Communicated by: Shunsuke Ishii
Identification of the interactive interface and phylogenic conservation of the Nrf2-Keap1 system
Article first published online: 7 AUG 2002
Genes to Cells
Volume 7, Issue 8, pages 807–820, August 2002
How to Cite
Kobayashi, M., Itoh, K., Suzuki, T., Osanai, H., Nishikawa, K., Katoh, Y., Takagi, Y. and Yamamoto, M. (2002), Identification of the interactive interface and phylogenic conservation of the Nrf2-Keap1 system. Genes to Cells, 7: 807–820. doi: 10.1046/j.1365-2443.2002.00561.x
- Issue published online: 7 AUG 2002
- Article first published online: 7 AUG 2002
- Received: 11 April 2002 Accepted: 7 May 2002
Background: The transcription factor Nrf2 and its negative regulator Keap1 play important roles in transcriptional induction of a set of detoxifying and anti-oxidant enzymes. To gain an insight into our present enigma as to how cells receive oxidative and electrophilic signals and transduce them to Nrf2, we have developed a zebrafish model system for molecular toxicological studies.
Results: We systematically cloned zebrafish cytoprotective enzyme cDNAs and found their expression to be efficiently induced by electrophilic agents. We consequently identified the presence of Nrf2 and Keap1 in zebrafish. Both loss- and gain-of-function analyses demonstrated that Nrf2 is the primary regulator of a subset of cytoprotective enzyme genes, while Keap1 suppresses Nrf2 activity in zebrafish. An ETGE motif, critical for the Nrf2–Keap1 interaction, was identified in the Neh2 domain of Nrf2 by reverse two-hybrid screening and found to be indispensable for the regulation of Nrf2 activity in zebrafish.
Conclusion: Taken together, these results indicate that the Nrf2-Keap1 system is highly conserved among vertebrates and that the interface between Nrf2 and Keap1 forms an important molecular basis of this regulatory system.
Mammalian cells have developed two primary lines of defence against electrophilic carcinogen metabolites and reactive oxygen species, the major causes of malignancy and cellular damage. These lines of defence consist of a high cellular level of anti-oxidants, such as glutathione (GSH), and a family of phase II detoxification enzymes, including glutathione S-transferases (GST) and NAD(P)H: quinone oxidoreductase (NQO1). Several lines of evidence indicate that an elevation in the levels of phase II enzymes and GSH protects against chemical carcinogens (Talalay et al. 1995). A wide variety of chemical agents can induce the transcription of phase II and anti-oxidant enzymes and genes encoding GSH biosynthetic enzymes, such as γ-glutamylcysteine synthetase (γGCS), which catalyses the rate-limiting reaction in de novo GSH synthesis. These inducers include oxidizable diphenols and quinones, Michael reaction acceptors, isothiocyanates, trivalent arsenicals, and hydroperoxides. Interestingly, these inducers do not share any substantial similarity in their structures, but they are all electrophiles capable of reacting with sulfhydryl groups.
Extensive biochemical analyses on the regulatory regions of genes encoding phase II enzymes revealed that the inducible expression is primarily mediated by a cis-acting regulatory sequence known as the anti-oxidant responsive element (ARE), or electrophile-responsive element (Primiano et al. 1997). Several years later, we discovered Nrf2 in our quest for transcription factors that bind to the ARE. Nrf2, a member of the Cap’n’Collar (CNC) family of transcription factors possessing a basic region-leucine zipper (bZip) structure, targets the ARE and transactivates genes encoding phase II enzymes (Itoh et al. 1999a; Hayes et al. 2000). This idea was supported by the severe impairment observed in the electrophile-induced activation of a battery of phase II enzymes and GSH biosynthetic proteins in viable Nrf2-deficient mice (Itoh et al. 1997; Ishii et al. 2000; McMahon et al. 2001). Moreover, it has recently been demonstrated that Nrf2-deficient mice are highly sensitive to carcinogen and/or oxidative stress. For instance, Nrf2-null mutant mice are susceptible to benzo[a]pyrene-induced neoplasia in the forestomach (Ramos-Gomez et al. 2001), diesel exhaust-induced hyperplasia and oxidative DNA adduct formation in the lung (Aoki et al. 2001), butylated hydroxytoluene-induced pulmonary injury (Chan & Kan 1999), and acetaminophen-induced hepatotoxicity (Enomoto et al. 2001; Chan et al. 2001).
Our recent studies suggest that Nrf2 activity is controlled by an interaction between Nrf2 and a cytoskeleton-associated protein called Keap1 (Kelch-like ECH Associating Protein 1)(Itoh et al. 1999b). Nrf2, which is normally localized in the cytoplasm, translocates to nuclei when cells are exposed to electrophiles. Even in the absence of an electrophilic exposure, forcing the expression of Nrf2 in cultured cells by-passed its cytoplasmic regulation, allowing Nrf2 to induce ARE-dependent gene expression. This induction was reduced by the simultaneous over-expression of Keap1 and de-repressed in the presence of electrophiles. These findings indicate that Nrf2 and Keap1 play critical roles in the induction of phase II enzymes and thus protection against malignancy.
Unravelling the molecular basis of the enzymatic induction defence mechanism will greatly accelerate the search for non-toxic cancer chemoprotective agents that potently induce phase II enzymes (Fahey & Talalay 1999; Kensler et al. 1999). The most important issue for solving this mechanism is a clarification of the regulatory mode of Nrf2 activation. For instance, what is the sensor molecule for a wide variety of electrophilic agents and how does this sensor transduce signals to Nrf2?
