The Nrf2-antioxidant response element signaling pathway and its activation by oxidative stress.

A major mechanism in the cellular defense against oxidative or electrophilic stress is activation of the Nrf2-antioxidant response element signaling pathway, which controls the expression of genes whose protein products are involved in the detoxication and elimination of reactive oxidants and electrophilic agents through conjugative reactions and by enhancing cellular antioxidant capacity. At the molecular level, however, the regulatory mechanisms involved in mediating Nrf2 activation are not fully understood. It is well established that Nrf2 activity is controlled, in part, by the cytosolic protein Keap1, but the nature of this pathway and the mechanisms by which Keap1 acts to repress Nrf2 activity remain to be fully characterized and are the topics of discussion in this minireview. In addition, a possible role of the Nrf2-antioxidant response element transcriptional pathway in neuroprotection will also be discussed.

The carboxy-terminal Neh3 domain of Nrf2 is required for transcriptional activation.

Nrf2 is a transcription factor critical for the maintenance of cellular redox homeostasis. We have previously found that Nrf2 is a labile protein, and its activation in cells under stress involves mechanisms leading to its stabilization. As a modular protein, Nrf2 possesses distinct transactivation and DNA binding domains essential for its transcriptional activity. In this study, we found that the C-terminal "Neh3" domain of Nrf2 is also important for its activity. Deletion of the last 16 amino acids of the protein completely abolishes its ability to activate both reporter and endogenous gene expression. Using site-directed mutagenesis, we have identified a stretch of amino acids within this region that are essential for its activity and that are found to be conserved across species and among other members of the CNC-bZIP family. Importantly, deletion of the final 16 amino acids of Nrf2 does not influence its dimerizing capability, DNA binding activity, or subcellular localization, although it does increase the half-life of the protein. In addition, this region was found to be important for interaction with CHD6 (a chromo-ATPase/helicase DNA binding protein) in a yeast two-hybrid screen. RNA interference-mediated knockdown of CHD6 reduced both the basal and tert-butylhydroquinone-inducible expression of NQO1, a prototypical Nrf2 target gene. These data suggest that the Neh3 domain may act as a transactivation domain and that it is possibly involved in interaction with components of the transcriptional apparatus to affect its transcriptional activity.

Nrf2 controls constitutive and inducible expression of ARE-driven genes through a dynamic pathway involving nucleocytoplasmic shuttling by Keap1.

Nrf2 regulates the expression of genes encoding antioxidant proteins involved in cellular redox homeostasis. Previous studies have suggested that activation of Nrf2 is mediated by mechanisms promoting its dissociation from Keap1, a cytosolic repressor that acts to sequester the transcription factor in the cytoplasm. As a short-lived protein, Nrf2 is also activated by mechanisms leading to its stabilization in cells under stress, and recent evidence indicates that Keap1 has an active role in the control of its stability. In this report, using immunocytochemistry, cell fractionation, and chromatin immunoprecipitation analyses, we found that Nrf2 is primarily a nuclear protein and that it is expressed and recruited to the chromatin constitutively to drive basal gene expression. Furthermore, we found evidence indicating that Keap1 may repress Nrf2 activity by transiently shuttling into the nucleus to promote its ubiquitylation. The data suggested that the steady-state level of Nrf2 is maintained by a dynamic pathway that balances its constitutive expression with a Keap1-regulated degradation process downstream of its role as a transcriptional activator. We suggest that the stabilization of Nrf2 in cells under stress represents the central regulatory response mediated by mechanisms that interfere with its interaction with Keap1, leading to the induction of antioxidant enzymes important to maintain cellular redox homeostasis.

The pathways and molecular mechanisms regulating Nrf2 activation in response to chemical stress.

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 cis-acting antioxidant response element (ARE) found in the promoter of the encoding genes. The transcription factor NF-E2-related 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.

Increased protein stability as a mechanism that enhances Nrf2-mediated transcriptional activation of the antioxidant response element. Degradation of Nrf2 by the 26 S proteasome.

