility shift assay of TCF11/Nrf1 and small Mafs to ARE elements; determination of ARE preference. (A) EMSA of puri¢ed
MBP-TCF11/Nrf1, MBP-MafG and MBP-MafK, each alone (lanes 2^4) or in combination (lanes 5 and 6), showing binding to the pARE34 probe.
(B) EMSA of puri¢ed MBP-TCF11/Nrf1 and MBP-MafG with the pARE34 probe in the absence (lane 2) or in the presence of the di¡erent competitors pARE3, pARE4 and mutated pARE4m (lane 3^5). (C) EMSA of puri¢ed MBP-TCF11/Nrf1 and MBP-MafG using three di¡erent probes pARE3
(lane 2), pARE4 (lane 4) and pARE4m (lane 9). With probe pARE4 the same three competitors as in B were used (lane 5^7).
single NF-E2 site [17]. To test whether TCF11/Nrf1-small
Maf heterodimerisation would enhance protein binding to
the di¡erent ARE sites we performed EMSA with bacterially synthesised TCF11/Nrf1 and Maf fusion proteins.
As heterodimers, both the combinations of TCF11/Nrf1/
MafG and TCF11/Nrf1/MafK show enhanced protein
binding to the pARE34 probe (Fig. 4A, lanes 5 and 6).
To further determine whether there were any di¡erences
between the binding a¤nities of the TCF11/Nrf1/MafG
heterodimer to the di¡erent ARE elements, two 33 bp
oligonucleotides each containing one ARE element
(named pARE3 and pARE4, Table 2) were used as competitors in EMSA. By including an excess of unlabelled
pARE3 or pARE4 (Fig. 4B, lanes 3 and 4) it is shown
that only the probe with the response element ARE4 is
able to compete out the shift formed by heterodimerisation of TCF11/Nrf1 and MafG to the pARE34 probe.
Unlabelled pARE3 used as a competitor does not seem
to have any e¡ect on the shift. To show that it was the
ARE4 element that was responsible for competing out the
shift, a new 33 bp oligonucleotide with a mutation in the
TCF11/Nrf1 half-site of the ARE4 response element
(named pARE4m, Table 2) was used as a competitor
(Fig. 4B, lane 5). This oligonucleotide was not able to
interfere with the shift.
This indicates that the TCF11/Nrf1/MafG heterodimer
has a higher a¤nity for ARE4 when both ARE3 and
ARE4 are present. To further con¢rm this, probes with
only a single ARE element were labelled. Using pARE3 as
the labelled probe, no detection of heterodimeric binding
was observed (Fig. 4C, lane 2). Using pARE4 as the
probe, a shift with the TCF11/Nrf1/MafG heterodimer
was obtained (Fig. 4C, lane 4). This shift was competed
out when an excess of unlabelled pARE4 was added (Fig.
4C, lane 6). On the contrary, adding an excess of either
unlabelled pARE3 or pARE4m did not interfere with the
shift (Fig. 4C, lanes 5 and 7). Using pARE4m as the
labelled probe, no heterodimeric binding was observed,
strongly suggesting that this single base pair mutation is
enough to completely abolish TCF11/Nrf1/MafG binding
to the response element. These results establish that
TCF11/Nrf1/Maf binds speci¢cally to the distal element
ARE4 in the GCSh promoter and that heterodimeric binding to ARE3 does not occur in vitro. Mulcahy et al. have
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shown that both constitutive and L-naphtho£avone induced GCSh expression was dependent upon the same
distal ARE4 site in the promoter [8].
