Drinking water contaminated with arsenic, a human carcinogen, is a worldwide health issue. An understanding of cellular signaling events in response to arsenic exposure and rational designing of strategies to reduce arsenic damages by modulating signaling events are important to fight against arsenic-induced diseases. Previously, we reported that activation of the Nrf2-mediated cellular defense pathway confers protection against toxic effects induced by sodium arsenite [As(III)] or monomethylarsonous acid [MMA(III)]. Paradoxically, arsenic has been reported to induce the Nrf2-dependent signaling pathway. Here, we report the unique mechanism of Nrf2 induction by arsenic. Similar to tert-butylhydroquinone (tBHQ) or sulforaphane (SF), arsenic induced the Nrf2-dependent response through enhancing Nrf2 protein levels by inhibiting Nrf2 ubiquitination and degradation. However, the detailed action of arsenic in Nrf2 induction is different from that of tBHQ or SF. Arsenic markedly enhanced the interaction between Keap1 and Cul3, subunits of the E3 ubiquitin ligase for Nrf2, which led to impaired dynamic assembly/disassembly of the E3 ubiquitin ligase and thus decreased its ligase activity. Furthermore, induction of Nrf2 by arsenic is independent of the previously identified C151 residue in Keap1 that is required for Nrf2 activation by tBHQ or SF. Distinct mechanisms of Nrf2 activation by seemingly harmful and beneficial reagents provide a molecular basis to design Nrf2-activating agents for therapeutic intervention.
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.
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.
Nrf2; Keap1; arsenic; arsenite; MMA(III); UROtsa
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.
Nrf2; Arsenic; Keap1; Oxidative stress; p62; Autophagy; Chemoprevention
The Nrf2-Keap1 signaling pathway is a protective mechanism promoting cell survival. Activation of the Nrf2 pathway by natural compounds has been proven to be an effective strategy for chemoprevention. Interestingly, a cancer-promoting function of Nrf2 has recently been observed in many types of tumors due to deregulation of the Nrf2-Keap1 axis, which leads to constitutive activation of Nrf2. Here, we report a novel mechanism of Nrf2 activation by arsenic that is distinct from that of chemopreventive compounds. Arsenic deregulates the autophagic pathway through blockage of autophagic flux, resulting in accumulation of autophagosomes and sequestration of p62, Keap1, and LC3. Thus, arsenic activates Nrf2 through a noncanonical mechanism (p62 dependent), leading to a chronic, sustained activation of Nrf2. In contrast, activation of Nrf2 by sulforaphane (SF) and tert-butylhydroquinone (tBHQ) depends upon Keap1-C151 and not p62 (the canonical mechanism). More importantly, SF and tBHQ do not have any effect on autophagy. In fact, SF and tBHQ alleviate arsenic-mediated deregulation of autophagy. Collectively, these findings provide evidence that arsenic causes prolonged activation of Nrf2 through autophagy dysfunction, possibly providing a scenario similar to that of constitutive activation of Nrf2 found in certain human cancers. This may represent a previously unrecognized mechanism underlying arsenic toxicity and carcinogenicity in humans.
Previous studies have proved that the environmental toxicant, inorganic arsenic, activates nuclear factor erythroid 2-related factor 2 (Nrf2) pathway in many different cell types. This study tried to explore the hepatic Nrf2 pathway upon arsenic treatment comprehensively, since liver is one of the major target organs of arsenical toxicity. Our results showed that inorganic arsenic significantly induced Nrf2 protein and mRNA expression in Chang human hepatocytes. We also observed a dose-dependent increase of antioxidant response element- (ARE-) luciferase activity. Both the mRNA and protein levels of NAD(P)H:quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1) were all upregulated dramatically. On the other hand, entry and accumulation of Nrf2 protein in the nucleus, while exportting the transcriptional repressor BTB and CNC homology 1 (Bach1) from nucleus to cytoplasm, were also confirmed by western blot and immunofluorescence assay. Our results therefore confirmed the arsenic-induced Nrf2 pathway activation in hepatocytes and also suggested that the translocation of Bach1 was associated with the regulation of Nrf2 pathway by arsenic. Hepatic Nrf2 pathway plays indispensable roles for cellular defenses against arsenic hepatotoxicity, and the interplay of Bach1 and Nrf2 may be helpful to understand the self-defensive responses and the diverse biological effects of arsenicals.
