Sustained Nrf2 Activation Research Articles

Live-cell imaging Unveils stimulus-specific dynamics of Nrf2 activation in UV-exposed melanoma cells: Implications for antioxidant compound screening

The transcription factor Nuclear factor e2-related factor 2 (Nrf2) is pivotal in orchestrating cellular antioxidant defense mechanisms, particularly in skin cells exposed to ultraviolet (UV) radiation and electrophilic phytochemicals. To comprehensively investigate Nrf2's role in maintaining cellular redox equilibrium following UV-induced stress, we engineered a novel Nrf2 fusion-based reporter system for real-time, live-cell quantification of Nrf2 activity in human melanoma cells. Utilizing live quantitative imaging, we dissected the kinetic profiles of Nrf2 activation in response to an array of stimuli, including UVA and UVB radiation, as well as a broad spectrum of phytochemicals including ferulic acid, gallic acid, hispidulin, p-coumaric acid, quercetin, resveratrol, tannic acid, and vanillic acid as well as well-known Nrf2 inducers, tert-butylhydroquinone (tBHQ) and sulforaphane (SFN). Intriguingly, we observed distinct dynamical patterns of Nrf2 activity contingent on the specific stimuli applied. Sustained activation of Nrf2 was empirically correlated with the increased antioxidant response element (ARE) activity. Our findings demonstrate the nuanced impact of different phenolic compounds on Nrf2 activity and the utility of our Nrf2-CTΔ16-YFP reporter in characterizing the dynamics of Nrf2 translocation in response to diverse stimuli. In summary, our innovative reporter system not only revealed compounds capable of modulating UVA-induced Nrf2 activity but also showcased its utility as a robust tool for future antioxidant compound screening efforts.

Inhibition of abnormal C/EBPβ/α-Syn signaling pathway through activation of Nrf2 ameliorates Parkinson's disease-like pathology.

Parkinson's disease (PD) is characterized by the formation of Lewy bodies (LBs) in the brain. These LBs are primarily composed of α-Synuclein (α-Syn), which has aggregated. A recent report proposes that CCAAT/enhancer-binding proteins β (C/EBPβ) may act as an age-dependent transcription factor for α-Syn, thereby initiating PD pathologies by regulating its transcription. Potential therapeutic approaches to address PD could involve targeting the regulation of α-Syn by C/EBPβ. This study has revealed that Nrf2, also known as nuclear factor (erythroid-derived 2)-like 2 (NFE2L2), suppresses the transcription of C/EBPβ in SH-SY5Y cells when treated with MPP+ . To activate Nrf2, sulforaphane, an Nrf2 activator, was administered. Additionally, C/EBPβ was silenced using C/EBPβ-DNA/RNA heteroduplex oligonucleotide (HDO). Both approaches successfully reduced abnormal α-Syn expression in primary neurons treated with MPP+ . Furthermore, sustained activation of Nrf2 via its activator or inhibition of C/EBPβ using C/EBPβ-HDO resulted in a reduction of aberrant α-Syn expression, thus leading to an improvement in the degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc) in mouse models induced by 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP) and those treated with preformed fibrils (PFFs). The data presented in this study illustrate that the activation of Nrf2 may provide a potential therapeutic strategy for PD by inhibiting the abnormal C/EBPβ/α-Syn signaling pathway.

Blockage of Nrf2 and autophagy by L-selenocystine induces selective death in Nrf2-addicted colorectal cancer cells through p62-Keap-1-Nrf2 axis

Persistent Nrf2 activation is typically noted in many cancers, including colorectal cancer (CRC), aiding cancer cells in overcoming growth stress and promoting cancer progression. Sustained Nrf2 activation, which is beneficial for cancer cells, is called “Nrf2 addiction”; it is closely associated with malignancy and poor prognosis in patients with cancer. However, Nrf2 inhibitors may have adverse effects on normal cells. Here, we found that the selenocompound l-selenocystine (SeC) is selectively cytotoxic in the Nrf2-addicted CRC cell line WiDr cells, but not in non–Nrf2-addicted mesenchymal stem cells (MSCs) and normal human colon cells. Another CRC cell line, C2BBe1, which harbored lower levels of Nrf2 and its downstream proteins were less sensitive to SeC, compared with the WiDr cells. We further demonstrated that SeC inhibited Nrf2 and autophagy activation in the CRC cells. Antioxidant GSH pretreatment partially rescued the CRC cells from SeC-induced cytotoxicity and Nrf2 and autophagy pathway inhibition. By contrast, SeC activated Nrf2 and autophagy pathway in non–Nrf2-addicted MSCs. Transfecting WiDr cells with Nrf2-targeting siRNA decreased persistent Nrf2 activation and alleviated SeC cytotoxicity. In KEAP1-knockdown C2BBe1 cells, Nrf2 pathway activation increased SeC sensitivity and cytotoxicity. In conclusion, SeC selectively attacks cancer cells with constitutively activated Nrf2 by reducing Nrf2 and autophagy pathway protein expression through the P62–Nrf2–antioxidant response element axis and eventually trigger cell death.