The zebrafish Danio rerio has emerged as an excellent model organism in which to study vertebrate biology. We hypothesized that zebrafish may serve as an excellent model system for addressing issues of toxicology and carcinogenesis, especially the molecular and genetic basis of Nrf2 activation. We know that GST genes can be induced by trans-stilbene oxide in flatfish (Leaver et al. 1993). Apart from that, however, little is known regarding the transcriptional regulation of phase II enzymes and GSH biosynthetic proteins in fish.
We assumed that fish also possess the Nrf2-Keap1 system for regulating enzymes that are cytoprotective against toxic electrophiles. In order to ascertain whether this regulatory mechanism does actually exist, we isolated zebrafish GSTπ, NQO1 and γGCS-h genes. We found these genes to be highly conserved in the zebrafish and its mammalian counterparts, indicating the importance of this defence mechanism for animal life. We also successfully isolated the zebrafish homologue genes of Nrf2 and Keap1. To our expectation, treatment with electrophiles and/or artificial Nrf2 expression induced gstp, nqo1 and γgcsh gene activation in zebrafish, but concomitant over-expression of Keap1 repressed Nrf2 from activating these genes. The important finding here is that the repressive function of Keap1 was diminished by a point mutation in Keap1 that abrogates its interaction with Nrf2. These results therefore unequivocally demonstrate that the Nrf2-Keap1 regulatory system of the phase II and anti-oxidant enzyme genes is highly conserved in vertebrates, from fish to mammals. The zebrafish system seems to be particularly beneficial for molecular mechanistic studies into the underlying toxicology and carcinogenesis in vivo.
Induction of detoxifying and anti-oxidant enzyme genes by electrophiles in zebrafish
Recent progress in zebrafish Expressed Sequence Tags (EST) projects led us to hypothesize that most of the detoxifying and anti-oxidant genes known in mammals may also exist in zebrafish. These genes include GST (α, µ, π and θ), NQO1, UDP-glucuronosyltransferase, and microsomal epoxide hydrolase. This knowledge further suggests that the regulatory mechanisms governing the expression of detoxifying and anti-oxidant genes may also be conserved between mammals and fish.
To address this important issue experimentally, we attempted a molecular cloning of several cDNAs encoding detoxifying and anti-oxidant enzymes in zebrafish. We first isolated gstp, a π-class GST gene of zebrafish, by exploiting EST database information to polymerase chain reaction (PCR)-amplify a zebrafish cDNA library. The percentage identity of deduced amino acid sequences was higher between zebrafish GSTπ and rat GSTπ (58%) than between zebrafish GSTπ and other classes of rat GST proteins (α, 29%; µ, 29%; σ, 27%; θ, 19%).
We carried out a gstp expression analysis in zebrafish larvae, either with or without electrophile treatment, by the whole mount in situ hybridization method. Albino fish were used in this analysis, because of their transparency during early larval development. A strong induction in gstp expression was observed in 7-day-old zebrafish larvae treated for 6 h with 30 µm of tert-butylhydroquinone (tBHQ), a synthetic anti-oxidant metabolized to an electrophilic quinone in cells (Fig. 1A, right panel). In contrast, no induction was observed in control larvae treated with vehicle alone (Fig. 1A, left panel). Induction in gstp was also observed in larvae at 4 days of age, but not in 24-h embryos (data not shown). Similarly, RNA blotting analyses indicated that gstp expression in larvae at 4 days old is markedly induced by tBHQ in a dose-dependent (Fig. 1B) and time-dependent (Fig. 1C) manner. These results, therefore indicate that gstp gene expression is inducible by tBHQ in zebrafish.
To test whether other electrophilic agents can induce gstp expression, we treated zebrafish larvae with diethylmaleate (DEM) 100 µm, and analysed the gene expression by RNA blot analysis. As expected, the GSTπ mRNA level was increased by DEM (Fig. 1D), suggesting that detoxifying and anti-oxidant enzymes in fish can be induced in response to a wide variety of electrophilic agents.
We also examined whether tBHQ can induce other detoxifying and anti-oxidant enzymes, including GSH biosynthetic enzymes. For this purpose, we isolated two additional zebrafish cDNAs encoding NQO1 and γGCS-h by a similar strategy used to isolate GSTπ cDNA. These zebrafish cDNAs showed a high amino acid sequence identity to rat NQO1 protein (51%) and the catalytic subunit of γGCS (71%). The expression of nqo1 and γgcsh genes was markedly induced by tBHQ in zebrafish larvae when examined by RT-PCR (Fig. 1E). These results further support the notion that the regulatory system responding to electrophilic agents by inducing the expression of detoxifying and anti-oxidant enzymes is common among vertebrates.
Identification of the zebrafish Nrf2
The induction of detoxifying and anti-oxidant genes by tBHQ and DEM in zebrafish implies that the Nrf2-Keap1 regulatory system exists in fish. Indeed, we found a cDNA related to Nrf2 in the EST database. To clarify this point therefore, we set about and successfully isolated full-length cDNA clones corresponding to the zebrafish EST clone. The deduced amino acid sequence identity between the zebrafish Nrf2-related clone and mouse Nrf2 is only 46.7%. Two highly conserved domains are present in the human and chicken Nrf2 proteins: the Neh2 domain (Nrf2-ECH homology)(Itoh et al. 1999b) and the CNC-type bZip (or Neh1) domain. We found the sequence identity between the Neh2 domains of zebrafish and mouse Nrf2 proteins to be 66% and that of the CNC-type bZip (Neh1) domains to be 71% (Fig. 2A). From this high sequence identity between the zebrafish and mouse Nrf2 domains, we concluded that the gene we isolated encodes zebrafish Nrf2 and we will refer to this gene as zebrafish nrf2. The functional analyses conducted in this study further supported this conclusion (below).
