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1.  Dysfunctional KEAP1–NRF2 Interaction in Non-Small-Cell Lung Cancer 
PLoS Medicine  2006;3(10):e420.
Background
Nuclear factor erythroid-2 related factor 2 (NRF2) is a redox-sensitive transcription factor that positively regulates the expression of genes encoding antioxidants, xenobiotic detoxification enzymes, and drug efflux pumps, and confers cytoprotection against oxidative stress and xenobiotics in normal cells. Kelch-like ECH-associated protein 1 (KEAP1) negatively regulates NRF2 activity by targeting it to proteasomal degradation. Increased expression of cellular antioxidants and xenobiotic detoxification enzymes has been implicated in resistance of tumor cells against chemotherapeutic drugs.
Methods and Findings
Here we report a systematic analysis of the KEAP1 genomic locus in lung cancer patients and cell lines that revealed deletion, insertion, and missense mutations in functionally important domains of KEAP1 and a very high percentage of loss of heterozygosity at 19p13.2, suggesting that biallelic inactivation of KEAP1 in lung cancer is a common event. Sequencing of KEAP1 in 12 cell lines and 54 non-small-cell lung cancer (NSCLC) samples revealed somatic mutations in KEAP1 in a total of six cell lines and ten tumors at a frequency of 50% and 19%, respectively. All the mutations were within highly conserved amino acid residues located in the Kelch or intervening region domain of the KEAP1 protein, suggesting that these mutations would likely abolish KEAP1 repressor activity. Evaluation of loss of heterozygosity at 19p13.2 revealed allelic losses in 61% of the NSCLC cell lines and 41% of the tumor samples. Decreased KEAP1 activity in cancer cells induced greater nuclear accumulation of NRF2, causing enhanced transcriptional induction of antioxidants, xenobiotic metabolism enzymes, and drug efflux pumps.
Conclusions
This is the first study to our knowledge to demonstrate that biallelic inactivation of KEAP1 is a frequent genetic alteration in NSCLC. Loss of KEAP1 function leading to constitutive activation of NRF2-mediated gene expression in cancer suggests that tumor cells manipulate the NRF2 pathway for their survival against chemotherapeutic agents.
Biallelic inactivation ofKEAP1, a frequent genetic alteration in NSCLC, is associated with activation of the NRF2 pathway which leads to expression of genes that contribute to resistance against chemotherapeutic drugs.
Editors' Summary
Background.
Lung cancer is the most common cause of cancer-related death worldwide. More than 150,000 people in the US alone die every year from this disease, which can be split into two basic types—small cell lung cancer and non-small-cell lung cancer (NSCLC). Four out of five lung cancers are NSCLCs, but both types are mainly caused by smoking. Exposure to chemicals in smoke produces changes (or mutations) in the genetic material of the cells lining the lungs that cause the cells to grow uncontrollably and to move around the body. In more than half the people who develop NSCLC, the cancer has spread out of the lungs before it is diagnosed, and therefore can't be removed surgically. Stage IV NSCLC, as this is known, is usually treated with chemotherapy—toxic chemicals that kill the fast-growing cancer cells. However, only 2% of people with stage IV NSCLC are still alive two years after their diagnosis, mainly because their cancer cells become resistant to chemotherapy. They do this by making proteins that destroy cancer drugs (detoxification enzymes) or that pump them out of cells (efflux pumps) and by making antioxidants, chemicals that protect cells against the oxidative damage caused by many chemotherapy agents.
Why Was This Study Done?
To improve the outlook for patients with lung cancer, researchers need to discover exactly how cancer cells become resistant to chemotherapy drugs. Detoxification enzymes, efflux pumps, and antioxidants normally protect cells from environmental toxins and from oxidants produced by the chemical processes of life. Their production is regulated by nuclear factor erythroid-2 related factor 2 (NRF2). The activity of this transcription factor (a protein that controls the expression of other proteins) is controlled by the protein Kelch-like ECH-associated protein 1 (KEAP1). KEAP1 holds NRF2 in the cytoplasm of the cell (the cytoplasm surrounds the cell's nucleus, where the genetic material is stored) when no oxidants are present and targets it for destruction. When oxidants are present, KEAP1 no longer interacts with NRF2, which moves into the nucleus and induces the expression of the proteins that protect the cell against oxidants and toxins. In this study, the researchers investigated whether changes in KEAP1 might underlie the drug resistance seen in lung cancer.
What Did the Researchers Do and Find?
The researchers looked carefully at the gene encoding KEAP1 in tissue taken from lung tumors and in several lung cancer cell lines—tumor cells that have been grown in a laboratory. They found mutations in parts of KEAP1 known to be important for its function in half the cell lines and a fifth of the tumor samples. They also found that about half of the samples had lost part of one copy of the KEAP1 gene—cells usually have two copies of each gene. Five of the six tumors with KEAP1 mutations had also lost one copy of KEAP1—geneticists call this biallelic inactivation. This means that these tumors should have no functional KEAP1. When the researchers checked this by staining the tumors for NRF2, they found that the tumor cells had more NRF2 than normal cells and that it accumulated in the nucleus. In addition, the tumor cells made more detoxification enzymes, efflux proteins, and antioxidants than normal cells. Finally, the researchers showed that lung cancer cells with KEAP1 mutations were more resistant to chemotherapy drugs than normal lung cells were.
What Do These Findings Mean?
These results indicate that biallelic inactivation of KEAP1 is a frequent genetic alteration in NSCLC and suggest that the loss of KEAP1 activity is one way that lung tumors can increase their NRF2 activity and develop resistance to chemotherapeutic drugs. More lung cancer samples need to be examined to confirm this result, and similar studies need to be done in other cancers to see whether loss of KEAP1 activity is a common mechanism by which tumors become resistant to chemotherapy. If such studies confirm that high NRF2 activity (either through mutation or by some other route) is often associated with a poor tumor response to chemotherapy, then the development of NRF2 inhibitors might help to improve treatment outcomes in patients with chemotherapy-resistant tumors.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030420.
US National Cancer Institute information on lung cancer and on cancer treatment
MedlinePlus entries on small cell lung cancer and NSCLC Cancer Research UK information on lung cancer
Wikipedia entries on lung cancer and chemotherapy (note that Wikipedia is a free online encyclopedia that anyone can edit)
doi:10.1371/journal.pmed.0030420
PMCID: PMC1584412  PMID: 17020408
2.  Different Electrostatic Potentials Define ETGE and DLG Motifs as Hinge and Latch in Oxidative Stress Response▿  
Molecular and Cellular Biology  2007;27(21):7511-7521.
