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1.  High levels of Nrf2 determine chemoresistance in type II endometrial cancer 
Cancer research  2010;70(13):5486-5496.
Type II endometrial cancer, which mainly presents as serous and clear cell types, has proved to be the most malignant and recurrent carcinoma among various female genital malignancies. The transcription factor, Nrf2, was first described as having chemopreventive activity. Activation of the Nrf2-mediated cellular defense response protects cells against the toxic and carcinogenic effects of environmental insults by upregulating an array of genes that detoxify reactive oxygen species (ROS) and restore cellular redox homeostasis. However, the cancer-promoting role of Nrf2 has recently been revealed. Nrf2 is constitutively upregulated in several types of human cancer tissues and cancer cell lines. Furthermore, inhibition of Nrf2 expression sensitizes cancer cells to chemotherapeutic drugs. In this study, the constitutive level of Nrf2 was compared in different types of human endometrial tumors. It was found that Nrf2 was highly expressed in endometrial serous carcinoma (ESC), whereas complex hyperplasia (CH) and endometrial endometrioid carcinoma (EEC) had no or marginal expression of Nrf2. Likewise, the ESC derived SPEC-2 cell line had a higher level of Nrf2 expression and was more resistant to the toxic effects of cisplatin and paclitaxel than that of the Ishikawa cell line, which was generated from EEC. Silencing of Nrf2 rendered SPEC-2 cells more susceptible to chemotherapeutic drugs while it had a limited effect on Ishikawa cells. Inhibition of Nrf2 expression by overexpressing Keap1 sensitized SPEC-2 cells or SPEC-2-derived xenografts to chemotherapeutic treatments using both cell culture and SCID mouse models. Collectively, we provide a molecular basis for the use of Nrf2 inhibitors to increase the efficacy of chemotherapeutic drugs and to combat chemoresistance, the biggest obstacle in chemotherapy.
doi:10.1158/0008-5472.CAN-10-0713
PMCID: PMC2896449  PMID: 20530669
Nrf2; chemoresistance; and endometrial cancer
2.  Ectodermal-Neural Cortex 1 Down-Regulates Nrf2 at the Translational Level 
PLoS ONE  2009;4(5):e5492.
The transcription factor Nrf2 is the master regulator of a cellular defense mechanism against environmental insults. The Nrf2-mediated antioxidant response is accomplished by the transcription of a battery of genes that encode phase II detoxifying enzymes, xenobiotic transporters, and antioxidants. Coordinated expression of these genes is critical in protecting cells from toxic and carcinogenic insults and in maintaining cellular redox homeostasis. Activation of the Nrf2 pathway is primarily controlled by Kelch-like ECH-associated protein 1 (Keap1), which is a molecular switch that turns on or off the Nrf2 signaling pathway according to intracellular redox conditions. Here we report our finding of a novel Nrf2 suppressor ectodermal-neural cortex 1 (ENC1), which is a BTB-Kelch protein and belongs to the same family as Keap1. Transient expression of ENC1 reduced steady-state levels of Nrf2 and its downstream gene expression. Although ENC1 interacted with Keap1 indirectly, the ENC1-mediated down-regulation of Nrf2 was independent of Keap1. The negative effect of ENC1 on Nrf2 was not due to a change in the stability of Nrf2 because neither proteasomal nor lysosomal inhibitors had any effects. Overexpression of ENC1 did not result in a change in the level of Nrf2 mRNA, rather, it caused a decrease in the rate of Nrf2 protein synthesis. These results demonstrate that ENC1 functions as a negative regulator of Nrf2 through suppressing Nrf2 protein translation, which adds another level of complexity in controlling the Nrf2 signaling pathway.
