Mechanisms of activation of the transcription factor Nrf2 by redox stressors, nutrient cues, and energy status and the pathways through which it attenuates degenerative disease

Abstract

Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) regulates the basal and stress-inducible expression of a battery of genes encoding key components of the glutathione-based and thioredoxin-based antioxidant systems, as well as aldo-keto reductase, glutathione S-transferase, and

Nad(p)h: quinone oxidoreductase-1 drug-metabolizing isoenzymes along with multidrug-resistance-associated efflux pumps. It therefore plays a pivotal role in both intrinsic resistance and cellular adaptation to reactive oxygen species (ROS) and xenobiotics. Activation of Nrf2 can, however, serve as a double-edged sword because some of the genes it induces may contribute to chemical carcinogenesis by promoting futile redox cycling of polycyclic aromatic hydrocarbon metabolites or confer resistance to chemotherapeutic drugs by increasing the expression of efflux pumps, suggesting its cytoprotective effects will vary in a context-specific fashion. In addition to cytoprotection, Nrf2 also controls genes involved in intermediary metabolism, positively regulating those involved in NADPH generation, purine biosynthesis, and the β-oxidation of fatty acids, while suppressing those involved in lipogenesis and gluconeogenesis. Nrf2 is subject to regulation at multiple levels. Its ability to orchestrate adaptation to oxidants and electrophiles is due principally to stress-stimulated modification of thiols within one of its repressors, the Kelch-like ECH-associated protein 1 (Keap1), which is present in the cullin-3 RING ubiquitin ligase (CRL) complex CRLKeap1. Thus modification of Cys residues in Keap1 blocks CRLKeap1 activity, allowing newly translated Nrf2 to accumulate rapidly and induce its target genes. The ability of Keap1 to repress Nrf2 can be attenuated by p62/sequestosome-1 in a mechanistic target of rapamycin complex 1 (mTORC1)-dependent manner, thereby allowing refeeding after fasting to increase Nrf2-target gene expression. In parallel with repression by Keap1, Nrf2 is also repressed by β-transducin repeat-containing protein (β-TrCP), present in the Skp1-cullin-1-F-box protein (SCF) ubiquitin ligase complex SCFβ-TrCP. The ability of SCFβ-TrCP to suppress Nrf2 activity is itself enhanced by prior phosphorylation of the transcription factor by glycogen synthase kinase-3 (GSK-3) through formation of a DSGIS-containing phosphodegron. However, formation of the phosphodegron in Nrf2 by GSK-3 is inhibited by stimuli that activate protein kinase B (PKB)/Akt. In particular, PKB/Akt activity can be increased by phosphoinositide 3-kinase and mTORC2, thereby providing an explanation of why antioxidant-responsive element-driven genes are induced by growth factors and nutrients. Thus Nrf2 activity is tightly controlled via CRLKeap1 and SCFβ-TrCP by oxidative stress and energy-based signals, allowing it to mediate adaptive responses that restore redox homeostasis and modulate intermediary metabolism. Based on the fact that Nrf2 influences multiple biochemical pathways in both positive and negative ways, it is likely its dose-response curve, in terms of susceptibility to certain degenerative disease, is U-shaped. Specifically, too little Nrf2 activity will lead to loss of cytoprotection, diminished antioxidant capacity, and lowered β-oxidation of fatty acids, while conversely also exhibiting heightened sensitivity to ROS-based signaling that involves receptor tyrosine kinases and apoptosis signal-regulating kinase-1. By contrast, too much Nrf2 activity disturbs the homeostatic balance in favor of reduction, and so may have deleterious consequences including overproduction of reduced glutathione and NADPH, the blunting of ROS-based signal transduction, epithelial cell hyperplasia, and failure of certain cell types to differentiate correctly. We discuss the basis of a putative U-shaped Nrf2 dose-response curve in terms of potentially competing processes relevant to different stages of tumorigenesis.

Keywords: Free radicals; GSK-3; Glutathione; Inflammation; Keap1; Lipid metabolism; Nrf2; Nutrient supply; Reactive oxygen species; Thioredoxin; aldo-keto reductase (AKR); β-TrCP.