Nrf2 is the regulator of the oxidative/electrophilic stress response. Its turnover is maintained by Keap1-mediated proteasomal degradation via a two-site substrate recognition mechanism in which two Nrf2-Keap1 binding sites form a hinge and latch. The E3 ligase adaptor Keap1 recognizes Nrf2 through its conserved ETGE and DLG motifs. In this study, we examined how the ETGE and DLG motifs bind to Keap1 in a very similar fashion but with different binding affinities by comparing the crystal complex of a Keap1-DC domain-DLG peptide with that of a Keap1-DC domain-ETGE peptide. We found that these two motifs interact with the same basic surface of either Keap1-DC domain of the Keap1 homodimer. The DLG motif works to correctly position the lysines within the Nrf2 Neh2 domain for efficient ubiquitination. Together with the results from calorimetric and functional studies, we conclude that different electrostatic potentials primarily define the ETGE and DLG motifs as a hinge and latch that senses the oxidative/electrophilic stress.
doi:10.1128/MCB.00753-07
PMCID: PMC2169061  PMID: 17785452
3.  KPNA6 (Importin α7)-Mediated Nuclear Import of Keap1 Represses the Nrf2-Dependent Antioxidant Response ▿  
Molecular and Cellular Biology  2011;31(9):1800-1811.
The transcription factor Nrf2 has emerged as a master regulator of cellular redox homeostasis. As an adaptive response to oxidative stress, Nrf2 activates the transcription of a battery of genes encoding antioxidants, detoxification enzymes, and xenobiotic transporters by binding the cis-antioxidant response element in the promoter regions of genes. The magnitude and duration of inducible Nrf2 signaling is delicately controlled at multiple levels by Keap1, which targets Nrf2 for redox-sensitive ubiquitin-mediated degradation in the cytoplasm and exports Nrf2 from the nucleus. However, it is not clear how Keap1 gains access to the nucleus. In this study, we show that Keap1 is constantly shuttling between the nucleus and the cytoplasm under physiological conditions. The nuclear import of Keap1 requires its C-terminal Kelch domain and is independent of Nrf1 and Nrf2. We have determined that importin α7, also known as karyopherin α6 (KPNA6), directly interacts with the Kelch domain of Keap1. Overexpression of KPNA6 facilitates Keap1 nuclear import and attenuates Nrf2 signaling, whereas knockdown of KPNA6 slows down Keap1 nuclear import and enhances the Nrf2-mediated adaptive response induced by oxidative stress. Furthermore, KPNA6 accelerates the clearance of Nrf2 protein from the nucleus during the postinduction phase, therefore promoting restoration of the Nrf2 protein to basal levels. These findings demonstrate that KPNA6-mediated Keap1 nuclear import plays an essential role in modulating the Nrf2-dependent antioxidant response and maintaining cellular redox homeostasis.
doi:10.1128/MCB.05036-11
PMCID: PMC3133232  PMID: 21383067
4.  Regulation of Nrf2 – An update 
Free radical biology & medicine  2013;66:10.1016/j.freeradbiomed.2013.02.008.
Nrf2:INrf2 (Keap1) are cellular sensors of oxidative and electrophilic stress. Nrf2 is a nuclear factor that controls the expression and coordinated induction of a battery of genes which encode detoxifying enzymes, drug transporters (MRPs), anti-apoptotic proteins and proteasomes. In the basal state, Nrf2 is constantly degraded in the cytoplasm by its inhibitor, INrf2. INrf2 functions as an adapter for Cul3/Rbx1 E3 ubiquitin ligase mediated degradation of Nrf2. Chemicals including antioxidants, tocopherols including α-tocopherol (vitamin E), phytochemicals and radiations antagonize the Nrf2:INrf2 interaction and leads to the stabilization and activation of Nrf2. The signaling events involve pre-induction, induction and post-induction responses that tightly control Nrf2 activation and repression back to the basal state. Oxidative/electrophilic signals activate unknown tyrosine kinase(s) in a pre-induction response which phosphorylates specific residues on Nrf2 negative-regulators, INrf2, Fyn and Bach1, leading to their nuclear export, ubiquitination and degradation. This prepares nuclei for unhindered import of Nrf2. Oxidative/electrophilic modification of INrf2cysteine151 followed by PKC phosphorylation of Nrf2serine40 in the induction response results in the escape or release of Nrf2 from INrf2. Nrf2 is thus stabilized and translocates to the nucleus resulting in a coordinated activation of gene expression. This is followed by a post-induction response that controls the ‘switching off’ of Nrf2-activated gene expression. GSK3β under the control of AKT and PI3K, phosphorylates Fyn leading to Fyn nuclear localization. Fyn phosphorylates Nrf2Y568 resulting in nuclear export and degradation of Nrf2. The activation and repression of Nrf2 provides protection against oxidative/electrophilic stress and associated diseases, including cancer. However, deregulation of INrf2 and Nrf2 due to mutations may lead to nuclear accumulation of Nrf2 that reduces apoptosis and promotes oncogenesis and drug resistance.
doi:10.1016/j.freeradbiomed.2013.02.008
PMCID: PMC3773280  PMID: 23434765
Nrf2; INrf2(Keap1); Antioxidants; Vitamins; Phytochemicals; ROS; Signaling; Regulation; Chemoprotection; Oncogenesis
5.  Keap1 Controls Postinduction Repression of the Nrf2-Mediated Antioxidant Response by Escorting Nuclear Export of Nrf2▿  
Molecular and Cellular Biology  2007;27(18):6334-6349.
