Dual Deletion of Keap1 and Rbpjκ Genes in Liver Leads to Hepatomegaly and Hypercholesterolemia.

The hepatic deletion of Rbpjκ (RbpjF/F::AlbCre) in the mouse leads to exhibition of the Alagille syndrome phenotype during early postnatal liver development with hyperlipidemia and cholestasis due to attenuated disruption of NOTCH signaling. Given the roles of NRF2 signaling in the regulation of lipid metabolism and bile ductal formation, it was anticipated that these symptoms could be alleviated by enhancing NRF2 signaling in the RbpjF/F::AlbCre mouse by hepatic deletion of Keap1 in compound Keap1F/F::RbpjF/F::AlbCre mice. Unexpectedly, these mice developed higher hepatic and plasma cholesterol levels with more severe cholestatic liver damage during the pre-weaning period than in the RbpjF/F::AlbCre mice. In addition, hypercholesterolemia and hepatic damage were sustained throughout the growth period unlike in the RbpjF/F::AlbCre mouse. These enhanced abnormalities in lipid metabolism appear to be due to NRF2-dependent changes in gene expression related to cholesterol synthetic and subsequent bile acid production pathways. Notably, the hepatic expression of Cyp1A7 and Abcb11 genes involved in bile acid homeostasis was significantly reduced in Keap1F/F::RbpjF/F::AlbCre compared to RbpjF/F::AlbCre mice. The accumulation of liver cholesterol and the weakened capacity for bile excretion during the 3 pre-weaning weeks in the Keap1F/F::RbpjF/F::AlbCre mice may aggravate hepatocellular damage level caused by both excessive cholesterol and residual bile acid toxicity in hepatocytes. These results indicate that a tuned balance of NOTCH and NRF2 signaling is of biological importance for early liver development after birth.

A Point Mutation at C151 of Keap1 of Mice Abrogates NRF2 Signaling, Cytoprotection in Vitro, and Hepatoprotection in Vivo by Bardoxolone Methyl (CDDO-Me).

Bardoxolone methyl (CDDO-Me) is an oleanane triterpenoid in late-stage clinical development for the treatment of patients with diabetic kidney disease. Preclinical studies in rodents demonstrate the efficacy of triterpenoids against carcinogenesis and other diseases, including renal ischemia-reperfusion injury, hyperoxia-induced acute lung injury, and immune hepatitis. Genetic disruption of Nrf2 abrogates protection by triterpenoids, suggesting that induction of the NRF2 pathway may drive this protection. Herein, we examined the effect of a point mutation (C151S) in KEAP1, a repressor of NRF2 signaling, at cysteine 151 in mouse embryo fibroblasts and mouse liver. Induction of target gene transcripts and enzyme activity by CDDO-Me was lost in C151S mutant fibroblasts compared with wild-type. Protection against menadione toxicity was also nullified in the mutant fibroblasts. In mouse liver, CDDO-Me evoked the nuclear translocation of NRF2, followed by increased transcript and activity levels of a prototypic target gene, Nqo1, in wild-type, but not C151S mutant, mice. To test the role of KEAP1 Cys151 in governing the broader pharmacodynamic action of CDDO-Me, wild-type and C151S mutant mice were challenged with concanavalin A to induce immune hepatitis. Strong protection was seen in wild-type but not C151S mutant mice. RNA-seq analysis of mouse liver from wild-type, C151S mutant, and Nrf2-knockout mice revealed a vigorous response of the NRF2 transcriptome in wild-type, but in neither C151S mutant nor Nrf2-knockout, mice. Activation of "off-target" pathways by CDDO were not observed. These data highlight the singular importance of the KEAP1 cysteine 151 sensor for activation of NRF2 signaling by CDDO-Me. SIGNIFICANCE STATEMENT: KEAP1 serves as a key sensor for induction of the cytoprotective signaling pathway driven by the transcription factor NRF2. Mutation of a single cysteine (C151) in KEAP1 abrogates the induction of NRF2 signaling and its downstream cytoprotective actions in vitro and in vivo by bardoxolone methyl (CDDO-Me), a drug in late-stage clinical development. Further, at these bioeffective concentrations/doses, activation of "off-target" pathways by CDDO-Me are not observed, highlighting the singular importance of NRF2 in its mode of action.

