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The pathways and molecular mechanisms regulating Nrf2 activation in response to chemical stress
Free Radical Biology & Medicine, Vol. 37, No. 4, pp. 433 – 441, 2004 Copyright D 2004 Elsevier Inc. Printed in the USA. All rights reserved 0891-5849/$-see front matter
doi:10.1016/j.freeradbiomed.2004.04.033
Serial Review: EpRE and Its Signaling Pathway Serial Review Editor: Henry J. Forman; Guest Co-Editor: Douglas Ruden THE PATHWAYS AND MOLECULAR MECHANISMS REGULATING NRF2 ACTIVATION IN RESPONSE TO CHEMICAL STRESS TRUYEN NGUYEN,*,y CHUNG S. YANG, y and CECIL B. PICKETT * *Schering – Plough Research Institute, Kenilworth, NJ 07033, USA; and y Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, NJ 08854, USA (Received 24 February 2004; Revised 26 April 2004; Accepted 28 April 2004) Available online 19 May 2004
Abstract—The induction of many antioxidant and phase II drug-metabolizing enzymes by phenolic antioxidants and electrophilic compounds is regulated at the transcriptional level. The response to these compounds is mediated by the cisacting antioxidant response element (ARE) found in the promoter of the encoding genes. The transcription factor NF-E2related factor 2, or Nrf2, has emerged as the central protein that binds to the ARE to activate gene transcription. Data from many studies indicate that Nrf2 is constitutively and ubiquitously expressed in a number of tissues and cell lines and is thus responsible for the low-level expression of its target genes observed under physiological conditions. However, in cells exposed to oxidative stress, Nrf2 activity is increased, further driving the transcriptional activation of genes whose expression is essential to control cellular redox homeostasis. Recent studies suggest that the activation of Nrf2 involves a coordinated process and is regulated at multiple levels. Nrf2 activity is believed to be repressed through the binding of the cytoskeleton-associated protein Keap1, and its activation involves mechanisms that interfere with this interaction. Activation of Nrf2 has also been demonstrated to be dependent on mechanisms that mediate its stabilization. In this review, the mechanisms controlling this activation process as reported in recent studies will be examined and discussed, with particular emphasis on those affecting Nrf2 stability at the molecular level. D 2004 Elsevier Inc. All rights reserved. Keywords—Free radicals
expressed at a low level under physiological conditions. However, their expression is increased in cells exposed to a diverse range of compounds, including oxidants, phenolic antioxidants, and electrophilic agents that can also undergo redox cycling to produce excess reactive oxygen species. And central to the induction of these enzymes is the transcription factor NF-E2-related factor 2 (Nrf2), which acts through the antioxidant response element (ARE) to activate gene transcription [1]. The involvement of Nrf2 in both the constitutive and the inducible expression of ARE-dependent genes has been well established and documented in numerous in vitro and in vivo studies [2 –14]. The molecular mechanisms and signaling pathways that regulate the activation of Nrf2 have thus been the focus of more recent studies. One of the mechanisms that have been widely studied is
INTRODUCTION
Chemical stress caused by environmental toxicants, and xenobiotic metabolism can lead to the pathogenesis of many inflammatory and degenerative diseases, including cancer, Alzheimer disease, and arthritis. To protect cells against oxidative damage by this chemical stress, many antioxidant and phase II drug-metabolizing enzymes act in concert to detoxify and eliminate these harmful chemicals and their metabolites. These enzymes are constitutively This article is part of a series of reviews on ‘‘EpRE and Its Signaling Pathway.’’ The full list of papers may be found on the home page of the journal. Address correspondence to: Cecil B. Pickett, Schering – Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA; Fax: (908) 740-7514; E-mail: [email protected]. 433
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that mediated by the repressor protein Keap1, which acts by directly interacting with Nrf2 and preventing its nuclear accumulation. Activation occurs when this interaction and repression is disrupted, allowing Nrf2 to translocate and accumulate in the nucleus [15]. The activation of Nrf2 has also been demonstrated to be mediated by mechanisms that lead to its stabilization, thus increasing levels of cellular Nrf2 and subsequent transcriptional activity [16,17]. Upregulation of Nrf2 gene expression at the transcriptional level has also been reported as another mechanism that contributes to increased amounts of Nrf2 in the cells in response to ARE inducers [18]. Thus, induction of the ARE gene battery is largely dependent on regulatory mechanisms acting in concert to increase the Nrf2 protein level and promote its subsequent accumulation in the nucleus to drive transcription. It should be noted that additional mechanisms affecting other activities of the transcription factor are also likely to occur during this activation process. These could include posttranslational modifications that affect its transactivational activity, DNA-binding affinity, or interaction with other transcription factors or with components of the transcription machinery. In this review, our discussion will be focused primarily on the posttranscriptional mechanisms that regulate Nrf2 activation, with particular emphasis on protein stabilization and the interaction with Keap1. There is now growing evidence suggesting a link between the inhibition of Keap1 activity and the stabilization of Nrf2, and we will briefly review key data on the molecular mechanisms that regulate these two processes. REGULATION OF NRF2 ACTIVATION
Protein stabilization as a mechanism for Nrf2 activation The activity of regulatory proteins such as transcription factors is often regulated posttranscriptionally and/or posttranslationally. Although transcription of the Nrf2 gene has been found to increase in murine keratinocytes in response to 3H-1,2-dithiole-3-thione [18], the Nrf2 mRNA levels were found to remain steady and unaffected by other inducers of ARE-dependent genes in other experimental systems [4,16,17,19]. These data suggest that posttranscriptional mechanisms are important in the regulation of Nrf2 activity. Indeed, data from cell labeling studies show that treatment of HepG2 cells with the phenolic antioxidant tert-butylhydroquinone (tert-BHQ) led to an increased level of Nrf2 phosphorylation that was accompanied by its nuclear accumulation [20]. In addition, tert-BHQ was also found to induce an overall increase in the level of cellular Nrf2 without affecting the apparent rate of Nrf2 transcription [16]. Of particular interest is that this posttranscriptional response is not unique to this phenolic antioxidant because similar effects
were observed in cells treated with other compounds, including h-naphthoflavone [16], cadmium [17], sulforaphane (Sul) [21], and diethylmaleate (DEM) [22], four structurally diverse inducers of the ARE-dependent gene expression. These observations provide evidence of a regulatory mechanism that increases the stability of Nrf2, resulting in its accumulation in the cell. This notion is confirmed by experimental results showing that the halflife of Nrf2 is extended in cells exposed to the same inducers. Because cycloheximide was used to block de novo protein synthesis in these experiments, these data provide evidence that a posttranslational mechanism is involved in increasing the Nrf2 protein level [16,17]. As the stability of intracellular proteins is often determined by the rate of its degradation by the ubiquitindependent proteasome, this proteolytic machinery became the prime candidate to be involved in the regulatory pathway that controls Nrf2 protein stability and hence its transcriptional activity. Evidence for a role for the proteasome was initially demonstrated in a study in which HepG2 cells treated with the proteasome inhibitor lactacystin had increased levels of the mRNA encoded by the ARE-dependent g-glutamylcysteine synthetase (GLCLC) gene [23]. That the 26S proteasome participates directly in the regulation of Nrf2 stability was examined further in other studies using lactacystin and another commonly used inhibitor of the proteasome, MG-132. Both of these inhibitors were found to increase the levels of Nrf2 protein and its ubiquitylated forms in HepG2 cells in a timedependent manner [16,17]. In addition, the elevated Nrf2 levels were accompanied by an increase in the expression of an ARE-driven reporter gene, two effects identical to those induced by tert-BHQ. These data are consistent with the notion that the activation of Nrf2 by chemical stress involves protein stabilization and is likely to be regulated by mechanisms that decrease the rate of its degradation by the ubiquitin-dependent proteasome. What has also become apparent is that, regardless of the mechanisms involved, an increase in cellular Nrf2 is essential for the induction of ARE-dependent genes. How this increase leads to an accumulation in the nucleus is not well understood, but it is presumed that Nrf2 can translocate readily into the nucleus and that this process is not necessarily regulated in a specific manner by the inducers. This presumption agrees well with experimental data showing a dose-dependent increase in the ARE-driven reporter gene activity in cells transfected with increasing amounts of an Nrf2-expressing vector without the need for treatment with an ARE inducer. Under these conditions, Nrf2 was apparently able to translocate into the nucleus to activate gene transcription in a nonregulated manner [2,4 – 6]. The fact that inhibitors of the proteasome not only stabilize Nrf2 but also increase the expression of an AREdriven reporter gene [16,17] or that of an endogenous gene