ARD1 stabilizes NRF2 through direct interaction and promotes colon cancer progression

AimsAberrant overactivation/overexpression of NRF2 is implicated as a driving event in tumor progression, which has been attributed to its mutation or inactivation of the inhibitory protein, KEAP1. However, alternative mechanisms responsible for sustained activation of NRF2 are less understood. Main methodsHuman colon cancer cell lines and tissues obtained from colorectal cancer (CRC) patients were used. To examine the expression levels of ARD1 and NRF2, Western blot and immunofluorescence analyses were performed. To investigate the potential relevance of NRF2 and ARD1 to human CRC, NRF2 and ARD1 were individually silenced in human colon cancer cells (HCT-116) by transfection with their specific small interfering RNA (siRNA). To determine the functional role of ARD1 in NRF2 regulation, in situ proximate ligation, co-immunoprecipitation, nano-LC-ESI MS/MS, and in vitro acetylation assays were performed. Key findingsARD1 knockdown in human colon cancer cell lines significantly reduced the protein levels of NRF2 without affecting its mRNA expression; however, silencing of NRF2 did not alter ARD1 protein expression. In addition, these two proteins were co-localized and physically interacted with each other both in human colon cancer cells (HCT-116) and human colon tumor tissues. Mechanistically, ARD1 overexpression increased the acetylation levels of NRF2. Moreover, an in vitro acetylation assay and mass spectrometric analysis demonstrated that ARD1 could directly acetylate NRF2. Ectopic expression of mutant forms of ARD1 with defective acetyltransferase activity reduced the stability of NRF2. SignificanceIn conclusion, ARD1 may potentiate the oncogenic function of NRF2 in human colon cancer by stabilizing this transcription factor.

Nrf2 induces malignant transformation of hepatic progenitor cells by inducing β-catenin expression

The Nrf2 signaling pathway prevents cancer initiation, but genetic mutations that activate this pathway are found in various types of cancer. The molecular mechanisms underlying this Janus-headed character are still not understood. Here, we show that sustained Nrf2 activation induces proliferation and dedifferentiation of a Wnt-responsive perivenular hepatic progenitor cell population, transforming them into metastatic cancer cells. The neoplastic lesions display many histological features known from human hepatoblastoma. We describe an Nrf2-induced upregulation of β-catenin expression and its activation as the underlying mechanism for the observed malignant transformation. Thus, we have identified the Nrf2–β-catenin axis promoting proliferation of hepatic stem cells and triggering tumorigenesis. These findings support the concept that different functional levels of Nrf2 control both the protection against various toxins as well as liver regeneration by activating hepatic stem cells. Activation of the hepatic stem cell compartment confers the observation that unbridled Nrf2 activation may trigger tumorigenesis.

Urolithin A protects against acetaminophen-induced liver injury in mice via sustained activation of Nrf2.

Acetaminophen overdose is a leading cause of acute live failure worldwide. N-acetylcysteine (NAC), as the only antidote, is limited due to its narrow therapeutic time window. Here we demonstrated that Urolithin A (UA), a metabolite of ellagitannin natural products in the gastrointestinal flora, protected against acetaminophen-induced liver injury (AILI) and is superior to NAC in terms of dosage and therapeutical time window. Transcriptomics assay revealed that UA promotes mitophagy and activated Nrf2/ARE signaling in the liver. Consistent with that, mitophagy and Nrf2/ARE signaling were activated, with less oxidative stress in UA-treated liver. Subsequently, molecular docking and dynamics simulation study revealed a binding mode between UA and Nrf-2/Keap1 including the hydrogen-bonding network among oxygen atoms in UA with the Nrf-2/Keap1 residues Arg 415, Ser 508 and Ser 602, which in turn trigger Nrf2 nuclear translocation, subsequently leading to activation of Nrf-2 target genes (HO-1, NQO1). Of note, mitophagy inhibition failed to prevent the protection of UA against AILI, which instead was compromised with Nrf2 gene silencing both in vivo and in vitro. Collectively, our data indicate that UA alleviated acetaminophen-induced oxidative stress and hepatic necrosis via activating Nrf2/ARE signaling pathway, highlighting a therapeutical potential of UA for AILI.

