ToxSci Advance Access originally published online on March 25, 2003
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Toxicological Sciences 73, 1-3 (2003)
Copyright © 2003 by the Society of Toxicology
TOXICOLOGICAL HIGHLIGHT |
Regulation of Nrf2-Mediated Induction of Glutamate Cysteine Ligase Modulatory Gene (GCLM) Expression: Considerations for Future Studies
Wadsworth Center, New York State Department of Health, Empire State Plaza, PO Box 509, Albany, New York 12201-0509
ABSTRACT
The article highlighted in this issue is "Erk Activation is Required for Nrf2 Nuclear Localization during Pyrrolidine Dithiocarbamate Induction of Glutamate Cysteine Ligase Modulatory Gene Expression in HepG2 cells," by Laurie M. Zipper and R. Timothy Mulcahy (pp. 124–134).
In a series of publications, Mulcahy and colleagues have described their continuing progress in characterizing regulation of the expression of human GCLM (formerly GLCLR) in response to various xenobiotics. In the latest report, contained in this issue, Zipper and Mulcahy address the role of phosphorylation in Nrf2-mediated induction of GCLM expression. The central role of the transcription factor Nrf2 in both basal and induced expression of a battery of phase II and cellular defense genes has been elucidated over the past 5 years by a number of investigators, as summarized by Zipper and Mulcahy. Nrf2-mediated upregulation of expression occurs through its binding to electrophile response elements (EpRE, also referred to as antioxidant response elements, or AREs) located in the promoter regions of these genes followed by Nrf2 transactivation. The most convincing evidence for the important role of Nrf2 in the cellular response to chemical inducers or oxidative stress comes from studies of nrf2 knockout mice. The Nrf2-deficient mice exhibit decreased expression of glutathione biosynthetic genes and glutathione S-transferases, decreased detoxification capability, decreased responsiveness to chemoprotective agents such as oltipraz, and increased sensitivity to benzo[a]pyrene-induced carcinogenesis.
Newly synthesized Nrf2 has several potential fates. It can translocate to the nucleus and, as a heterodimer with a second b-ZIP transcription factor, can regulate gene expression via EpRE elements in the targeted promoters. It can be degraded via a ubiquitin-26S proteosome pathway, or it can be sequestered within the cytoplasm and protected from degradation through its physical interaction with a member of the kelch repeat superfamily of proteins. This widely expressed tethering protein is designated as Keap1, INrf2, and KIAA0132 for the mouse, rat, and human homologues, respectively. Keap1 and its homologues are thought to sequester Nrf2 in the cytoplasm via an association with actin, a conclusion based on its possession of a double glycine repeat (DGR) moiety (also known as the Kelch motif) and on an observed colocalization with actin. Sekhar et al.(2002b)
hypothesize that in HepG2 cells basal Nrf2-EpRE-mediated gene expression occurs via Nrf2 molecules, which are in controlled excess relative to the levels of KIAA0132 (a balance maintained by de novo synthesis and degradation of Nrf2).
A number of investigators have postulated that the Keap1–Nrf2 complex (the stoichiometry of which has not been determined) constitutes a critical sensor of cellular oxidative stress (Dhakshinamoorthy and Jaiswal, 2001; Itoh et al., 1999). On exposure to an inducing agent, the sequestered cytoplasmic pool of Nrf2 is released to provide immediate transactivation of regulated genes which encode protective phase II and antioxidant-related enzymes. As discussed by Zipper and Mulcahy, two potential mechanisms for the release of Nrf2 from Keap1 have been proposed. One is that Keap1 is a direct sensor of oxidative stress, with its structural conformation being altered upon thiol modification. This possibility, as discussed by Dinkova-Kostova et al.(2002), is attractive; it provides for a direct sensing mechanism via reaction of inducing agents with a critical Keap1 cysteine(s) leading to release of Nrf2. It also provides a mechanism for reestablishing homeostasis through induction of antioxidant-related enzymes such as glutamate cysteine ligase, which ultimately leads to regeneration of the Nrf-2-binding Keap1 conformation. The second proposed mechanism involves oxidative stress-induced kinase activation that results in phosphorylation of key residues of Nrf2 or Keap1 and results in dissociation of the Nrf2-Keap1 complex. Zipper and Mulcahy (2000) have previously reported that inhibition of Erk and p38 mitogen-activated protein (MAP) kinases inhibits Nrf2-mediated induction of GCLM expression. These results are consistent with the observation that Erk pathway inhibition abrogated accumulation of GCLM mRNA following treatment with indomethacin or resveratrol (Sekhar et al., 2002a).