Nrf2 through Aryl Hydrocarbon Receptor Regulates IL-22 Response in CD4+ T Cells.

IL-17A and IL-22 derived from Th17 cells play a significant role in mucosal immunity and inflammation. TGF-ß and IL-6 promote Th17 differentiation; however, these cytokines have multiple targets. The identification and screening of additional molecules that regulate IL-17A and IL-22 responses in certain inflammatory conditions is of great clinical significance. In this study, we show that CDDO-Im, a specific Nrf2 activator, promotes IL-17A and IL-22 responses in murine Th17 cells. In contrast, CDDO-Im inhibits IL-17A response in multiple sclerosis patient-derived PBMCs. However, Nrf2 specifically regulates IL-22 response in vivo. Nrf2 acts through the regulation of antioxidant response element (ARE) binding motifs in target genes to induce or repress transcription. Promoter analysis revealed that Il17a, Rorc, and Ahr genes have several ARE motifs. We showed that Nrf2 bound to ARE repressor (ARE-R2) of Rorc and inhibited Rorc-dependent IL-17A transactivation. The luciferase reporter assay data showed that CDDO-Im regulated Ahr promoter activity. Chromatin immunoprecipitation quantitative PCR data showed that Nrf2 bound to ARE of AhR. Finally, we confirmed that the CDDO-Im-mediated induction of IL-22 production in CD4+ T cells was abrogated in CD4-specific Ahr knockout mice (AhrCD4 ). CH-223191, a specific AhR antagonist, inhibits CDDO-Im-induced IL-22 production in CD4+ T cells, which further confirmed the AhR-dependent regulation. Collectively, our data showed that Nrf2 via AhR pathways regulated IL-22 response in CD4+ T cells.

Forced Hepatic Expression of NRF2 or NQO1 Impedes Hepatocyte Lipid Accumulation in a Lipodystrophy Mouse Model.

Lipodystrophy is a disorder featuring loss of normal adipose tissue depots due to impaired production of normal adipocytes. It leads to a gain of fat deposition in ectopic tissues such as liver and skeletal muscle that results in steatosis, dyslipidemia, and insulin resistance. Previously, we established a Rosa NIC/NIC::AdiCre lipodystrophy model mouse. The lipodystrophic phenotype that included hepatomegaly accompanied with hepatic damage due to higher lipid accumulation was attenuated substantially by amplified systemic NRF2 signaling in mice with hypomorphic expression of Keap1; whole-body Nrf2 deletion abrogated this protection. To determine whether hepatic-specific NRF2 signaling would be sufficient for protection against hepatomegaly and fatty liver development, direct, powerful, transient expression of Nrf2 or its target gene Nqo1 was achieved by administration through hydrodynamic tail vein injection of pCAG expression vectors of dominant-active Nrf2 and Nqo1 in Rosa NIC/NIC::AdiCre mice fed a 9% fat diet. Both vectors enabled protection from hepatic damage, with the pCAG-Nqo1 vector being the more effective as seen with a ~50% decrease in hepatic triglyceride levels. Therefore, activating NRF2 signaling or direct elevation of NQO1 in the liver provides new possibilities to partially reduce steatosis that accompanies lipodystrophy.

NRF2/ARE pathway negatively regulates BACE1 expression and ameliorates cognitive deficits in mouse Alzheimer's models.

BACE1 is the rate-limiting enzyme for amyloid-ß peptides (Aß) generation, a key event in the pathogenesis of Alzheimer's disease (AD). By an unknown mechanism, levels of BACE1 and a BACE1 mRNA-stabilizing antisense RNA (BACE1-AS) are elevated in the brains of AD patients, implicating that dysregulation of BACE1 expression plays an important role in AD pathogenesis. We found that nuclear factor erythroid-derived 2-related factor 2 (NRF2/NFE2L2) represses the expression of BACE1 and BACE1-AS through binding to antioxidant response elements (AREs) in their promoters of mouse and human. NRF2-mediated inhibition of BACE1 and BACE1-AS expression is independent of redox regulation. NRF2 activation decreases production of BACE1 and BACE1-AS transcripts and Aß production and ameliorates cognitive deficits in animal models of AD. Depletion of NRF2 increases BACE1 and BACE1-AS expression and Aß production and worsens cognitive deficits. Our findings suggest that activation of NRF2 can prevent a key early pathogenic process in AD.