The transcription factor Nrf2 regulates cellular redox homeostasis. Under basal conditions, Keap1 recruits Nrf2 into the Cul3-containing E3 ubiquitin ligase complex for ubiquitin conjugation and subsequent proteasomal degradation. Oxidative stress triggers activation of Nrf2 through inhibition of E3 ubiquitin ligase activity, resulting in increased levels of Nrf2 and transcriptional activation of Nrf2-dependent genes. In this study, we identify Keap1 as a key postinduction repressor of Nrf2 and demonstrate that a nuclear export sequence (NES) in Keap1 is required for termination of Nrf2-antioxidant response element (ARE) signaling by escorting nuclear export of Nrf2. We provide evidence that ubiquitination of Nrf2 is carried out in the cytosol. Furthermore, we show that Keap1 nuclear translocation is independent of Nrf2 and the Nrf2-Keap1 complex does not bind the ARE. Collectively, our results suggest the following mechanism of postinduction repression: upon recovery of cellular redox homeostasis, Keap1 translocates into the nucleus to dissociate Nrf2 from the ARE. The Nrf2-Keap1 complex is then transported out of the nucleus by the NES in Keap1. Once in the cytoplasm, the Keap1-Nrf2 complex associates with the E3 ubiquitin ligase, resulting in degradation of Nrf2 and termination of the Nrf2 signaling pathway. Hence, postinduction repression of the Nrf2-mediated antioxidant response is controlled by the nuclear export function of Keap1 in alliance with the cytoplasmic ubiquitination and degradation machinery.
doi:10.1128/MCB.00630-07
PMCID: PMC2099624  PMID: 17636022
6.  Acetylation of Nrf2 by p300/CBP Augments Promoter-Specific DNA Binding of Nrf2 during the Antioxidant Response▿ †  
Molecular and Cellular Biology  2009;29(10):2658-2672.
To maintain intracellular redox homeostasis, genes encoding many antioxidants and detoxification enzymes are transcriptionally upregulated upon deleterious oxidative stress through the cis antioxidant responsive elements (AREs) in their promoter regions. Nrf2 is the critical transcription factor responsible for ARE-dependent transcription. We and others have previously demonstrated that Nrf2 is targeted for ubiquitin-mediated degradation by Keap1 in a redox-sensitive manner through modifications of distinct cysteine residues of Keap1. Here, we report that p300/CBP directly acetylates Nrf2 in response to arsenite-induced stress. We have identified multiple acetylated lysine residues within the Nrf2 Neh1 DNA-binding domain. Combined lysine-to-arginine mutations on the acetylation sites, with no effects on Nrf2 protein stability, compromised the DNA-binding activity of Nrf2 in a promoter-specific manner. These findings demonstrated that acetylation of Nrf2 by p300/CBP augments promoter-specific DNA binding of Nrf2 and established acetylation as a novel regulatory mechanism that functions in concert with Keap1-mediated ubiquitination in modulating the Nrf2-dependent antioxidant response.
doi:10.1128/MCB.01639-08
PMCID: PMC2682049  PMID: 19273602
7.  Kinetic, Thermodynamic, and Structural Characterizations of the Association between Nrf2-DLGex Degron and Keap1 
Molecular and Cellular Biology  2014;34(5):832-846.
Transcription factor Nrf2 (NF-E2-related factor 2) coordinately regulates cytoprotective gene expression, but under unstressed conditions, Nrf2 is degraded rapidly through Keap1 (Kelch-like ECH-associated protein 1)-mediated ubiquitination. Nrf2 harbors two Keap1-binding motifs, DLG and ETGE. Interactions between these two motifs and Keap1 constitute a key regulatory nexus for cellular Nrf2 activity through the formation of a two-site binding hinge-and-latch mechanism. In this study, we determined the minimum Keap1-binding sequence of the DLG motif, the low-affinity latch site, and defined a new DLGex motif that covers a sequence much longer than that previously defined. We have successfully clarified the crystal structure of the Keap1-DC-DLGex complex at 1.6 Å. DLGex possesses a complicated helix structure, which interprets well the human-cancer-derived loss-of-function mutations in DLGex. In thermodynamic analyses, Keap1-DLGex binding is characterized as enthalpy and entropy driven, while Keap1-ETGE binding is characterized as purely enthalpy driven. In kinetic analyses, Keap1-DLGex binding follows a fast-association and fast-dissociation model, while Keap1-ETGE binding contains a slow-reaction step that leads to a stable conformation. These results demonstrate that the mode of DLGex binding to Keap1 is distinct from that of ETGE structurally, thermodynamically, and kinetically and support our contention that the DLGex motif serves as a converter transmitting environmental stress to Nrf2 induction as the latch site.
doi:10.1128/MCB.01191-13
PMCID: PMC4023822  PMID: 24366543
8.  A Noncanonical Mechanism of Nrf2 Activation by Autophagy Deficiency: Direct Interaction between Keap1 and p62▿  
Molecular and Cellular Biology  2010;30(13):3275-3285.
In response to stress, cells can utilize several cellular processes, such as autophagy, which is a bulk-lysosomal degradation pathway, to mitigate damages and increase the chances of cell survival. Deregulation of autophagy causes upregulation of p62 and the formation of p62-containing aggregates, which are associated with neurodegenerative diseases and cancer. The Nrf2-Keap1 pathway functions as a critical regulator of the cell's defense mechanism against oxidative stress by controlling the expression of many cellular protective proteins. Under basal conditions, Nrf2 is ubiquitinated by the Keap1-Cul3-E3 ubiquitin ligase complex and targeted to the 26S proteasome for degradation. Upon induction, the activity of the E3 ubiquitin ligase is inhibited through the modification of cysteine residues in Keap1, resulting in the stabilization and activation of Nrf2. In this current study, we identified the direct interaction between p62 and Keap1 and the residues required for the interaction have been mapped to 349-DPSTGE-354 in p62 and three arginines in the Kelch domain of Keap1. Accumulation of endogenous p62 or ectopic expression of p62 sequesters Keap1 into aggregates, resulting in the inhibition of Keap1-mediated Nrf2 ubiquitination and its subsequent degradation by the proteasome. In contrast, overexpression of mutated p62, which loses its ability to interact with Keap1, had no effect on Nrf2 stability, demonstrating that p62-mediated Nrf2 upregulation is Keap1 dependent. These findings demonstrate that autophagy deficiency activates the Nrf2 pathway in a noncanonical cysteine-independent mechanism.
doi:10.1128/MCB.00248-10
PMCID: PMC2897585  PMID: 20421418
9.  Keap1 Recruits Neh2 through Binding to ETGE and DLG Motifs: Characterization of the Two-Site Molecular Recognition Model 
Molecular and Cellular Biology  2006;26(8):2887-2900.
