Service of Endocrinology, Diabetology, Metabolism, Lausanne University Hospital, 1011 Lausanne, Switzerland (SDM, PGZ, GPS); Faculty of Biology, Medicine, University of Lausanne, 1011 Lausanne, Switzerland (GPS)
miRNA3128 is a non-coding RNA that is the transcript product of a region in the first intron of NFE2L2 (chr. 2: 177255945-177256010, complement). It is not known whether it has a role in the regulation of NFE2L2 expression. Micro RNAs species reported to suppress NFE2L2 expression include miR-27a, miR-28, miR-34a, miR-93, miR-142-5p, miR-144, and miR-153 (Filipowicz et al., 2008; Cheng et al., 2013; Hayes and Dinkova-Kostova, 2014).
Oxidative stress and the antioxidant transcriptional response mediated by Nrf2
Cells and tissues are constantly exposed to various oxidative substances and electrophilic chemicals, including reactive oxygen species (ROS) and reactive nitrogen species (RNS), derived from both endogenous and exogenous sources. To adapt to the oxidative environment, cells have developed elaborate and highly efficient antioxidant machineries. When pro-oxidant and electrophilic challenges overwhelm the cells antioxidant and detoxification proteins, cells experience oxidative stress. Oxidative stress conditions can cause damage to cellular structures, including lipids, proteins, and nucleic acids. Among other injuries, this can lead to mutations and epigenetic perturbations by damaging DNA and proteins that modify chromatin. Thus, oxidative stress can be a causative or exacerbating factor in a range of diseases, including, for example, respiratory and metabolic disorders, neurodegenerative diseases, and cancer.
In order to maintain homeostasis in the face of oxidative insults, cells possess signalling pathways that can sense oxidative stress and launch adaptive responses. Multiple ways of managing the intracellular oxidative load have been identified over the last two decades; among them, it has been recognized that gene transcription can be regulated by redox reactions. Prominent among the redox-sensitive pathways of gene activation is the Nrf2 system. The core of this pathway comprises the transcription factor Nrf2 and its negative regulator Keap1. In addition, small Maf proteins serve as dimerization partners of Nrf2 to facilitate its binding to DNA on special sequences termed antioxidant response elements (AREs) or electrophile response elements (EpREs) in the regulatory regions of the many Nrf2-regulated genes, including the genes encoding glutamate cysteine ligase catalytic subunit (GCLC), heme oxygenase-1 (HO-1), NADP(H) quinone oxidoreductase-1 (NQO1), microsomal glutathione-S-transferases such as MGST1 and MGST2, and multi-drug resistance-associated proteins such as ATP-binding cassette, subfamily C (CFTR/MRP), member 1 (ABCC1) (Magesh et al., 2012; Hayes and Dinkova-Kostova, 2014).
Activity and regulation of Nrf2
The Nrf2 pathway responds to oxidative stress by inducing the transcriptional upregulation of a broad range of cytoprotective genes. The Nrf2 system responds to both endogenous reactive molecules, such as reactive oxygen species (ROS) and reactive nitrogen species (RNS), and to exogenous substances. The sensing mechanisms comprise oxidation or alkylation of critical Keap1 cysteine residues (including Cys151, Cys273 and Cys288), and phosphorylation of Nrf2 on amino acids Ser40 and Tyr568 (Zhang and Hannink, 2003; Yamamoto et al., 2008; Magesh et al., 2012).
When redox homeostasis is restored, Nrf2 activity is repressed via export from the nucleus back into the cytoplasm and degradation via a Cullin-RING ligase 3 - Keap1 complex (CRLkeap1 complex). Nrf2 can also trigger a feedback loop of increased expression of ARE-dependent genes including Keap1 and Cul3, which then promote Nrf2 degradation and thus participate in resetting Nrf2 activity at its basal level (Rachakonda et al., 2008; Eggler et al., 2009; Baird and Dinkova-Kostova, 2011).