Nrf2 deletion from adipocytes, but not hepatocytes, potentiates systemic metabolic dysfunction after long-term high-fat diet-induced obesity in mice.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a canonical regulator of cytoprotective gene expression, but evidence of its cross talk with other pathways, including metabolic ones, is ever increasing. Pharmacologic or systemic genetic activation of the Nrf2 pathway partially protects from obesity in mice and ameliorates fasting hyperglycemia in mice and humans. However, systemic Nrf2 deletion also protects from diet-induced obesity and insulin resistance in mice. To further investigate the effect of the disruption of Nrf2 on obesity in a tissue-specific manner, we focused on adipocytes and hepatocytes with targeted deletion of Nrf2. To this end, mice with cell-specific deletion of Nrf2 in adipocytes (ANKO) or hepatocytes (HeNKO) were fed a high-fat diet (HFD) for 6 mo and showed similar increases in body weight and body fat content. ANKO mice showed a partially deteriorated glucose tolerance, higher fasting glucose levels, and higher levels of cholesterol and nonesterified fatty acids compared with their Control counterparts. The HeNKO mice, though, had lower insulin levels and trended toward improved insulin sensitivity without having any difference in liver triglyceride accumulation. This study compared for the first time two conditional Nrf2 knockout models in adipocytes and in hepatocytes during HFD-induced obesity. None of these models could completely recapitulate the unexpected protection against obesity observed in the whole body Nrf2 knockout mice, but this study points out the differential roles that Nrf2 may play, beyond cytoprotection, in different target tissues and rather suggests systemic activation of the Nrf2 pathway as an effective means of prevention and treatment of obesity and type 2 diabetes.

Keap1/Nrf2 pathway activation leads to a repressed hepatic gluconeogenic and lipogenic program in mice on a high-fat diet.

The Keap1/Nrf2 pathway, known to regulate the expression of a series of cytoprotective and antioxidant genes, has been studied in the context of obesity and type 2 diabetes; diseases that are characterized by chronic oxidative stress. There is increasing evidence, however, that the transcription factor Nrf2 can crosstalk with pathways not directly related to cytoprotection. Our present work focuses on the effect of Nrf2 on hepatic gluconeogenesis and lipogenesis, two metabolic processes which are dysregulated in the obese/diabetic state. To this end, a genetic mouse model of Nrf2 pathway activation was used (Keap1-hypo; both Keap1 alleles are hypomorphic) and was exposed to a 3-month high-fat diet along with the relevant control wild-type mice. The Keap1-hypo mice were partially protected from obesity, had lower fasting glucose and insulin levels and developed less liver steatosis compared to the wild-type. Key gluconeogenic and lipogenic enzymes were repressed in the Keap1-hypo livers with concomitant activated Ampk signaling. Primary Keap1-hypo hepatocyte cultures also show increased Ampk signaling and repressed glucose production. In conclusion, increased Keap1/Nrf2 signaling in the liver is accompanied by repressed gluconeogenesis and lipogenesis that can, at least partially, explain the ameliorated diabetic phenotype in the Keap1-hypo mice.

Keap1/Nrf2 pathway in the frontiers of cancer and non-cancer cell metabolism.

Cancer cells adapt their metabolism to their increased needs for energy and substrates for protein, lipid and nucleic acid synthesis. Nuclear erythroid factor 2-like 2 (Nrf2) pathway is usually activated in cancers and has been suggested to promote cancer cell survival mainly by inducing a large battery of cytoprotective genes. This mini review focuses on metabolic pathways, beyond cytoprotection, which can be directly or indirectly regulated by Nrf2 in cancer cells to affect their survival. The pentose phosphate pathway (PPP) is enhanced by Nrf2 in cancers and aids their growth. PPP has also been found to be up-regulated in non-cancer tissues and other pathways, such as de novo lipogenesis, have been found to be repressed after activation of the Nrf2 pathway. The importance of these Nrf2-regulated metabolic pathways in cancer compared with non-cancer state remains to be determined. Last but not least, the importance of context about Nrf2 and cancer is highlighted as the Nrf2 pathway may be activated in cancers but its pharmacological activators are useful in chemoprevention.