In response to oxidative stress, the transcription factor NF-E2-related factor 2 (Nrf2) controls the fate of cells through transcriptional upregulation of antioxidant response element (ARE)-bearing genes, including those encoding endogenous antioxidants, phase II detoxifying enzymes, and transporters. Expression of the Nrf2-dependent proteins is critical for ameliorating or eliminating toxicants/carcinogens to maintain cellular redox homeostasis. As a result, activation of the Nrf2 pathway, by naturally-occurring compounds or synthetic chemicals at sub-toxic doses, confers protection against subsequent toxic/carcinogenic exposure. Thus, the use of dietary compounds or synthetic chemicals to boost the Nrf2-dependent adaptive response to counteract environmental insults has emerged to be a promising strategy for cancer prevention. Interestingly, recent emerging data has revealed the “dark” side of Nrf2. Nrf2 and its downstream genes are overexpressed in many cancer cell lines and human cancer tissues, giving cancer cells an advantage for survival and growth. Furthermore, Nrf2 is upregulated in resistant cancer cells and is thought to be responsible for acquired chemoresistance. Therefore, it may be necessary to inhibit the Nrf2 pathway during chemotherapy. This review is primarily focused on the role of Nrf2 in cancer, with emphasis on the recent findings indicating the cancer promoting function of Nrf2 and its role in acquired chemoresistance.
Keap1/Nrf2 signaling defends organisms against the detrimental effects of oxidative stress, and has been suggested to abate its consequences, including aging-associated diseases like neurodegeneration, chronic inflammation, and cancer. Nrf2 is a prominent target for drug discovery, and Nrf2-activating agents are in clinical trials for cancer chemoprevention. However, aberrant activation of Nrf2 by keap1 somatic mutations may contribute to carcinogenesis and promote resistance to chemotherapy. To evaluate potential functions of Keap1 and Nrf2 for organismal homeostasis, we characterized the pathway in Drosophila. We demonstrate that Keap1/Nrf2 signaling in the fruitfly is activated by oxidants, induces antioxidant and detoxification responses, and confers increased tolerance to oxidative stress. Importantly, keap1 loss-of-function mutations extend the lifespan of Drosophila males, supporting a role for Nrf2 signaling in the regulation of longevity. Interestingly, cancer chemopreventive drugs potently stimulate Drosophila Nrf2 activity, suggesting the fruitfly as an experimental system to identify and characterize such agents.
Nuclear factor erythroid 2–related factor 2 (Nrf2) is a transcription factor that is important in protection against oxidative stress. This study was designed to determine the role of Nrf2 signaling in transcriptional activation of detoxifying and antioxidant genes in an in vivo mouse fetal alcohol syndrome model. Maternal ethanol treatment was found to increase both Nrf2 protein levels and Nrf2-ARE binding in mouse embryos. It also resulted in a moderate increase in the mRNA expression of Nrf2 downstream target detoxifying and antioxidant genes as well as an increase in the expression of antioxidant proteins. Pretreatment with the Nrf2 inducer, 3H-1,2 dithiole-3-thione (D3T), significantly increased Nrf2 protein levels and Nrf2-ARE binding, and strongly induced the mRNA expression of Nrf2 downstream target genes. It also increased the expression of antioxidant proteins and the activities of the antioxidant enzymes. Additionally, D3T pretreatment resulted in a significant decrease in ethanol-induced reactive oxygen species generation and apoptosis in mouse embryos. These results demonstrate that Nrf2 signaling is involved in the induction of antioxidant response in ethanol-exposed embryos. In addition, the potency of D3T in inducing antioxidants as well as in diminishing ethanol-induced apoptosis suggests that further exploration of the antiteratogenic effect of this compound will be fruitful. Antioxid. Redox Signal. 11, 2023–2033.