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[23] also clearly links Nrf2 stabilization with its activity in the nucleus. In addition, direct analysis of fractionated cell lysates demonstrates that treatment of cells with these inhibitors leads to an increased level of Nrf2 in the nucleus rather than in the cytosolic fraction [22]. Thus, Nrf2 stabilization alone seems to be sufficient to trigger its nuclear accumulation. The similar effects induced by lactacystin and MG132 raise an important question about whether the ARE inducers act by inhibiting the proteasome activity to stabilize Nrf2 and thus increase its transcriptional activation of target genes. Current evidence, however, argues against this possibility. Conditions of oxidative stress seem to increase rather than inhibit the overall activity of the ubiquitin-dependent proteasome, a response that is apparently necessitated due to the need to remove damaged proteins from the cells [24]. More recently, upregulation of genes encoding several subunits of the proteasome complex has also been observed in mice treated with a dithiolethione [25]. These data suggest that ARE inducers are unlikely to act as proteasome inhibitors but rather through distinct pathways to stabilize Nrf2. Together, these studies provide compelling evidence that protein stabilization is an integral part of the major regulatory mechanisms mediating Nrf2 activation by many inducers of the ARE-dependent genes. Phosphorylation and Nrf2 stability The degradation of proteins by the ubiquitin-dependent 26S proteasome is a highly regulated process that involves the specific recognition and targeting of the substrate molecule for ubiquitylation by an E3 ubiquitin ligase before degradation by the proteasome complex. How target proteins are selected for degradation is based primarily on the presence of specific structures known as degrons that are generally composed of a short and specific amino acid sequence and/or a particular conformational structure [26,27]. These degrons serve as recognition motifs for the E3 ligase and the interaction between them is believed to represent the critical, ratelimiting step that regulates the stability of the target protein. The stabilization of Nrf2 by ARE inducers may be brought about by a decrease in the rate of its degradation, and this could be mediated by mechanisms that prevent or weaken the recognition/interaction of Nrf2 by its E3 ligase or by directly inhibiting the E3 ligase activity itself. As protein modification by phosphorylation is the major posttranslational mechanism in signaling processes, it is believed to have a central role in regulating the stability of the Nrf2 protein. Recent data showing that tert-BHQ increases Nrf2 protein phosphorylation in HepG2 cells [20] provide evidence for this possibility. Further support for this mechanism is the finding that the
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phosphatase inhibitor okadaic acid, which induces cellular hyperphosphorylation [28], has positive effects on both the Nrf2 accumulation and the transcriptional activation of an ARE-driven reporter gene [16]. Although these data do not provide conclusive evidence, they do suggest that stabilization of Nrf2 is intimately linked to protein phosphorylation. In other such cases, phosphorylation commonly occurs at one or more sites close to, or within, the degrons to impair the protein recognition by the proteasome. This mechanism would be reminiscent of that controlling p53 stability. Under physiological conditions, p53 is bound and targeted for ubiquitindependent degradation by the Mdm2 protein, an E3 ligase; in response to stimulating signals such as DNA damage by ionizing radiation or UV light, p53 is phosphorylated at Ser-20 by the activated ATM/ATR kinases, which weakens its interaction with Mdm2 and thus leads to its stabilization [29 –31]. Though phosphorylation of Nrf2 seems to be important for its stabilization the identity of the protein kinase(s) involved has yet to be determined. In a recent study, inhibitors of the MAPK/ERK signaling pathway were found to interfere with the inducing effects of tertBHQ on the level of Nrf2 in HepG2 cells [16], suggesting a possible role for this pathway in the regulation of its stability. Consistent with this observation is the finding that treatment of HepG2 or Hepa1c1c7 cells with either tert-BHQ or Sul led to ERK2 activation as well as the induction of ARE-dependent genes and that both of these inducing effects were impaired by the MEK inhibitor PD98059 [32]. It is not known from these studies whether ERK2 itself or other downstream kinases are responsible for directly phosphorylating Nrf2. What has been demonstrated is that Nrf2 can be phosphorylated by members of the protein kinase C (PKC) family (discussed below), and this was shown to interfere with its interaction with Keap1 and promote its nuclear accumulation [33,34]. Phosphorylation by these kinases, however, is not directly responsible for Nrf2 stabilization because inhibition of their activity by staurosporine did not have any effects on the tert-BHQ-induced accumulation of Nrf2 in HepG2 cells [16,35]. More recently, Nrf2 was reported to be phosphorylated by PERK, a kinase activated in response to stress in the endoplasmic reticulum. Similar to effects produced by PKC, phosphorylation by this kinase was shown to induce the dissociation of Nrf2 from Keap1 and increase its nuclear import [36]. Because protein stability was not examined in this study, it is unclear if PERK contributes to the phosphorylation-dependent stabilization of Nrf2. Apart from the aforementioned kinases, the phosphatidylinositol 3-kinase (PI3K) cascade has also been implicated in the regulation of ARE-dependent genes in H4II rat hepatoma cells [37,38] as well as in IMR-32
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human neuroblastoma cells [39,40], but whether kinases of this pathway act directly on Nrf2 to affect its stability is not known. It has recently been reported that PI3K mediates the induction of Nrf2 nuclear accumulation by hemin in SH-SY5Y human neuroblastoma cells, as treatment with either wortmannin or LY 294002 was demonstrated by immunoblot analysis to antagonize this hemin-induced effect [41]. In a separate study, LY294002 was shown to attenuate the stabilizing effects of the phytochemical carnosol on the cellular Nrf2, suggesting a possible role for PI3K in the regulation of Nrf2 stability [42]. The nature of the molecular mechanisms mediated by this pathway awaits further study. Together these studies demonstrate that phosphorylation is important for Nrf2 activation and it involves different protein kinases, each of which may be associated with regulating a specific activity of the transcription factor. With the exception of PKC, which phosphorylates Nrf2 at Ser-40 [33 –35], the target sites for other kinases have yet to be identified. Because many of these studies focused their analysis on the effects of inducers and kinase activity on the accumulation of Nrf2 in the nucleus, as opposed to the total cellular Nrf2 level, it is of particular interest to determine whether the nuclear accumulation observed in these studies was the result of an enhanced nuclear translocation process or a direct result of its stabilization. A distinction between these two processes would provide a better understanding of the role of each protein kinase cascade in the regulation of Nrf2 activity. Another critical question is whether Nrf2 is phosphorylated simultaneously by all these different protein kinases in response to all ARE inducers or whether its phosphorylation is mediated only by specific kinases determined by the nature of the inducers and cell types. Given that compounds capable of inducing the Nrf2/ARE-dependent gene expression are structurally diverse and work in a wide variety of cell types, this would suggest the latter possibility. Thus, whereas the stabilization of Nrf2 might depend on phosphorylation by certain kinases in one cell type, this mechanism could be mediated by different kinases in another cell type. The activation of specific kinase cascades may similarly be determined by the nature of the inducing compounds. Future studies focusing on the identification of phosphorylation sites will facilitate the determination of the role of specific kinases involved in various mechanisms regulating the activation of Nrf2. Keap1 and regulation of Nrf2 activation The identification of Keap1, and its rat homolog termed INrf2 [43], as a repressor of Nrf2 activity defined the current regulatory pathways for Nrf2 activation [15]. Keap1 was initially isolated in a yeast two-hybrid screening experiment to identify potential proteins that interact with Nrf2. Biochemical and localization studies
indicate that Keap1, being anchored in the cytoplasm, functions by binding and preventing nuclear accumulation of Nrf2 and thus repressing its transcriptional activity (Fig. 1). This repressing activity is lost in cells under oxidative stress, as DEM, a compound with the propensity to react with sulfhydryl groups and induce oxidative stress, was shown to induce the dissociation of Nrf2 from Keap1 and promote nuclear accumulation of the transcription factor. For this reason, the Keap1/Nrf2 complex was proposed to constitute a cellular redox sensor through which the activation of ARE-dependent genes in response to oxidative stress is regulated [15]. This hypothesis is also supported by the observation that the Keap1 protein contains a large number of cysteine residues, some of which are thought to represent target sites for direct attack by electrophilic compounds to cause the release of the Nrf2 protein. Indeed, four potential cysteines at residues 257, 273, 288, and 297 have recently been identified by in vitro labeling and competition studies in which their reactivity to several alkylating compounds was measured and demonstrated [44]. The role of these cysteine residues was explored further in a cell-based study to define the biological function of Keap1 [45]. A significant finding in this report demonstrated that Keap1 not only acts to retain Nrf2 in the cytoplasm but also actively promotes its ubiquitylation and subsequent degradation by the proteasome. Two cysteine residues in Keap1, C273 and C288,
Fig. 1. Repression of Nrf2 activity by Keap1. (A) Under the current model, repression of Nrf2 has been proposed to be mediated by a mechanism by which Keap1 acts to retain the transcription factor in the cytoplasm and Nrf2 activation occurs when this interaction is disrupted by inducers of the ARE-dependent genes, allowing Nrf2 to translocate into the nucleus to effect its transcriptional activity. (B) Based on its homology with the Kelch protein, the structure of Keap1 is defined as consisting of two distinct domains known as the BTB and Kelch domains, separated by an intervening region (Linker) and flanked by two regions at the NH2-terminus (N) and COOH-terminus (C) as described [15,45]. Keap1 interacts with Nrf2 by binding to its Neh2 domain. The ETGE motif within the Neh2 domain important for this interaction [49] is indicated. Cysteine residues in the Keap1 protein that are important for its activity are also indicated.