Fig. 1

Mechanism of action of endogenous small protein antioxidants. (A) The biosynthesis of glutathione involves two steps. First (i), glutamate-cysteine ligase (GCL) conjugates cysteine (Cys) with glutamate (Glu), in a rate-limiting reaction that requires ATP, to produce γ-glutamyl-cysteine (γGC). Second (ii), glutathione synthetase (GSS) attaches glycine (Gly) to the C-terminal cysteine of γGC to produce the tripeptide glutathione (GSH). In turn, GSH may be oxidized by ROS (shown as H₂O₂), generating a disulfide bridge between two glutathione molecules, resulting in the formation of GSSG. The GSSG can be reduced back to two GSH molecules by the action of GSR1, an enzyme that utilizes NADPH as the electron donor. (B) The small protein dithiol TXN is oxidized, producing an intermolecular disulfide that is reduced by the flavoprotein TXNRD1, using NADPH as the electron donor. (C) The typical 2-Cys PRDX isoenzymes 1, 2, 3, and 4 reduce H₂O₂ through a catalytic cycle (shown on the left-hand side) that involves oxidation of the – SH group in an active-site Cys residue to sulfenic acid (–S–OH) in one subunit of the dimeric proteins. The oxidized thiol then forms an intermolecular disulfide bridge with a Cys residue in the other subunit before it is reduced by TXN; the resulting oxidized TXN is reduced by TXNRD1 in an NADPH-dependent manner. During reduction of H₂O₂, the active-site Cys in a small fraction of PRDX is hyperoxidized to sulfinic acid (–SO₂H) (shown on the right-hand side). Overoxidation of the peroxidatic Cys to sulfinic acid inactivates PRDX, but it can be reactivated by SRXN1 through a mechanism that involves a transient covalent linkage between the two proteins, followed by a thiol-mediated reduction that is likely to involve GSH.

Fig. 2

Chemical structures of dietary antioxidant compounds. Polyphenol family members that exhibit indirect antioxidant activity include resveratrol, a stilbenoid found in red wine; quercetin, a flavonol found in red and yellow onions; curcumin, a curcuminoid found in turmeric and mustard; and luteolin, a flavone found in celery, parsley, and thyme. Glucosinolate breakdown products that have indirect antioxidant properties include the isothiocyanates sulforaphane and phenethyl isothiocyanate and the epithionitriles 1-cyano-2,3-epithiopropane and 1-cyano-3,4-epithiobutane: glucosinolates that give rise to these compounds are found in cruciferous vegetables, such as broccoli.

Fig. 3

Domain structure of human Nrf2. The relative positions of the Neh domains of transcription factor Nrf2 are shown. The degrons responsible for targeting of Nrf2 protein by Keap1 and β-TrCP for proteasomal degradation are indicated above the domains, and the region of Nrf2 through which it is inhibited by RXRα is indicated. The numbering of amino acids is based on the human sequence and is shown below the cartoon.

Fig. 4

Structure of CNC-bZIP family members. By definition, all family members contain both CNC and bZIP sequences, which together comprise the Neh1 domain. The Neh2 or the Neh2-like (Neh2L) domain is present in mammalian Nrf1 and Nrf2 as well as the Drosophila CNC protein. The Neh3, Neh5, and Neh6 domains are common to all family members. The Neh4 domain is represented only in Nrf2 and CNC. The Neh7 domain has to date been identified only in Nrf2. An N-terminal domain (NTD) is found in Nrf1, Nrf3, and CNC, which directs them to the endoplasmic reticulum.

Fig. 5

The antioxidant response element. The characteristics of ARE sequences across human and rodent genes, and Nrf2-binding profiles, have been assessed using bioinformatics. (A) The ARE consensus sequence as usually reported in the literature on the basis of mutation analyses of gene reporter plasmids based primarily on rat, mouse, and human GST and NQO1 genes. (B and C) The positional matrices for the human and murine ARE sequences, Jasper IDs MA0150.1 and MA0150.2, respectively, which have been generated by the frequency at which these sites have been found experimentally to be occupied by Nrf2.