In this issue, Zipper and Mulcahy address pyrrolidine dithiocarbamate (PDTC) induction of GCLM in human hepatocellular carcinoma derived HepG2 cells, concentrating their analysis on the effects of MAPK involvement in this induced expression. Prompted by in vitro studies showing that purified Nrf2 protein is phosphorylated by immunoprecipitated Erk kinases and by the presence of multiple conserved MAPK phosphorylation sites within Nrf2, mutated versions of Nrf2 were produced to examine the effects of elimination of the relevant serine residues. Five Nrf2 potential MAPK phosphorylation sites conserved in the mouse, rat, and human and one site conserved in the human and rat were mutated from serine to alanine. In cotransfection studies, the effect of mutation of the conserved consensus MAPK phosphorylation sites in Nrf2, individually or in the tested combinations, was examined. It is concluded that it is unlikely that Nrf2 itself is a direct target for MAPK phosphorylation because the mutated forms can act like the wild type Nrf2 with respect to interaction with or induced release from Keap1, translocation to the nucleus and transactivation of the cotransfected GCLM-luciferase gene. In these studies, human Nrf2 was coexpressed with mouse Keap1; this is likely to be quite legitimate since there is 94% amino acid sequence identity between the human and mouse homologues of Keap1. To further address the issue of nuclear translocalization, Hep-G2 cells were treated with MAPK inhibitors (PD98059 ± SB202190) prior to PDTC exposure and accumulation of Nrf2 in nuclei was assessed by Western analysis. When the cells were treated with the inhibitors, nuclear translocation of Nrf2 was reduced in the PDTC-treated cells and apparently in non–PDTC-treated cells as well. Zipper and Mulcahy present a model for potential phosphorylation-sensitive steps required for Nrf2 translocalization in which MAPK phosphorylation of a Nrf2 binding protein or chaperone is required for nuclear translocation.
There are differing opinions on whether Nrf2 expression is upregulated in response to xenobiotic inducing agents, and this may reflect differences in the species, cell type, or inducing agents studied. Kwak et al. (2002) propose that Nrf2 can bind EpRE-like sequences in its own promoter and auto-upregulate its expression, saturating Keap1 and leading to a sustained signaling of Nrf2-regulated gene expression, such as was observed in mouse keratinocytes with an ICR mouse Nrf2 promoter and induction by 3H-1,2-dithiole-3-thione. This raises the possibility of a role for MAPK activity in the induced de novo expression of Nrf2. Studies examining Nrf2 expression in HepG2 cells, however, have not revealed evidence for autoregulation (Dhakshinamoorthy and Jaiswal, 2001; Wild et al., 1999). As discussed above, Nrf2 forms heterodimers with other proteins, especially members of the small maf class and other bZIP transcription factors such as c-Jun. Data indicate that, depending on the system studied and the identity of the Nrf2 binding partner, the resulting heterodimer can have promoter activating or repressing activities. For example, Kwak et al. (2002) observed that cotransfection with Nrf2 and MafK increased Nrf2 promoter activity 6-fold while others have observed repression by MafK (Nguyen et al., 2000). In addition to potentially influencing Nrf2 promoter regulating activities, the Nrf2 binding partner could be involved in induction of Nrf2. It has been suggested that c-Jun, as the Nrf2 binding partner, could be the oxidant sensor, since it, along with Nrf2 and INrf2, contains several potential conserved phosphorylation and redox regulation sites (Dhakshinamoorthy and Jaiswal, 2001). Zipper and Mulcahy address the possible need for phosphorylation of the Nrf2 binding partner to allow association of the two heterodimer components and translocalization to the nuclear compartment. Such an association may lead to a conformational change in Nrf2 revealing its nuclear localization signal. As with Nrf2, however, it is also possible that MAPK activity is required for upregulated expression of the gene encoding the Nrf2 binding partner. Transcription of c-Jun is induced in response to some phase II inducers.