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were found to be essential for this function, as mutation of either residue to serine abolishes this ubiquitylating activity. In addition, these Keap1 mutants not only fail to repress Nrf2 but also actually increase its half-life and transcriptional activity, suggesting that loss of Keap1’s ability to repress is important for Nrf2 stabilization. The importance of these two cysteine residues in the Keap1dependent repression of Nrf2 has also been independently reported in another study [46]. A third cysteine residue, C151, has been identified and found to be critical for the inducible release of Nrf2 from Keap1 repression and for its stabilization by tert-BHQ and Sul. Release from this repression seems to be linked to Keap1 undergoing a posttranslational modification involving the C151 residue. The molecular nature of this modification and how it affects the stabilization of Nrf2 are not well understood. Binding of the electrophilic lipid 15-deoxyD12,14-prostaglandin J2 to sulfhydryl groups of Keap1 in HEK293 cells has been linked to activation of the ARE, but whether this binding involves any of these cysteines was not established [46]. Thus, although mutation of these cysteine residues clearly affects Keap1 activity, it remains to be determined if they serve as sites of direct attack by ARE inducers or their reactive metabolites in the cells. Nevertheless, the data provide strong evidence supporting the proposal that Keap1 might function as part of an E3 ubiquitin ligase complex or perhaps even as the E3 ligase itself [45]. This proposal is supported by a number of studies demonstrating the involvement of Keap1 and the proteasome in the regulation of Nrf2 stability [16,17,21,22]. The ability of Keap1 to repress Nrf2 also seems to depend upon it being able to form a homodimeric complex. This was reported in a study in which the retarded electrophoretic mobility of the Keap1 dimer could be detected on polyacrylamide gels under native conditions [47]. In cells treated with the pro-oxidant compound pyrrolidine dithiocarbamate, the relative amount of the slow-migrating Keap1 dimer was found to be significantly reduced compared to untreated cells, suggesting that Keap1 dimerization might have been affected. A conserved serine residue at position 104 in the Keap1 protein has been identified and found to be important for this dimerization process, as mutation of this residue to alanine prevented the formation of the retarded form of Keap1. The effect of this mutation on the ability of Keap1 to repress Nrf2 transcriptional activity, however, was not examined [47]. It is also not understood if Ser-104 constitutes a target site for regulation by a phosphorylation-dependent mechanism during the response to chemical inducers. Regardless, the regulation of Keap1 activity by a dimerization process thus adds another dimension to the complexity of the regulatory network controlling Nrf2 activation.
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In addition to the mechanisms directly affecting Keap1, the stimulated release of Nrf2 from Keap1 also depends on its phosphorylation, particularly at sites within the Keap1-interacting Neh2 domain near the Nterminus of the protein. Indeed, the rapid increase (<10 min) in the level of Nrf2 phosphorylation in HepG2 cells exposed to tert-BHQ [20] suggests that this modification may be part of the early response leading to its activation. Nrf2 is believed to be phosphorylated at multiple sites by different protein kinases and, among these sites, that mediated by members of the PKC family promotes its nuclear accumulation [33,34]. Phosphorylation by these kinases occurs on a serine residue at position 40 of Nrf2 [33 –35], a site located in the Neh2 domain, suggesting a possible role for this residue in regulating the Nrf2 interaction with Keap1. In transfection experiments, repression of the Nrf2 transcriptional activity by Keap1 was found to be more pronounced in tert-BHQ-treated cells transfected with an Nrf2-expressing plasmid bearing an alanine substitution at Ser-40 (S40A) than with a wildtype Nrf2. This is presumably because the S40A mutant could not dissociate readily from Keap1 after tert-BHQ treatment, as demonstrated by coimmunoprecipitation experiments, and thus failed to accumulate in the nucleus to activate gene transcription [33]. These data are consistent with observations made in an independent study showing that the S40A mutant is localized primarily in the cytoplasm and that treatment with an inducer (in this case, hemin) did not promote its nuclear accumulation. By contrast, a substitution of Ser-40 with glutamic acid, which mimics a phosphorylated site, causes the Nrf2 protein to be localized predominantly in the nucleus, even in the presence of overexpressed Keap1 [34]. The role of Ser-40 phosphorylation in the dissociation of Nrf2 from INrf2 has been similarly observed [35]. As discussed previously, phosphorylation by PKC does not directly lead to Nrf2 stabilization because inhibition of PKC activity has no effect on the tert-BHQ-induced accumulation of cellular Nrf2 (as opposed to nuclear Nrf2). Thus, despite the fact that Keap1 promotes its degradation [21,22], the stabilization of Nrf2 does not depend only on its dissociation from Keap1 but is likely to require the involvement of additional mechanisms. Phosphorylation of Nrf2 by Perk also induces its release from Keap1 and subsequent nuclear accumulation [36]. In transfection experiments, tunicamycin, which induces stress in the ER, was found to promote Nrf2 accumulation in the nucleus as well as cause an f2-fold increase in the ARE-driven reporter activity. That Perk was involved in mediating these effects was confirmed by experiments using fibroblasts with a targeted disruption of the Perk locus. Thus, after tunicamycin exposure, whereas Nrf2 was found accumulating in the nucleus of wild-type cells, it remains localized in the cytoplasm of
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Perk/ cells. An interesting finding reported in this study is the demonstration that N-acetylcysteine did not have any effects on the tunicamycin-induced nuclear accumulation of Nrf2, suggesting a mechanism of action independent of oxidative stress. This implies that the induced release of Nrf2 from Keap1 by tunicamycin is mediated primarily by the Perk-dependent phosphorylation of Nrf2, irrespective of the Keap1 protein. Providing support for this notion are data showing that Perk can phosphorylate free Nrf2 as well as Nrf2 complexed with Keap1 and that this is sufficient to prevent the association or reassociation of the two proteins, respectively [36]. Thus, at least in vitro, direct Nrf2 phosphorylation rather than Keap1 modification can lead to complex dissociation and subsequent Nrf2 activation. Although the target site has not been established, the fact that Perk can induce effects similar to those mediated by PKC phosphorylation of Nrf2 at Ser-40 [33 – 35] raises the possibility that this residue may represent the common site for both kinases. It is noteworthy to point out that the involvement of Perk and PKC in mediating a similar mechanism of Nrf2 activation illustrates an example of the importance of the nature of the chemical inducers in determining the signaling pathway responsible for the activation of ARE-dependent genes. The interaction between Nrf2 and Keap1 and how it might be regulated by the ARE inducers leading to Nrf2 stabilization is summarized and depicted in Fig. 2. Regulation of Nrf2 stability by other factors
Fig. 2. Regulation of the Nrf2 – Keap1 interaction. The various mechanisms involved in regulating the Nrf2 – Keap1 interaction in response to the ARE inducers are summarized in this schematic representation. Under physiological conditions Keap1 is believed to actively and continually promote Nrf2 degradation. Stabilization of Nrf2 is brought about when ARE inducers or their reactive intermediates stimulate Nrf2 phosphorylation through various protein kinases, which triggers its dissociation from Keap1 and prevents/ decreases its rate of degradation by the proteasome. In addition, the reactive intermediates may also cause direct modifications of Keap1, inhibiting its activity and thus contributing to the stabilization of Nrf2. The nature of these modifications is not understood, but it may involve the alkylation of cysteine residues critical for Keap1 activity.