The expression of the phase 2 detoxification enzymes and antioxidant proteins is induced at the transcriptional level by Nrf2 and negatively regulated at the posttranslational level by Keap1 through protein-protein interactions with and subsequent proteolysis of Nrf2. We found that the Neh2 domain of Nrf2 is an intrinsically disordered but biologically active regulatory domain containing a 33-residue central α-helix followed by a mini antiparallel β-sheet. Isothermal calorimetry analysis indicated that one Neh2 molecule interacts with two molecules of Keap1 via two binding sites, the stronger binding ETGE motif and the weaker binding DLG motif. Nuclear magnetic resonance titration study showed that these two motifs of the Neh2 domain bind to an overlapping site on the bottom surface of the β-propeller structure of Keap1. In contrast, the central α-helix of the Neh2 domain does not have any observable affinity to Keap1, suggesting that this region may serve as a bridge connecting the two motifs for the association with the two β-propeller structures of a dimer of Keap1. Based on these observations, we propose that Keap1 recruits Nrf2 by the ETGE motif and that the DLG motif of the Neh2 domain locks its lysine-rich central α-helix in a correct position to benefit ubiquitin signaling.
doi:10.1128/MCB.26.8.2887-2900.2006
PMCID: PMC1446969  PMID: 16581765
10.  Proteomic analysis of ubiquitin ligase KEAP1 reveals associated proteins that inhibit NRF2 ubiquitination 
Cancer research  2013;73(7):2199-2210.
Somatic mutations in the KEAP1 ubiquitin ligase or its substrate NRF2 (NFE2L2) commonly occur in human cancer, resulting in constitutive NRF2-mediated transcription of cytoprotective genes. However, many tumors display high NRF2 activity in the absence of mutation, supporting the hypothesis that alternative mechanisms of pathway activation exist. Previously, we and others discovered that via a competitive binding mechanism, the proteins WTX (AMER1), PALB2 and SQSTM1 bind KEAP1 to activate NRF2. Proteomic analysis of the KEAP1 protein interaction network revealed a significant enrichment of associated proteins containing an ETGE amino acid motif, which matches the KEAP1 interaction motif found in NRF2. Like WTX, PALB2, and SQSTM1, we found that the dipeptidyl peptidase 3 (DPP3) protein binds KEAP1 via an ‘ETGE’ motif to displace NRF2, thus inhibiting NRF2 ubiquitination and driving NRF2-dependent transcription. Comparing the spectrum of KEAP1 interacting proteins with the genomic profile of 178 squamous cell lung carcinomas characterized by The Cancer Genome Atlas revealed amplification and mRNA over-expression of the DPP3 gene in tumors with high NRF2 activity but lacking NRF2 stabilizing mutations. We further show that tumor-derived mutations in KEAP1 are hypomorphic with respect to NRF2 inhibition and that DPP3 over-expression in the presence of these mutants further promotes NRF2 activation. Collectively, our findings further support the competition model of NRF2 activation and suggest that ‘ETGE’-containing proteins like DPP3 contribute to NRF2 activity in cancer.
doi:10.1158/0008-5472.CAN-12-4400
PMCID: PMC3618590  PMID: 23382044
11.  Distinct Cysteine Residues in Keap1 Are Required for Keap1-Dependent Ubiquitination of Nrf2 and for Stabilization of Nrf2 by Chemopreventive Agents and Oxidative Stress 
Molecular and Cellular Biology  2003;23(22):8137-8151.
A common feature of diverse chemopreventive agents is the ability to activate expression of a genetic program that protects cells from reactive chemical species that, if left unchecked, would cause mutagenic DNA damage. The bZIP transcription factor Nrf2 has emerged as a key regulator of this cancer-preventive genetic program. Nrf2 is normally sequestered in the cytoplasm by a protein known as Keap1. Chemopreventive agents allow Nrf2 to escape from Keap1-mediated repression, although the molecular mechanism(s) responsible for activation of Nrf2 is not understood. In this report, we demonstrate that Keap1 does not passively sequester Nrf2 in the cytoplasm but actively targets Nrf2 for ubiquitination and degradation by the proteosome under basal culture conditions. We have identified two critical cysteine residues in Keap1, C273 and C288, that are required for Keap1-dependent ubiquitination of Nrf2. Both sulforaphane, a chemopreventive isothiocyanate, and oxidative stress enable Nrf2 to escape Keap1-dependent degradation, leading to stabilization of Nrf2, increased nuclear localization of Nrf2, and activation of Nrf2-dependent cancer-protective genes. We have identified a third cysteine residue in Keap1, C151, that is uniquely required for inhibition of Keap1-dependent degradation of Nrf2 by sulforaphane and oxidative stress. This cysteine residue is also required for a novel posttranslational modification to Keap1 that is induced by oxidative stress. We propose that Keap1 is a component of a novel E3 ubiquitin ligase complex that is specifically targeted for inhibition by both chemopreventive agents and oxidative stress.
doi:10.1128/MCB.23.22.8137-8151.2003
PMCID: PMC262403  PMID: 14585973
12.  Targeted Deletion of Nrf2 Impairs Lung Development and Oxidant Injury in Neonatal Mice 
Antioxidants & Redox Signaling  2012;17(8):1066-1082.
Abstract
Aims: Nrf2 is an essential transcription factor for protection against oxidant disorders. However, its role in organ development and neonatal disease has received little attention. Therapeutically administered oxygen has been considered to contribute to bronchopulmonary dysplasia (BPD) in prematurity. The current study was performed to determine Nrf2-mediated molecular events during saccular-to-alveolar lung maturation, and the role of Nrf2 in the pathogenesis of hyperoxic lung injury using newborn Nrf2-deficient (Nrf2−/−) and wild-type (Nrf2+/+) mice. Results: Pulmonary basal expression of cell cycle, redox balance, and lipid/carbohydrate metabolism genes was lower while lymphocyte immunity genes were more highly expressed in Nrf2−/− neonates than in Nrf2+/+ neonates. Hyperoxia-induced phenotypes, including mortality, arrest of saccular-to-alveolar transition, and lung edema, and inflammation accompanying DNA damage and tissue oxidation were significantly more severe in Nrf2−/− neonates than in Nrf2+/+ neonates. During lung injury pathogenesis, Nrf2 orchestrated expression of lung genes involved in organ injury and morphology, cellular growth/proliferation, vasculature development, immune response, and cell–cell interaction. Bioinformatic identification of Nrf2 binding motifs and augmented hyperoxia-induced inflammation in genetically deficient neonates supported Gpx2 and Marco as Nrf2 effectors. Innovation: This investigation used lung transcriptomics and gene targeted mice to identify novel molecular events during saccular-to-alveolar stage transition and to elucidate Nrf2 downstream mechanisms in protection from hyperoxia-induced injury in neonate mouse lungs. Conclusion: Nrf2 deficiency augmented lung injury and arrest of alveolarization caused by hyperoxia during the newborn period. Results suggest a therapeutic potential of specific Nrf2 activators for oxidative stress-associated neonatal disorders including BPD. Antioxid. Redox Signal. 00, 000–000.