Nrf2 (NF-E2-related factor 2) is a central transcription factor involved in the transcriptional activation of many genes encoding phase II drug-metabolizing enzymes via the antioxidant response element. Nrf2 has previously been found to undergo nuclear translocation by a phosphorylation-dependent mechanism mediated by protein kinase C in HepG2 cells treated with tert-butylhydroquinone, beta-naphthoflavone, or 12-O-tetradecanoylphorbol-13-acetate. In the present report, we have found that the levels of Nrf2 were increased in cells treated with tert-butylhydroquinone or beta-naphthoflavone by a post-transcriptional mechanism. Treatment of HepG2 cells with cycloheximide resulted in the loss of Nrf2 within 30 min. By contrast, treatment with the proteasome inhibitors (lactacystin or MG-132) caused an accumulation of Nrf2 as well as an induction of reporter gene activity in cells transfected with the GSTA2 antioxidant response element-chloramphenicol acetyl transferase construct. Similarly, the protein phosphatase inhibitor okadaic acid also caused an accumulation of Nrf2, whereas the reverse effects were observed with PD 98059 and U 0126, two compounds that block the activation of the MAPK/ERK signaling cascade. These data suggest that Nrf2 is degraded by the ubiquitin-dependent pathway and that phosphorylation of Nrf2 leads to an increase in its stability and subsequent transactivation activity.

Regulatory mechanisms controlling gene expression mediated by the antioxidant response element.

The expression of genes encoding antioxidative and Phase II detoxification enzymes is induced in cells exposed to electrophilic compounds and phenolic antioxidants. Induction of these enzymes is regulated at the transcriptional level and is mediated by a specific enhancer, the antioxidant response element or ARE, found in the promoter of the enzyme's gene. The transcription factor Nrf2 has been implicated as the central protein that interacts with the ARE to activate gene transcription constitutively or in response to an oxidative stress signal. This review focuses on the molecular mechanisms whereby the transcriptional activation mediated by the interaction between the ARE and NF-E2-related factor 2 (Nrf2) is regulated. Recent studies suggest that the sequence context of the ARE, the nature of the chemical inducers, and the cell type are important for determining the activity of the enhancer in a particular gene.

Phosphorylation of Nrf2 at Ser-40 by protein kinase C regulates antioxidant response element-mediated transcription.

Nrf2, a basic leucine zipper transcription factor, is an essential activator of the coordinated transcription of genes encoding antioxidant enzymes and phase II detoxifying enzymes through the regulatory sequence termed antioxidant response element (ARE). Recently we reported evidence for the involvement of protein kinase C (PKC) in phosphorylating Nrf2 and triggering its nuclear translocation in response to oxidative stress. We show here that phosphorylation of purified rat Nrf2 by the catalytic subunit of PKC was blocked by a synthetic peptide mimicking one of the potential PKC sites. Accordingly, Nrf2 bearing a Ser to Ala mutation at amino acid 40 (S40A) could not be phosphorylated by PKC. The S40A mutation did not affect in vitro binding of Nrf2/MafK to the ARE. However, it partially impaired Nrf2 activation of ARE-driven transcription in a reporter gene assay when Keap1 was overexpressed. In vitro transcribed/translated Keap1 could be coimmunoprecipitated with Nrf2. Phosphorylation of wild-type Nrf2 by PKC promoted its dissociation from Keap1, whereas the Nrf2-S40A mutant remained associated. These findings together with our prior studies suggest that the PKC-catalyzed phosphorylation of Nrf2 at Ser-40 is a critical signaling event leading to ARE-mediated cellular antioxidant response.

Human survivin is negatively regulated by wild-type p53 and participates in p53-dependent apoptotic pathway.

Survivin is an inhibitor of apoptosis protein, which is over-expressed in most tumors. Aberrant expression of survivin and loss of wild-type p53 in many tumors prompted us to investigate a possible link between these two events. Here we show that wild-type p53 represses survivin expression at both mRNA and protein levels. Transient transfection analyses revealed that the expression of wild-type p53, but not mutant p53, was associated with strong repression of the survivin promoter in various cell types. The over-expression of exogenous survivin protein rescues cells from p53-induced apoptosis in a dose-dependent manner, suggesting that loss of survivin mediates, at least, in part the p53-dependent apoptotic pathway. In spite of the presence of two putative p53-binding sites in the survivin promoter, deletion and mutation analyses suggested that neither site is required for transcriptional repression of survivin expression. This was confirmed by chromatin immunoprecipitation assays. Further analyses suggested that the modification of chromatin within the survivin promoter could be a molecular explanation for silencing of survivin gene transcription by p53.