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Nrf1 overexpression increases the intracellular glutathione level and can transactivate the γ-glutamylcysteine synthetase (GCS) heavy subunit promoter
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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
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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¢-
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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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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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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.
References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12]
J.D. Hayes, L.I. McLellan, Free Radic. Res. 31 (1999) 273^300. M.E. Anderson, Chem.-Biol Interact. 111^112 (1998) 1^14. P.G. Richman, A. Meister, J. Biol. Chem. 250 (1975) 1422^1426. C.S. Huang, M.E. Anderson, A. Meister, J. Biol. Chem. 268 (1993) 20578^20583. C.S. Huang, L.S. Chang, M.E. Anderson, A. Meister, J. Biol. Chem. 268 (1993) 19675^19680. N. Yan, A. Meister, J. Biol. Chem. 265 (1990) 1588^1593. S.C. Lu, FASEB J. 13 (1999) 1169^1183. R.T. Mulcahy, M.A. Wartman, H.H. Bailey, J.J. Gipp, J. Biol. Chem. 272 (1997) 7445^7454. L.V. Favreau, C.B. Pickett, J. Biol. Chem. 266 (1991) 4556^4561. T. Nguyen, C.B. Pickett, J. Biol. Chem. 267 (1992) 13535^13539. T. Prestera, P. Talalay, J. Alam, Y.I. Ahn, P.J. Lee, A.M. Choi, Mol. Med. 1 (1995) 827^837. W.W. Wasserman, W.E. Fahl, Proc. Natl. Acad. Sci. USA 94 (1997) 5361^5366.
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M.C.W. Myhrstad et al. / Biochimica et Biophysica Acta 1517 (2001) 212^219 [13] N.C. Andrews, H. Erdjument-Bromage, M.B. Davidson, P. Tempst, S.H. Orkin, Nature 362 (1993) 722^728. [14] A. Kobayashi, E. Ito, T. Toki, K. Kogame, S. Takahashi, K. Igarashi, N. Hayashi, M. Yamamoto, J. Biol. Chem. 274 (1999) 6443^ 6452. [15] L. Luna, O. Johnsen, A.H. Skartlien, F. Pedeutour, C. Turc-Carel, H. Prydz, A.B. Kolsto, Genomics 22 (1994) 553^562. [16] P. Moi, K. Chan, I. Asunis, A. Cao, Y.W. Kan, Proc. Natl. Acad. Sci. USA 91 (1994) 9926^9930. [17] O. Johnsen, N. Skammelsrud, L. Luna, M. Nishizawa, H. Prydz, A.B. Kolsto, Nucleic Acids Res. 24 (1996) 4289^4297. [18] O. Johnsen, P. Murphy, H. Prydz, A.B. Kolsto, Nucleic Acids Res. 26 (1998) 512^520. [19] R. Venugopal, A.K. Jaiswal, Proc. Natl. Acad. Sci. USA 93 (1996) 14960^14965. [20] R. Venugopal, A.K. Jaiswal, Oncogene 17 (1998) 3145^3156. [21] A.C. Wild, H.R. Moinova, R.T. Mulcahy, J. Biol. Chem. 274 (1999) 33627^33636. [22] K. Itoh, T. Chiba, S. Takahashi, T. Ishii, K. Igarashi, Y. Katoh, T. Oyake, N. Hayashi, K. Satoh, I. Hatayama, M. Yamamoto, Y. Nabeshima, Biochem. Biophys. Res. Commun. 236 (1997) 313^322.
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[23] M. Kwong, Y.W. Kan, J.Y. Chan, J. Biol. Chem. 274 (1999) 37491^ 37498. [24] T. Wirth, L. Staudt, D. Baltimore, Nature 329 (1987) 174^178. [25] V. Natarajan, K.B. Holven, S. Reppe, R. Blomho¡, J.O. Moskaug, Biochem. Biophys. Res. Commun. 221 (1996) 374^379. [26] E.E. Prieschl, V. Novotny, R. Csonga, D. Jaksche, A. Elbe-Burger, W. Thumb, M. Auer, G. Stingl, T. Baumruker, Nucleic Acids Res. 26 (1998) 2291^2297. [27] H.R. Moinova, R.T. Mulcahy, J. Biol. Chem. 273 (1998) 14683^ 14689. [28] J.J. Gipp, H.H. Bailey, R.T. Mulcahy, Biochem. Biophys. Res. Commun. 206 (1995) 584^589. [29] Y. Kang, V. Viswanath, N. Jha, X. Qiao, J.Q. Mo, J.K. Andersen, J. Neurosci. Res. 58 (1999) 436^441. [30] H.R. Moinova, R.T. Mulcahy, Biochem. Biophys. Res. Commun. 261 (1999) 661^668. [31] J.Y. Chan, M. Kwong, R. Lu, J. Chang, B. Wang, T.S. Yen, Y.W. Kan, EMBO J. 17 (1998) 1779^1787.