Fig. 6

Model to explain the influence of Nrf2 on ROS-dependent ASK1 signaling. Intracellular levels of ROS represent a major regulator of ASK1 activity, as the kinase is repressed by reduced TXN but not by oxidized TXN. Transcription factor Nrf2 is predicted to modulate the sensitivity of ASK1 to ROS-dependent activation by its ability to increase GSH-based and TXN-based antioxidant systems, its ability to suppress production of ROS by modulating mitochondrial function, and possibly also its ability to repress the expression of NOX2 and NOX4. (A) In wild-type cells, Nrf2 negatively controls expression of NOX2 and NOX4, which produce ROS (top), with inhibition depicted by a blunt-headed arrow. Moreover, Nrf2 supports inactivation of ROS by increasing the expression of GCL (upper right-hand side), which catalyzes the rate-limiting step in GSH synthesis; by increasing the expression of GPX2, which reduces H₂O₂ using GSH as a cofactor; and by increasing the expression of PRDX1, which reduces H₂O₂ in a TXN-dependent manner (all are shown as arrows). Nrf2 maintains TXN in a reduced state by increasing expression of TXNRD1 and SRXN1 (middle and lower right-hand side), and TXN, TXNRD1, and SRXN1 contribute to the reduction of H₂O₂ by PRDX1 (Fig. 1). Last, by improving the efficiency of oxidative phosphorylation, Nrf2 limits production of ROS by mitochondria (left-hand side). (B) In Nrf2-null cells, production of ROS by NOX2 and NOX4 is increased (top), as is the production by mitochondria (left-hand side). In addition, the GSH-based antioxidant and TXN-based antioxidant systems are diminished through decreased expression of GPX2, GCL, PRDX1, TXNRD1, and SRXN1 that results from loss of Nrf2 (right-hand side). The increased production of ROS and the diminished antioxidant capacity in Nrf2-null cells combine to cause the redox status of TXN to be shifted toward oxidation, and therefore its inhibition of ASK1 will be less effective. As a consequence, the downstream p38^MAPK and JNK kinases are more readily activated in Nrf2-null cells. Moreover, the increase in ROS in Nrf2-null cells will increase inhibition of MKP enzymes, and this will decrease the ability of the phosphatases to inhibit JNK and p38^MAPK.

Fig. 7

Domain structure of human Keap1. The Keap1 protein can be divided into five domains, the N- and C-terminal NTR and CTR sequences and the BTB, IVR, and DGR sequences. The BTB domain is responsible for dimerization and also the recruitment of cullin-3 to the CRL^Keap1 complex. The DGR domain along with the CTR form the β-propeller structure to which Nrf2 binds, with the DLG motif in the Neh2 domain of Nrf2 docking onto one Keap1 subunit and the ETGE motif docking onto the other Keap1 subunit. Cysteine residues that are functionally important or unusual are highlighted: Cys-151 is crucial for the ability of tBHQ and SFN to inhibit the substrate adaptor activity of Keap1 [250], and it forms a transient disulfide bridge with Cys-151 in the other Keap1 subunit [251]; Cys-273 and Cys-288 recognize alkenals and cyclopentanone prostaglandins [252, 253]; Cys-434 recognizes 8-nitro-cGMP [254]; Cys-226 and Cys-613 recognize metals such as Zn²⁺, Cd²⁺, As³⁺, and Se⁴⁺[253], and they form a transient disulfide bridge upon exposure to H₂O₂ [113, 251], which is represented by a horizontal two-headed arrow. Mutation of Cys-23 to tyrosine has been shown to impair the ability of Keap1 to ubiquitylate Nrf2 in breast cancer cells [255, 256]. The three amino acids at the C-terminus of human, mouse, and rat Keap1 comprises a distinctive CTC motif but its functional significance is obscure.

Fig. 8

Inducers of Nrf2-target genes. Many small molecules that induce ARE-driven gene expression are indirect antioxidants or share structural similarity with indirect antioxidants. Examples shown include the metabolite of butylated hydroxyanisole, tert-butyl hydroquinone (tBHQ), the diethyl ester of fumaric acid and the related compound diethyl maleate, the synthetic triterpenoid 1-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]imidazolide (CDDO-Im), the acetylenic tricyclic bis (cyanoenone) designated TBE-31, and 15-deoxy-Δ^12,14-prostaglandin J₂.

Fig. 9

Regulation of Nrf2 by Keap1 under basal and stressed conditions. Under unstressed conditions, newly synthesized Nrf2 protein is short lived, being sequestered in the cytoplasm by Keap1 and targeted immediately for proteasomal degradation by CRL^Keap1. Under such circumstances, Nrf2 is rapidly ubiquitylated and degraded, leaving the Keap1 homodimer free to sequester and process newly translated Nrf2 for degradation. A small fraction of the Nrf2 escapes degradation and translocates to the nucleus, maintaining a low basal level of ARE-driven gene expression even under normal homeostatic circumstances. Under stressed conditions, modification of Keap1 causes a conformational change that prevents the CRL^Keap1 protein complex from ubiquitylating Nrf2. In this situation, Keap1 is unable to turn over bound Nrf2, and as “free-Keap1” cannot be regenerated, it is unable to bind newly translated Nrf2. Thus under stressed conditions, newly synthesized Nrf2 evades Keap1 capture and translocates directly to the nucleus where it heterodimerizes with sMaf proteins, and together they bind ARE sequences to transactivate target genes.