4. Discussion
Expression of TCF11/Nrf1 in COS-1 cells led to an increase in the intracellular glutathione level. Recently, it
was reported that ¢broblasts from Nrf1 knockout mice
showed a decreased level of glutathione and enhanced
sensitivity to oxidants as compared to cells from wild
type mice [23]. In the Nrf1 knockout ¢broblasts the
GCSl mRNA level was reduced, whereas the GCSh
mRNA level was not noticeably changed. Our data suggest that TCF11/Nrf1 can also transactivate the GCSh
promoter since we show a clear but low level transactivation of the GCSh promoter in transfection assays. Kwong
et al. did not ¢nd a detectable di¡erence in GCSh levels by
Northern blot analysis in cells from Nrf1 null mice whereas GCSl levels were induced to a lesser extent as compared
to wild type cells following stress induction. These observations may be reconciled if TCF11/Nrf1 has a relatively
moderate in£uence on the GCSh locus. Alternatively, Nrf2
or other transcription factors may be able to compensate
for the loss of TCF11/Nrf1 at the GCSh locus whereas it/
they cannot do so at the GCSl locus.
The transactivating e¡ect of TCF11/Nrf1 on the GCSh
promoter was largely mediated through the distal portion,
where two response elements (ARE3 and ARE4) that perfectly match the ARE consensus sequence, are located.
Yet only ARE4 was able to bind TCF11/Nrf1/MafG heterodimers in EMSA analyses. Therefore the presence of
consensus ARE motifs in the GCSh promoter, or elsewhere, does not necessarily constitute a binding site for
TCF11/Nrf1. This is in accordance with the ¢ndings of
Johnsen et al. [18] where the optimal TCF11/Maf binding
site was found to be TGCTgaGTCAt, with GTCAT representing the TCF11/Nrf1 half-site. This is a more limited
de¢nition of a response element than the ARE sequence
and a better indication of a possible site for TCF11/Nrf1
binding. It further emphasises that not all AREs are qualitatively equal ; TCF11/Nrf1 can bind to a subclass of
ARE elements while other potential AREs, if functional,
may be regulated by other factors or other combinations
of factors. A single base mutation in the ARE4 core to
TGCTgaGTCCt totally abolished the binding of Nrf1/
MafG in our EMSA analyses. The same single base mutation in the ARE4 sequence diminished both constitutive
and L-naphtho£avone induced expression of GCSh [8].
The ARE4 element may therefore represent a key regulatory element in the regulation of GCSh .
The GCSh promoter contains four ARE motifs in addition to two AP-1 sites and one NF-UB site [8]. This indicates that a tight regulation of the GCSh level, including
positive and negative in£uences, may be important. The
limited response to TCF11/Nrf1 we observe may be due to
this type of complex regulation. The GCSl promoter has
so far only one identi¢ed ARE element [27], which again
might indicate that a tight regulation through ARE elements is of less importance for this gene. Indeed, it has
been shown that the individual levels of mRNA for the
two subunits vary between di¡erent tissues [28,29].
Nrf2 can bind to and transactivate through ARE sites in
both the heavy and the light chain GCS promoters [21,30].
Nrf1 was found to be present (or detected) in nuclear
extracts capable of binding the ARE element in the
GCSl promoter, although Nrf2 was more prominent [30].
Both small Mafs and JunD are also implicated in the
regulation of the GCS genes [21,30]. The relatively large
number of possible bZIP dimer combinations may therefore contribute to a £exible but tightly regulated response
system of the GCS genes. We propose here that the low,
but signi¢cant level of transactivation by TCF11/Nrf1
through the GCSh promoter is important to achieve the
right level of the GCS protein complex in the cells.
A common function of the CNC bZIP factors TCF11/
Nrf1 and Nrf2 may be the regulation of the cellular response to chemical and oxidative stress mediated via ARE
motifs. Yet, it is clear that the two transcription factors do
not have entirely redundant functions, as they cannot
compensate for each other in knockout models
[22,23,31], both mutants su¡ering from reduced protection
against oxidative stress. Based on our ¢ndings we propose
that TCF11/Nrf1 plays a role in the regulation of GSH
synthesis. GSH is, however, the end point in a complex
metabolic pathway involving numerous proteins, including
GCSl and glutathione synthetase as well as GCSh . We
therefore suggest that TCF11/Nrf1 has the ability to activate transcription of several genes including GCSh , and
that regulation of the cellular antioxidant defence system
may be mediated, at least in part, through regulation of
TCF11/Nrf1 activity.
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