doi:10.1371/journal.pone.0005492
PMCID: PMC2675063  PMID: 19424503
3.  Nrf2 protects against As(III)-induced damage in mouse liver and bladder 
Arsenic compounds are classified as toxicants and human carcinogens. Environmental exposure to arsenic imposes a big health issue worldwide. Arsenic elicits its toxic efforts through many mechanisms, including generation of reactive oxygen species (ROS). Nrf2 is the primary transcription factor that controls expression of a main cellular antioxidant response, which is required for neutralizing ROS and thus defending cells from exogenous insults. Previously, we demonstrated a protective role of Nrf2 against arsenic-induced toxicity using a cell culture model. In this report, we present evidence that Nrf2 protects against liver and bladder injury in response to six-weeks of arsenic exposure in a mouse model. Nrf2−/− mice displayed more severe pathological changes in the liver and bladder, compared to Nrf2+/+ mice. Furthermore, Nrf2−/− mice were more sensitive to arsenic-induced DNA hypomethylation, oxidative DNA damage, and apoptotic cell death. These results indicate a protective role of Nrf2 against arsenic toxicity in vivo. Hence, this work demonstrates the feasibility of using dietary compounds that target activation of the Nrf2 signaling pathway to alleviate arsenic-induced damage.
doi:10.1016/j.taap.2009.06.010
PMCID: PMC2739886  PMID: 19538980
4.  Nrf2 enhances resistance of cancer cells to chemotherapeutic drugs, the dark side of Nrf2 
Carcinogenesis  2008;29(6):1235-1243.
Drug resistance during chemotherapy is the major obstacle to the successful treatment of many cancers. Here, we report that inhibition of NF-E2-related factor 2 (Nrf2) may be a promising strategy to combat chemoresistance. Nrf2 is a critical transcription factor regulating a cellular protective response that defends cells against toxic insults from a broad spectrum of chemicals. Under normal conditions, the low constitutive amount of Nrf2 protein is maintained by the Kelch-like ECH-associated protein1 (Keap1)-mediated ubiquitination and proteasomal degradation system. Upon activation, this Keap1-dependent Nrf2 degradation mechanism is quickly inactivated, resulting in accumulation and activation of the antioxidant response element (ARE)-dependent cytoprotective genes. Since its discovery, Nrf2 has been viewed as a ‘good’ transcription factor that protects us from many diseases. In this study, we demonstrate the dark side of Nrf2: stable overexpression of Nrf2 resulted in enhanced resistance of cancer cells to chemotherapeutic agents including cisplatin, doxorubicin and etoposide. Inversely, downregulation of the Nrf2-dependent response by overexpression of Keap1 or transient transfection of Nrf2–small interfering RNA (siRNA) rendered cancer cells more susceptible to these drugs. Upregulation of Nrf2 by the small chemical tert-butylhydroquinone (tBHQ) also enhanced the resistance of cancer cells, indicating the feasibility of using small chemical inhibitors of Nrf2 as adjuvants to chemotherapy to increase the efficacy of chemotherapeutic agents. Furthermore, we provide evidence that the strategy of using Nrf2 inhibitors to increase efficacy of chemotherapeutic agents is not limited to certain cancer types or anticancer drugs and thus can be applied during the course of chemotherapy to treat many cancer types.
doi:10.1093/carcin/bgn095
PMCID: PMC3312612  PMID: 18413364
5.  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
6.  Nrf2 promotes neuronal cell differentiation 
Free radical biology & medicine  2009;47(6):867-879.
The transcription factor Nrf2 has emerged as a master regulator for the endogenous antioxidant response, which is critical in defending cells against environmental insults and in maintaining intracellular redox balance. However, whether Nrf2 has any role in neuronal cell differentiation is largely unknown. In this report, we have examined the effects of Nrf2 on cell differentiation using a neuroblastoma cell line, SH-SY5Y. Retinoic acid (RA) and 12-O-tetradecanoylphorbol-13-acetate (TPA), two well-studied inducers for neuronal differentiation, are able to induce Nrf2 and its target gene NAD(P)H quinone oxidoreductase 1 (NQO1) in a dose- and time- dependent manner. RA-induced Nrf2 up-regulation is accompanied by neurite outgrowth and an induction of two neuronal differentiation markers, neurofilament-M (NF-M) and microtubule-associated protein 2 (MAP-2). Overexpression of Nrf2 in SH-SY5Y cells promotes neuronal differentiation whereas inhibition of endogenous Nrf2 expression inhibited neuronal differentiation. More remarkably, the positive role of Nrf2 in neuronal differentiation was verified ex vivo in primary neuron culture. Primary neurons isolated from Nrf2-null mice showed a retarded progress in differentiation, compared to that from wild-type mice. Collectively, our data demonstrate a novel role for Nrf2 in promoting neuronal cell differentiation, which will open new perspectives for therapeutic uses of Nrf2 activators in patients with neurodegenerative diseases.