It has become evident that the activation of Nrf2 involves multiple mechanisms by which the ARE inducers act to promote its stabilization and accumulation in the nucleus. Because of its involvement in the degradation of Nrf2 [21,22,45], Keap1 has a prominent role in the regulation of Nrf2 stability. The importance of this protein is further confirmed by recent in vivo data showing the increased level of Nrf2 and the increased expression of some of the Nrf2-dependent, phase II enzymes in Keap1/ mice compared to wild-type animals [48]. However, it is important to note that Keap1 might not be the sole regulator of Nrf2 stability and that additional mechanisms mediated by other factors are involved. This is supported by experiments using an Nrf2 mutant lacking the Keap1-interacting motif ETGE in its Neh2 domain (see Fig. 1) [49], Nrf2DETGE, in which the transcription factor was observed to accumulate rapidly in the presence of MG-132, suggesting that it is still subject to degradation by the proteasome but via a Keap1independent pathway [21]. Though the estimated half-life of Nrf2DETGE (t1/2 = f30 min) is approximately twice that of wild-type Nrf2 (t1/2 = f10 – 15 min), there is no difference in the half-life of a Gal4 protein fused either
with the Neh2DETGE domain (Gal4-mNeh2DETGE) or with the wild-type Neh2 domain (Gal4-mNeh2). At least two conclusions can be drawn from these data: (i) the stability of Nrf2 is regulated by at least two degrons, one found in the Neh2 domain and the second elsewhere in the protein, and (ii) the degron in the Neh2 domain continues to be active regardless of its affinity for Keap1. The stabilization of Nrf2 might therefore depend on mechanisms acting simultaneously on both degrons to efficiently decrease the rate of its degradation, a notion consistent with the earlier observation that disruption of Nrf2 interaction with Keap1 by phosphorylation of Ser-40 is not sufficient to increase its stability [16]. We have indeed found that the half-life of the Nrf2 S40A mutant is virtually identical to that of wild-type Nrf2 and that this mutant protein retains its responsiveness to the stabilizing effects of tert-BHQ in HepG2 cells (our unpublished observation). In addition, the destabilizing effects of the Neh2DETGE domain on the Gal4 protein suggest that, in addition to Keap1, other proteins may act through this region to regulate Nrf2 stability. This is particularly important for maintaining the Nrf2 steady-state level in
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cells in which Keap1 is not expressed or is expressed at very low levels. CONCLUDING REMARKS
Significant progress has been made on the transcriptional regulation of ARE-dependent genes in response to oxidative stress. Expression of these genes is important to maintain cellular redox homeostasis and prevent the pathogenesis of many inflammatory and related diseases. The Nrf2 transcription factor has become the focus of recent research studies into the molecular mechanisms that regulate this process of gene activation. Data from these studies have provided compelling evidence of Nrf2 as an unstable protein and that its steady-state level is maintained by a constant rate of synthesis countered by a similar rate of active degradation. Although its regulation is likely to be complex and involves numerous pathways, a consensus has emerged that Nrf2 activation is mediated in large part by mechanisms by which Nrf2 becomes stabilized and accumulates in the nucleus. However, much remains to be learned about how Nrf2 is regulated at the molecular level. For example, most recent studies have suggested that the nuclear accumulation of Nrf2 is the direct result of an increase in the nuclear translocation process after its dissociation from Keap1. As current evidence demonstrates an active role for Keap1 in promoting Nrf2 degradation, the inevitable question arises as to the precise mechanism by which Keap1 sequesters and retains Nrf2 in the cytoplasm without causing its degradation. Given the short half-life of Nrf2, the purpose of its sequestration also needs to be clarified. In addition, the Nrf2-mediated constitutive expression of a number of ARE-dependent genes [8,9] implies a continuous presence of this transcription factor in the nucleus under physiological conditions. How this Nrf2 population escapes Keap1 repression to regulate the basal expression of these genes is another question that needs to be reconciled. Because of these conflicting questions, an alternative role for Keap1 in the regulation of Nrf2 activity is suggested. Keap1 might act more as a negative feedback regulator to return Nrf2, and the expression of its target genes, to its steady-state level after activation. A hint of this possibility is the recent observation that Keap1 expression is upregulated by tert-BHQ [40]. Accordingly, this would entail repression of Nrf2 by Keap1 only after Nrf2 has exerted its transcriptional activity in the nucleus (see Fig. 3). In addition to the Neh2 domain, there seems to be a second region in the Nrf2 protein involved in the regulation of its stability. Whether this region is regulated independently or in conjunction with the Neh2 domain through Keap1 remains to be determined. In addition, other proteins might also be involved in the regulation of Nrf2 stability and it would be of interest to determine if
Fig. 3. An alternative model for the regulation of Nrf2 activity. Growing evidence from recent studies on the active role of Keap1 in promoting Nrf2 degradation suggests that the repression of Nrf2 activity is more likely to occur after its transcriptional function in the nucleus. According to this model, cellular Nrf2 is maintained at a steady-state level by a constant rate of synthesis countered by a similar rate of degradation. After its synthesis Nrf2 translocates directly into the nucleus, activates transcription of ARE-dependent genes constitutively, and is then targeted for degradation by Keap1. Upon exposure to ARE inducers, Nrf2 becomes stabilized as a result of its Keap1-dependent degradation being inhibited and, combined with its de novo synthesis, accumulates in the nucleus to increase the rate of transcription of its target genes. Although a feedback regulatory role of Keap1 has yet to be confirmed, an increase in its expression in response to an ARE inducer has been reported [40] and this provides a potential mechanism to downregulate and return Nrf2 to its steady-state level.
they synergize with Keap1. Nrf2 phosphorylation clearly is important for its stabilization. The target site and how its phosphorylation leads to Nrf2 stabilization have yet to be identified and the mechanisms elucidated. These are some of the issues that are currently being addressed so as to provide a better understanding into the regulatory mechanisms controlling Nrf2 activation in response to chemical stress. REFERENCES [1] Nguyen, T.; Sherratt, P. J.; Pickett, C. B. Regulatory mechanisms controlling gene expression mediated by the antioxidant response element. Annu. Rev. Pharmacol. Toxicol. 43:233 – 260; 2003. [2] Venugopal, R.; Jaiswal, A. K. Nrf1 and Nrf2 positively and c-Fos and Fra1 negatively regulate the human antioxidant response element-mediated expression of NAD(P)H:quinone oxidoreductase1 gene. Proc. Natl. Acad. Sci. USA 93:14960 – 14965; 1996. [3] Venugopal, R.; Jaiswal, A. K. 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; 1998. [4] Wild, A. C.; Moinova, H. R.; Mulcahy, R. T. Regulation of g-glutamylcysteine synthetase regulatory subunit gene expression by the transcription factor Nrf2. J. Biol. Chem. 274: 33627 – 33636; 1999. [5] Alam, J.; Stewart, D.; Touchard, C.; Boinapally, S.; Choi, A. M. K.; Cook, J. L. Nrf2, a Cap’n’Collar transcription factor, regulates induction of the heme oxigenase-1 gene. J. Biol. Chem. 274: 26071 – 26078; 1999. [6] Nguyen, T.; Huang, H. C.; Pickett, C. B. Transcriptional regulation of the antioxidant response element: activation by Nrf2 and repression by MafK. J. Biol. Chem. 275:15466 – 15473; 2000.