Fig. 10

Degradation of Nrf2 by β-TrCP-mediated ubiquitylation. In addition to ubiquitylation of Nrf2 by CRL^Keap1, Nrf2 is also targeted for proteasomal degradation by SCF^β-TrCP through sequences in its Neh6 domain. (A) Sequence alignment of human, mouse, and rat Nrf2 (hNrf2, mNrf2, and rNrf2, respectively) using Clustal Omega shows that the two β-TrCP binding sites, and the surrounding residues in the Neh6 domain of Nrf2, are highly conserved across species. (B) The SCF complex mediates ubiquitylation of Nrf2 via the substrate receptor β-TrCP. Through its WD40 domain, β-TrCP binds to the DSGIS and DSAPGS peptide sequences in Nrf2. Phosphorylation of at least one of the Ser residues in the DSGIS motif by GSK-3 leads to increased activity of the degron.

Fig. 11

Posttranslational mechanisms that regulate Nrf2. The Nrf2 transcription factor is repressed under normal homeostatic conditions by the dual actions of β-TrCP and Keap1, thereby ensuring that the expression of antioxidant proteins and detoxication systems is restricted when the cell is not exposed to stress. However, numerous signaling processes allow derepression of Nrf2 by β-TrCP and Keap1, and this ensures the induction of ARE-driven genes to enable the elimination of electrophiles and oxidative stressors as well as metabolic adaptation to nutrients and growth cues. Thus, from the top left, the binding of insulin and growth factors to their cognate receptors will activate PI3K, which in turn increases Akt and mTORC2 activity while decreasing that of mTORC1 (not shown, but see Fig. 12). Increases in the PI3K–Akt–mTORC2 signaling system will inhibit GSK-3 activity, causing a decrease in the rate of β-TrCP-mediated Nrf2 turnover through reduced formation of the DSGIS-containing phosphodegron. In addition to insulin and growth factor signaling, the activity of mTORC1 is also decreased under conditions of low nutrient availability by the activation of AMPK. As shown top center, the activity of mTORC1 increases competitive inhibition of Keap1 by p62/SQSTM1 because it phosphorylates the STGE motif in the autophagy cargo receptor, which increases the affinity of p62/SQSTM1 for Keap1 and stimulates elimination of Keap1 by autophagy. Thus, during fasting, phosphorylation by mTORC1 of the STGE motif in p62/SQSTM1 is diminished and Keap1 is able to repress Nrf2 more effectively. As shown top right, signaling from autophagy, mitochondria, and inflammation pathways will diminish Keap1-mediated degradation of Nrf2 via competitive binding by p62/SQSTM1, PGAM5, and IKKβ to Keap1. Electrophiles and ROS also antagonize Keap1 substrate adaptor activity by modifying its sensor Cys-151, Cys-273, Cys-288, Cys-226/Cys-613, and Cys-434 residues. When small-molecule inducers inactivate Keap1, the conformation and/or orientation of the Keap1 dimer relative to other proteins within the Cul3–RBX1 complex is altered, producing a stalling of ligase activity that traps bound Nrf2 within CRL^Keap1. As a consequence, newly translated Nrf2 evades degradation by CRL^Keap1. This free fraction of Nrf2 accumulates and translocates to the nucleus where it heterodimerizes with sMaf proteins before binding to the ARE sequences in the promoters of target genes, causing induction of cytoprotective proteins. The increased expression of ARE-driven genes results in the synthesis and recycling of antioxidants, detoxification of harmful agents, and excretion of drugs. The physiological consequence of the induction of ARE-driven genes is the restoration of cellular redox homeostasis, through a negative feedback loop that ultimately allows Keap1 repression of Nrf2 to be reinstated through a variety of mechanisms that may include reactivation of oxidized Keap1 by TXNRD1, the induction of p62/SQSTM1 leading to increased degradation of electrophile-modified Keap1 by autophagy, and increased synthesis of Keap1.