Abstract 551: Distinct Transcriptional Roles for Wildtype vs. Truncated Nrf2 in the Heart of Transgenic Mice

Background: Nuclear erythroid-2 like factor-2 (Nrf2), a redox-sensitive transcriptional regulator of cytoprotective and antioxidant genes. Despite the basal transcription of antioxidants/redox genes, forced activation of Nrf2 may lead to “reductive stress” (RS). In this study, we hypothesized that controlled vs. sustained activation of Nrf2 differentially regulates the myocardial transcriptome, which elucidates the transition of a physiological adaptation to pathological process. Methods: Cardiac specific Nrf2 transgenic mice expressing full length Nrf2 (FL-TG) and truncated Nrf2 (TR-TG), and their non-transgenic littermates (NTG) in the C57/BL6J backround at the age of 6-8 months were used for this study. Next generation RNA sequencing was performed using the myocardial mRNA obtained from FL-TG, TR-TG and NTG mice (n=3/group). Validation of the NGS data was carried out by qPCR analysis using specific primers for the targeted genes(n=4 to 6/group). Results: Analysis of the NGS data uncovered eccentric genomic profiles in the myocardium of mice with a 310 differentially regulated genes (DEGs) in FL-TG ( vs. NTG) and 472 DEGs in TR-TG ( vs. NTG). A closer evaluation reveals 397 and 237 DEGs commonly shared between FL-TG and TR-TG, respectively. STRING analysis revealed genomic networks involve in oxidoreductase, antioxidant and glutathione transferase were differentially altered between TL-TG vs. TR-TG groups. For instance, within the glutathione synthesis pathway, Gclm transcript was upregulated 6.0 fold (FL-TG) and 8.0 fold (TR-TG) when compared to NTG. Indeed, a significant douwnregulation of stress response genes Hspa1b (5.0 & 2.0 fold) and Hsph1 (2.0 & 2.2 fold) was apparent in between the groups. An eccentric cytoskeletal disarrangement ( Efnb3, Map1a, Acta ), an impaired protein quality control ( Bin1,Myl4 ) and a reduced cardiac function ( Timp4) were significantly downregulated in both the groups. Conclusion: These findings conclude that transgenic expression of wild-type vs. mutant forms of Nrf2 in the heart uniquely regulate the myocardial transcriptome, which might result in independent pathological processes.

Mitochondrial biology in airway pathogenesis and the role of NRF2

A constant improvement in understanding of mitochondrial biology has provided new insights into mitochondrial dysfunction in human disease pathogenesis. Impaired mitochondrial dynamics caused by various stressors are characterized by structural abnormalities and leakage, compromised turnover, andreactive oxygen species overproduction in mitochondria as well as increased mitochondrial DNA mutation frequency, which leads to modified energy production and mitochondria-derived cell signaling. The mitochondrial dysfunction in airway epithelial, smooth muscle, and endothelial cells has been implicated in diseases including chronic obstructive lung diseases and acute lung injury. Increasing evidence indicates that the NRF2-antioxidant response element (ARE) pathway not only enhances redox defense but also facilitates mitochondrial homeostasis and bioenergetics. Identification of functional or potential AREs further supports the role for Nrf2 in mitochondrial dysfunction-associated airway disorders. While clinical reports indicate mixed efficacy, NRF2 agonists acting on respiratory mitochondrial dynamics are potentially beneficial. In lung cancer, growth advantage provided by sustained NRF2 activation is suggested to be through increased cellular antioxidant defense as well as mitochondria reinforcement and metabolic reprogramming to the preferred pathways to meet the increased energy demands of uncontrolled cell proliferation. Further studies are warranted to better understand NRF2 regulation of mitochondrial functions as therapeutic targets in airway disorders.

Dysregulation of Nrf2 in Hepatocellular Carcinoma: Role in Cancer Progression and Chemoresistance.