The Keap1–Nrf2 [Kelch-like ECH-associated protein 1–nuclear factor (erythroid-derived 2)-like 2] pathway plays a central role in the protection of cells against oxidative and xenobiotic stresses. Nrf2 is a potent transcription activator that recognizes a unique DNA sequence known as the antioxidant response element (ARE). Under normal conditions, Nrf2 binds to Keap1 in the cytoplasm, resulting in proteasomal degradation. Following exposure to electrophiles or reactive oxygen species, Nrf2 becomes stabilized, translocates into the nucleus, and activates the transcription of various cytoprotective genes. Increasing attention has been paid to the role of Nrf2 in cancer cells because the constitutive stabilization of Nrf2 has been observed in many human cancers with poor prognosis. Recent studies have shown that the antioxidant and detoxification activities of Nrf2 confer chemo- and radio-resistance to cancer cells. In this review, we provide an overview of the Keap1–Nrf2 system and discuss its role under physiological and pathological conditions, including cancers. We also introduce the results of our recent study describing Nrf2 function in the metabolism of cancer cells. Nrf2 likely confers a growth advantage to cancer cells through enhancing cytoprotection and anabolism. Finally, we discuss the possible impact of Nrf2 inhibitors on cancer therapy.
stress response; redox homeostasis; transcription; purine nucleotide; glutathione
Methylmercury (MeHg) is a potent neurotoxicant and preferentially induces oxidative injury in astrocytes. In neuronal tissues, nuclear factor erythroid 2–related factor 2 (Nrf2) is a key factor determining the protective antioxidant response against various environmental toxicants. Nrf2 is subjected to regulation by many other signaling pathways. The purpose of this study is to characterize its interaction with the phosphatidylinositol 3 (PI3) kinase in cultured rat neonatal primary astrocytes. The results showed that at pathologically relevant concentrations, exposure of primary astrocytes to MeHg led to Nrf2 activation and upregulation of its downstream antioxidant genes. Inhibition of the PI3 kinase resulted in decreased Nrf2 activity, decreased cellular glutathione, and increased cell death to high-dose MeHg. The functional interaction between the two signaling pathways underlined an important mechanism for astrocyte protection against MeHg toxicity. Modulation of Nrf2 by pharmacological modalities should afford a treatment to attenuate MeHg-induced neurotoxicity.
methylmercury; astrocyte; glutathione; Nrf2; phosphatidylinositol 3 kinase
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.
Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that positively regulates the basal and inducible expression of a large battery of cytoprotective genes. These gene products include proteins that catalyze reduction reactions (NAD(P)H:quinone oxidoreductase 1, Nqo1), conjugation reactions (glutathione-S-transferases, Gsts and UDP-glucuronosyltransferases, Ugts), as well as the efflux of potentially toxic xenobiotics and xenobiotic conjugates (multidrug resistance-associated proteins, Mrps). The significance of Nrf2 in the liver has been established, as livers of Nrf2-null mice are more susceptible to various oxidative/electrophilic stress-induced pathologies than wild-type mice. In contrast, both pharmacological and genetic models of hepatic Nrf2 activation are protective against oxidative/electrophilic stress. Furthermore, because certain Nrf2-target genes in the liver could affect the distribution, metabolism, and excretion of xenobiotics, the effects of Nrf2 on the kinetics of drugs and other xenobiotics should also be considered, with a special emphasis on metabolism and excretion. Therefore, this review highlights the research that has contributed to the understanding of the importance of Nrf2 in toxicodynamics and toxicokinetics, especially that which pertains to the liver.
Genetic disruption of Nrf2 greatly enhances susceptibility to prooxidant- and carcinogen-induced experimental models of various human disorders; but the mechanisms by which this transcription factor confers protection are unclear. Using Nrf2-proficient (Nrf2+/+) and Nrf2-deficient (Nrf2−/−) primary epithelial cultures as a model, we now show that Nrf2 deficiency leads to oxidative stress and DNA lesions, accompanied by impairment of cell-cycle progression, mainly G2/M-phase arrest. Both N-acetylcysteine and glutathione (GSH) supplementation ablated the DNA lesions and DNA damage–response pathways in Nrf2−/− cells; however only GSH could rescue the impaired colocalization of mitosis-promoting factors and the growth arrest. Akt activation was deregulated in Nrf2−/− cells, but GSH supplementation restored it. Inhibition of Akt signaling greatly diminished the GSH-induced Nrf2−/− cell proliferation and wild-type cell proliferation. GSH depletion impaired Akt signaling and mitosis-promoting factor colocalization in Nrf2+/+ cells. Collectively, our findings uncover novel functions for Nrf2 in regulating oxidative stress-induced cell-cycle arrest, especially G2/M-checkpoint arrest, and proliferation, and GSH-regulated redox signaling and Akt are required for this process.