As indicated by Zipper and Mulcahy, the mechanism by which Nrf2 becomes released from Keap-1 may be specific to the cell type and/or inducing agent studied, and although there is a high degree of homology between the human, rat, and mouse homologues of Nrf2 and Keap1, the possibility of species-specific differences also exists. Moreover, the significance of species-specific variation in the GCLM promoter is unknown at this time. A consideration of the system is therefore always necessary when trying to interpret studies designed to delineate regulatory pathways. Even within a species, the significance of interindividual genetic variation in the studied genes should be considered. This is especially relevant for the study of GCLM regulation by Nrf2 in light of two recent publications. In a study using hyperoxia as a model for oxidant injury, linkage analysis has been performed to identify candidate genes conferring susceptibility to hyperoxia-induced lung injury in mice (Cho et al., 2002). Nrf2 polymorphism was implicated, and further analysis revealed differences between susceptible and resistant mouse strains in both basal and induced expression of nrf2 mRNA and cosegregation of a single nucleotide polymorphism (T substituted for C at -336) in the nrf2 promoter with susceptibility in F2 mice. This raises the possibility that functionally significant Nrf2 polymorphism, which influences Nrf2 expression, may exist within the human population as well. Additionally, and of great interest, a recent study has been reported in which a single nucleotide polymorphism at -588 in human GCLM has been characterized as influencing the induction of GCLM expression in response to oxidants (Nakamura et al., 2002). The -588T genotype was significantly associated with both decreased plasma glutathione levels and myocardial infarction. This was true for -588 CT heterozygotes as well as for -588 TT homozygotes, and supports a role for GCLM as a susceptibility gene for coronary artery disease in which oxidative stress is thought to influence pathogenesis, suggesting its possible involvement in other pathologies associated with oxidative stress. (Evidence for functionally significant genetic variation of human GCLC [formerly named GLCLC] has also been reported [Walsh et al., 2001]). The -588 GCLM position is not associated with any defined promoter element, underscoring the need for continued investigation of the promoter region and transcription factors involved in regulation of GCLM. The sequence of the GCLM 5'-flanking region reported by Moinova and Mulcahy (derived from a human P1 genomic library clone from Genome Systems, Inc.) (Moinova and Mulcahy, 1998) shows the wild type sequence (–588C/–23G), while the sequence reported by Galloway et al.(1999) (derived from a human P1 genomic library clone from Resourcenzentrum im Deutschen Humangenomprojekt am Max-Planck-Institut für Molekulare Genetik, Berlin-Charlottenburg, Germany) shows the variant genotype (–588T/–23T) of GCLM. Thus the use of constructs derived from the corresponding DNA clones to assess induction of this gene may yield varying but ultimately explainable results. Efforts to define regulatory pathways will be greatly enhanced if investigators include in their publications information as to the specific source of the genetic material studied. The data reported can then be appropriately reinterpreted when relevant sequence variations are revealed as functionally significant in the future.
The important position that Nrf2 activation plays in mobilizing cellular defenses to detoxify potentially damaging chemicals makes it a target for design of chemoprotective agents. Such agents may improve on natural substances which are known to induce expression of Nrf2 transactivation activity (e.g., resveratrol, sulforaphane, and 6-HITC derived from red wine, broccoli, and wasabi, respectively), and may provide a source of such substances, which is more palatable to those who object to consumption of broccoli. An exciting area of study will be the continued delineation of functionally significant genetic variations, in Nrf2 or in key target genes of Nrf2, with a potential role in conferring increased susceptibility or resistance to carcinogenic or oxidant producing substances. Information derived from such studies may ultimately be used to indicate which individuals in the population would particularly benefit from dietary or pharmacological supplementation of various chemoprotective agents.
NOTES
1 For correspondence via fax: (703) 516-2393. E-mail: jrodricks{at}environcorp.com. 
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