Activation of Nrf2 pathway
A. Transcriptional induction of the NFE2L2 gene
A1. Nrf2 autoregulation
It has been shown that the promoter of the mouse orthologue of the NFE2L2 gene includes two antioxidant response element-like sequences (ARE-L1 and ARE-L2), which are located at -492 bp and -754 bp from the transcription start site, respectively. It has been proposed that under stress conditions newly translated Nrf2 protein escapes Keap1-mediated degradation and binds to the ARE-L1 and ARE-L2 sequences to induce NFE2L2 gene transcription in a feed-forward manner (Shin et al., 2007; Hayes and Dinkova-Kostova, 2014).
A2. NFE2L2 transcription induced by the oncoproteins K-Ras and B-Raf
It has been demonstrated that oncogene-directed increased expression of the NFE2L2 gene can be an alternative mechanism of Nrf2 activation. K-Ras and B-Raf, which operate in the mitogen-activated protein kinase (MAPK) pathway, have been shown to increase NFE2L2 transcription via activation of Jun or/and Myc. It has been proposed that via this mechanism oncogenic signaling may modulate redox homeostasis during tumirogenesis (DeNicola et al., 2011).
A3. Cross-talk of Nrf2 with the NF-κB and AhR signaling pathways
In general, the effects of the intracellular events induced by Nrf2 activation lead to NF-κB suppression, and vice versa; thus, overall, Nrf2 signaling antagonises NF-κB signaling. Nevertheless, in acute myeloid leukemia it has been reported that Nrf2 is upregulated by NF-κB-mediated transactivation of the NFE2L2 gene by direct binding of NF-κB to the NFE2L2 promoter (Rushworth et al., 2012).
The AhR/ARNT complex (aryl hydrocarbon receptor / AhR nuclear translocator) regulates gene transcription in response to xenobiotics, such as polycyclic aromatic hydrocarbons, via binding to xenobiotic response elements (XREs). Three XRE-like elements have been identified in the mouse Nfe2l2 promoter; via these elements, the AhR/XRE pathway can control Nrf2/ARE signaling (Miao et al., 2005).
A4. BRCA1 / ARNT-mediated induction of NFE2L2 gene transcription
The transcription factor BRCA1 (breast cancer 1, early onset) has been reported to increase the transcription of the NFE2L2 gene. As BRCA1 has the ability to interact with ARNT, it is possible that BRCA1 induces Nrf2 expression in an ARNT-dependent manner (Kang et al., 2006).
B. Post-translational activation of Nrf2
B1. The "hinge and latch" and "quaternary" models
At basal conditions (meaning the absence of oxidative stress) the NFE2L2 gene is constantly transcribed and Nrf2 protein is constantly synthesized, but Nrf2 protein abundance and activity are maintained at low levels due to the negative regulation of Nrf2 by Keap1 through the CRLkeap1 complex.
The "hinge and latch" model has been proposed as a mechanistic model that accounts for the interaction between Nrf2 and Keap1 and provides as structural basis for the Keap1-dependent polyubiquitination and degradation of Nrf2. This model posits an interaction of one Nrf2 molecule with a Keap1 homodimer, in which the high affinity binding of the ETGE motif of the Neh2 domain of Nrf2 functions as a "hinge" to fix Nrf2 to one of two Keap1 molecules, whereas the low affinity binding of the DLG motif of the Neh2 domain of Nrf2 functions as a "latch" to lock down the Neh2 domain to the other Keap1 molecule of the homodimer. The fixation of the Neh2 domain between the two Keap1 molecules thus facilitates its ubiquitination and the subsequent degradation of Nrf2 by the 26S proteasome. A competing structural model is the "quaternary complex" model, which proposes that a Keap1 dimer binds two molecules of substrate through high-affinity interactions with ETGE motifs. Specifically, a Keap1 dimer can bind two Nrf2 molecules, or one Nrf2 molecule and one PGAM5 molecule. PGAM5 possesses an N-terminal membrane targeting signal through which the Nrf2-Keap1-PGAM complex is tethered to the cytosolic surface of the outer mitochondrial membrane (Tong et al., 2006; Sykiotis and Bohmann, 2010; Kansanen et al., 2012), potentially to allow Nrf2 to be activated in response to mitochondrial leakage of ROS.