oxidative stress; Nrf2; cell cycle; G2/M-checkpoint; Akt
Nuclear factor erythroid-2 related factor-2 (Nrf2) is a redox-sensitive transcription factor that regulates the expression of electrophile and xenobiotic detoxification enzymes and efflux proteins, which confer cytoprotection against oxidative stress and apoptosis in normal cells. Loss of function mutations in the Nrf2 inhibitor, Kelch-like ECH-associated protein (Keap1), results in constitutive activation of Nrf2 function in non-small-cell lung cancer (NSCLC). In this study, we demonstrate that constitutive activation of Nrf2 in lung cancer cells promotes tumorigenicity and contributes to chemoresistance by upregulation of glutathione, thioredoxin and the drug efflux pathways involved in detoxification of electrophiles and broad spectrum of drugs. RNAi-mediated reduction of Nrf2 expression in lung cancer cells induces generation of reactive oxygen species, suppresses tumor growth and results in increased sensitivity to chemotherapeutic drug induced cell death in vitro and in vivo. Inhibiting Nrf2 expression using naked siRNA duplexes in combination with carboplatin significantly inhibits tumor growth in a subcutaneous model of lung cancer. Thus, targeting Nrf2 activity in lung cancers, particularly those with Keap1 mutations, could be a promising strategy to inhibit tumor growth and circumvent chemoresistance.
Nrf2; Keap1; lung cancer; drug resistance; ROS; RNAi
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.
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.
Exposure to arsenic is associated with an increased risk of lung disease. Novel strategies are needed to reduce the adverse health effects associated with arsenic exposure in the lung. Nrf2, a transcription factor that mediates an adaptive cellular defense response, is effective in detoxifying environmental insults and prevents a broad spectrum of diseases induced by environmental exposure to harmful substances. In this report, we tested whether Nrf2 activation protects mice from arsenic-induced toxicity. We used an in vivo arsenic inhalation model that is highly relevant to low environmental human exposure to arsenic-containing dusts. Two-week exposure to arsenic-containing dust resulted in pathological alterations, oxidative DNA damage, and mild apoptotic cell death in the lung; all of which were blocked by sulforaphane (SF) in an Nrf2-dependent manner. Mechanistically, SF-mediated activation of Nrf2 alleviated inflammatory responses by modulating cytokine production. This study provides strong evidence that dietary intervention targeting Nrf2 activation is a feasible approach to reduce adverse health effects associated with arsenic exposure.
Nrf2; Keap1; Arsenic; Antioxidant response
Many phytochemicals function as noxious agents that protect plants against insects and other damaging organisms. However, at subtoxic doses the same phytochemicals may activate adaptive cellular stress response pathways that can protect cells against a variety of adverse conditions. We screened a panel of botanical pesticides using cultured human and rodent neuronal cell models, and identified plumbagin as a novel potent activator of the nuclear factor E2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway. In vitro, plumbagin increases nuclear localization and transcriptional activity of Nrf2, and induces the expression of the Nrf2/ARE-dependent genes, such as heme oxygenase 1 in human neuroblastoma cells. Plumbagin specifically activates the Nrf2/ARE pathway in primary mixed cultures from ARE-human placental alkaline phosphatase reporter mice. Exposure of neuroblastoma cells and primary cortical neurons to plumbagin provides protection against subsequent oxidative and metabolic insults. The neuroprotective effects of plumbagin are abolished by RNA interference-mediated knockdown of Nrf2 expression. In vivo, administration of plumbagin significantly reduces the amount of brain damage and ameliorates associated neurological deficits in a mouse model of focal ischemic stroke. Our findings establish precedence for the identification and characterization of neuroprotective phytochemicals based upon their ability to activate adaptive cellular stress response pathways.