doi:10.1089/ars.2011.4288
PMCID: PMC3423869  PMID: 22400915
13.  Genetic or Pharmacologic Amplification of Nrf2 Signaling Inhibits Acute Inflammatory Liver Injury in Mice 
Oxidative stress-mediated destruction of normal parenchymal cells during hepatic inflammatory responses contributes to the pathogenesis of immune-mediated hepatitis and is implicated in the progression of acute inflammatory liver injury to chronic inflammatory liver disease. The transcription factor NF-E2-related factor 2 (Nrf2) regulates the expression of a battery of antioxidative enzymes and Nrf2 signaling can be activated by small-molecule drugs that disrupt Keap1-mediated repression of Nrf2 signaling. Therefore, genetic and pharmacologic approaches were used to activate Nrf2 signaling to assess protection against inflammatory liver injury. Profound increases in ind of cell death were observed in both Nrf2 wild-type (Nrf2-WT) mice and Nrf2-disrupted (Nrf2-KO) mice 24-hr following intravenous injection of concanavalin A (12.5 mg/kg, ConA), a model for T cell-mediated acute inflammatory liver injury. However, hepatocyte-specific conditional Keap1 null (Alb-Cre:Keap1flox/−, cKeap1-KO) mice with constitutively enhanced expression of Nrf2-regulated antioxidative genes as well as Nrf2-WT mice but not Nrf2-KO mice pretreated with three daily doses of a triterpenoid that potently activates Nrf2 (30 µmole/kg, CDDO-Im) were highly resistant to ConA-mediated inflammatory liver injury. CDDO-Im pretreatment of both Nrf2-WT and Nrf2-KO mice resulted in equivalent suppression of serum pro-inflammatory soluble proteins suggesting that the hepatoprotection afforded by CDDO-Im pretreatment of Nrf2-WT mice but not Nrf2-KO mice was not due to suppression of systemic pro-inflammatory signaling, but instead was due to activation of Nrf2 signaling in the liver. Enhanced hepatic expression of Nrf2-regulated antioxidative genes inhibited inflammation-mediated oxidative stress, thereby preventing hepatocyte necrosis. Attenuation of hepatocyte death in cKeap1-KO mice and CDDO-Im pretreated Nrf2-WT mice resulted in decreased late-phase pro-inflammatory gene expression in the liver thereby diminishing the sustained influx of inflammatory cells initially stimulated by the ConA challenge. Taken together, these results clearly illustrate that targeted cytoprotection of hepatocytes through Nrf2 signaling during inflammation prevents the amplification of inflammatory responses in the liver.
doi:10.1093/toxsci/kfn079
PMCID: PMC2435415  PMID: 18417483
Liver inflammation; Nrf2; Keap1; antioxidative enzymes; cytoprotection; triterpenoid
14.  Nrf2 Signaling and Cell Survival 
Nrf2:INrf2 acts as a sensor for oxidative/electrophilic stress. INrf2 serves as an adaptor to link Nrf2 to the ubiquitin ligase Cul3-Rbx1 complex that ubiquitinate and degrade Nrf2. Under basal conditions, cytosolic INrf2/Cul3-Rbx1 is constantly degrading Nrf2. When a cell encounters stress Nrf2 dissociates from the INrf2 and translocates into the nucleus. Oxidative/electrophilic stress induced modification of INrf2Cysteine151 and/or protein kinase C (PKC)-mediated phosporylation of Nrf2Serine40 controls Nrf2 release from INrf2 followed by stabilization and nuclear translocation of Nrf2. Nrf2 binds to the antioxidant response element (ARE) and activates a myriad of genes that protect cells against oxidative/electrophilic stress and neoplasia. A delayed response of oxidative/electrophilic stress activates GSK-3β that phosphorylates Fyn at unknown threonine residue(s). Phosphorylated Fyn translocates to the nucleus and phosphorylates Nrf2Tyrosine568 that leads to nuclear export and degradation of Nrf2. Prothymosin-α mediated nuclear translocation of INrf2 also degrades nuclear Nrf2. The degradation of Nrf2 both in cytosol and nuclear compartments rapidly brings down its levels to normal resulting in suppression of Nrf2 downstream gene expression. An autoregulatory loop between Nrf2 and INrf2 controls their cellular abundance. Nrf2 regulates INrf2 by controlling its transcription, and INrf2 controls Nrf2 by degrading it. In conclusion, switching on and off of Nrf2 combined with promoting an autoregulatory loop between them regulates activation/deactivation of defensive genes leading to protection of cells against adverse effects of oxidative and electrophilic stress and promote cell survival.
doi:10.1016/j.taap.2009.06.009
PMCID: PMC2837794  PMID: 19538984
Nrf; INrf2 (keap1); Oxidative/electrophilic stress; Defensive gene expression; Cell signaling; Cell survival
15.  Nrf2:INrf2(Keap1) Signaling in Oxidative Stress 
Free radical biology & medicine  2009;47(9):1304-1309.