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doi:10.1016/j.freeradbiomed.2004.04.033
Serial Review: EpRE and Its Signaling Pathway Serial Review Editor: Henry J. Forman; Guest Co-Editor: Douglas Ruden THE PATHWAYS AND MOLECULAR MECHANISMS REGULATING NRF2 ACTIVATION IN RESPONSE TO CHEMICAL STRESS TRUYEN NGUYEN,*,y CHUNG S. YANG, y and CECIL B. PICKETT * *Schering – Plough Research Institute, Kenilworth, NJ 07033, USA; and y Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, NJ 08854, USA (Received 24 February 2004; Revised 26 April 2004; Accepted 28 April 2004) Available online 19 May 2004
Abstract—The induction of many antioxidant and phase II drug-metabolizing enzymes by phenolic antioxidants and electrophilic compounds is regulated at the transcriptional level. The response to these compounds is mediated by the cisacting antioxidant response element (ARE) found in the promoter of the encoding genes. The transcription factor NF-E2related factor 2, or Nrf2, has emerged as the central protein that binds to the ARE to activate gene transcription. Data from many studies indicate that Nrf2 is constitutively and ubiquitously expressed in a number of tissues and cell lines and is thus responsible for the low-level expression of its target genes observed under physiological conditions. However, in cells exposed to oxidative stress, Nrf2 activity is increased, further driving the transcriptional activation of genes whose expression is essential to control cellular redox homeostasis. Recent studies suggest that the activation of Nrf2 involves a coordinated process and is regulated at multiple levels. Nrf2 activity is believed to be repressed through the binding of the cytoskeleton-associated protein Keap1, and its activation involves mechanisms that interfere with this interaction. Activation of Nrf2 has also been demonstrated to be dependent on mechanisms that mediate its stabilization. In this review, the mechanisms controlling this activation process as reported in recent studies will be examined and discussed, with particular emphasis on those affecting Nrf2 stability at the molecular level. D 2004 Elsevier Inc. All rights reserved. Keywords—Free radicals
expressed at a low level under physiological conditions. However, their expression is increased in cells exposed to a diverse range of compounds, including oxidants, phenolic antioxidants, and electrophilic agents that can also undergo redox cycling to produce excess reactive oxygen species. And central to the induction of these enzymes is the transcription factor NF-E2-related factor 2 (Nrf2), which acts through the antioxidant response element (ARE) to activate gene transcription [1]. The involvement of Nrf2 in both the constitutive and the inducible expression of ARE-dependent genes has been well established and documented in numerous in vitro and in vivo studies [2 –14]. The molecular mechanisms and signaling pathways that regulate the activation of Nrf2 have thus been the focus of more recent studies. One of the mechanisms that have been widely studied is
INTRODUCTION
Chemical stress caused by environmental toxicants, and xenobiotic metabolism can lead to the pathogenesis of many inflammatory and degenerative diseases, including cancer, Alzheimer disease, and arthritis. To protect cells against oxidative damage by this chemical stress, many antioxidant and phase II drug-metabolizing enzymes act in concert to detoxify and eliminate these harmful chemicals and their metabolites. These enzymes are constitutively This article is part of a series of reviews on ‘‘EpRE and Its Signaling Pathway.’’ The full list of papers may be found on the home page of the journal. Address correspondence to: Cecil B. Pickett, Schering – Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA; Fax: (908) 740-7514; E-mail: [email protected]. 433
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that mediated by the repressor protein Keap1, which acts by directly interacting with Nrf2 and preventing its nuclear accumulation. Activation occurs when this interaction and repression is disrupted, allowing Nrf2 to translocate and accumulate in the nucleus [15]. The activation of Nrf2 has also been demonstrated to be mediated by mechanisms that lead to its stabilization, thus increasing levels of cellular Nrf2 and subsequent transcriptional activity [16,17]. Upregulation of Nrf2 gene expression at the transcriptional level has also been reported as another mechanism that contributes to increased amounts of Nrf2 in the cells in response to ARE inducers [18]. Thus, induction of the ARE gene battery is largely dependent on regulatory mechanisms acting in concert to increase the Nrf2 protein level and promote its subsequent accumulation in the nucleus to drive transcription. It should be noted that additional mechanisms affecting other activities of the transcription factor are also likely to occur during this activation process. These could include posttranslational modifications that affect its transactivational activity, DNA-binding affinity, or interaction with other transcription factors or with components of the transcription machinery. In this review, our discussion will be focused primarily on the posttranscriptional mechanisms that regulate Nrf2 activation, with particular emphasis on protein stabilization and the interaction with Keap1. There is now growing evidence suggesting a link between the inhibition of Keap1 activity and the stabilization of Nrf2, and we will briefly review key data on the molecular mechanisms that regulate these two processes. REGULATION OF NRF2 ACTIVATION
Protein stabilization as a mechanism for Nrf2 activation The activity of regulatory proteins such as transcription factors is often regulated posttranscriptionally and/or posttranslationally. Although transcription of the Nrf2 gene has been found to increase in murine keratinocytes in response to 3H-1,2-dithiole-3-thione [18], the Nrf2 mRNA levels were found to remain steady and unaffected by other inducers of ARE-dependent genes in other experimental systems [4,16,17,19]. These data suggest that posttranscriptional mechanisms are important in the regulation of Nrf2 activity. Indeed, data from cell labeling studies show that treatment of HepG2 cells with the phenolic antioxidant tert-butylhydroquinone (tert-BHQ) led to an increased level of Nrf2 phosphorylation that was accompanied by its nuclear accumulation [20]. In addition, tert-BHQ was also found to induce an overall increase in the level of cellular Nrf2 without affecting the apparent rate of Nrf2 transcription [16]. Of particular interest is that this posttranscriptional response is not unique to this phenolic antioxidant because similar effects
were observed in cells treated with other compounds, including h-naphthoflavone [16], cadmium [17], sulforaphane (Sul) [21], and diethylmaleate (DEM) [22], four structurally diverse inducers of the ARE-dependent gene expression. These observations provide evidence of a regulatory mechanism that increases the stability of Nrf2, resulting in its accumulation in the cell. This notion is confirmed by experimental results showing that the halflife of Nrf2 is extended in cells exposed to the same inducers. Because cycloheximide was used to block de novo protein synthesis in these experiments, these data provide evidence that a posttranslational mechanism is involved in increasing the Nrf2 protein level [16,17]. As the stability of intracellular proteins is often determined by the rate of its degradation by the ubiquitindependent proteasome, this proteolytic machinery became the prime candidate to be involved in the regulatory pathway that controls Nrf2 protein stability and hence its transcriptional activity. Evidence for a role for the proteasome was initially demonstrated in a study in which HepG2 cells treated with the proteasome inhibitor lactacystin had increased levels of the mRNA encoded by the ARE-dependent g-glutamylcysteine synthetase (GLCLC) gene [23]. That the 26S proteasome participates directly in the regulation of Nrf2 stability was examined further in other studies using lactacystin and another commonly used inhibitor of the proteasome, MG-132. Both of these inhibitors were found to increase the levels of Nrf2 protein and its ubiquitylated forms in HepG2 cells in a timedependent manner [16,17]. In addition, the elevated Nrf2 levels were accompanied by an increase in the expression of an ARE-driven reporter gene, two effects identical to those induced by tert-BHQ. These data are consistent with the notion that the activation of Nrf2 by chemical stress involves protein stabilization and is likely to be regulated by mechanisms that decrease the rate of its degradation by the ubiquitin-dependent proteasome. What has also become apparent is that, regardless of the mechanisms involved, an increase in cellular Nrf2 is essential for the induction of ARE-dependent genes. How this increase leads to an accumulation in the nucleus is not well understood, but it is presumed that Nrf2 can translocate readily into the nucleus and that this process is not necessarily regulated in a specific manner by the inducers. This presumption agrees well with experimental data showing a dose-dependent increase in the ARE-driven reporter gene activity in cells transfected with increasing amounts of an Nrf2-expressing vector without the need for treatment with an ARE inducer. Under these conditions, Nrf2 was apparently able to translocate into the nucleus to activate gene transcription in a nonregulated manner [2,4 – 6]. The fact that inhibitors of the proteasome not only stabilize Nrf2 but also increase the expression of an AREdriven reporter gene [16,17] or that of an endogenous gene