Modulation of nitro-fatty acid signaling: prostaglandin reductase-1 is a nitroalkene reductase.

Inflammation, characterized by the activation of both resident and infiltrated immune cells, is accompanied by increased production of oxidizing and nitrating species. Nitrogen dioxide, the proximal nitrating species formed under these conditions, reacts with unsaturated fatty acids to yield nitroalkene derivatives. These electrophilic products modulate protein function via post-translational modification of susceptible nucleophilic amino acids. Nitroalkenes react with Keap1 to instigate Nrf2 signaling, activate heat shock response gene expression, and inhibit NF-κB-mediated signaling, inducing net anti-inflammatory and tissue-protective metabolic responses. We report the purification and characterization of a NADPH-dependent liver enzyme that reduces the nitroalkene moiety of nitro-oleic acid, yielding the inactive product nitro-stearic acid. Prostaglandin reductase-1 (PtGR-1) was identified as a nitroalkene reductase by protein purification and proteomic studies. Kinetic measurements, inhibition studies, immunological and molecular biology approaches as well as clinical analyses confirmed this identification. Overexpression of PtGR-1 in HEK293T cells promoted nitroalkene metabolism to inactive nitroalkanes, an effect that abrogated the Nrf2-dependent induction of heme oxygenase-1 expression by nitro-oleic acid. These results situate PtGR-1 as a critical modulator of both the steady state levels and signaling activities of fatty acid nitroalkenes in vivo.

Hydrogen gas reduces hyperoxic lung injury via the Nrf2 pathway in vivo.

Hyperoxic lung injury is a major concern in critically ill patients who receive high concentrations of oxygen to treat lung diseases. Successful abrogation of hyperoxic lung injury would have a huge impact on respiratory and critical care medicine. Hydrogen can be administered as a therapeutic medical gas. We recently demonstrated that inhaled hydrogen reduced transplant-induced lung injury and induced heme oxygenase (HO)-1. To determine whether hydrogen could reduce hyperoxic lung injury and investigate the underlying mechanisms, we randomly assigned rats to four experimental groups and administered the following gas mixtures for 60 h: 98% oxygen (hyperoxia), 2% nitrogen; 98% oxygen (hyperoxia), 2% hydrogen; 98% balanced air (normoxia), 2% nitrogen; and 98% balanced air (normoxia), 2% hydrogen. We examined lung function by blood gas analysis, extent of lung injury, and expression of HO-1. We also investigated the role of NF-E2-related factor (Nrf) 2, which regulates HO-1 expression, by examining the expression of Nrf2-dependent genes and the ability of hydrogen to reduce hyperoxic lung injury in Nrf2-deficient mice. Hydrogen treatment during exposure to hyperoxia significantly improved blood oxygenation, reduced inflammatory events, and induced HO-1 expression. Hydrogen did not mitigate hyperoxic lung injury or induce HO-1 in Nrf2-deficient mice. These findings indicate that hydrogen gas can ameliorate hyperoxic lung injury through induction of Nrf2-dependent genes, such as HO-1. The findings suggest a potentially novel and applicable solution to hyperoxic lung injury and provide new insight into the molecular mechanisms and actions of hydrogen.

Keap1-null mutation leads to postnatal lethality due to constitutive Nrf2 activation.

Transcription factor Nrf2 (encoded by Nfe2l2) regulates a battery of detoxifying and antioxidant genes, and Keap1 represses Nrf2 function. When we ablated Keap1, Keap1-deficient mice died postnatally, probably from malnutrition resulting from hyperkeratosis in the esophagus and forestomach. Nrf2 activity affects the expression levels of several squamous epithelial genes. Biochemical data show that, without Keap1, Nrf2 constitutively accumulates in the nucleus to stimulate transcription of cytoprotective genes. Breeding to Nrf2-deficient mice reversed the phenotypic Keap1 deficiencies. These experiments show that Keap1 acts upstream of Nrf2 in the cellular response to oxidative and xenobiotic stress.

Transcriptional Regulation of Math1 by Aryl Hydrocarbon Receptor: Effect on Math1+ Progenitor Cells in Mouse Small Intestine.