Nrf2:INrf2(Keap1) are cellular sensors of chemical and radiation induced oxidative and electrophilic stress. Nrf2 is a nuclear transcription factor that controls the expression and coordinated induction of a battery of defensive genes encoding detoxifying enzymes and antioxidant proteins. This is a mechanism of critical importance for cellular protection and cell survival. Nrf2 is retained in the cytoplasm by an inhibitor INrf2. INrf2 functions as an adapter for Cul3/Rbx1 mediated degradation of Nrf2. In response to oxidative/electrophilic stress, Nrf2 is switched on and then off by distinct early and delayed mechanisms. Oxidative/electrophilic modification of INrf2cysteine151 and/or PKC phosphorylation of Nrf2serine40 results in the escape or release of Nrf2 from INrf2. Nrf2 is stabilized and translocates to the nucleus, forms heterodimers with unknown proteins, and binds antioxidant response element (ARE) that leads to coordinated activation of gene expression. It takes less than fifteen minutes from the time of exposure to switch on nuclear import of Nrf2. This is followed by activation of a delayed mechanism that controls switching off of Nrf2 activation of gene expression. GSK3β phosphorylates Fyn at unknown threonine residue(s) leading to nuclear localization of Fyn. Fyn phosphorylates Nrf2tyrosine568 resulting in nuclear export of Nrf2, binding with INrf2 and degradation of Nrf2. The switching on and off of Nrf2 protect cells against free radical damage, prevents apoptosis and promotes cell survival.
doi:10.1016/j.freeradbiomed.2009.07.035
PMCID: PMC2763938  PMID: 19666107
16.  Dihydro-CDDO-Trifluoroethyl Amide (dh404), a Novel Nrf2 Activator, Suppresses Oxidative Stress in Cardiomyocytes 
PLoS ONE  2009;4(12):e8391.
Targeting Nrf2 signaling appears to be an attractive approach for the treatment of maladaptive cardiac remodeling and dysfunction; however, pharmacological modulation of the Nrf2 pathway in the cardiovascular system remains to be established. Herein, we report that a novel synthetic triterpenoid derivative, dihydro-CDDO-trifluoroethyl amide (dh404), activates Nrf2 and suppresses oxidative stress in cardiomyocytes. Dh404 interrupted the Keap1-Cul3-Rbx1 E3 ligase complex-mediated Nrf2 ubiquitination and subsequent degradation saturating the binding capacity of Keap1 to Nrf2, thereby rendering more Nrf2 to be translocated into the nuclei to activate Nrf2-driven gene transcription. A mutant Keap1 protein containing a single cysteine-to-serine substitution at residue 151 within the BTB domain of Keap1 was resistant to dh404-induced stabilization of Nrf2 protein. In addition, dh404 did not dissociate the interaction of Nrf2 with the Keap1-Cul3-Rbx1 E3 ligase complex. Thus, it is likely that dh404 inhibits the ability of Keap1-Cul3-Rbx1 E3 ligase complex to target Nrf2 for ubiquitination and degradation via modifying Cys-151 of Keap1 to change the conformation of the complex. Moreover, dh404 was able to stabilize Nrf2 protein, to enhance Nrf2 nuclear translocation, to activate Nrf2-driven transcription, and to suppress angiotensin II (Ang II)-induced oxidative stress in cardiomyocytes. Knockdown of Nrf2 almost blocked the anti-oxidative effect of dh404. Dh404 activated Nrf2 signaling in the heart. Taken together, dh404 appears to be a novel Nrf2 activator with a therapeutic potential for cardiac diseases via suppressing oxidative stress.
doi:10.1371/journal.pone.0008391
PMCID: PMC2791441  PMID: 20027226
17.  Keap1 Is a Redox-Regulated Substrate Adaptor Protein for a Cul3-Dependent Ubiquitin Ligase Complex 
Molecular and Cellular Biology  2004;24(24):10941-10953.
The bZIP transcription factor Nrf2 controls a genetic program that protects cells from oxidative damage and maintains cellular redox homeostasis. Keap1, a BTB-Kelch protein, is the major upstream regulator of Nrf2 and controls both the subcellular localization and steady-state levels of Nrf2. In this report, we demonstrate that Keap1 functions as a substrate adaptor protein for a Cul3-dependent E3 ubiquitin ligase complex. Keap1 assembles into a functional E3 ubiquitin ligase complex with Cul3 and Rbx1 that targets multiple lysine residues located in the N-terminal Neh2 domain of Nrf2 for ubiquitin conjugation both in vivo and in vitro. Keap1-dependent ubiquitination of Nrf2 is inhibited following exposure of cells to quinone-induced oxidative stress and sulforaphane, a cancer-preventive isothiocyanate. A mutant Keap1 protein containing a single cysteine-to-serine substitution at residue 151 within the BTB domain of Keap1 is markedly resistant to inhibition by either quinone-induced oxidative stress or sulforaphane. Inhibition of Keap1-dependent ubiquitination of Nrf2 correlates with decreased association of Keap1 with Cul3. Neither quinone-induced oxidative stress nor sulforaphane disrupts association between Keap1 and Nrf2. Our results suggest that the ability of Keap1 to assemble into a functional E3 ubiquitin ligase complex is the critical determinant that controls steady-state levels of Nrf2 in response to cancer-preventive compounds and oxidative stress.
doi:10.1128/MCB.24.24.10941-10953.2004
PMCID: PMC533977  PMID: 15572695
18.  Structural insights into the similar modes of Nrf2 transcription factor recognition by the cytoplasmic repressor Keap1 
Journal of Synchrotron Radiation  2008;15(Pt 3):273-276.
The structure of mouse Keap1-DC complexed with the DLG motif peptide of Nrf2 transcription factor was determined at 1.9 Å resolution. The structure showed that the peptide binds to Keap1-DC at the bottom region of the β-propeller domain.
The cytoplasmic repressor Keap1 regulates the function of transcription factor Nrf2 which plays critical roles in oxidative and xenobiotic stresses. The Neh2 domain of Nrf2 interacts with Keap1 at the bottom region of the Kelch/β-propeller domain which is formed by double-glycine repeat and C-terminal region domains (Keap1-DC). The structure of Keap1-DC complexed with an Nrf2 peptide containing a conserved DLG motif has been determined at 1.9 Å resolution. The Keap1-bound DLG peptide possesses a hairpin conformation, and it binds to the Keap1 protein at the bottom region of the β-propeller domain. The intermolecular interaction occurs through their complementary electrostatic interactions. Comparison of the present structure with the recently reported Keap1-DC complex structure suggests that the DLG and ETGE motifs of Neh2 in Nrf2 bind to Keap1 in a similar manner but with different binding potencies.
doi:10.1107/S090904950705114X
PMCID: PMC2394801  PMID: 18421157
oxidative stress; Nrf2 transcription factor; Keap1; β-propeller domain; structure of the complex
19.  Molecular Mechanisms of Nrf2-Mediated Antioxidant Response 
Molecular carcinogenesis  2009;48(2):91-104.