doi:10.1016/j.freeradbiomed.2009.06.029
PMCID: PMC2748111  PMID: 19573594
Nrf2; Keap1; Oxidative Stress; Neuronal differentiation; SH-SY5Y; NQO1
7.  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
8.  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
9.  Targeted Deletion of Nrf2 Reduces Urethane-Induced Lung Tumor Development in Mice 
PLoS ONE  2011;6(10):e26590.
Nrf2 is a key transcription factor that regulates cellular redox and defense responses. However, permanent Nrf2 activation in human lung carcinomas promotes pulmonary malignancy and chemoresistance. We tested the hypothesis that Nrf2 has cell survival properties and lack of Nrf2 suppresses chemically-induced pulmonary neoplasia by treating Nrf2+/+ and Nrf2-/- mice with urethane. Airway inflammation and injury were assessed by bronchoalveolar lavage analyses and histopathology, and lung tumors were analyzed by gross and histologic analysis. We used transcriptomics to assess Nrf2-dependent changes in pulmonary gene transcripts at multiple stages of neoplasia. Lung hyperpermeability, cell death and apoptosis, and inflammatory cell infiltration were significantly higher in Nrf2-/- mice compared to Nrf2+/+ mice 9 and 11 wk after urethane. Significantly fewer lung adenomas were found in Nrf2-/- mice than in Nrf2+/+ mice at 12 and 22 wk. Nrf2 modulated expression of genes involved cell-cell signaling, glutathione metabolism and oxidative stress response, and immune responses during early stage neoplasia. In lung tumors, Nrf2-altered genes had roles in transcriptional regulation of cell cycle and proliferation, carcinogenesis, organismal injury and abnormalities, xenobiotic metabolism, and cell-cell signaling genes. Collectively, Nrf2 deficiency decreased susceptibility to urethane-induced lung tumorigenesis in mice. Cell survival properties of Nrf2 were supported, at least in part, by reduced early death of initiated cells and heightened advantage for tumor cell expansion in Nrf2+/+ mice relative to Nrf2-/- mice. Our results were consistent with the concept that Nrf2 over-activation is an adaptive response of cancer conferring resistance to anti-cancer drugs and promoting malignancy.
doi:10.1371/journal.pone.0026590
PMCID: PMC3198791  PMID: 22039513
10.  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
11.  The Antioxidant Defense System Keap1-Nrf2 Comprises a Multiple Sensing Mechanism for Responding to a Wide Range of Chemical Compounds▿ †  
Molecular and Cellular Biology  2008;29(2):493-502.
Animals have evolved defense systems for surviving in a chemically diverse environment. Such systems should demonstrate plasticity, such as adaptive immunity, enabling a response to even unknown chemicals. The antioxidant transcription factor Nrf2 is activated in response to various electrophiles and induces cytoprotective enzymes that detoxify them. We report here the discovery of a multiple sensing mechanism for Nrf2 activation using zebrafish and 11 Nrf2-activating compounds. First, we showed that six of the compounds tested specifically target Cys-151 in Keap1, the ubiquitin ligase for Nrf2, while two compounds target Cys-273. Second, in addition to Nrf2 and Keap1, a third factor was deemed necessary for responding to three of the compounds. Finally, we isolated a zebrafish mutant defective in its response to seven compounds but not in response to the remaining four. These results led us to categorize Nrf2 activators into six classes and hypothesize that multiple sensing allows enhanced plasticity in the system.