The liver executes versatile functions and is the chief organ for metabolism of toxicants/xenobiotics. Hepatocellular carcinoma (HCC) is the most common primary liver malignancy and the third foremost cause of cancer death worldwide. Oxidative stress is a key factor related with the development and progression of HCC. Nuclear factor erythroid 2 [NF-E2]-related factor 2 (Nrf2) is a cytosolic transcription factor, which regulates redox homeostasis by activating the expression of an array of antioxidant response element-dependent genes. Nrf2 displays conflicting roles in normal, healthy liver and HCC; in the former, Nrf2 offers beneficial effects, whereas in the latter it causes detrimental effects favouring the proliferation and survival of HCC. Sustained Nrf2 activation has been observed in HCC and facilitates its progression and aggressiveness. This review summarizes the role and mechanism(s) of action of Nrf2 dysregulation in HCC and therapeutic options that can be employed to modulate this transcription factor.

Aurothioglucose does not improve alveolarization or elicit sustained Nrf2 activation in C57BL/6 models of bronchopulmonary dysplasia.

We previously showed that the thioredoxin reductase-1 (TrxR1) inhibitor aurothioglucose (ATG) improves alveolarization in hyperoxia-exposed newborn C3H/HeN mice. Our data supported a mechanism by which the protective effects of ATG are mediated via sustained nuclear factor E2-related factor 2 (Nrf2) activation in hyperoxia-exposed C3H/HeN mice 72 h after ATG administration. Given that inbred mouse strains have differential sensitivity and endogenous Nrf2 activation by hyperoxia, the present studies utilized two C57BL/6 exposure models to evaluate the effects of ATG on lung development and Nrf2 activation. The first model (0-14 days) was used in our C3H/HeN studies and the 2nd model (4-14 days) is well characterized in C57BL/6 mice. ATG significantly inhibited lung TrxR1 activity in both models; however, there was no effect on parameters of alveolarization in C57BL/6 mice. In sharp contrast to C3H/HeN mice, there was no effect of ATG on pulmonary NADPH quinone oxidoreductase-1 ( Nqo1) and heme oxygenase-1 ( Hmox1) at 72 h in either C57BL/6 model. In conclusion, although ATG inhibited TrxR1 activity in the lungs of newborn C57BL/6 mice, effects on lung development and sustained Nrf2-dependent pulmonary responses were blunted. These findings also highlight the importance of strain-dependent hyperoxic sensitivity in evaluation of potential novel therapies.

Arsenic-induced Metabolic Reprogramming: A Role for Autophagy, NRF2, and OGlcNAcylation

Metabolic reprogramming, originally used in the context of a cancer cell's ability to utilize glycolysis over oxidative phosphorylation to support rapid growth, refers to a shift away from normal homeostatic metabolism. This metabolic shift can arise from a number of sources, including genetic mutations, aging, or exposure to environmental factors. For example, prolonged exposure to environmental toxicants, such as the heavy metal arsenic, has been linked to an increased risk of cardiovascular disease, kidney disease, type II diabetes, and certain types of cancer. While arsenic is known to increase the risk of developing these diseases, the exact mechanisms underlying arsenic-related pathogenesis remain unclear. Previous work in our lab has shown that arsenic exposure results in inhibition of the autophagy-lysosome pathway, resulting in prolonged activation of the critical antioxidant transcription factor NRF2, a process termed non-canonical activation of NRF2. Both autophagy inhibition and sustained activation of NRF2 can significantly alter not only the overall redox state of the cell, but also the manner in which key metabolites and metabolic pathways are utilized. Interestingly, our results indicate that along with autophagy inhibition and increased activation of NRF2, arsenic treatment also results in increased global protein OGlcNAcylation, alterations to mitochondrial structure and function, changes in oxidative phosphorylation and glycolytic flux, as well as decreased production of reactive oxygen species (ROS). Thus, these phenotypes, coupled with non-canonical activation of NRF2, could significantly contribute to the metabolic reprogramming seen in diseases associated with prolonged exposure to arsenic. Whether or not these phenotypes are a result of prolonged autophagic dysfunction, sustained activation of NRF2 and its downstream targets, or other contributing factors is an area of active interest in our lab.