Nrf2 is the key transcription factor regulating the antioxidant response. Nrf2 signaling is repressed by Keap1 at basal condition and induced by oxidative stress. Keap1 is recently identified as a Cullin 3-dependent substrate adaptor protein. A two-sites binding “hinge & latch” model vividly depicts how Keap1 can efficiently present Nrf2 as substrate for ubiquitination. Oxidative perturbation can impede Keap1-mediated Nrf2 ubiquitination but fail to disrupt Nrf2/Keap1 binding. Nrf2 per se is a redox-sensitive transcripon factor. A new Nrf2-mediated redox signaling model is proposed based on these new discoveries. Free floating Nrf2 protein functions as a redox-sensitive probe. Keap1 instead functions as a gate keeper to control the availability of Nrf2 probes and thus regulates the overall sensitivity of the redox signaling.
doi:10.1002/mc.20465
PMCID: PMC2631094  PMID: 18618599
Nrf2; Keap1; redox
20.  Cross-Regulations among NRFs and KEAP1 and Effects of their Silencing on Arsenic-Induced Antioxidant Response and Cytotoxicity in Human Keratinocytes 
Environmental Health Perspectives  2012;120(4):583-589.
Background: Nuclear factor E2-related factors (NRFs), including NRF2 and NRF1, play critical roles in mediating the cellular adaptive response to oxidative stress. Human exposure to inorganic arsenic, a potent oxidative stressor, causes various dermal disorders, including hyperkeratosis and skin cancer.
Objective: We investigated the cross-regulations among NRF2, NRF1, and KEAP1, a cullin-3–adapter protein that allows NRF2 to be ubiquinated and degraded by the proteasome complex, in arsenic-induced antioxidant responses.
Results: In human keratinocyte HaCaT cells, selective knockdown (KD) of NRF2 by lentiviral short hairpin RNAs (shRNAs) significantly reduced the expression of many antioxidant enzymes and sensitized the cells to acute cytotoxicity of inorganic arsenite (iAs3+). In contrast, silencing KEAP1 led to a dramatic resistance to iAs3+-induced apoptosis. Pretreatment of HaCaT cells with NRF2 activators, such as tert-butylhydroquinone, protects the cells against acute iAs3+ toxicity in an NRF2-dependent fashion. Consistent with the negative regulatory role of KEAP1 in NRF2 activation, KEAP1-KD cells exhibited enhanced transcriptional activity of NRF2 under nonstressed conditions. However, deficiency in KEAP1 did not facilitate induction of NRF2-target genes by iAs3+. In addition, NRF2 silencing reduced the expression of KEAP1 at transcription and protein levels but increased the protein expression of NRF1 under the iAs3+-exposed condition. In contrast, silencing KEAP1 augmented protein accumulation of NRF2 under basal and iAs3+-exposed conditions, whereas the iAs3+-induced protein accumulation of NRF1 was attenuated in KEAP1-KD cells.
Conclusions: Our studies suggest that NRF2, KEAP1, and NRF1 are coordinately involved in the regulation of the cellular adaptive response to iAs3+-induced oxidative stress.
doi:10.1289/ehp.1104580
PMCID: PMC3339469  PMID: 22476201
antioxidant response; arsenic; cytotoxicity; KEAP1; keratinocyte; NRF1; NRF2
21.  Roles Nrf2 Plays in Myeloid Cells and Related Disorders 
The Keap1-Nrf2 system protects animals from oxidative and electrophilic stresses. Nrf2 is a transcription factor that induces the expression of genes essential for detoxifying reactive oxygen species (ROS) and cytotoxic electrophiles. Keap1 is a stress sensor protein that binds to and ubiquitinates Nrf2 under unstressed conditions, leading to the rapid proteasomal degradation of Nrf2. Upon exposure to stress, Keap1 is modified and inactivated, which allows Nrf2 to accumulate and activate the transcription of a battery of cytoprotective genes. Antioxidative and detoxification activities are important for many types of cells to avoid DNA damage and cell death. Accumulating lines of recent evidence suggest that Nrf2 is also required for the primary functions of myeloid cells, which include phagocytosis, inflammation regulation, and ROS generation for bactericidal activities. In fact, results from several mouse models have shown that Nrf2 expression in myeloid cells is required for the proper regulation of inflammation, antitumor immunity, and atherosclerosis. Moreover, several molecules generated upon inflammation activate Nrf2. Although ROS detoxification mediated by Nrf2 is assumed to be required for anti-inflammation, the entire picture of the Nrf2-mediated regulation of myeloid cell primary functions has yet to be elucidated. In this review, we describe the Nrf2 inducers characteristic of myeloid cells and the contributions of Nrf2 to diseases.
doi:10.1155/2013/529219
PMCID: PMC3684031  PMID: 23819012
22.  Tanshinone I Activates the Nrf2-Dependent Antioxidant Response and Protects Against As(III)-Induced Lung Inflammation In Vitro and In Vivo 
Antioxidants & Redox Signaling  2013;19(14):1647-1661.
Abstract
Aims: The NF-E2 p45-related factor 2 (Nrf2) signaling pathway regulates the cellular antioxidant response and activation of Nrf2 has recently been shown to limit tissue damage from exposure to environmental toxicants, including As(III). In an attempt to identify improved molecular agents for systemic protection against environmental insults, we have focused on the identification of novel medicinal plant-derived Nrf2 activators. Results: Tanshinones [tanshinone I (T-I), tanshinone IIA, dihydrotanshinone, cryptotanshinone], phenanthrenequinone-based redox therapeutics derived from the medicinal herb Salvia miltiorrhiza, have been tested as experimental therapeutics for Nrf2-dependent cytoprotection. Using a dual luciferase reporter assay overexpressing wild-type or mutant Kelch-like ECH-associated protein-1 (Keap1), we demonstrate that T-I is a potent Keap1-C151-dependent Nrf2 activator that stabilizes Nrf2 by hindering its ubiquitination. In human bronchial epithelial cells exposed to As(III), T-I displays pronounced cytoprotective activity with upregulation of Nrf2-orchestrated gene expression. In Nrf2 wild-type mice, systemic administration of T-I attenuates As(III) induced inflammatory lung damage, a protective effect not observed in Nrf2 knockout mice. Innovation: Tanshinones have been identified as a novel class of Nrf2-inducers for antioxidant tissue protection in an in vivo As(III) inhalation model, that is relevant to low doses of environmental exposure. Conclusion: T-I represents a prototype Nrf2-activator that displays cytoprotective activity upon systemic administration targeting lung damage originating from environmental insults. T-I based Nrf2-directed systemic intervention may provide therapeutic benefit in protecting other organs against environmental insults. Antioxid. Redox Signal. 19, 1647–1661.
doi:10.1089/ars.2012.5117
PMCID: PMC3809600  PMID: 23394605
23.  Regulation of NF-E2-Related Factor 2 Signaling for Cancer Chemoprevention: Antioxidant Coupled with Antiinflammatory 
Antioxidants & Redox Signaling  2010;13(11):1679-1698.