doi:10.1128/MCB.01080-08
PMCID: PMC2612520  PMID: 19001094
12.  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
13.  Conditioned Media Downregulates Nuclear Expression of Nrf2 
Nuclear factor erythroid 2-related factor-2 (Nrf2) is a redox-sensitive transcription factor that activates several antioxidant and cytoprotective genes in response to oxidative stress. The role of Nrf2 activators and the intracellular regulation of Nrf2 have been studied extensively. In comparison, little is known about the self-regulation of Nrf2 due to experimental techniques commonly used to synchronize cellular signaling. Here we report that endogenous Nrf2 was downregulated in the nucleus of HeLa and MDA-MB-231 cells serum starved for 24hrs. Nrf2 expression was rescued by the addition of unconditioned media irrespective of its serum content. No concomitant change was observed in the expression of the primary inhibitor of Nrf2, Kelch-like ECH-associated protein-1 (Keap1). Nrf2 was upregulated by tert-butyl hydroquinone, although there was limited increase in Nrf2 in conditioned media-treated cells as compared to unconditioned media-treated cells. Decreasing the fraction of conditioned media in culture resulted in a dose-dependent increase in Nrf2 protein level. Taken together, our data suggests the existence of a complex self-regulatory mechanism for endogenous Nrf2 signaling.
doi:10.1007/s12195-013-0272-0
PMCID: PMC3780449  PMID: 24073021
Nrf2; Keap1; conditioned media; starvation; serum-free media; cell synchronization
14.  Disruption of Nrf2, a Key Inducer of Antioxidant Defenses, Attenuates ApoE-Mediated Atherosclerosis in Mice 
PLoS ONE  2008;3(11):e3791.
Background
Oxidative stress and inflammation are two critical factors that drive the formation of plaques in atherosclerosis. Nrf2 is a redox-sensitive transcription factor that upregulates a battery of antioxidative genes and cytoprotective enzymes that constitute the cellular response to oxidative stress. Our previous studies have shown that disruption of Nrf2 in mice (Nrf2−/−) causes increased susceptibility to pulmonary emphysema, asthma and sepsis due to increased oxidative stress and inflammation. Here we have tested the hypothesis that disruption of Nrf2 in mice causes increased atherosclerosis.
Principal Findings
To investigate the role of Nrf2 in the development of atherosclerosis, we crossed Nrf2−/− mice with apoliporotein E-deficient (ApoE−/−) mice. ApoE−/− and ApoE−/− Nrf2−/− mice were fed an atherogenic diet for 20 weeks, and plaque area was assessed in the aortas. Surprisingly, ApoE−/− Nrf2−/− mice exhibited significantly smaller plaque area than ApoE−/− controls (11.5% vs 29.5%). This decrease in plaque area observed in ApoE−/− Nrf2−/− mice was associated with a significant decrease in uptake of modified low density lipoproteins (AcLDL) by isolated macrophages from ApoE−/− Nrf2−/− mice. Furthermore, atherosclerotic plaques and isolated macrophages from ApoE−/− Nrf2−/− mice exhibited decreased expression of the scavenger receptor CD36.
Conclusions
Nrf2 is pro-atherogenic in mice, despite its antioxidative function. The net pro-atherogenic effect of Nrf2 may be mediated via positive regulation of CD36. Our data demonstrates that the potential effects of Nrf2-targeted therapies on cardiovascular disease need to be investigated.
doi:10.1371/journal.pone.0003791
PMCID: PMC2582492  PMID: 19023427
15.  Pseudomonas aeruginosa Pyocyanin Activates NRF2-ARE-Mediated Transcriptional Response via the ROS-EGFR-PI3K-AKT/MEK-ERK MAP Kinase Signaling in Pulmonary Epithelial Cells 
PLoS ONE  2013;8(8):e72528.