Under conditions of oxidative stress, it is believed that the oxidative modification of certain cysteine residues of Keap1 leads to conformational changes of the Keap1 dimer. In the "hinge and latch" model, this results in dissociation of the DLG motif from Keap1, wuch that Nrf2 cannot be properly presented for ubiquitination by the CRLkeap1 complex and thus escapes proteosomal degradation. The stabilized Nrf2 accumulates in the nucleus where it heterodimerizes with small Maf proteins and binds to AREs, leading to transcription of ARE-dependent cytoprotective genes (reviewed in Sykiotis and Bohmann, 2010; Hayes and Dinkova-Kostova, 2014).
B2. Phosphorylation of Nrf2 by PKC
Several protein kinases, including protein kinase C (PKC), have been implicated in the upstream regulation of Nrf2 pathway. Specifically, phosphorylation of Nrf2 by PKC induces nuclear translocation of this transcription factor and activation of the ARE in response to oxidative stress. Furthermore, it has been found that PKC phosphorylates Nrf2 at Ser-40 facilitating its release from Keap1-mediated inhibition (Wakabayashi et al., 2010; Stepkowski and Kruszewski, 2011).
B3. The redox signaling "model of two Nrf2 pools"
Multiple NLS/NES (Nuclear localisation signal/ nuclear export signal) motifs have been identified in the Nrf2 sequence. These include three NLS motifs (bNLS, NLSN and NLSc) and two NES motifs (NESTA and NESZIP) but only the NESTA motif has been found to be redox-sensitive. Specifically, the NESTA motif has been shown to display a graded response to oxidative stress, implying that it can not only sense the presence of reactive oxidative species, but it also has the ability to transmit the oxidative stress "intensity" to the nucleus in order to up-regulate the transcription of ARE-genes accordingly.
Based on these observations, Nrf2 has been proposed as a direct redox-sensor. Specifically, under basal condition a dynamic balance can be observed as the combined nuclear exporting forces of NESTA and NESZIP counteract the combined nuclear importing force of the bNLS, NLSN and NLSc leading to a whole-cell distribution of Nrf2. However, under oxidative stress the NESTA is functionally disabled, and the driving force of NLSs becomes dominant and favors the nuclear localization of Nrf2.
The NLS/NES motifs and their role in activation of Nrf2 have led to the hypothesis for Keap1-independent Nrf2 signaling. Nevertheless, this model does not exclude Keap1 involvement in redox signaling. Consequently, a new model has been proposed that encompasses both Keap1-dependent and Keap1-independent Nrf2 signaling. This model proposes that in cells there may exist a free-floating pool of Nrf2 (fNrf2) and a Keap1-bound pool of Nrf2 (kNrf2). Under homeostatic conditions there is an equilibrium between synthesis and degradation of Nrf2, such that the fNrf2 pool remains small. But when cells are exposed to oxidative stress, the Nrf2-binding capacity of Keap1 is diminished and the fNrf2 pool is enlarged. As the NESTA of the fNrf2 redox-sensitive pool is disabled by the stress, nuclear localization of Nrf2 is favored (Li and Kong, 2009).
B4. Competitors of Nrf2 for binding to Keap1
It has been demonstrated that the ability of Keap1 to repress Nrf2 can be modulated by proteins that also possess ETGE motifs and thereby compete with Nrf2 for the same binding site in Keap1. For example, dipeptidyl-peptidase 3 (DPP3), IκB kinase β (IKKβ), partner and localizer of BRCA2 (PALB2), phosphoglycerate mutase 5 (PGAM5) and Wilms tumor gene on X chromosome (WTX) contain ETGE motifs that enable them to bind Keap1 and act as competitors of Nrf2 (Hayes and Dinkova-Kostova, 2014).
B5. mTOR signaling and p62-dependent degradation of Keap1
There is also cross-talk between Nrf2-Keap1 signaling and autophagy. It has been shown that in normal cells this interaction serves as a host defence mechanism leading to expression of antioxidant enzymes as well as elimination of cytotoxic products. Keap1 can bind the autophagy cargo receptor p62, which contains an STGE motif similar to the ETGE motif of Nrf2. Following phosphorylation of Ser 351 within its STGE motif by the mammalian target of rapamycin complex (mTORC1), p62 becomes a potent inhibitor of Keap1. p62 is phosphorylated by mTORC1 in the presence of ubiquitinated autophagic cargos, which can occur under oxidative conditions; this in turn favors the binding of Keap1 to the phosphorylated STGE motifs. As a result, Keap1 is sequestrated in autophagy cargos in a p62-dependent manner, allowing Nrf2 to be stabilized and to accumulate in the nucleus to induce cytoprotective enzymes (Komatsu et al., 2010; Ichimura et al., 2013; Lamming and Sabatini, 2013).