P 075 - Modulation of oxidative stress response in neurodevelopment disorders. The case of the Rett syndrome variants: MECP2 and CDKL5

Rett syndrome (RTT) variants are rare neurodevelopmental disorders caused by mutations in the X-linked MECP2 or CDKL5 genes. Because CDKL5 mediates MeCP2 phosphorylation and CDKL5 is a MeCP2-repressed target gene, it is possible that these proteins share similar molecular pathways. Altered redox status and subclinical inflammation seem to be common denominators between the two RTT variants: increased systemic levels of 4-HNE protein adducts and dysregulated cytokine profiles were reported. The altered redox homeostasis appears to be a critical player in the posttranslational modifications of SRB1, a specific high-density lipoproteins receptor, leading to altered serum lipid profiles in both variants. Although these parallels, Nrf2 system shows an impaired function in both disorders, but with some divergent aspects. CDKL5 show an inability to induce a proper defensive response with a significantly low nuclear Nrf2 translocation after oxidative challenge, while in MECP2 a sustained Nrf2 activation observed in basaline and upon oxidative stress stimulus. These findings reveal common defective pathways related to redox homeostasis between MECP2 and CDKL5. At present, the molecular basis of the discrepancy in the Nrf2 is unknown and requires further investigation in the light of the possibility to develop new therapeutic strategies for these devastating disorders without a cure.

A Novel Notch/NRF2 Mechanism Mediates Reductive Stress in Acute Myeloblastic Leukemia (AML): A Therapeutic Strategy to Overcome Chemoresistance

Background: Chronic oxidative stress (COS) is the consequence of prolonged elevation of reactive oxygen species (ROS). In the context of AML, COS leads to sustained induction of antioxidant pathways to compensate for COS. The presence of abundant antioxidants may contribute to chemoresistance in AML. NRF2 is a master regulator of the antioxidant response, is induced by oxidative stress and functions as a cyto-protective mechanism in acute oxidative stress. In AML, sustained NRF2-activation can be viewed as a pathological maladaptation as it leads to elevated reductive metabolite formation, termed reductive stress (RS). In this study we describe a novel link between NRF2-induced reductive stress and the Notch pathway in AML.Approach: Redox balance was measured in AML patient samples in the setting of Notch activation (via DLL1 ligand) and/or NRF2 inhibition (via Brusatol).Results: AML samples demonstrate increased reductive metabolites (i.e. reductive stress) compared to controls and Notch activation decreases aberrant reductive metabolite levels to control baseline (GSH/GSSG ratio, panel A). Mechanistically, Notch signaling induced increased ROS formation, decreased NRF2 protein levels, and decreased expression of multiple NRF2-responsive antioxidants (data not shown). Importantly, Notch activation also enhanced the efficacy of an NRF2 inhibitor Brusatol (from *P≤0.05 to **P≤0.01) in combination with ROS-producing chemotherapeutic doxorubicin, eradicating >98% of AML cells within 48 hours (** P≤0.01; panel B).Conclusions: We describe the aberrant accumulation of reductive metabolites in human AML as a potential cytoprotective maladaptation, i.e. reductive stress. Furthermore, we demonstrate a novel mechanism whereby Notch activation regulates the antioxidant response via suppression of the master regulator NRF2. These data establish the pivotal role of Notch/NRF2 in the regulation of antioxidant responses in AML, which have potential clinical significance for ROS-inducing chemotherapies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Sustained NRF2 activation in hereditary leiomyomatosis and renal cell cancer (HLRCC) and in hereditary tyrosinemia type 1 (HT1).

The nuclear erythroid 2-like 2 transcription factor (NRF2), is a major regulator of cellular redox balance. Although NRF2 activation is generally regarded as beneficial to human health, recent studies have identified that sustained NRF2 activation is over-represented in many cancers. This raises the question regarding the role of NRF2 activation in the development and progression of those cancers. This review focuses on the mechanisms and the effects of NRF2 activation in two hereditary cancer predisposition syndromes: hereditary leiomyomatosis and renal cell cancer (HLRCC) and hereditary tyrosinemia type 1 (HT1). Because the cancer initiating mutations in these hereditary syndromes are well defined, they offer a unique opportunity to explore the roles of NRF2 activation in the early stages of carcinogenesis. Over the years, a variety of approaches have been utilized to study the biology of HLRCC and HT1. In HLRCC, in vitro studies have demonstrated the importance of NRF2 activation in sustaining cancer cell proliferation. In the mouse model of HT1 however, NRF2 activation seems to protect cells from malignant transformation. In both HT1 and HLRCC, NRF2 activation promotes the clearance of electrophilic metabolites, enabling cells to survive cancer-initiating mutations. Biological insights gained from the hereditary syndromes' studies may shed light on to the roles of NRF2 activation in sporadic tumours.