Phylogenetic trees based on comparisons among the CNC-bZip domain structures unequivocally indicated that zebrafish Nrf2 belongs to the Nrf2 subfamily rather than to the other CNC subfamilies, such as NF-E2 p45, Nrf1 or Nrf3 (Fig. 2B). Intriguingly, zebrafish Nrf2 contains a Neh4 domain (Fig. 2A), a domain conserved only among the Nrf2 subfamily, with 45% identity to the mouse Neh4 sequence (Katoh et al. 2001).
Nrf2 is essential for gstp induction by tBHQ
Recently, an in vivo gene targeting strategy using morpholino phosphorodiamidate oligonucleotide (MO) was established and has been successfully utilized in zebrafish (Ekker & Larson 2001). To examine whether Nrf2 is responsible for the inducible expression of detoxifying and anti-oxidant enzymes by electrophiles in zebrafish larvae, we exploited MO to reduce the endogenous expression level of Nrf2. We designed a specific MO for zebrafish Nrf2 mRNA (MO-nrf2) and injected it into zebrafish embryos to examine its effect on the expression of Nrf2-target genes (Fig. 3A). Day 4 larvae developed from MO-nrf2 or mock-injected embryos were treated with tBHQ (30 µm) or vehicle alone for 6 h and after the tBHQ treatment, the gstp expression in the larvae was analysed by RNA blotting analysis (Fig. 3B) or by in situ hybridization (Fig. 3C). In both cases, gstp expression was efficiently abolished by the MO-nrf2 treatment of embryos. These results indicated that Nrf2 is essential for the inducible expression of gstp by tBHQ.
The important findings from this analysis can be summarized into three points. Firstly, based on both structural and functional criteria, zebrafish Nrf2 is an authentic homologue of mammalian Nrf2. Secondly, Nrf2 regulation of detoxifying and anti-oxidant genes appears to be highly conserved among vertebrates. Thirdly, Nrf2 might not be crucial for early morphogenesis in zebrafish, as most of the embryos injected with MO-nrf2 executed normal development (Fig. 3C and data not shown). This observation is consistent with studies in rodents, in which Nrf2-deficient mice develop normally and are fertile (Itoh et al. 1997; Chan et al. 1996).
Nrf2 is a transcriptional activator in zebrafish embryos
We examined the transactivation activity of zebrafish Nrf2 by co-injection of firefly luciferase (Luc) reporter DNA and synthetic capped RNA providing zebrafish Nrf2 expression. We used pRBGP2 as a testing reporter, which contains three copies of ARE (or NF-E2 binding sequence) tandemly upstream of the rabbit β-globin basal promoter (Igarashi et al. 1994). After co-injecting pRBGP2 and Nrf2 mRNA into zebrafish embryos at the one-cell stage, the Luc activities of the whole cell extracts were measured at mid-gastrula. Luc expression was dramatically enhanced in embryos over-expressing Nrf2 compared to that in embryos injected with the reporter gene alone (approximately 75-fold; Fig. 4). This activation was not observed when we used pRBGP3, a reporter construct lacking the ARE sequences.
We recently showed that the Neh4 and Neh5 domains (see Fig. 2A) of the mouse Nrf2 protein bind cooperatively to the transcriptional co-activator CBP (CREB binding protein) and act synergistically to attain a maximum transcriptional activation in mouse hepatoma cells (Katoh et al. 2001). Since zebrafish Nrf2 possess both Neh4 and Neh5 domains (data not shown), the latter of which includes the FXE/DXXXL sequence known as a CBP binding motif in E1A protein (O’Connor et al. 1999), it is quite plausible that these two domains also play a role in transactivation in zebrafish. The contribution of the Neh4 and Neh5 domains to the transactivation activity in zebrafish embryos was assessed using synthetic mutant mRNA for zebrafish Nrf2 in which the Neh4 and Neh5 domains were deleted (ΔN4N5). Over-expression of ΔN4N5 resulted in insignificant transactivation of the pRBGP2 reporter gene (Fig. 4), indicating that the Neh4 and Neh5 domains of zebrafish Nrf2 are fundamentally important for transcriptional activation.
Nrf2 over-expression induces detoxifying and anti-oxidant enzymes in zebrafish embryos
It has been difficult to monitor the transactivation of endogenous target genes by Nrf2 in cultured cell lines (unpublished observations). One possible explanation is that cultured cells continuously suffer from oxidative stress, such that the basal expression level of Nrf2 target genes are already constitutively enhanced. We envisaged that this problem might be overcome by using the zebrafish system, in which we may be able to recapitulate the in vivo situation of gene expression in the absence of artificial oxidative stress.
We examined the effect of exogenous Nrf2 on gstp expression in zebrafish embryos. In situ hybridization analysis was performed using 6, 8 and 10 h embryos, injected either with or without Nrf2 mRNA at the one-cell stage. A remarkable expression of gstp in the whole body was observed in the Nrf2-over-expressing embryos at all three stages, but not in the control embryos (Fig. 5A). This induction was confirmed by RNA blot analysis (Fig. 5B). Deletion of the Neh4 and Neh5 domains eliminated this activity, indicating that these domains were actually required for transactivation of target genes, as was the case in the Luc reporter gene analysis. We also examined the induction of other Nrf2 target genes by RT-PCR analysis. The expression of both nqo1 and γgcsh was induced by the over-expression of Nrf2 in a dose-dependent manner (Fig. 5C), suggesting that Nrf2 is a master regulator of a subset of detoxifying and anti-oxidant enzymes.