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[23] also clearly links Nrf2 stabilization with its activity in the nucleus. In addition, direct analysis of fractionated cell lysates demonstrates that treatment of cells with these inhibitors leads to an increased level of Nrf2 in the nucleus rather than in the cytosolic fraction [22]. Thus, Nrf2 stabilization alone seems to be sufficient to trigger its nuclear accumulation. The similar effects induced by lactacystin and MG132 raise an important question about whether the ARE inducers act by inhibiting the proteasome activity to stabilize Nrf2 and thus increase its transcriptional activation of target genes. Current evidence, however, argues against this possibility. Conditions of oxidative stress seem to increase rather than inhibit the overall activity of the ubiquitin-dependent proteasome, a response that is apparently necessitated due to the need to remove damaged proteins from the cells [24]. More recently, upregulation of genes encoding several subunits of the proteasome complex has also been observed in mice treated with a dithiolethione [25]. These data suggest that ARE inducers are unlikely to act as proteasome inhibitors but rather through distinct pathways to stabilize Nrf2. Together, these studies provide compelling evidence that protein stabilization is an integral part of the major regulatory mechanisms mediating Nrf2 activation by many inducers of the ARE-dependent genes. Phosphorylation and Nrf2 stability The degradation of proteins by the ubiquitin-dependent 26S proteasome is a highly regulated process that involves the specific recognition and targeting of the substrate molecule for ubiquitylation by an E3 ubiquitin ligase before degradation by the proteasome complex. How target proteins are selected for degradation is based primarily on the presence of specific structures known as degrons that are generally composed of a short and specific amino acid sequence and/or a particular conformational structure [26,27]. These degrons serve as recognition motifs for the E3 ligase and the interaction between them is believed to represent the critical, ratelimiting step that regulates the stability of the target protein. The stabilization of Nrf2 by ARE inducers may be brought about by a decrease in the rate of its degradation, and this could be mediated by mechanisms that prevent or weaken the recognition/interaction of Nrf2 by its E3 ligase or by directly inhibiting the E3 ligase activity itself. As protein modification by phosphorylation is the major posttranslational mechanism in signaling processes, it is believed to have a central role in regulating the stability of the Nrf2 protein. Recent data showing that tert-BHQ increases Nrf2 protein phosphorylation in HepG2 cells [20] provide evidence for this possibility. Further support for this mechanism is the finding that the
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phosphatase inhibitor okadaic acid, which induces cellular hyperphosphorylation [28], has positive effects on both the Nrf2 accumulation and the transcriptional activation of an ARE-driven reporter gene [16]. Although these data do not provide conclusive evidence, they do suggest that stabilization of Nrf2 is intimately linked to protein phosphorylation. In other such cases, phosphorylation commonly occurs at one or more sites close to, or within, the degrons to impair the protein recognition by the proteasome. This mechanism would be reminiscent of that controlling p53 stability. Under physiological conditions, p53 is bound and targeted for ubiquitindependent degradation by the Mdm2 protein, an E3 ligase; in response to stimulating signals such as DNA damage by ionizing radiation or UV light, p53 is phosphorylated at Ser-20 by the activated ATM/ATR kinases, which weakens its interaction with Mdm2 and thus leads to its stabilization [29 –31]. Though phosphorylation of Nrf2 seems to be important for its stabilization the identity of the protein kinase(s) involved has yet to be determined. In a recent study, inhibitors of the MAPK/ERK signaling pathway were found to interfere with the inducing effects of tertBHQ on the level of Nrf2 in HepG2 cells [16], suggesting a possible role for this pathway in the regulation of its stability. Consistent with this observation is the finding that treatment of HepG2 or Hepa1c1c7 cells with either tert-BHQ or Sul led to ERK2 activation as well as the induction of ARE-dependent genes and that both of these inducing effects were impaired by the MEK inhibitor PD98059 [32]. It is not known from these studies whether ERK2 itself or other downstream kinases are responsible for directly phosphorylating Nrf2. What has been demonstrated is that Nrf2 can be phosphorylated by members of the protein kinase C (PKC) family (discussed below), and this was shown to interfere with its interaction with Keap1 and promote its nuclear accumulation [33,34]. Phosphorylation by these kinases, however, is not directly responsible for Nrf2 stabilization because inhibition of their activity by staurosporine did not have any effects on the tert-BHQ-induced accumulation of Nrf2 in HepG2 cells [16,35]. More recently, Nrf2 was reported to be phosphorylated by PERK, a kinase activated in response to stress in the endoplasmic reticulum. Similar to effects produced by PKC, phosphorylation by this kinase was shown to induce the dissociation of Nrf2 from Keap1 and increase its nuclear import [36]. Because protein stability was not examined in this study, it is unclear if PERK contributes to the phosphorylation-dependent stabilization of Nrf2. Apart from the aforementioned kinases, the phosphatidylinositol 3-kinase (PI3K) cascade has also been implicated in the regulation of ARE-dependent genes in H4II rat hepatoma cells [37,38] as well as in IMR-32
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human neuroblastoma cells [39,40], but whether kinases of this pathway act directly on Nrf2 to affect its stability is not known. It has recently been reported that PI3K mediates the induction of Nrf2 nuclear accumulation by hemin in SH-SY5Y human neuroblastoma cells, as treatment with either wortmannin or LY 294002 was demonstrated by immunoblot analysis to antagonize this hemin-induced effect [41]. In a separate study, LY294002 was shown to attenuate the stabilizing effects of the phytochemical carnosol on the cellular Nrf2, suggesting a possible role for PI3K in the regulation of Nrf2 stability [42]. The nature of the molecular mechanisms mediated by this pathway awaits further study. Together these studies demonstrate that phosphorylation is important for Nrf2 activation and it involves different protein kinases, each of which may be associated with regulating a specific activity of the transcription factor. With the exception of PKC, which phosphorylates Nrf2 at Ser-40 [33 –35], the target sites for other kinases have yet to be identified. Because many of these studies focused their analysis on the effects of inducers and kinase activity on the accumulation of Nrf2 in the nucleus, as opposed to the total cellular Nrf2 level, it is of particular interest to determine whether the nuclear accumulation observed in these studies was the result of an enhanced nuclear translocation process or a direct result of its stabilization. A distinction between these two processes would provide a better understanding of the role of each protein kinase cascade in the regulation of Nrf2 activity. Another critical question is whether Nrf2 is phosphorylated simultaneously by all these different protein kinases in response to all ARE inducers or whether its phosphorylation is mediated only by specific kinases determined by the nature of the inducers and cell types. Given that compounds capable of inducing the Nrf2/ARE-dependent gene expression are structurally diverse and work in a wide variety of cell types, this would suggest the latter possibility. Thus, whereas the stabilization of Nrf2 might depend on phosphorylation by certain kinases in one cell type, this mechanism could be mediated by different kinases in another cell type. The activation of specific kinase cascades may similarly be determined by the nature of the inducing compounds. Future studies focusing on the identification of phosphorylation sites will facilitate the determination of the role of specific kinases involved in various mechanisms regulating the activation of Nrf2. Keap1 and regulation of Nrf2 activation The identification of Keap1, and its rat homolog termed INrf2 [43], as a repressor of Nrf2 activity defined the current regulatory pathways for Nrf2 activation [15]. Keap1 was initially isolated in a yeast two-hybrid screening experiment to identify potential proteins that interact with Nrf2. Biochemical and localization studies
indicate that Keap1, being anchored in the cytoplasm, functions by binding and preventing nuclear accumulation of Nrf2 and thus repressing its transcriptional activity (Fig. 1). This repressing activity is lost in cells under oxidative stress, as DEM, a compound with the propensity to react with sulfhydryl groups and induce oxidative stress, was shown to induce the dissociation of Nrf2 from Keap1 and promote nuclear accumulation of the transcription factor. For this reason, the Keap1/Nrf2 complex was proposed to constitute a cellular redox sensor through which the activation of ARE-dependent genes in response to oxidative stress is regulated [15]. This hypothesis is also supported by the observation that the Keap1 protein contains a large number of cysteine residues, some of which are thought to represent target sites for direct attack by electrophilic compounds to cause the release of the Nrf2 protein. Indeed, four potential cysteines at residues 257, 273, 288, and 297 have recently been identified by in vitro labeling and competition studies in which their reactivity to several alkylating compounds was measured and demonstrated [44]. The role of these cysteine residues was explored further in a cell-based study to define the biological function of Keap1 [45]. A significant finding in this report demonstrated that Keap1 not only acts to retain Nrf2 in the cytoplasm but also actively promotes its ubiquitylation and subsequent degradation by the proteasome. Two cysteine residues in Keap1, C273 and C288,
Fig. 1. Repression of Nrf2 activity by Keap1. (A) Under the current model, repression of Nrf2 has been proposed to be mediated by a mechanism by which Keap1 acts to retain the transcription factor in the cytoplasm and Nrf2 activation occurs when this interaction is disrupted by inducers of the ARE-dependent genes, allowing Nrf2 to translocate into the nucleus to effect its transcriptional activity. (B) Based on its homology with the Kelch protein, the structure of Keap1 is defined as consisting of two distinct domains known as the BTB and Kelch domains, separated by an intervening region (Linker) and flanked by two regions at the NH2-terminus (N) and COOH-terminus (C) as described [15,45]. Keap1 interacts with Nrf2 by binding to its Neh2 domain. The ETGE motif within the Neh2 domain important for this interaction [49] is indicated. Cysteine residues in the Keap1 protein that are important for its activity are also indicated.