Fig. 12

Complexity of the direct and indirect effects of mTOR on signaling pathways. Metabolic signaling through insulin receptor substrate-1 (IRS) leads to activation of PI3K and subsequent recruitment of phosphoinositide-dependent kinase-1 (PDK1) to the cell membrane. Here, PDK1 phosphorylates Akt at Thr-308, which in turn triggers phosphorylation of Akt at Ser-473 by mTORC2. Upon its activation, Akt becomes capable of inhibiting the TSC1/2 complex, leading to the stimulation of mTORC1 and its downstream targets 4EBP1 and p70^S6K1. The resulting stimulation of p70^S6K1 activity can then cause negative feedback of the mTOR signaling cascade at several points of the pathway. Thus, increased p70^S6K1 activity can lead to phosphorylation of IRS, leading to degradation of the receptor. Active p70^S6K1 also negatively regulates Akt signaling through inhibition of PDGFR and ERK/MAPK signaling. The two mTOR complexes are also able to regulate each other in an inter-complex feedback loop, as activation of p70^S6K1 stimulates phosphorylation of the RICTOR subunit of mTORC2, causing a decrease in Akt signaling thorough impaired phosphorylation of Ser-473. In addition, mTORC2, via p70^S6K1, also inhibits IRS, leading to a reduction in Akt signaling.

Fig. 13

Potential role of PPARα in the activation of Nrf2 in response to fasting. During fasting, triglyceride stores are mobilized and broken down to glycerol and free fatty acids (FFA). Once formed, the FFA act as ligands for PPARα, activating it and causing transcription of genes involved in their β-oxidation. Upon activation, PPARα could induce the NFE2L2 gene encoding Nrf2, which could in turn suppress PPARα mRNA levels. Another possibility is that the β-oxidation of fatty acids, which occurs in a PPARα-dependent manner, causes the production of ROS and lipid peroxides that indirectly cause activation of Nrf2 by antagonizing Keap1 and/or β-TrCP. It is also possible that a combination of both mechanisms is responsible for the activation of Nrf2 seen during fasting.

Fig. 14

Somatic mutations that upregulate Nrf2. Mutations that result in gain of function are depicted in octagonal shapes outlined in blue, and those that result in loss of function are outlined in red. (A) Transcription of the NFE2L2 gene is increased by mutant KRAS and Myc. (B) Inhibition of GSK-3 and failure to form the DSGIS phosphodegron is likely to occur from mutations in EGFR, PIK3CA and PTEN. (C) Mutations in Keap1 or mutations in the DLG or ETGE motifs in Nrf2 result in failure of CRL^Keap1 to ubiquitylate and repress Nrf2. (D) Accumulation of Nrf2 in tumors induces ARE genes, leading to increases in ROS scavenging, increases in metabolic detoxication, and efflux of endogenous electrophiles.

Fig. 15

Metabolic activation of carcinogens by inducible human reductases encoded by genes that are regulated via ARE sequences. AKR1C family members and NQO1 are capable of activating carcinogens. The AhR and Nrf2 transcription factors are depicted in red letters (toward the left-hand side) with an arrow pointing to their cognate cis-elements through which they regulate their target genes; a blunted red arrow above the transcription factors indicates circumstances when it might be desirable to inhibit their activity. To the right of the enzymes (depicted in blue boxes, shown slightly left of center), reactions representing the activation of carcinogens are shown linked to individual isoenzymes, and the consequences of carcinogen activation on DNA adduct formation are presented on the far right-hand side. The blue lines leading from carcinogen metabolites and ROS toward AhR and Nrf2 refer to pathways that result in transcription factor activation. The individual pathways are described below. (A) PAHs, e.g., tobacco and environmental carcinogens, are activated by AKR1C isoenzymes through NADP⁺-dependent oxidation of PAH-trans-dihydrodiols to their PAH ortho-quinones. In turn, PAH ortho-quinones are unstable and spontaneously generate ROS. Furthermore, PAH ortho-quinones are ligands for the AhR, and both PAH ortho-quinones and ROS induce Nrf2-target gene expression, which further exacerbates activation of PAHs. (B) NNK is a tobacco carcinogen that is converted by AKR1C isoenzymes to S-NNAL, which cannot be glucuronidated and as a consequence provides an additional source of DNA adducts. The induction of Nrf2-target gene expression by tobacco smoke further exacerbates the conversion of NNK to S-NNAL. (C) Nitroarenes, a carcinogenic component of diesel exhaust, undergo an NQO1-catalyzed six-electron reduction to yield aminoarenes, resulting in DNA adducts and ROS formation. The generation of ROS can induce Nrf2-target gene expression, which further exacerbates nitroarene activation. It is likely that Nrf2 is activated by diesel exhaust exposure.