B6. Cross-talk between p53/p21 and the Nrf2 pathway
The p53 tumor suppressor protein regulates several intracellular procedures including gene transcription and induction of apoptosis. It has been demonstrated that p53 is implicated in the regulation of the Nrf2-mediated oxidative response in a dual manner: under low or mild levels of oxidative stress, p53 promotes the stabilization of Nrf2 and its subsequent nuclear accumulation through the transcriptional activation of p21, and as a result reduces the oxidative burden to promote cell survival. p21 stabilizes Nrf2 due to the existence of a KRR motif within the p21 sequence which interacts with the DLG motif of Nrf2 inhibiting its binding to Keap1. On the other hand, under conditions of high or sustained levels of oxidative stress, Nrf2-mediated cell survival is suppressed, and high activity levels of p53 induce apoptosis to prevent tumorigenesis (Chen et al., 2009; Chen W et al., 2012).
B7. Competitive binding of BRCA1 to Nrf2
The ability of Keap1 to repress Nrf2 can be diminished by the competitive binding of breast cancer protein BRAC1, thereby preventing Keap1 from simultaneously binding to the ETGE motif of Nrf2 (Gorrini et al., 2013).
B8. Acetylation of Nrf2 by p300/CBP
It has been found that acetylation of the Neh1 domain of Nrf2 can increase the binding affinity of Nrf2-Maf heterodimers for ARE sequences. p300/CBP acetylates lysine residues of the Neh1 domain and enhances the interaction between Nrf2 and ARE sequence of antioxidant genes promoter resulting in induction of the respective genes transcription (Sun et al., 2009).
Other mechanistic models which have been proposed for Nrf2 stabilization and activation include the oxidation-induced dissociation of the CRLkeap1 complex, and the nucleocytoplasmic shuttling of Keap1 (Rachakonda et al., 2008; Eggler et al., 2009; Baird and Dinkova-Kostova, 2011).
Repression of Nrf2 signaling
A. Transcriptional repression of the NFE2L2 gene
A CpG island has been identified in the 5 flanking region of the NFE2L2 gene that extends to position -1175. The first 5 CpGs in this CpG island are found to be hypermethylated in prostate cancer samples and prostate cancer cell lines compared to normal prostate issues and cells. This hypermethylation leads to repression of NFE2L2 gene expression, potentially favouring tumorigenesis (Yu et al., 2010).
B. Post-transcriptional repression of Nrf2
At the post-transcriptional level, various micro RNAs (miRNAs) have been identified to interact with the Nrf2 mRNA resulting in repression of Nrf2 expression, including miR-27a, miR-28, miR-93, miR-142-5p, miR144 and miR-153 (Hayes and Dinkova-Kostova, 2014).
C. Post-translational repression of Nrf2
C1. CRLkeap1 complex-mediated degradation of Nrf2
As mentioned, the CRLkeap1 complex is responsible for the ubiquitination and 26S degradation of Nrf2 under normal conditions. Keap1 acts as an adaptor protein to mediate the interaction between Nrf2 and the Cul3 E3-ligase enyme, resulting in ubiquitination of lysines residues of the region located between the ETGE and DLG motifs in the Neh2 domain. Thereafter, ubiquitinated Nrf2 undergoes degradation by the 26S proteosome. It has been reported that the Nedd8 molecule serves as a factor of stabilization of the CRLkeap1 complex, and that the removal of Nedd8 by the CSN signalosome causes disruption of the complex and inhibition of Nrf2 ubiquitination. CAND1 is a mediator protein that can also block the degradation process of Nrf2 (Villeneuve et al., 2010).