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were found to be essential for this function, as mutation of either residue to serine abolishes this ubiquitylating activity. In addition, these Keap1 mutants not only fail to repress Nrf2 but also actually increase its half-life and transcriptional activity, suggesting that loss of Keap1’s ability to repress is important for Nrf2 stabilization. The importance of these two cysteine residues in the Keap1dependent repression of Nrf2 has also been independently reported in another study [46]. A third cysteine residue, C151, has been identified and found to be critical for the inducible release of Nrf2 from Keap1 repression and for its stabilization by tert-BHQ and Sul. Release from this repression seems to be linked to Keap1 undergoing a posttranslational modification involving the C151 residue. The molecular nature of this modification and how it affects the stabilization of Nrf2 are not well understood. Binding of the electrophilic lipid 15-deoxyD12,14-prostaglandin J2 to sulfhydryl groups of Keap1 in HEK293 cells has been linked to activation of the ARE, but whether this binding involves any of these cysteines was not established [46]. Thus, although mutation of these cysteine residues clearly affects Keap1 activity, it remains to be determined if they serve as sites of direct attack by ARE inducers or their reactive metabolites in the cells. Nevertheless, the data provide strong evidence supporting the proposal that Keap1 might function as part of an E3 ubiquitin ligase complex or perhaps even as the E3 ligase itself [45]. This proposal is supported by a number of studies demonstrating the involvement of Keap1 and the proteasome in the regulation of Nrf2 stability [16,17,21,22]. The ability of Keap1 to repress Nrf2 also seems to depend upon it being able to form a homodimeric complex. This was reported in a study in which the retarded electrophoretic mobility of the Keap1 dimer could be detected on polyacrylamide gels under native conditions [47]. In cells treated with the pro-oxidant compound pyrrolidine dithiocarbamate, the relative amount of the slow-migrating Keap1 dimer was found to be significantly reduced compared to untreated cells, suggesting that Keap1 dimerization might have been affected. A conserved serine residue at position 104 in the Keap1 protein has been identified and found to be important for this dimerization process, as mutation of this residue to alanine prevented the formation of the retarded form of Keap1. The effect of this mutation on the ability of Keap1 to repress Nrf2 transcriptional activity, however, was not examined [47]. It is also not understood if Ser-104 constitutes a target site for regulation by a phosphorylation-dependent mechanism during the response to chemical inducers. Regardless, the regulation of Keap1 activity by a dimerization process thus adds another dimension to the complexity of the regulatory network controlling Nrf2 activation.
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In addition to the mechanisms directly affecting Keap1, the stimulated release of Nrf2 from Keap1 also depends on its phosphorylation, particularly at sites within the Keap1-interacting Neh2 domain near the Nterminus of the protein. Indeed, the rapid increase (<10 min) in the level of Nrf2 phosphorylation in HepG2 cells exposed to tert-BHQ [20] suggests that this modification may be part of the early response leading to its activation. Nrf2 is believed to be phosphorylated at multiple sites by different protein kinases and, among these sites, that mediated by members of the PKC family promotes its nuclear accumulation [33,34]. Phosphorylation by these kinases occurs on a serine residue at position 40 of Nrf2 [33 –35], a site located in the Neh2 domain, suggesting a possible role for this residue in regulating the Nrf2 interaction with Keap1. In transfection experiments, repression of the Nrf2 transcriptional activity by Keap1 was found to be more pronounced in tert-BHQ-treated cells transfected with an Nrf2-expressing plasmid bearing an alanine substitution at Ser-40 (S40A) than with a wildtype Nrf2. This is presumably because the S40A mutant could not dissociate readily from Keap1 after tert-BHQ treatment, as demonstrated by coimmunoprecipitation experiments, and thus failed to accumulate in the nucleus to activate gene transcription [33]. These data are consistent with observations made in an independent study showing that the S40A mutant is localized primarily in the cytoplasm and that treatment with an inducer (in this case, hemin) did not promote its nuclear accumulation. By contrast, a substitution of Ser-40 with glutamic acid, which mimics a phosphorylated site, causes the Nrf2 protein to be localized predominantly in the nucleus, even in the presence of overexpressed Keap1 [34]. The role of Ser-40 phosphorylation in the dissociation of Nrf2 from INrf2 has been similarly observed [35]. As discussed previously, phosphorylation by PKC does not directly lead to Nrf2 stabilization because inhibition of PKC activity has no effect on the tert-BHQ-induced accumulation of cellular Nrf2 (as opposed to nuclear Nrf2). Thus, despite the fact that Keap1 promotes its degradation [21,22], the stabilization of Nrf2 does not depend only on its dissociation from Keap1 but is likely to require the involvement of additional mechanisms. Phosphorylation of Nrf2 by Perk also induces its release from Keap1 and subsequent nuclear accumulation [36]. In transfection experiments, tunicamycin, which induces stress in the ER, was found to promote Nrf2 accumulation in the nucleus as well as cause an f2-fold increase in the ARE-driven reporter activity. That Perk was involved in mediating these effects was confirmed by experiments using fibroblasts with a targeted disruption of the Perk locus. Thus, after tunicamycin exposure, whereas Nrf2 was found accumulating in the nucleus of wild-type cells, it remains localized in the cytoplasm of
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Perk/ cells. An interesting finding reported in this study is the demonstration that N-acetylcysteine did not have any effects on the tunicamycin-induced nuclear accumulation of Nrf2, suggesting a mechanism of action independent of oxidative stress. This implies that the induced release of Nrf2 from Keap1 by tunicamycin is mediated primarily by the Perk-dependent phosphorylation of Nrf2, irrespective of the Keap1 protein. Providing support for this notion are data showing that Perk can phosphorylate free Nrf2 as well as Nrf2 complexed with Keap1 and that this is sufficient to prevent the association or reassociation of the two proteins, respectively [36]. Thus, at least in vitro, direct Nrf2 phosphorylation rather than Keap1 modification can lead to complex dissociation and subsequent Nrf2 activation. Although the target site has not been established, the fact that Perk can induce effects similar to those mediated by PKC phosphorylation of Nrf2 at Ser-40 [33 – 35] raises the possibility that this residue may represent the common site for both kinases. It is noteworthy to point out that the involvement of Perk and PKC in mediating a similar mechanism of Nrf2 activation illustrates an example of the importance of the nature of the chemical inducers in determining the signaling pathway responsible for the activation of ARE-dependent genes. The interaction between Nrf2 and Keap1 and how it might be regulated by the ARE inducers leading to Nrf2 stabilization is summarized and depicted in Fig. 2. Regulation of Nrf2 stability by other factors
Fig. 2. Regulation of the Nrf2 – Keap1 interaction. The various mechanisms involved in regulating the Nrf2 – Keap1 interaction in response to the ARE inducers are summarized in this schematic representation. Under physiological conditions Keap1 is believed to actively and continually promote Nrf2 degradation. Stabilization of Nrf2 is brought about when ARE inducers or their reactive intermediates stimulate Nrf2 phosphorylation through various protein kinases, which triggers its dissociation from Keap1 and prevents/ decreases its rate of degradation by the proteasome. In addition, the reactive intermediates may also cause direct modifications of Keap1, inhibiting its activity and thus contributing to the stabilization of Nrf2. The nature of these modifications is not understood, but it may involve the alkylation of cysteine residues critical for Keap1 activity.