The physiological roles of aryl hydrocarbon receptor (AhR) in the small intestine have been revealed as immunomodulatory and barrier functions. However, its contributions to cell fate regulation are incompletely understood. The Notch-activated signaling cascade is a central component of intestinal cell fate determinations. The lateral inhibitory mechanism governed by Notch directs cell fates toward distinct cell lineages (i.e., absorptive and secretory cell lineages) through its downstream effector, mouse atonal homolog 1 (MATH1). An investigation employing cell lines and intestinal crypt cells revealed that AhR regulates Math1 expression in a xenobiotic response element (XRE)-dependent manner. The AhR-Math1 axis was further addressed using intestinal organoids, where AhR-Math1 and HES1-Math1 axes appeared to coexist within the underlying Math1 transcriptional machinery. When the HES1-Math1 axis was pharmacologically suppressed, ß-naphthoflavone-mediated AhR activation increased the number of goblet and Math1+ progenitor cells in the organoids. The same pharmacological dissection of the AhR-Math1 axis was applied in vivo, demonstrating an enhanced number of Math1+ progenitor cells in the small intestine following AhR activation. We report here that AhR-Math1 is a direct transcriptional axis with effects on Math1+ progenitor cells in the small intestine, highlighting a novel molecular basis for fine-tuning Notch-mediated cell fate regulation.

NRF2 and the Moirai: Life and Death Decisions on Cell Fates.

Significance: The transcription factor NRF2 (NF-E2-related factor 2) plays an important role as a master regulator of the cellular defense system by activating transcriptional programs of NRF2 target genes encoding multiple enzymes related to cellular redox balance and xenobiotic detoxication. Comprehensive transcriptional analyses continue to reveal an ever-broadening range of NRF2 target genes, demonstrating the sophistication and diversification of NRF2 biological signatures beyond its canonical cytoprotective roles. Recent Advances: Accumulating evidence indicates that NRF2 has a strong association with the regulation of cell fates by influencing key processes of cellular transitions in the three major phases of the life cycle of the cell (i.e., cell birth, cell differentiation, and cell death). The molecular integration of NRF2 signaling into this regulatory program occurs through a wide range of NRF2 target genes encompassing canonical functions and those manipulating cell fate pathways. Critical Issues: A singular focus on NRF2 signaling for dissecting its actions limits in-depth understanding of its intersection with the molecular machinery of cell fate determinations. Compensatory responses of downstream pathways governed by NRF2 executed by a variety of transcription factors and multifactorial signaling crosstalk require further exploration. Future Directions: Further investigations using optimized in vivo models and active engagement of overarching approaches to probe the interplay of widespread pathways are needed to study the properties and capabilities of NRF2 signaling as a part of a large network within the cell fate regulatory domain. Antioxid. Redox Signal. 38, 684-708.

Global mapping of binding sites for Nrf2 identifies novel targets in cell survival response through ChIP-Seq profiling and network analysis.

The Nrf2 (nuclear factor E2 p45-related factor 2) transcription factor responds to diverse oxidative and electrophilic environmental stresses by circumventing repression by Keap1, translocating to the nucleus, and activating cytoprotective genes. Nrf2 responses provide protection against chemical carcinogenesis, chronic inflammation, neurodegeneration, emphysema, asthma and sepsis in murine models. Nrf2 regulates the expression of a plethora of genes that detoxify oxidants and electrophiles and repair or remove damaged macromolecules, such as through proteasomal processing. However, many direct targets of Nrf2 remain undefined. Here, mouse embryonic fibroblasts (MEF) with either constitutive nuclear accumulation (Keap1(-/-)) or depletion (Nrf2(-/-)) of Nrf2 were utilized to perform chromatin-immunoprecipitation with parallel sequencing (ChIP-Seq) and global transcription profiling. This unique Nrf2 ChIP-Seq dataset is highly enriched for Nrf2-binding motifs. Integrating ChIP-Seq and microarray analyses, we identified 645 basal and 654 inducible direct targets of Nrf2, with 244 genes at the intersection. Modulated pathways in stress response and cell proliferation distinguish the inducible and basal programs. Results were confirmed in an in vivo stress model of cigarette smoke-exposed mice. This study reveals global circuitry of the Nrf2 stress response emphasizing Nrf2 as a central node in cell survival response.