Identification of zebrafish Keap1
Keap1, an actin-cytoskeleton binding protein, binds to and retains Nrf2 in the cytoplasm during unstressed conditions. Keap1 consequently represses the transcriptional activation of ARE-regulated genes by Nrf2 (Itoh et al. 1999b). Exposure of the cells to electrophiles disrupts this interaction, allowing Nrf2 to translocate into nuclei, resulting in de-repression of a subset of detoxifying and anti-oxidant genes. As we now understand that Nrf2 exists in zebrafish, it is of interest to identify the zebrafish Keap1. Therefore, we searched an EST database and found a candidate clone for zebrafish Keap1. A lambda phage cDNA library was screened in order to isolate cDNA clones encoding full-length zebrafish Keap1 protein. The deduced amino acid sequence of the cDNA product shows 49% and 55% identity to the BTB and double glycine repeat (DGR) domains, respectively, of the mouse Keap1 protein (Fig. 6A). A phylogenetic tree of Kelch family proteins based on the amino acid sequences of their DGR domains indicated that zebrafish Keap1 belongs to the Keap1 subfamily rather than to the other Kelch family proteins such as IPP, Mayven, KLHL3a, KLHL4 or NS1-BP (Fig. 6B).
The DGR domain of mouse Keap1 has been shown to interact with the Neh2 domain of mouse Nrf2 by yeast two hybrid and BIAcore interaction analyses (Itoh et al. 1999b). To test our prediction that zebrafish Keap1 and Nrf2 interact, we examined the interaction between the DGR domain of zebrafish Keap1 and the Neh2 domain of zebrafish Nrf2 by yeast two-hybrid analysis. Figure 6C shows yeast cells containing both the DGR domain of Keap1 and the Neh2 domain of Nrf2 grown in amino acid deficient media, while those containing Nrf2 or Keap1 alone could not grow in these culture conditions. These results demonstrated that the Keap1 and Nrf2 of zebrafish do indeed interact with each other.
To elucidate whether zebrafish Keap1 acts to repress the inducible function of Nrf2, we tested the extent of its repression on the Nrf2-mediated inducible expression of endogenous gstp in zebrafish embryos. The capped mRNA of zebrafish Keap1 was synthesized in vitro and co-injected with Nrf2 mRNA (100 pg) into zebrafish embryos at the one-cell stage. At midgastrula, total RNA was isolated from the injected embryos and gstp expression was analysed by RNA blot analysis. The inducible expression of gstp by Nrf2 was repressed by co-expression of Keap1 in a dose-dependent manner (Fig. 6D). This repression was also observed by whole mount in situ hybridization analysis (data not shown). Importantly, over-expression of Keap1 alone showed no obvious effect on zebrafish development, even when the amount of injected mRNA was increased to 200 pg (data not shown). These results unequivocally demonstrate that the mechanism by which Nrf2 activates cytoprotective gene expression is highly conserved between mammals and fish.
Identification of a motif in the Neh2 domain critical for Keap1 binding
In our effort to characterize amino acid residues in the Neh2 domain which are critical for the interaction between Nrf2 and Keap1, we screened for Neh2 mutants of Nrf2 that fail to interact with Keap1. To this end, we utilized the yeast reverse two-hybrid screening approach (Vidal 1997) using mouse Nrf2 and Keap1. We fused β-galactosidase to the C-terminal end of Neh2 to eliminate Neh2 clones to which nonsense mutations were introduced. Of the eight clones isolated in this screening, six clones were single point mutants. These six were classified into four groups: 82Glu to Gly (E82G), 80Thr to Ala (T80A), 79Glu to Val (E79V), and 82Glu to Val (E82V) (Fig. 7A and B). Since two clones were isolated for both T80A and E82V, the screening could be saturated. The results were quite intriguing, since the mutation sites are concentrated in the C-terminal region of the Neh2 domain, particularly at the ETGE sequence. This ETGE motif is completely conserved among vertebrate Nrf2 proteins, including the zebrafish Nrf2 identified in the present study (Fig. 7A).
We then examined how these point mutations affect the repressive activity of Keap1 on Nrf2. The Neh2-GFP construct containing the Neh2 domain fused to green fluorescence protein (GFP) has been shown to localize both in the cytoplasm and the nucleus when expressed in mouse NIH3T3 cells (Fig. 7C, C = N). However, when Neh2-GFP was co-transfected with Keap1, it was retained in the cytoplasm in a dose dependent manner (C > N). The important observation here is that the point mutation at 82Glu (E82G) greatly reduced Nrf2 translocation into the nucleus by Keap1, indicating that the ETGE motif is essential for the Keap1–Neh2 interaction, hence activation of Nrf2, in the cells.
Sequence alignment analysis revealed that two regions, the C-terminal stretch and the N-terminal stretch including the hydrophobic region, are conserved among vertebrate Neh2 domains (Fig. 7A, shaded amino acids). Strikingly, the ETGE motif in the C-terminal stretch also exists in the Neh2-related region of human and mouse Nrf1 (Fig. 7A). It is therefore plausible that Keap1 binds to Nrf1 and regulates its activation. The C-type, but not the A or B isoform, of Drosophila cap’n’collar protein (dCNC-C)(McGinnis et al. 1998) also has an ETGE motif in the N-terminal region (Fig. 7A). Interestingly, the dCNC-C protein possesses a stretch of amino acid residues similar to the hydrophobic region of Nrf2 on the N-terminal side of the ETGE motif (Fig. 7A). This observation suggests that the ETGE motif and the hydrophobic region may have some cooperative functions.
ETGE motif is essential for Keap1 to repress the Nrf2 activity
To elucidate whether the ETGE motif is critical for the Keap1 regulation of Nrf2 activity, various mutations were introduced into the Neh2 domain of mouse Nrf2 and the transactivation activity of these mutants was analysed using an ARE-linked reporter gene transfected into QT6 cells, either with or without Keap1 co-expression. All mutants showed comparable transactivation activity of the ARE-linked reporter gene with wild-type Nrf2, demonstrating that the ETGE motif is not important for the transcriptional activation per se (data not shown). Co-expression of Keap1 repressed the Nrf2-mediated induction of the ARE-Luc reporter gene in a dose dependent manner. To our expectation, mutations in the ETGE motif markedly diminished this repression (Fig. 8A). These results indicate that the ETGE motif is a key site where Keap1 acts to repress Nrf2 activity.