The redox-active pyocyanin (PCN) secreted by the respiratory pathogen Pseudomonas aeruginosa generates reactive oxygen species (ROS) and causes oxidative stress to pulmonary epithelial cells. Nuclear factor (erythroid-derived 2)-like 2 (NRF2) confers protection against ROS-mediated cell death by inducing the expression of detoxifying enzymes and proteins via its binding to the cis-acting antioxidant response element (ARE). However, a clear relationship between NRF2 and PCN-mediated oxidative stress has not been established experimentally. In this study, we investigated the induction of NRF2-ARE response by PCN in the pulmonary epithelial cells. We analyzed the effect of PCN on NRF2 expression and nuclear translocation in cultured human airway epithelial cells, and in a mouse model of chronic PCN exposure. NRF2-dependent transcription of antioxidative enzymes was also assessed. Furthermore, we used inhibitors to examine the involvement of EGFR and its downstream signaling components that mediate NRF2-ARE-activation in response to PCN. PCN enhances the nuclear NRF2 accumulation and activates the transcription of ARE-mediated antioxidant genes. Furthermore, PCN activates NRF2 by inducing the EGFR-phosphoinositide-3-kinase (PI3K) signaling pathway and its main downstream effectors, AKT and MEK1/2-ERK1/2 MAP kinases. Inhibition of the EGFR-PI3K signaling markedly attenuates PCN-stimulated NRF2 accumulation in the nucleus. We demonstrate for the first time that PCN-mediated oxidative stress activates the EGFR-PI3K-AKT/MEK1/2-ERK1/2 MAP kinase signaling pathway, leading to nuclear NRF2 translocation and ARE responsiveness in pulmonary epithelial cells.
doi:10.1371/journal.pone.0072528
PMCID: PMC3755003  PMID: 24015256
16.  Targeting the Transcription Factor Nrf2 to Ameliorate Oxidative Stress and Inflammation in Chronic Kidney Disease 
Kidney international  2013;83(6):1029-1041.
Oxidative stress and inflammation are mediators in the development and progression of chronic kidney disease (CKD) and its complications, and they are inseparably linked as each begets and amplifies the other. CKD-associated oxidative stress is due to increased production of reactive oxygen species (ROS) and diminished antioxidant capacity. The latter is largely caused by impaired activation of Nrf2, the transcription factor that regulates genes encoding antioxidant and detoxifying molecules. Protective effects of Nrf2 are evidenced by amelioration of oxidative stress, inflammation, and kidney disease in response to natural Nrf2 activators in animal models, while Nrf2 deletion amplifies these pathogenic pathways and leads to autoimmune nephritis. Given the role of impaired Nrf2 activity in CKD-induced oxidative stress and inflammation, interventions aimed at restoring Nrf2 may be effective in retarding CKD progression. Clinical trials of the potent Nrf2 activator bardoxolone methyl showed significant improvement in renal function in CKD patients with type 2 diabetes. Results of the ongoing BEACON trial investigating the effect of this drug on time to end-stage renal disease or cardiovascular death will help further characterize the efficacy of Nrf2 pharmacological modulation in CKD. This article provides an overview of the role of impaired Nrf2 activity in the pathogenesis of CKD-associated oxidative stress and inflammation and the potential utility of targeting Nrf2 in the treatment of CKD.
doi:10.1038/ki.2012.439
PMCID: PMC3633725  PMID: 23325084
Oxidative stress; Inflammation; CKD progression; Cardiovascular disease; ESRD; Antioxidant therapy; bardoxolone methyl
17.  Discovery of potent, novel Nrf2 inducers via quantum modeling, virtual screening and in vitro experimental validation 
Chemical biology & drug design  2012;80(6):810-820.
Nuclear factor erythroid 2-related factor 2 (Nrf2) is the master transcription factor of the antioxidant response element (ARE) pathway, coordinating the induction of detoxifying and antioxidant enzymes. Nrf2 is normally sequestered in the cytoplasm by Kelch-like ECH associating protein 1 (Keap1). To identify novel small molecules that will disturb Nrf2:Keap1 binding and promote activation of the Nrf2-ARE pathway, we generated a quantum model based on the structures of known Nrf2-ARE activators. We used the quantum model to perform in silico screening on over 18 million commercially available chemicals to identify the structures predicted to activate the Nrf2-ARE pathway based on the quantum model. The top hits were tested in vitro and half of the predicted hits activated the Nrf2-ARE pathway significantly in primary cell culture. In addition, we identified a new family of Nrf2-ARE activating structures that all have comparable activity to tBHQ and protect against oxidative stress and dopaminergic toxins in vitro. The improved ability to identify potent activators of Nrf2 through the combination of in silico and in vitro screening described here improves the speed and cost associated with screening Nrf2-ARE activating compounds for drug development.
doi:10.1111/cbdd.12040
PMCID: PMC3484224  PMID: 22925725
18.  Activation of the Nrf2-ARE pathway by siRNA knockdown of Keap1 reduces oxidative stress and provides partial protection from MPTP-mediated neurotoxicity 
Neurotoxicology  2012;33(3):272-279.
Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that binds to the antioxidant response element, a cis-acting regulatory element that increases expression of detoxifying enzymes and antioxidant proteins. Kelch-like ECH associating protein 1 (Keap1) protein is a negative regulator of Nrf2. Previous work has shown that genetic overexpression of Nrf2 is protective in vitro and in vivo. To modulate the Nrf2-ARE system without overexpressing Nrf2, we used short interfering RNA (siRNA) directed against Keap1. Keap1 siRNA administration in primary astrocytes increased the levels of Nrf2-ARE driven genes and protected against oxidative stress. Moreover, Keap1 siRNA resulted in a persistent upregulation of the Nrf2-ARE pathway and protection against oxidative stress in primary astrocytes. Keap1 siRNA injected into the striatum was also modestly protective against MPTP-induced dopaminergic terminal damage. These data indicate that activation of endogenous intracellular levels of Nrf2 is sufficient to protect in models of oxidative stress and Parkinson's disease.
doi:10.1016/j.neuro.2012.01.015
PMCID: PMC3521526  PMID: 22342405
19.  Arsenic-Mediated Activation of the Nrf2-Keap1 Antioxidant Pathway 
Arsenic is present in the environment and has become a worldwide health concern due to its toxicity and carcinogenicity. However, the specific mechanism(s) by which arsenic elicits its toxic effects has yet to be fully elucidated. The transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) has been recognized as the master regulator of a cellular defense mechanism against toxic insults. This review highlights studies demonstrating that arsenic activates the Nrf2-Keap1 antioxidant pathway by a distinct mechanism from that of natural compounds such as sulforaphane (SF) found in broccoli sprouts or tert-butylhyrdoquinone (tBHQ), a natural antioxidant commonly used as a food preservative. Evidence also suggests that arsenic prolongs Nrf2 activation and may mimic constitutive activation of Nrf2, which has been found in several human cancers due to disruption of the Nrf2-Keap1 axis. The current literature strongly suggests that activation of Nrf2 by arsenic potentially contributes to, rather than protects against, arsenic toxicity and carcinogenicity. The mechanism(s) by which known Nrf2 activators, such as the natural chemopreventive compounds SF and lipoic acid, protect against the deleterious effects caused by arsenic will also be discussed. These findings will provide insight to further understand how arsenic promotes a prolonged Nrf2 response, which will lead to the identification of novel molecular markers and development of rational therapies for the prevention or intervention of arsenic-induced diseases. The National Institute of Environmental Health Science (NIEHS) Outstanding New Environmental Scientist (ONES) award has provided the opportunity to review the progress both in the fields of arsenic toxicology and Nrf2 biology. Much of the funding has led to (1) the novel discovery that arsenic activates the Nrf2 pathway by a mechanism different to that of other Nrf2 activators, such as sulforaphane and tert-butylhydroquinone, (2) activation of Nrf2 by chemopreventive compounds protects against arsenic toxicity and carcinogenicity both in vitro and in vivo, (3) constitutive activation of Nrf2 by disrupting Keap1-mediated negative regulation contributes to cancer and chemoresistance, (4) p62-mediated sequestration of Keap1 activates the Nrf2 pathway, and (5) arsenic-mediated Nrf2 activation may be through a p62-dependent mechanism. All of these findings have been published and are discussed in this review. This award has laid the foundation for my laboratory to further investigate the molecular mechanism(s) that regulate the Nrf2 pathway and how it may play an integral role in arsenic toxicity. Moreover, understanding the biology behind arsenic toxicity and carcinogenicity will help in the discovery of potential strategies to prevent or control arsenic-mediated adverse effects.
doi:10.1002/jbt.21463
PMCID: PMC3725327  PMID: 23188707
Nrf2; Arsenic; Keap1; Oxidative stress; p62; Autophagy; Chemoprevention
20.  Oridonin Confers Protection against Arsenic-Induced Toxicity through Activation of the Nrf2-Mediated Defensive Response 
Environmental Health Perspectives  2008;116(9):1154-1161.