Nrf2/ARE; stroke; neuroprotection; phytochemicals
Human exposure to inorganic arsenic leads to various dermal disorders, including hyperkeratosis and skin cancer. Curcumin is demonstrated to induce remarkable antioxidant activity in a variety of cells and tissues. The present study aimed at identifying curcumin as a potent activator of nuclear factor erythroid 2-related factor 2 (NRF2) and demonstrating its protective effect against inorganic arsenite- (iAs3+-) induced cytotoxicity in human keratinocytes. We found that curcumin led to nuclear accumulation of NRF2 protein and increased the expression of antioxidant response element- (ARE-) regulated genes in HaCaT keratinocytes in concentration- and time-dependent manners. High concentration of curcumin (20 μM) also increased protein expression of long isoforms of NRF1. Treatment with low concentrations of curcumin (2.5 or 5 μM) effectively increased the viability and survival of HaCaT cells against iAs3+-induced cytotoxicity as assessed by the MTT assay and flow cytometry and also attenuated iAs3+-induced expression of cleaved caspase-3 and cleaved PARP protein. Selective knockdown of NRF2 or KEAP1 by lentiviral shRNAs significantly diminished the cytoprotection conferred by curcumin, suggesting that the protection against iAs3+-induced cytotoxicity is dependent on the activation of NRF2. Our results provided a proof of the concept of using curcumin to activate the NRF2 pathway to alleviate arsenic-induced dermal damage.
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.
Nrf; INrf2 (keap1); Oxidative/electrophilic stress; Defensive gene expression; Cell signaling; Cell survival
Genetic and biochemical analyses have uncovered an essential role for nuclear factor erythroid 2-related factor 2 (Nrf2) in regulating phase II xenobiotic metabolism and antioxidant response. Here we show that Nrf2 protects against the ovarian toxicity of 4-vinylcyclohexene diepoxide (VCD) in mice. Nrf2−/− female mice exposed to VCD exhibit an age-dependent decline in reproduction leading to secondary infertility accompanied by hypergonadotropic hypogonadism after 30 weeks of age. VCD is shown to selectively destroy small ovarian follicles, resulting in early depletion of functional follicles. Treatment with VCD induces apoptotic death in cultured cells and in ovarian follicles, suggesting apoptosis as a mechanism of follicle loss. Loss of Nrf2 function blocks the basal and inducible expression of microsomal epoxide hydrolase, a key enzyme in the detoxification of VCD, and increases the oxidative stress in cells that is further exacerbated by VCD. Foxo3a, a repressor in the early stages of follicle activation, displays reduced expression in Nrf2−/− ovaries, causing accelerated growth of follicles in the absence of exposure to exogenous chemicals. Furthermore, Foxo3a is degraded through the 26S proteasome pathway in untreated cells and is induced by VCD via both Nrf2-dependent transcription and protein stabilization. This study demonstrates that Nrf2 serves as an essential sensor and regulator of chemical homeostasis in ovarian cells, protecting the cells from toxic chemicals by controlling metabolic detoxification, reactive oxygen species defense, and Foxo3a expression. In addition, these findings raise the possibility that exposure to environmental or occupational ovotoxicants plays a role in the premature ovarian failure commonly associated with infertility and premature aging in women.
Rationale: Aberrant airway smooth muscle cell (ASMC) function and overexpression of transforming growth factor (TGF)-β, which modulates ASMC proliferative and inflammatory function and induces oxidant release, are features of asthma. Nuclear factor E2-related factor 2 (Nrf2) activates antioxidant genes conferring protection against oxidative stress.
Objectives: To determine the role of Nrf2 in ASMCs and its modulation by TGF-β, and compare Nrf2 activity in ASMCs from subjects with severe and nonsevere asthma and healthy subjects.
Methods: ASMCs were cultured from airways of subjects without asthma, and from airway biopsies from patients with severe and nonsevere asthma. We studied Nrf2 activation on antioxidant gene expression and proliferation, the effect of TGF-β on Nrf2 transcriptional activity, and the impact of Nrf2 activation on TGF-β–mediated proliferation and IL-6 release. Nrf2–antioxidant response elements binding and Nrf2-dependent antioxidant gene expression was determined in asthmatic ASMCs.