To assess whether the ETGE motif is actually important in regulating the Nrf2-mediated induction of endogenous detoxifying and anti-oxidant enzymes under physiological conditions, we introduced an E81G mutation corresponding to the E82G mutation in mouse Nrf2, into zebrafish Nrf2. We examined the effect of this ETGE mutation on gstp expression in zebrafish embryos by co-over-expression analysis. RNA blot analysis demonstrated that the E81G mutation in Nrf2 prevented repression of the inducible expression by Keap1, whereas Keap1 was able to repress wild-type Nrf2 (Fig. 8B). The same conclusion was obtained by analysing gstp induction by whole mount in situ hybridization (data not shown). Since the E81G mutation was less effective compared to a complete deletion of the Neh2 domain (data not shown), we surmise that E81G mutated Nrf2 can still weakly interact with Keap1. These results indicate that the ETGE motif is critical for the repression of Nrf2 activity by Keap1 in vivo.
To confirm our contention that the Neh2 mutations affected the Keap1–Nrf2 interaction, and provoked a de-repression of the Nrf2 activity, we prepared a Keap1 mutant molecule in which the DGR domain was deleted (ΔDGR) and examined the functional repression of this mutant on the activity Nrf2. The results demonstrated that Keap1ΔDGR could not repress the Nrf2-mediated inducible expression of the gstp gene (Fig. 8C). In conclusion, the ability of Keap1 to bind to Nrf2 through the ETGE motif is crucial to the function of Keap1 as a repressor of Nrf2 activity and this molecular mechanism is widely conserved among vertebrates.
We decided to use zebrafish to address the question as to how vertebrate cells sense oxidative and electrophilic stress signals and transduce them into an activation of Nrf2. In order to develop this animal model system, we isolated cDNA clones encoding zebrafish Nrf2 and Keap1, along with those encoding a number of detoxifying and anti-oxidant enzymes. The results of this study gave rise to conclusive evidence supporting the phylogenic conservation of the Nrf2-Keap1 regulatory system for a subset of defence enzymes within vertebrates. We also discovered a critical ETGE motif within Nrf2 that is necessary for its interaction with Keap1 and showed that the introduction of a point mutation in this motif abolished the repressive activity of Keap1 in vivo. Furthermore, our study established the zebrafish system as a valuable animal model system in which to study the regulation of cellular defence genes.
Conservation of the Nrf2-Keap1 regulatory system among vertebrates
In zebrafish, electrophile treatment induces the transcription of three genes: gstp, nqo1 and γgcsh, while this induction is eliminated by nrf2 gene knockdown with MO. Since these genes encode representative cytoprotective enzymes in zebrafish, the results suggest that Nrf2 is essential for the electrophile-induced transactivation of a subset of cytoprotective genes in fish. Indeed, over-expression of Nrf2 in zebrafish embryos transactivates these three genes in vivo. Already, we have thoroughly investigated the Nrf2-Keap1 system regulating detoxifying and anti-oxidant enzymes in the mammalian system (Itoh et al. 1999b; Ishii et al. 2000) and from our present study conclude that the Nrf2-Keap1 system also functions in fish.
Accumulation of GST following electrophile treatment has been reported in the flatfish Plaice (Leaver et al. 1993). ARE-like motifs were found in the promoter region of the plaice GST gene (Leaver et al. 1997). Although the molecular mechanism of GST gene induction in these fish remains to be elucidated, we suppose that these fish express Nrf2 (or a related molecule) that acts in the ARE-mediated induction of detoxifying enzymes. The present study, along with previous observations, suggests the conservation of the Nrf2-Keap1 regulatory system in a wide range of vertebrates. Indeed, the chicken Nrf2 homologue has been identified (Itoh et al. 1995) and EST clones highly homologous to Nrf2 were found in Xenopus (data not shown). The phylogenic conservation of the Nrf2-Keap1 regulatory system in mammals and fish further infers that protection against electrophilic chemicals and oxidative stress is critical in vertebrates, regardless of whether they are aquatic or land animals.
Regulatory mechanism of Nrf2 activation
Two regulatory mechanisms of Nrf2 activation have been proposed: subcellular localization (Itoh et al. 1999b) and protein phosphorylation (Huang et al. 2000). Translocation of Nrf2 to the nucleus in response to electrophiles is likely to represent a more significant regulatory step in Nrf2 activation. Indeed, electrophile-induced accumulation of Nrf2 in the nucleus (Itoh et al. 1999b) has been repeatedly observed in a number of laboratories (Ishii et al. 2000; Huang et al. 2000; Kwak et al. 2001; Ramos-Gomez et al. 2001). We reported previously that Keap1 interacts physically with Nrf2 and retains it in the cytoplasm of cells in the non-induced condition. Forced expression of Keap1 represses the transactivation activity of both mouse and chicken Nrf2 in fibroblasts (Itoh et al. 1999b).
We showed in this study that a physical interaction does occur between Nrf2 and Keap1 in zebrafish. The repression of Nrf2 activity by Keap1 was observed in various embryonic tissues and cells of the zebrafish. These results gave rise to the idea that Keap1 may be the major critical suppressor of Nrf2 in most vertebrate cells in the non-induced state, by physically interacting with and retaining Nrf2 in the cytoplasm. Our recent observation in Keap1 gene knockout mice substantiates this idea (unpublished data); the basal levels of both GSTπ and NQO1, and the abundance of Nrf2 in the nuclei were significantly elevated in the livers of Keap1-null mutant mice in comparison to the wild-type. According to this context, the interaction of Nrf2 with Keap1 might constitute a part of the sensor system responding to electrophiles or their downstream signals.