C2. Crm1-dependent nuclear export and β-TrCP-dependent degradation of Nrf2
Nrf2 degradation by Keap1 is mediated by interaction via the Nrf2 Neh2 ETGE and DLG motifs. Nevertheless, in Nrf2 proteins mutant for the ETGE and DLG motifs, it has been observed that the Neh6 domain accounts for some of the residual instability of Nrf2 in a Keap1-independent way. Specifically, it has been shown that the DSGIS and DSAPGS motifs located within the Neh6 domain serve as binding sites through which Nrf2 binds with β-TrCP. β-TrCP has the ability to target Nrf2 for ubiquitination and degradation through a Skp1-Cul1-Rbx1/ Roc1 ubiquitin ligase complex; in vitro experiments with fibroblasts where β-TrCP is knocked down have shown increased Nrf2 protein levels.
This mechanism participates in the post-induction regulation of Nrf2 activity. The serine/threonine kinase GSK-3 controls the activity of the nuclear kinase Fyn which in turn phosphorylates Tyr568 of Nrf2 and promotes its Crm1 (exportin)-mediated export from the nucleus. GSK-3-mediated Fyn phosphorylation also causes an increase of the DSGIS degron activity in the Neh6 domain. The latter results in β-TrCP binding to the Neh6 domain of Nrf2 and consequently in β-TrCP-mediated Nrf2 degradation (Jain and Jaiswal, 2007; Chowdhry et al., 2013).
C3. Repression of Nrf2 by CRIF1, SIAH2 and RNF4
The CR6-interacting factor 1 (CRIF1) can promote the ubiquitination of Nrf2 through its interaction with both the N-terminal Neh2 and C-terminal Neh3 domains of Nrf2. The physiological circumstances when CRIF1 represses Nrf2 activity remain obscure.
During hypoxia, it has been observed that SIAH2 can lead to Nrf2 ubiquitination in a Neh2-independent manner. Further work is required to elucidate the basis of interaction between SIAH2 and Nrf2 and the conditions that regulate it.
It has been reported that small ubiquitin-like modifiers 1 and 2 (SUMO-1, SUMO-2) polysumoylate Nrf2 in promyelocytic leukemia nuclear bodies. The polysumoylated Nrf2 (pNrf2) translocates into the nucleus where SUMO-specific RING finger protein 4 (RNF4) ubiquitinates the pNrf2 leading it to degradation within the nucleus (Hayes and Dinkova-Kostova, 2014).
C4. Negative feedback loops regulating Nrf2
In vitro experiments have shown that antioxidant treatment can induce the expression of Keap1, suggesting a possible role of Nrf2 in the regulation of Keap1 expression. Keap1 has three ARE sequences within its promoter, of which one ARE on the reverse strand (position -46) has been demonstrated to be functional in facilitating KEAP1 gene transcription. Thus, it has been suggested that Nrf2 can control its own degradation by binding to the Keap1 ARE(-46) and thereby inducing KEAP1 transcription. In other words, there exists an autoregulatory loop in which Nrf2 controls Keap1 at the transcriptional level and Keap1 regulates Nrf2 at the post-translational level (Lee et al., 2007).
Similarly, it has been observed that Nrf2 regulates the expression of the Cul3 and Rbx1 genes. The Cul3 and Rbx1 proteins are constituents of the CRLkeap1 complex which is responsible for the ubiquitination of Nrf2. Specifically, it has been found that both the Cul3 gene promoter and the Rbx1 gene promoter contain one functional ARE, and that Nrf2 acts in an autoregulatory way by binding to these AREs to regulate the expression of the Cul3 and Rbx1 genes (Kaspar and Jaiswal, 2010).
In addition, there is evidence that Nrf2 is implicated in the expression of genes encoding 26S proteasome subunits, presumably in order to increase the proteasome-dependent removal of oxidatively damaged proteins. Therefore, it has been proposed that Nrf2 may regulate this negative autoregulatory feedback loop via the proteasome to restore its levels to the basal state after the removal of oxidative stimuli (Chapple et al., 2012).
Furthermore, it is known that Bach1 competes with Nrf2 for binding to the ARE-sequence of Nrf2-regulated genes. It has been demonstrated that Bach1 transcript variant 2 has an intronic ARE sequence (position +1411) and can be a transcriptional target gene of Nrf2 (negative autoregulatory feedback mechanism) (Jyrkkänen et al., 2011).