Abstract 218: Chemical Induced Reductive Stress Causes Cardiomyocyte Hypertrophy

Background: Progressive accumulation of misfolded or unfolded proteins is a symbol of impaired proteostasis and proteotoxicity. Such a chronic proteotoxicity is amenable to cell types that are post mitotically matured with lack of further differentiation or proliferation. Our recent discovery using a mouse model of familial human cardiac disease displayed protuberant shift in the redox state towards reductive stress (RS) in association with accumulation of toxic protein aggregates. Further, sustained trans-activation of Nrf2/antioxidant signaling caused RS in the myopathy hearts. Accordingly, we hypothesized that whether profound activation of Nrf2/antioxidant signaling and subsequent RS may cause pathological remodeling in cardiomyocyte. The aim of this study was to investigate the effect of sustained pharmacological activation of Nrf2 on cardiac remodeling. Methods: HL1 cardiomyocytes were used as an in vitro model to study the RS-mediated cardiac remodeling. They were treated with 2-10 μM of potential Nrf2-inducers; sulforaphane (SF), di-methyl fumarate (DMF) and novel small molecules (C-38, C-50, C-63 and C-66) to establish RS by sustained activation of Nrf2/antioxidant signaling. Next, we investigated the implications of RS in cardiomyocyte remodeling by analyzing transcriptional and translational mechanisms using immunoblotting, qPCR, immunofluorescence, GSH and NADPH redox measurements in HL1 cells. Results: Dose dependent effects for individual small molecules including known Nrf2 inducers (SF and DMF) revealed distinct pro-reductive and reductive intracellular (i.e. reductive stress) environments. In fact, the obligatory activation of Nrf2 signaling was associated with significant upregulation of antioxidant enzymes and small molecular thiols including glutathione (GSH). Surprisingly, while pro-reductive condition in HL1 cells was subdued, the RS induced cardiomyocyte hypertrophy was evident from microscopic examination and molecular signature (increased expression of ANF and BNF) after 24-48 hrs of Nrf2 activation. Conclusion: In summary, the chemical induced sustained activation of Nrf2 leading to formation of reductive stress showed hypertrophic remodeling in HL1 cardiomyocytes.

Arsenic Inhibits Autophagic Flux, Activating the Nrf2-Keap1 Pathway in a p62-Dependent Manner

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.

Nrf2 inhibits hepatic iron accumulation and counteracts oxidative stress-induced liver injury in nutritional steatohepatitis

The transcription factor nuclear factor-E2-related factor-2 (Nrf2) is a key regulator for induction of hepatic antioxidative stress systems. We aimed to investigate whether activation of Nrf2 protects against steatohepatitis. Wild-type mice (WT), Nrf2 gene-null mice (Nrf2-null) and Keap1 gene-knockdown mice (Keap1-kd), which represent the sustained activation of Nrf2, were fed a methionine- and choline-deficient diet (MCDD) for 13 weeks and analyzed. In Keap1-kd fed an MCDD, steatohepatitis did not develop over the observation periods; however, in Nrf2-null fed an MCDD, the pathological state of the steatohepatitis was aggravated in terms of fatty change, inflammation, fibrosis and iron accumulation. In WT mice fed an MCDD, Nrf2 and antioxidative stress genes regulated by Nrf2 were potently activated in the livers, and in Keap1-kd, their basal levels were potently activated. Oxidative stress was significantly increased in the livers of the Nrf2-null and suppressed in the livers of the Keap1-kd compared to that of WT, based on the levels of 4-hydroxy-2-nonenal and malondialdehyde. Iron accumulation was greater in the livers of the Nrf2-null mice compared to those of the WT mice, and it was not observed in Keap1-kd. Further, the iron release from the isolated hepatocyte of Nrf2-null mice was significantly decreased. Sulforaphane, an activator of Nrf2, suppressed the pathological states and oxidative stress in the livers. Nrf2 has protective roles against nutritional steatohepatitis through inhibition of hepatic iron accumulation and counteraction against oxidative stress-induced liver injury. Nrf2 activation by pharmaceutical intervention could be a new option for the prevention and treatment of steatohepatitis.