We wish to determine what triggers Nrf2 to dissociate from Keap1 in response to electrophiles. One plausible candidate is the phosphorylation of Nrf2 by protein kinase C (PKC) or mitogen-activated protein kinases (MAPK). Supporting this scenario, potential phosphorylation sites are conserved among vertebrate Nrf2 molecules, such as sites for PKC in the Neh2 domain (Fig. 7A, asterisk) or sites for MAPK in the Neh3 and Neh6 domains (data not shown). The PKC phosphorylation site is of particular interest, as it is located within the Neh2 domain that binds to Keap1, and because Nrf2 was shown to be phosphorylated by PKC both in vivo and in vitro (Huang et al. 2000). In addition, inhibitors of PKC were shown to suppress not only the transactivation of ARE-linked reporter genes, but also tBHQ-induced nuclear translocation of Nrf2 in human hepatoma cells (Huang et al. 2000). However, a strong reservation exists regarding PKC, based on several lines of evidence. For instance, co-transfection analysis in cultured cells (Itoh et al. 1999b) and the present mRNA injection analysis in zebrafish embryos indicate that a forced expression of naive Nrf2 circumvents the activating signalling pathway which may require post-transcriptional modification. PKC inhibitors did not completely suppress tBHQ-induced transactivation of the ARE-linked reporter gene, although it did completely suppress Nrf2 phosphorylation (Huang et al. 2000). Activation of PKC by phorbol esters in human erythroid cells induced the nuclear translocation of Jun and Fos, but not that of Nrf2 (Kim et al. 2001). Thus, our current hypothesis is that Nrf2 phosphorylation by PKC may facilitate the electrophile-induced dissociation of the Nrf2–Keap1 interaction, but not the trigger per se.
Identification of the ETGE motif
In this study, we identified the ETGE motif in Nrf2, a motif essential for the interaction of Nrf2 with Keap1 and for the cytoplasmic localization of Nrf2 during non-stressed conditions. In addition, an amino acid alignment analysis of the Neh2 domains indicated that the ETGE motif is also conserved in a subset of Nrf1 homologues (Fig. 7A). Nrf1 has been shown to positively regulate the expression of ARE-linked reporter genes (Venugopal & Jaiswal 1998) and fibroblasts from Nrf1-null mice showed a decreased level of glutathione and enhanced sensitivity to oxidants, as compared to cells from wild-type mice (Kwong et al. 1999). These observations imply that Nrf1 and Nrf2 may share a common activation mechanism for regulating cytoprotective genes.
Furthermore, we also found that the ETGE motif resides in the Drosophila CNC-C protein, together with its upstream hydrophobic region. CNC-C represents the longest of the three different CNC isoforms known to be required for the formation of mandibular and labral structures in the fly (McGinnis et al. 1998). Genomic sequence information on Drosophila indicates that the hypothetical gene CG3962 is a potential candidate for Drosophila Keap1 (see Fig. 6). It would be intriguing to know whether or not CNC-C interacts with CG3962 and, if so, the functions and regulatory mechanisms of the interaction should be clarified.
Isolation of cDNAs
Partial cDNA fragments encoding zebrafish GSTπ, NQO1, γGCS-h, Nrf2 and Keap1 were prepared by PCR using specific primers designed based on the corresponding EST sequences. The β-actin cDNA was a kind gift from Dr Higashijima (Higashijima et al. 1997). A cDNA library of 1-month-old zebrafish (Clontech) was screened using the partial cDNA clones as probes to isolate full-length Nrf2 and Keap1 cDNA clones. Probes were labelled using an AlkPhos Direct DNA labelling kit and positive plaques on the membrane filters were detected with CDP-Star as substrate, according to the manufacturer’s instruction (Amersham Pharmacia Biotech).
Fish and inducer treatment
Zebrafish embryos and larvae were obtained by natural mating. Albino fish were a kind gift from Dr Yuba (University Osaka). Stock solutions of tBHQ and DEM were prepared using dimethyl sulphoxide (DMSO) as a solvent. For the induction studies, larvae were placed in culture dishes containing inducers diluted with E3 medium (Haffter et al. 1996).
The plasmids pCS2nrf2 and pCS2keap1 were constructed by subcloning cDNAs for the open reading frame (ORF) regions of zebrafish Nrf2 and Keap1, respectively, into pCS2 vector (Rupp et al. 1994). For the construction of pCS2nrf2ΔN4N5, the cDNA fragment encoding 124Gln–215Ser of Nrf2 was deleted from pCS2nrf2. The point mutation GAG to GGG, corresponding to a Glu-to-Gly mutation at 81Glu of Nrf2, was introduced into pCS2nrf2 to give pCS2nrf2E81G. The construct pCS2keap1ΔDGR was made by replacing the cDNA encoding 281Phe to 601Met with that encoding Asp-Leu-Pro-stop. The PCR fragment corresponding to 1Met–89Leu of zebrafish Nrf2 was inserted into pGBT9 (Clontech) to give pGBDzNeh2. To make the pGADzDGR, cDNA corresponding to 280Ile–601Met of zebrafish Keap1 was ligated into pGADT7. To construct yGADNeh2LacZ, a LacZ cDNA fragment was terminally ligated to yGADNeh2 (Katoh et al. 2001). GFP cDNA from pEGFP-N1 (Clontech) was inserted into pcDNA3 vector (Invitrogen) to give pcDNA3/GFP. In turn, the mammalian expression plasmid pNeh2-GFP was constructed by cloning a cDNA corresponding to the Neh2 domain (1Met–99Ser) of mouse Nrf2 into pcDNA3/GFP. To give pNeh2E82G-GFP, a Glu-to-Gly point mutation was introduced by PCR at 82Glu of the mouse Nrf2 sequence in pNeh2-GFP. Mammalian expression plasmids for Nrf2 containing ETGE mutations were prepared by replacing the BglII–SalI fragment of pcDNA3/Nrf2 (Katoh et al. 2001) with those of yeast reverse two-hybrid plasmids containing ETGE mutations. The construct pcDNA3/Keap1 was designed by cloning the cDNA corresponding to the ORF region of mouse Keap1 into pcDNA3. The sequence of each construct was verified by DNA sequencing.