Abstract
Cancer chemoprevention is a process of using either natural or synthetic compounds to reduce the risk of developing cancer. Observations that NF-E2-related factor 2 (Nrf2)-deficient mice lack response to some chemopreventive agents point to the important role of Nrf2 in chemoprevention. Nrf2 is a member of basic-leucine zipper transcription factor family and has been shown to regulate gene expression by binding to a response element, antioxidant responsive element. It is generally believed that activation of Nrf2 signaling is an adaptive response to the environmental and endogenous stresses. Under homeostatic conditions, Nrf2 is suppressed by association with Kelch-like ECH-associated protein 1 (Keap1), but is stimulated upon exposure to oxidative or electrophilic stress. Once activated, Nrf2 translocates into nuclei and upregulates a group of genes that act in concert to combat oxidative stress. Nrf2 is also shown to have protective function against inflammation, a pathological process that could contribute to carcinogenesis. In this review, we will discuss the current progress in the study of Nrf2 signaling, in particular, the mechanisms of Nrf2 activation by chemopreventive agents. We will also discuss some of the potential caveats of Nrf2 in cancer treatment and future opportunity and challenges on regulation of Nrf2-mediated antioxidant and antiinflammatory signaling in the context of cancer prevention. Antioxid. Redox Signal. 13, 1679–1698.
doi:10.1089/ars.2010.3276
PMCID: PMC2966483  PMID: 20486765
24.  Optimization of fluorescently labeled Nrf2 peptide probes and the development of a fluorescence polarization assay for the discovery of inhibitors of Keap1-Nrf2 interaction 
Journal of Biomolecular Screening  2011;17(4):435-447.
Activation of the antioxidant response element (ARE) up-regulates enzymes involved in detoxification of electrophiles and reactive oxygen species. The induction of ARE genes is regulated by the interaction between redox sensor protein, Keap1, and the transcription factor, Nrf2. Fluorescently labeled Nrf2 peptides containing the ETGE motif were synthesized and optimized as tracers in the development of a fluorescence polarization (FP) assay to identify small molecule inhibitors of Keap1-Nrf2 interaction. The tracers were optimized to increase the dynamic range of the assay and their binding affinities to the Keap1 Kelch domain. The binding affinities of Nrf2 peptide inhibitors obtained in our FP assay using FITC-9mer Nrf2 peptide amide as the probe were in good agreement with those obtained previously by a surface plasmon resonance (SPR) assay. The FP assay exhibits considerable tolerance towards DMSO and produced a Z'-factor greater than 0.6 in a 384-well format. Further optimization of the probe led to cyanine-labeled 9mer Nrf2 peptide amide, which can be used along with the FITC-9mer Nrf2 peptide amide in a high throughput screening (HTS) assay to discover small molecule inhibitors of Keap1-Nrf2 interaction.
doi:10.1177/1087057111430124
PMCID: PMC3309107  PMID: 22156223
Nrf2; Keap1; ARE; fluorescence polarization; high throughput screening; oxidative response
25.  Transcription Factor Nrf2-Mediated Antioxidant Defense System in the Development of Diabetic Retinopathy 
Purpose.
Increase in reactive oxygen species (ROS) is one of the major retinal metabolic abnormalities associated with the development of diabetic retinopathy. NF-E2–related factor 2 (Nrf2), a redox sensitive factor, provides cellular defenses against the cytotoxic ROS. In stress conditions, Nrf2 dissociates from its cytosolic inhibitor, Kelch like-ECH-associated protein 1 (Keap1), and moves to the nucleus to regulate the transcription of antioxidant genes including the catalytic subunit of glutamylcysteine ligase (GCLC), a rate-limiting reduced glutathione (GSH) biosynthesis enzyme. Our aim is to understand the role of Nrf2-Keap1-GCLC in the development of diabetic retinopathy.
Methods.
Effect of diabetes on Nrf2-Keap1-GCLC pathway, and subcellular localization of Nrf2 and its binding with Keap1 was investigated in the retina of streptozotocin-induced diabetic rats. The binding of Nrf2 at GCLC was quantified by chromatin immunoprecipitation technique. The results were confirmed in isolated retinal endothelial cells, and also in the retina from human donors with diabetic retinopathy.
Results.
Diabetes increased retinal Nrf2 and its binding with Keap1, but decreased DNA-binding activity of Nrf2 and also its binding at the promoter region of GCLC. Similar impairments in Nrf2-Keap1-GCLC were observed in the endothelial cells exposed to high glucose and in the retina from donors with diabetic retinopathy. In retinal endothelial cells, glucose-induced impairments in Nrf2-GCLC were prevented by Nrf2 inducer tBHQ and also by Keap1-siRNA.
Conclusions.
Due to increased binding of Nrf2 with Keap1, its translocation to the nucleus is compromised contributing to the decreased GSH levels. Thus, regulation of Nrf2-Keap1 by pharmacological or molecular means could serve as a potential adjunct therapy to combat oxidative stress and inhibit the development of diabetic retinopathy.
Diabetes increases retinal Nrf2 levels, but decreases its DNA binding activity. Due to increased binding of Nrf2 with its inhibitor, the recruitment of Nrf2 at the promoter of GCLC, a rate-limiting enzyme in GSH biosynthesis, is decreased, resulting in subnormal antioxidant defense system.
doi:10.1167/iovs.13-11598
PMCID: PMC3676188  PMID: 23633659
antioxidant defense; diabetic retinopathy; Nrf2

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