Measurements and Main Results: Activation of Nrf2 led to up-regulation of the antioxidant genes heme oxygenase (HO)-1, NAD(P)H:quinone oxidoreductase, and manganese superoxide dismutase, and a reduction in proliferation. TGF-β reduced Nrf2-mediated antioxidant gene transcription through induction of activating transcription factor-3 expression. Nrf2 activation attenuated TGF-β–mediated reduction in HO-1, ASMC proliferation, and IL-6 release. Nrf2–antioxidant response elements binding was reduced in ASMCs from patients with severe asthma compared with ASMCs from patients with nonsevere asthma and normal subjects. HO-1 expression was reduced in ASMCs from patients with both nonsevere and severe asthma compared with healthy subjects.
Conclusions: Nrf2 regulates antioxidant responses and proliferation in ASMCs and is inactivated by TGF-β. Nrf2 reduction may underlie compromised antioxidant protection and aberrant ASM function in asthma.
asthma; airway smooth muscle; nuclear factor E2-related factor 2; transforming growth factor-β; antioxidant
Keap1-Nrf2-ARE pathway represents one of the most important cellular defense mechanisms against oxidative stress and xenobiotic damage. Activation of Nrf2 signaling induces the transcriptional regulation of ARE-dependent expression of various detoxifying and antioxidant defense enzymes and proteins. Keap1-Nrf2-ARE signaling has become an attractive target for the prevention and treatment of oxidative stress-related diseases and conditions including cancer, neurodegenerative, cardiovascular, metabolic and inflammatory diseases. Over the last few decades, numerous Nrf2 inducers have been developed and some of them are currently undergoing clinical trials. Recently, over-activation of Nrf2 has been implicated in cancer progression as well as in drug resistance to cancer chemotherapy. Thus, Nrf2 inhibitors could potentially be used to improve the effectiveness of cancer therapy. Herein, we review the signaling mechanism of Keap1-Nrf2-ARE pathway, its disease relevance, and currently known classes of small molecule modulators. We also discuss several aspects of Keap1-Nrf2 interaction, Nrf2-based peptide inhibitor design, and the screening assays currently used for the discovery of direct inhibitors of Keap1-Nrf2 interaction.
Oxidative stress; Keap1 (Kelch-like ECH-associated protein 1); Nrf2 (Nuclear factor erythroid 2-related factor 2); ARE (antioxidant response element); antioxidant inflammation modulator (AIM)
Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) is the primary transcription factor protecting cells from oxidative stress by regulating cytoprotective genes, including the antioxidant glutathione (GSH) pathway. GSH maintains cellular redox status and affects redox signaling, cell proliferation, and death. GSH homeostasis is regulated by de novo synthesis as well as GSH redox state; previous studies have demonstrated that Nrf2 regulates GSH homeostasis by affecting de novo synthesis. We report that Nrf2 modulates the GSH redox state by regulating glutathione reductase (GSR). In response to oxidants, lungs and embryonic fibroblasts (MEFs) from Nrf2-deficient (Nrf2−/−) mice showed lower levels of GSR mRNA, protein, and enzyme activity relative to wild type (Nrf2+/+). Nrf2−/− MEFs exhibited greater accumulation of glutathione disulfide and cytotoxicity compared to Nrf2+/+ MEFs in response to t-butylhydroquinone, which was rescued by restoring GSR. Microinjection of glutathione disulfide induced greater apoptosis in Nrf2−/− MEFs compared to Nrf2+/+ MEFs. In silico promoter analysis of the GSR gene revealed three putative antioxidant-response elements (ARE1, −44; ARE2, −813; ARE3, −1041). Reporter analysis, site-directed mutagenesis, and chromatin immunoprecipitation assays demonstrated binding of Nrf2 to two AREs distal to the transcription start site. Overall, Nrf2 is critical for maintaining the GSH redox state via transcriptional regulation of GSR and protecting cells against oxidative stress.
Nrf2; Oxidative stress; Glutathione; Glutathione reductase; Cigarette smoke; COPD; Emphysema; Free radicals