Total RNA was prepared from the whole bodies of 40–100 embryos or larvae using RNAzol (Tel.Test). For the RNA blot analysis, isolated RNA (5 µg for each lane) was separated by agarose gel electrophoresis, transferred to ZetaProbe GT membrane (Bio-Rad) and hybridized at 65 °C to 32P-labelled probes. For reverse transcriptase-PCR (RT-PCR) analysis, first-strand cDNA was synthesized by incubation at 42 °C for 1 h with AMV reverse-transcriptase (Roche) and 0.05–0.2 µL of this reaction mixture (total 20 µL) was used for the PCR. Sequences of the primers used for RT-PCR analysis were as follows: gstp, 5′-CTTCACTCAGCGCTACAAC and 5′-TGGCCAGAACATTTTCAAAGC; nqo1, 5′-AACGCCGCTGCGCGAGATGTTG and 5′-TCCATTCCTCCAGTGGTGAAGG; γgcsh, 5′-CCACATAACGTGAAACCGG and 5′-GTTCCCTCGATCATGTAGC; β-actin, 5′-TCCGTGACATCAAGGAGAAG and 5′-GCAATGCCAGGGTACATGG. Whole-mount in situ hybridization was performed as previously described (Kobayashi et al. 2001a).
Microinjection into zebrafish embryos
DNA constructs containing the Luc reporter gene were injected into the blastomeres of early one-cell stage embryos as previously described (Kobayashi et al. 2001b). For RNA injection, synthetic capped RNA was made using the SP6 mMESSAGE mMACHINE in vitro transcription kit (Ambion) using the linearized DNA of the pCS2 derivatives. MO-nrf2 was purchased from Gene-Tools. RNA and MO were injected into yolk at the one-cell stage and consequently spread out into the whole bodies.
QT6 and NIH3T3 cells were maintained in Dulbecco’s modified Eagle’s medium, supplemented with 10% foetal bovine serum. Cells were transfected with plasmids using the calcium phosphate precipitation method, as previously described (Itoh et al. 1999b). The subcellular localization of GFP-fusion proteins was examined by optical observation using a Leica DMIRBE microscope.
For the zebrafish embryos, 50 pg of reporter constructs were co-injected with 100 pg of wild-type or mutant Nrf2 mRNA into yolk at the one-cell stage. Embryos were harvested at mid-gastrula and the Luc activity in five embryos for each condition was determined. For the cultured cells, reporter constructs were co-transfected with Nrf2s and/or Keap1 expression plasmids and a Luc assay was performed 48 h after transfection, as previously described (Itoh et al. 1999b). All Luc activities were analysed using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s instruction. Transfection efficiencies were routinely normalized to the activity of a Renilla Luc expression plasmid, pRL-TK. Three independent experiments, each carried out in duplicate, were performed.
Yeast two-hybrid and reverse two-hybrid analyses
Yeast two-hybrid analysis was performed using the Matchmaker Two-hybrid System (Clontech) according to the manufacturer’s instructions. After transformation of Saccharomyces cerevisiae HF7c with each of the two pGBT9 and pGADT7 derivatives, cells viable on synthetic media lacking Leu and Trp were plated on to Trp−Leu−His− media containing 50 mm 3-amino-1,2,4-triazole and grown at 30 °C for four overnights. For the reverse two-hybrid screening, mouse cDNA for the Neh2 domain was amplified by mutagenic PCR (Vidal 1997). PCR products were mixed with StuI-linearized yGADNeh2LacZ and co-transformed into MAV203 yeast cells (Gibco BRL) with pGBT9Keap1 (Itoh et al. 1999b). Cells were plated on to Trp−Leu− media containing 0.1% 5-fluoroorotic acid and viable clones were segregated in Leu− media for 2 days to obtain leucine auxotrophs. The β-galactosidase activity of each Leu auxotroph was analysed as previously described (Katoh et al. 2001).
We thank Ms Toshiko Arai and Ayako Hayashi for help in the fish maintenance and Mr Toshiaki Nakamura for technical assistance. We also thank Dr Shunsuke Yuba for the albino fish, Dr Sin-ichi Higashijima for a probe, Dr Tomonori Hosoya for technical advice, and Drs Igor B. Dawid, Kazuhiko Igarashi, Tetsuro Ishii, Tania O’Connor and Nobunao Wakabayashi for critical reading of the manuscript. This work was supported by the Japanese Society for Promotion of Sciences, and the Ministry of Education, Science, Sports and Culture of Japan.