Finally, it has been recently discovered in cancer cell lines that retinoid X receptor α (RXRα) serves as an inhibitor of Nrf2 that regulates Nrf2 activity through a direct interaction with Neh7 domain, where a RXRα-binding site has been mapped. As the activation of Nrf2 results in upregulation of RXRα, this can form another negative feedback loop for Nrf2 regulation (Wang et al., 2013).
Specific polymorphisms associated with disease risk*
Heterozygosity (T/G) for rs6721961 (T/C/G) has been associated with increased risk of acute lung injury (ALI) in patients with major trauma in Caucasian/African-American and Japanese populations. Paradoxically, in a Japanese cohort, the haplotype (rs2001350T/rs6726395A/ rs1962142A/rs2364722A/rs6721961T) containing the homozygous SNP rs6721961 TT has been correlated with lower annual decline in forced expiratory volume in one second (FEV1), a measure of pulmonary function. In contrast, the rs6726395 G allele showed association with higher annual decline of FEV1 induced by cigarette smoking in Japanese. Furthermore, a haplotype containing rs35652124 C, rs6706649 C, rs6721961 G and GGC4 (a repeat polymorphism) has been proposed as a predictor factor of increased respiratory failure development in German patients with chronic obstructive pulmonary disease (COPD). A further study in a Netherlands population showed correlations between rs1806649 C and reduced COPD mortality, and between the rs2364723 CC and reduced FEV1. In a Hungarian population of childhood asthma, rs6721961 T and rs2588882 G have been inversely correlated with the infection-induced asthma.
The rs6721961 TT genotype has been associated with higher systolic and diastolic blood pressure in Japanese haemodialysis patients than the CC or CT genotypes. Similarly, haemodialysis patients with the rs35652124 TT genotype had higher diastolic blood pressure and higher cardiovascular mortality than CC or CT carriers. Finally, in a Netherlands population, it has been demonstrated that carriers of the rs2364723 (G/C) minor G allele showed lower triglyceride levels and reduced risk of cardiovascular mortality.
The rs6706649 C and rs35652124 C SNPs have higher frequency in Japanese patients with ulcerative colitis, and their presence has been correlated with a chronic continuous disease phenotype. In Helicobacter pylori-infected patients, the rs6706649C/rs35652124C and rs6706649C/rs35652124T haplotypes have been correlated with increased and decreased risk, respectively, of CpG methylation; rs6706649C/rs35652124T carriers with negative Helicobacter pylori test showed reduced risk of gastric cancer.
In a Mexican Mestizo population it was found that lupus nephritis in women was significantly associated with presence of the heterozygous rs35652124 (C/T).
Homozygosity for rs6721961 (TT) or rs2706110 (TT) has been associated with increased risk of breast cancer in a Finish population. Moreover, presence of the rs6721961 T allele together with the intronic rs1962142 A allele was associated with reduced Nrf2 expression in breast cancer tissue. In a study of a Finish population, Nrf2 rs2886182 (T/C) rare homozygous genotype TT has been significantly associated with poorer survival and recurrence-free survival in patients with breast cancer that had received adjuvant chemotherapy, and with poorer survival in patients with breast cancer that had undergone postoperative radiotherapy.
In postmenopausal women the rs6721961 (T allele) increased the risk of venous thromboembolism after oral estrogen therapy.
In Swedish populations, a protective effect against Parkinsons disease has been detected for a haplotype containing promoter SNPs rs7557529C/ rs35652124T/ rs6706649C/ rs6721961G as well as intronic SNPs rs2886161T/ rs1806649T/ rs2001350T/ rs10183914T) (Yamamoto et al., 2004; Marzec et al., 2007; Arisawa et al., 2008; Siedlinski et al., 2009; Masuko et al., 2011; Hartikainen et al., 2012; Cho, 2013; Figarska et al., 2014; Shimoyama et al., 2014).
* The nucleotides for each SNP correspond to the map on chr. 2, and are thus complementary to the gene sequence (NFE2L2 lies on the reverse strand).
Stavroula D Manolakou ; Panos G Ziros ; Gerasimos P Sykiotis
NFE2L2 (nuclear factor, erythroid 2-like 2)
Atlas Genet Cytogenet Oncol Haematol. 2014-09-01
Online version: http://atlasgeneticsoncology.org/gene/44284/nfe2l2