Abstract P184: Rescue of Reductive Stress by Nrf2 Deficiency in Protein Aggregation Cardiomyopathy Mice

Background: We recently discovered that the overexpression of human mutant αB-crystallin (hR120GCryAB) in mouse resulted in protein aggregation cardiomyopathy (PAC) and reductive stress (RS) in the heart tissue (Rajasekaran et.al Cell, 2007). Further, we identified that sustained activation of Nrf2 (nuclear erythroid 2 related factor 2)/ARE signaling as a causal mechanism for RS in the MPAC mice. We hypothesize that abolishing Nrf2 could prevent RS in R120GTG mice and thereby rescue them from cardiomyopathy and heart failure. Methods: To elucidate loss-of-function mechanisms for Nrf2 on RS and MPAC, we produced (1) NTG/WT, (2) R120GTG/WT, (3) R120GTG/Nrf2+/- and (4) R120GTG/Nrf2-/- mice by intercrossing R120GTG X Nrf2-KO. Primarily, we determined the rate of survival and performed echocardiography to understand the degree of pathogenesis and cardiac function over time (n=6-10). Next, we analyzed the redox state and protein/mRNA expression for major antioxidants including enzymes that are involved in glutathione metabolism in the heart. Results: Disruption of Nrf2 prolonged the survival of MPAC mice along with no signs of cardiac dysfunction (by ECHO). The R120GTG mice with Nrf2 deficiency (either Nrf2+/- or Nrf2-/-; n=4) had significantly decreased glutathione (but equal to NTG) levels when compared with R120GTG/WT cohorts. Such a decline in GSH resulted in quenching of “reducing power” to prevent “reductive stress” and facilitated redox homeostasis in the myocardium. Interestingly, the intercross mice showed no cardiac hypertrophy even at 12 months while the R120GTG exhibited 90% mortality due to PAC/heart failure. Further, protein and mRNA levels for major antioxidants were found to be normal in the R120GTG/Nrf2+/- while there was significant down regulation of these parameters in the R120GTG/Nrf2-/- mice when compared to NTG/WT suggesting that Nrf2-independent pathways are inadequate to maintain the antioxidant capacity under stressed conditions. Conclusions: These results demonstrate a critical role for Nrf2 underlying antioxidant potential in the myocardium. Abrogating RS via Nrf2 might be highly relevant to cure the protein aggregation diseases that are coupled with elevated intracellular reducing potential.

Correction: TNF Mediates the Sustained Activation of Nrf2 in Human Monocytes

Rushworth, S. A., S. Shah, and D. J. MacEwan. 2011. TNF mediates the sustained activation of Nrf2 in human monocytes. J. Immunol. 187: [702–707][1]. In [Fig. 4 D ][2], the incorrect immunoblot for the loading control was mistakenly used. The correct immunoblot has now been used in the updated [

Acute exercise stress activates Nrf2/ARE signaling and promotes antioxidant mechanisms in the myocardium

Oxidative stress has been implicated in the pathogenesis of cardiovascular diseases, including myocardial hypertrophy and infarction. Although impairment of antioxidant defense mechanisms has been thought to provoke oxidative stress-induced myocardial dysfunction, it has been difficult to clearly demonstrate. Nuclear erythroid 2 p45-related factor 2 (Nrf2) is a redox-sensitive, basic leucine zipper protein that regulates the transcription of several antioxidant genes. We previously reported that sustained activation of Nrf2 upregulates transcription of a number of endogenous antioxidants in the heart. Here, we show that acute exercise stress (AES) results in activation of Nrf2/ARE (antioxidant response element) signaling and subsequent enhancement of antioxidant defense pathways in wild-type (WT) mouse hearts, while oxidative stress, along with blunted defense mechanisms, was observed in Nrf2−/− mice. We also find that AES is associated with increased trans-activation of ARE-containing genes in exercised animals when compared to age-matched sedentary WT mice. However, enhanced oxidative stress in response to AES was observed in Nrf2−/− mice due to lower basal expression and marked attenuation of the transcriptional induction of several antioxidant genes. Thus, AES induces ROS and promotes Nrf2 function, but disruption of Nrf2 increases susceptibility of the myocardium to oxidative stress. Our findings suggest the basis for a nonpharmacological approach to activate Nrf2/ARE signaling, which might be a potential therapeutic target to protect the heart from oxidative stress-induced cardiovascular complications.