Background
Groundwater contaminated with arsenic imposes a big challenge to human health worldwide. Using natural compounds to subvert the detrimental effects of arsenic represents an attractive strategy. The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) is a critical regulator of the cellular antioxidant response and xenobiotic metabolism. Recently, activation of the Nrf2 signaling pathway has been reported to confer protection against arsenic-induced toxicity in a cell culture model.
Objectives
The goal of the present work was to identify a potent Nrf2 activator from plants as a chemopreventive compound and to demonstrate the efficacy of the compound in battling arsenic-induced toxicity.
Results
Oridonin activated the Nrf2 signaling pathway at a low subtoxic dose and was able to stabilize Nrf2 by blocking Nrf2 ubiquitination and degradation, leading to accumulation of the Nrf2 protein and activation of the Nrf2-dependent cytoprotective response. Pretreatment of UROtsa cells with 1.4 μM oridonin significantly enhanced the cellular redox capacity, reduced formation of reactive oxygen species (ROS), and improved cell survival after arsenic challenge.
Conclusions
We identified oridonin as representing a novel class of Nrf2 activators and illustrated the mechanism by which the Nrf2 pathway is activated. Furthermore, we demonstrated the feasibility of using natural compounds targeting Nrf2 as a therapeutic approach to protect humans from various environmental insults that may occur daily.
doi:10.1289/ehp.11464
PMCID: PMC2535615  PMID: 18795156
antioxidant responsive element; antitumor; ARE; arsenic; chemoprevention; diterpenoid; Keap1; Nrf2; oridonin; oxidative stress; rubescensin
21.  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
22.  The role of the antioxidant and longevity-promoting Nrf2 pathway in metabolic regulation 
Purpose of Review
The vertebrate cap’n’collar family transcription factor Nrf2 and its invertebrate homologs SKN-1 (in worms) and CncC (in flies) function as master mediators of antioxidant and detoxification responses and regulators of the cellular redox state. Nrf2 controls gene expression programs that defend various tissues against diverse electrophilic stressors and oxidative insults, thus protecting the organism from pathologies that are caused or exacerbated by such stresses. Moreover, studies in model organisms implicate the Nrf2 pathway in the prevention of aging-related diseases, and suggest that SKN-1- and CncC-regulated gene expression can promote longevity. These facets of Nrf2 signaling have been thoroughly reviewed. This article discusses another aspect of the Nrf2 pathway’s function that has not yet received the same degree of attention but emerges as a topic of increasing interest and potential clinical impact: its role in metabolic regulation and its interaction with central signaling systems that respond to nutritional inputs.
Recent findings
Recent evidence identifies Nrf2 signaling as a mediator of the salutary effects of caloric restriction. Nrf2 signaling also cross-talks with metabolic signaling systems such as the insulin/Akt pathway as well as with the metabolism of lipids. Moreover, Nrf2 has a protective role in models of diabetic nephropathy.
Summary
The emerging role of Nrf2 as an effector of metabolic and longevity signals offers new therapeutic perspectives. The potential impact of pharmacological manipulation of Nrf2 signaling as a strategy for the prevention and treatment of metabolic disease can be envisioned.
doi:10.1097/MCO.0b013e32834136f2
PMCID: PMC3092636  PMID: 21102319
Nrf2; calorie restriction; aging; insulin signaling; obesity; diabetic nephropathy
23.  Expression of stress-response ATF3 is mediated by Nrf2 in astrocytes 
Nucleic Acids Research  2009;38(1):48-59.