- 2001) Accelerated DNA adduct formation in the lung of the Nrf2 knockout mouse exposed to diesel exhaust. Toxicol. Appl. Pharmacol. 173, 154–160. , , , , & (
- 2001) An important function of Nrf2 in combating oxidative stress: detoxification of acetaminophen. Proc. Natl. Acad. Sci. USA 98, 4611–4616. , & (
- 1999) Nrf2 is essential for protection against acute pulmonary injury in mice. Proc. Natl. Acad. Sci. USA 96, 12731–12736. & (
- 1996) NRF2, a member of the NFE2 family of transcription factors, is not essential for murine erythropoiesis, growth, and development. Proc. Natl. Acad. Sci. USA 93, 13943–13948. , , & (
- 2001) Morphant technology in model developmental systems. Genesis 30, 89–93. & (
- 2001) High sensitivity of Nrf2 knockout mice to acetaminophen hepatotoxicity associated with decreased expression of ARE-regulated drug metabolizing enzymes and antioxidant genes. Toxicol. Sci. 59, 169–177. , , , et al. (
- 1999) Antioxidant functions of sulforaphane: a potent inducer of Phase II detoxication enzymes. Food Chem. Toxicol. 37, 973–979. & (
- 1996) The identification of genes with unique and essential functions in the development of the zebrafish, Danio rerio. Development 123, 1–36. , , , et al. (
- 2000) The Nrf2 transcription factor contributes both to the basal expression of glutathione S-transferases in mouse liver and to their induction by the chemopreventive synthetic antioxidants, butylated hydroxyanisole and ethoxyquin. Biochem. Soc. Trans. 28, 33–41. , , , et al. (
- 1997) High-frequency generation of transgenic zebrafish which reliably express GFP in whole muscles or the whole body by using promoters of zebrafish origin. Dev. Biol. 192, 289–299. , , , & (
- 2000) Regulation of the antioxidant response element by protein kinase C-mediated phosphorylation of NF-E2-related factor 2. Proc. Natl. Acad. Sci. USA 97, 12475–12480. , & (
- 1994) Regulation of transcription by dimerization of erythroid factor NF-E2 p45 with small Maf proteins. Nature 367, 568–572. , , , , & (
- 2000) Transcription factor Nrf2 coordinately regulates a group of oxidative stress-inducible genes in macrophages. J. Biol. Chem. 275, 16023–16029. , , , et al. (
- 1997) An Nrf2/small Maf heterodimer mediates the induction of phase II detoxifying enzyme genes through antioxidant response elements. Biochem. Biophys. Res. Commun. 236, 313–322. , , , et al. (
- 1995) Cloning and characterization of a novel erythroid cell-derived CNC family transcription factor heterodimerizing with the small Maf family proteins. Mol. Cell. Biol. 15, 4184–4193. , , , & (
- 1999a) Regulatory mechanisms of cellular response to oxidative stress. Free Rad. Res. 31, 319–324. , , & (
- 1999b) Keap1 represses nuclear activation of antioxidant responsive elements by Nrf2 through binding to the amino-terminal Neh2 domain. Genes Dev. 13, 76–86. , , , et al. (
- 2001) Two domains of Nrf2 cooperatively bind CBP, a CREB binding protein, and synergistically activate transcription. Genes Cells 6, 857–868. , , , , & (
- 1999) Development of cancer chemopreventive agents: oltipraz. as a paradigm. Chem. Res. Toxicol. 12, 113–126. , , , & (
- 2001) Hemin-induced activation of the thioredoxin gene by Nrf2: a differential regulation of the antioxidant responsive element by a switch of its binding factors. J. Biol. Chem. 276, 18399–18406. , , , , & (
- 2001a) The homeobox protein Six3 interacts with the Groucho corepressor and acts as a transcriptional repressor in eye and forebrain formation. Dev. Biol. 232, 315–326. , , & (
- 2001b) Hematopoietic regulatory domain of gata1 gene is positively regulated by GATA1 protein in zebrafish embryos. Development 128, 2341–2350. , & (
- 2001) Role of transcription factor Nrf2 in the induction of hepatic phase 2 and antioxidative enzymes in vivo by the cancer chemoprotective agent, 3H-1, 2-dimethiole-3-thione. Mol. Med. 7, 135–145. , , , & (
- 1999) The CNC basic leucine zipper factor, Nrf1, is essential for cell survival in response to oxidative stress-inducing agents: role for Nrf1 in γ-gcs and gss expression in mouse fibroblasts. J. Biol. Chem. 274, 37491–37498. , & (
- 1993) Cloning and characterization of the major hepatic glutathione S-transferase from a marine teleost flatfish, the plaice (Pleuronectes platessa), with structural similarities to plant, insect and mammalian Theta class isoenzymes. Biochem. J. 292, 189–195. , & (
- 1997) Structure and expression of a cluster of glutathione S-transferase genes from a marine fish, the plaice (Pleuronectes platessa). Biochem. J. 321, 405–412. , & (
- 1998) A cap ‘n’ collar protein isoform contains a selective Hox repressor function. Development 125, 4553–4564. , , & (
- 2001) The Cap’n’Collar basic leucine zipper transcription factor Nrf2 (NF-E2 p45-related factor 2) controls both constitutive and inducible expression of intestinal detoxification and glutathione biosynthetic enzymes. Cancer Res. 61, 3299–3307. , , , et al. (
- 1999) Characterization of an E1A–CBP interaction defines a novel transcriptional adapter motif (TRAM) in CBP/p300. J. Virol. 73, 3574–3581. , , , & (
- 1997) Antioxidant-inducible genes. Adv. Pharmacol. 38, 293–328. , & (
- 2001) Sensitivity to carcinogenesis is increased and chemoprotective efficacy of enzyme inducers is lost in nrf2 transcription factor-deficient mice. Proc. Natl. Acad. Sci. USA 98, 3410–3415. , , , et al. (
- 1994) Xenopus embryos regulate the nuclear localization of XMyoD. Genes Dev. 8, 1311–1323. , & (
- 1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425. & (
- 1995) Chemoprotection against cancer by Phase 2 enzyme induction. Toxicol. Lett. 82–83, 173–179. , , , & (
- 1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucl. Acids Res. 22, 4673–4680. , & (
- 1998) Nrf2 and Nrf1 in association with Jun proteins regulate antioxidant response element-mediated expression and coordinated induction of genes encoding detoxifying enzymes. Oncogene 17, 3145–3156. & (
- 1997) The reverse two-hybrid system. In: The Yeast Two-Hybrid System (eds P.L.Bartel & S.Fields), pp. 109–147. New York, NY: Oxford University Press. (