It has become evident that the activation of Nrf2 involves multiple mechanisms by which the ARE inducers act to promote its stabilization and accumulation in the nucleus. Because of its involvement in the degradation of Nrf2 [21,22,45], Keap1 has a prominent role in the regulation of Nrf2 stability. The importance of this protein is further confirmed by recent in vivo data showing the increased level of Nrf2 and the increased expression of some of the Nrf2-dependent, phase II enzymes in Keap1/ mice compared to wild-type animals [48]. However, it is important to note that Keap1 might not be the sole regulator of Nrf2 stability and that additional mechanisms mediated by other factors are involved. This is supported by experiments using an Nrf2 mutant lacking the Keap1-interacting motif ETGE in its Neh2 domain (see Fig. 1) [49], Nrf2DETGE, in which the transcription factor was observed to accumulate rapidly in the presence of MG-132, suggesting that it is still subject to degradation by the proteasome but via a Keap1independent pathway [21]. Though the estimated half-life of Nrf2DETGE (t1/2 = f30 min) is approximately twice that of wild-type Nrf2 (t1/2 = f10 – 15 min), there is no difference in the half-life of a Gal4 protein fused either
with the Neh2DETGE domain (Gal4-mNeh2DETGE) or with the wild-type Neh2 domain (Gal4-mNeh2). At least two conclusions can be drawn from these data: (i) the stability of Nrf2 is regulated by at least two degrons, one found in the Neh2 domain and the second elsewhere in the protein, and (ii) the degron in the Neh2 domain continues to be active regardless of its affinity for Keap1. The stabilization of Nrf2 might therefore depend on mechanisms acting simultaneously on both degrons to efficiently decrease the rate of its degradation, a notion consistent with the earlier observation that disruption of Nrf2 interaction with Keap1 by phosphorylation of Ser-40 is not sufficient to increase its stability [16]. We have indeed found that the half-life of the Nrf2 S40A mutant is virtually identical to that of wild-type Nrf2 and that this mutant protein retains its responsiveness to the stabilizing effects of tert-BHQ in HepG2 cells (our unpublished observation). In addition, the destabilizing effects of the Neh2DETGE domain on the Gal4 protein suggest that, in addition to Keap1, other proteins may act through this region to regulate Nrf2 stability. This is particularly important for maintaining the Nrf2 steady-state level in
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cells in which Keap1 is not expressed or is expressed at very low levels. CONCLUDING REMARKS
Significant progress has been made on the transcriptional regulation of ARE-dependent genes in response to oxidative stress. Expression of these genes is important to maintain cellular redox homeostasis and prevent the pathogenesis of many inflammatory and related diseases. The Nrf2 transcription factor has become the focus of recent research studies into the molecular mechanisms that regulate this process of gene activation. Data from these studies have provided compelling evidence of Nrf2 as an unstable protein and that its steady-state level is maintained by a constant rate of synthesis countered by a similar rate of active degradation. Although its regulation is likely to be complex and involves numerous pathways, a consensus has emerged that Nrf2 activation is mediated in large part by mechanisms by which Nrf2 becomes stabilized and accumulates in the nucleus. However, much remains to be learned about how Nrf2 is regulated at the molecular level. For example, most recent studies have suggested that the nuclear accumulation of Nrf2 is the direct result of an increase in the nuclear translocation process after its dissociation from Keap1. As current evidence demonstrates an active role for Keap1 in promoting Nrf2 degradation, the inevitable question arises as to the precise mechanism by which Keap1 sequesters and retains Nrf2 in the cytoplasm without causing its degradation. Given the short half-life of Nrf2, the purpose of its sequestration also needs to be clarified. In addition, the Nrf2-mediated constitutive expression of a number of ARE-dependent genes [8,9] implies a continuous presence of this transcription factor in the nucleus under physiological conditions. How this Nrf2 population escapes Keap1 repression to regulate the basal expression of these genes is another question that needs to be reconciled. Because of these conflicting questions, an alternative role for Keap1 in the regulation of Nrf2 activity is suggested. Keap1 might act more as a negative feedback regulator to return Nrf2, and the expression of its target genes, to its steady-state level after activation. A hint of this possibility is the recent observation that Keap1 expression is upregulated by tert-BHQ [40]. Accordingly, this would entail repression of Nrf2 by Keap1 only after Nrf2 has exerted its transcriptional activity in the nucleus (see Fig. 3). In addition to the Neh2 domain, there seems to be a second region in the Nrf2 protein involved in the regulation of its stability. Whether this region is regulated independently or in conjunction with the Neh2 domain through Keap1 remains to be determined. In addition, other proteins might also be involved in the regulation of Nrf2 stability and it would be of interest to determine if
Fig. 3. An alternative model for the regulation of Nrf2 activity. Growing evidence from recent studies on the active role of Keap1 in promoting Nrf2 degradation suggests that the repression of Nrf2 activity is more likely to occur after its transcriptional function in the nucleus. According to this model, cellular Nrf2 is maintained at a steady-state level by a constant rate of synthesis countered by a similar rate of degradation. After its synthesis Nrf2 translocates directly into the nucleus, activates transcription of ARE-dependent genes constitutively, and is then targeted for degradation by Keap1. Upon exposure to ARE inducers, Nrf2 becomes stabilized as a result of its Keap1-dependent degradation being inhibited and, combined with its de novo synthesis, accumulates in the nucleus to increase the rate of transcription of its target genes. Although a feedback regulatory role of Keap1 has yet to be confirmed, an increase in its expression in response to an ARE inducer has been reported [40] and this provides a potential mechanism to downregulate and return Nrf2 to its steady-state level.
they synergize with Keap1. Nrf2 phosphorylation clearly is important for its stabilization. The target site and how its phosphorylation leads to Nrf2 stabilization have yet to be identified and the mechanisms elucidated. These are some of the issues that are currently being addressed so as to provide a better understanding into the regulatory mechanisms controlling Nrf2 activation in response to chemical stress. REFERENCES [1] Nguyen, T.; Sherratt, P. J.; Pickett, C. B. Regulatory mechanisms controlling gene expression mediated by the antioxidant response element. Annu. Rev. Pharmacol. Toxicol. 43:233 – 260; 2003. [2] Venugopal, R.; Jaiswal, A. K. Nrf1 and Nrf2 positively and c-Fos and Fra1 negatively regulate the human antioxidant response element-mediated expression of NAD(P)H:quinone oxidoreductase1 gene. Proc. Natl. Acad. Sci. USA 93:14960 – 14965; 1996. [3] Venugopal, R.; Jaiswal, A. K. 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; 1998. [4] Wild, A. C.; Moinova, H. R.; Mulcahy, R. T. Regulation of g-glutamylcysteine synthetase regulatory subunit gene expression by the transcription factor Nrf2. J. Biol. Chem. 274: 33627 – 33636; 1999. [5] Alam, J.; Stewart, D.; Touchard, C.; Boinapally, S.; Choi, A. M. K.; Cook, J. L. Nrf2, a Cap’n’Collar transcription factor, regulates induction of the heme oxigenase-1 gene. J. Biol. Chem. 274: 26071 – 26078; 1999. [6] Nguyen, T.; Huang, H. C.; Pickett, C. B. Transcriptional regulation of the antioxidant response element: activation by Nrf2 and repression by MafK. J. Biol. Chem. 275:15466 – 15473; 2000.
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