Activating Transcription Factor 3 (ATF3), a member of the ATF/CREB family, is induced rapidly by various stresses. Its induction mechanism and role in response to changes in cellular redox status, however, have not been elucidated. Here, we found that NF-E2-related factor 2 (Nrf2), a transcription factor known to bind to antioxidant response element (ARE) in promoters, transcriptionally upregulated ATF3 expression in astrocytes. Treatment with Nrf2 activators and oxidants provoked ATF3 induction in astrocytes, whereas its expression was reduced in Nrf2-depleted cells. We further demonstrated that the consensus ARE in the ATF3 promoter is critical for Nrf2-mediation by promoter analyses using an ATF3 promoter-driven luciferase construct and a chromatin immunoprecipitation assay. In addition, we found that Nrf2-dependent ATF3 induction contributed to the antioxidative and cytoprotective functions of Nrf2 in astrocytes. Taken together, our findings suggest that ATF3 is a new target for Nrf2 and has a cytoprotective function in astrocytes.
doi:10.1093/nar/gkp865
PMCID: PMC2800224  PMID: 19864258
24.  Phosphorylation of Nrf2 at Multiple Sites by MAP Kinases Has a Limited Contribution in Modulating the Nrf2-Dependent Antioxidant Response 
PLoS ONE  2009;4(8):e6588.
The bZIP transcription factor Nrf2 has emerged as a pivotal regulator of intracellular redox homeostasis by controlling the expression of many endogenous antioxidants and phase II detoxification enzymes. Upon oxidative stress, Nrf2 is induced at protein levels through redox-sensitive modifications on cysteine residues of Keap1, a component of the E3 ubiquitin ligase that targets Nrf2 for ubiquitin-dependent degradation. The mitogen activated protein kinases (MAPKs) have previously been proposed to regulate Nrf2 in response to oxidative stress. However, the exact role of MAPKs and the underlying molecular mechanism remain poorly defined. Here we report the first evidence that Nrf2 is phosphorylated in vivo by MAPKs. We have identified multiple serine/threonine residues as major targets of MAPK-mediated phosphorylation. Combined alanine substitution on those residues leads to a moderate decrease in the transcriptional activity of Nrf2, most likely due to a slight reduction in its nuclear accumulation. More importantly, Nrf2 protein stability, primarily controlled by Keap1, is not altered by Nrf2 phosphorylation in vivo. These data indicate that direct phosphorylation of Nrf2 by MAPKs has limited contribution in modulating Nrf2 activity. We suggest that MAPKs regulate the Nrf2 signaling pathway mainly through indirect mechanisms.
doi:10.1371/journal.pone.0006588
PMCID: PMC2719090  PMID: 19668370
25.  Nrf2 protects human bladder urothelial cells from arsenite and monomethylarsonous acid toxicity 
Toxicology and applied pharmacology  2007;225(2):206-213.
Arsenic is widely spread in our living environment and imposes a big challenge on human health worldwide. Arsenic damages biological systems through multiple mechanisms including the generation of reactive oxygen species. The transcription factor Nrf2 regulates the cellular antioxidant response that protects cells from various insults. In this study, the protective role of Nrf2 in arsenic toxicity was investigated in a human bladder urothelial cell line, UROtsa. Using an UROtsa cell line stably infected with Nrf2-siRNA, we clearly demonstrate that compromised Nrf2 expression sensitized the cells to As(III)- and MMA(III)-induced toxicity. On the other hand, the activation of the Nrf2 pathway by tert-butylhydroquinone (tBHQ) and sulforaphane (SF), the known Nrf2-inducers, rendered UROtsa cells more resistant to As(III)- and MMA(III). Furthermore, the wild type mouse embryo fibroblast (WT-MEF) cells were protected from As(III)- and MMA(III)-induced toxicity following Nrf2 activation by tBHQ or SF whereas neither tBHQ nor SF conferred protection in the Nrf2−/−-MEF cells, demonstrating that tBHQ- or SF-mediated protection against As(III)- and MMA(III)-induced toxicity depends on Nrf2 activation. These results, obtained by both loss of function and gain of function analyses, clearly demonstrate the protective role of Nrf2 in arsenic-induced toxicity. The current work lays the groundwork for using Nrf2 activators for therapeutic and dietary interventions against adverse effects of arsenic.
doi:10.1016/j.taap.2007.07.016
PMCID: PMC2610476  PMID: 17765279
Nrf2; Keap1; arsenic; arsenite; MMA(III); UROtsa

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