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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¢-
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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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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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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.
References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12]
J.D. Hayes, L.I. McLellan, Free Radic. Res. 31 (1999) 273^300. M.E. Anderson, Chem.-Biol Interact. 111^112 (1998) 1^14. P.G. Richman, A. Meister, J. Biol. Chem. 250 (1975) 1422^1426. C.S. Huang, M.E. Anderson, A. Meister, J. Biol. Chem. 268 (1993) 20578^20583. C.S. Huang, L.S. Chang, M.E. Anderson, A. Meister, J. Biol. Chem. 268 (1993) 19675^19680. N. Yan, A. Meister, J. Biol. Chem. 265 (1990) 1588^1593. S.C. Lu, FASEB J. 13 (1999) 1169^1183. R.T. Mulcahy, M.A. Wartman, H.H. Bailey, J.J. Gipp, J. Biol. Chem. 272 (1997) 7445^7454. L.V. Favreau, C.B. Pickett, J. Biol. Chem. 266 (1991) 4556^4561. T. Nguyen, C.B. Pickett, J. Biol. Chem. 267 (1992) 13535^13539. T. Prestera, P. Talalay, J. Alam, Y.I. Ahn, P.J. Lee, A.M. Choi, Mol. Med. 1 (1995) 827^837. W.W. Wasserman, W.E. Fahl, Proc. Natl. Acad. Sci. USA 94 (1997) 5361^5366.
BBAEXP 93489 15-1-01
M.C.W. Myhrstad et al. / Biochimica et Biophysica Acta 1517 (2001) 212^219 [13] N.C. Andrews, H. Erdjument-Bromage, M.B. Davidson, P. Tempst, S.H. Orkin, Nature 362 (1993) 722^728. [14] A. Kobayashi, E. Ito, T. Toki, K. Kogame, S. Takahashi, K. Igarashi, N. Hayashi, M. Yamamoto, J. Biol. Chem. 274 (1999) 6443^ 6452. [15] L. Luna, O. Johnsen, A.H. Skartlien, F. Pedeutour, C. Turc-Carel, H. Prydz, A.B. Kolsto, Genomics 22 (1994) 553^562. [16] P. Moi, K. Chan, I. Asunis, A. Cao, Y.W. Kan, Proc. Natl. Acad. Sci. USA 91 (1994) 9926^9930. [17] O. Johnsen, N. Skammelsrud, L. Luna, M. Nishizawa, H. Prydz, A.B. Kolsto, Nucleic Acids Res. 24 (1996) 4289^4297. [18] O. Johnsen, P. Murphy, H. Prydz, A.B. Kolsto, Nucleic Acids Res. 26 (1998) 512^520. [19] R. Venugopal, A.K. Jaiswal, Proc. Natl. Acad. Sci. USA 93 (1996) 14960^14965. [20] R. Venugopal, A.K. Jaiswal, Oncogene 17 (1998) 3145^3156. [21] A.C. Wild, H.R. Moinova, R.T. Mulcahy, J. Biol. Chem. 274 (1999) 33627^33636. [22] K. Itoh, T. Chiba, S. Takahashi, T. Ishii, K. Igarashi, Y. Katoh, T. Oyake, N. Hayashi, K. Satoh, I. Hatayama, M. Yamamoto, Y. Nabeshima, Biochem. Biophys. Res. Commun. 236 (1997) 313^322.
219
[23] M. Kwong, Y.W. Kan, J.Y. Chan, J. Biol. Chem. 274 (1999) 37491^ 37498. [24] T. Wirth, L. Staudt, D. Baltimore, Nature 329 (1987) 174^178. [25] V. Natarajan, K.B. Holven, S. Reppe, R. Blomho¡, J.O. Moskaug, Biochem. Biophys. Res. Commun. 221 (1996) 374^379. [26] E.E. Prieschl, V. Novotny, R. Csonga, D. Jaksche, A. Elbe-Burger, W. Thumb, M. Auer, G. Stingl, T. Baumruker, Nucleic Acids Res. 26 (1998) 2291^2297. [27] H.R. Moinova, R.T. Mulcahy, J. Biol. Chem. 273 (1998) 14683^ 14689. [28] J.J. Gipp, H.H. Bailey, R.T. Mulcahy, Biochem. Biophys. Res. Commun. 206 (1995) 584^589. [29] Y. Kang, V. Viswanath, N. Jha, X. Qiao, J.Q. Mo, J.K. Andersen, J. Neurosci. Res. 58 (1999) 436^441. [30] H.R. Moinova, R.T. Mulcahy, Biochem. Biophys. Res. Commun. 261 (1999) 661^668. [31] J.Y. Chan, M. Kwong, R. Lu, J. Chang, B. Wang, T.S. Yen, Y.W. Kan, EMBO J. 17 (1998) 1779^1787.
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