Transcription factor Nrf2 maintains the basal expression of Mdm2: An implication of the regulation of p53 signaling by Nrf2.

Co-operated regulation of oxidative stress-response transcription factors would be an important issue for animals to determine the cell fate under environmental stress. This notion raises a possibility that NF-E2-related factor 2 (Nrf2), which confers cytoprotection against oxidative stress, and p53 can have a direct co-regulation network. In the current study, we have indentified that the expression of murine double minute 2 (Mdm2) is repressed in nrf2-deleted murine embryonic fibroblasts (MEFs). This was confirmed by microarray, RT-PCR, and immunoblot analyses, and further promoter analysis showed that Nrf2 is directly involved in the basal expression of Mdm2 through the antioxidant response element, which is located in the first intron of this gene. This linkage between Nrf2 and Mdm2 appears to cause the accumulation of p53 protein in nrf2-deficent MEFs. In addition, we show that ovarian carcinoma A2780 cells with Nrf2 shRNA expression displayed higher levels of p53 activation in response to hydrogen peroxide treatment, leading to increased cell death. Collectively, our results suggest novel evidence that the inhibition of Nrf2 can suppress Mdm2 expression, which may result in p53 signaling modulation. In addition, this observation supports the concept that Nrf2 inhibition in cancer cells can facilitate apoptotic response upon environmental stress.

Constitutive Activation of Nrf2 in Mice Expands Enterogenesis in Small Intestine Through Negative Regulation of Math1.

BACKGROUND & AIMS: Notch signaling coordinates cell differentiation processes in the intestinal epithelium. The transcription factor Nrf2 orchestrates defense mechanisms by regulating cellular redox homeostasis, which, as shown previously in murine liver, can be amplified through signaling crosstalk with the Notch pathway. However, interplay between these 2 signaling pathways in the gut is unknown. METHODS: Mice modified genetically to amplify Nrf2 in the intestinal epithelium (Keap1f/f::VilCre) were generated as well as pharmacological activation of Nrf2 and subjected to phenotypic and cell lineage analyses. Cell lines were used for reporter gene assays together with Nrf2 overexpression to study transcriptional regulation of the Notch downstream effector. RESULTS: Constitutive activation of Nrf2 signaling caused increased intestinal length along with expanded cell number and thickness of enterocytes without any alterations of secretory lineage, outcomes abrogated by concomitant disruption of Nrf2. The Nrf2 and Notch pathways in epithelium showed inverse spatial profiles, where Nrf2 activity in crypts was lower than villi. In progenitor cells of Keap1f/f::VilCre mice, Notch downstream effector Math1, which regulates a differentiation balance of cell lineage through lateral inhibition, showed suppressed expression. In vitro results demonstrated Nrf2 negatively regulated Math1, where 6 antioxidant response elements located in the regulatory regions contributed to this repression. CONCLUSIONS: Activation of Nrf2 perturbed the dialog of the Notch cascade though negative regulation of Math1 in progenitor cells, leading to enhanced enterogenesis. The crosstalk between the Nrf2 and Notch pathways could be critical for fine-tuning intestinal homeostasis and point to new approaches for the pharmacological management of absorptive deficiencies.

Nrf2: friend or foe for chemoprevention?

Health reflects the ability of an organism to adapt to stress. Stresses--metabolic, proteotoxic, mitotic, oxidative and DNA-damage stresses--not only contribute to the etiology of cancer and other chronic degenerative diseases but are also hallmarks of the cancer phenotype. Activation of the Kelch-like ECH-associated protein 1 (KEAP1)-NF-E2-related factor 2 (NRF2)-signaling pathway is an adaptive response to environmental and endogenous stresses and serves to render animals resistant to chemical carcinogenesis and other forms of toxicity, whilst disruption of the pathway exacerbates these outcomes. This pathway can be induced by thiol-reactive small molecules that demonstrate protective efficacy in preclinical chemoprevention models and in clinical trials. However, mutations and epigenetic modifications affecting the regulation and fate of NRF2 can lead to constitutive dominant hyperactivation of signaling that preserves rather than attenuates cancer phenotypes by providing selective resistance to stresses. This review provides a synopsis of KEAP1-NRF2 signaling, compares the impact of genetic versus pharmacologic activation and considers both the attributes and concerns of targeting the pathway in chemoprevention.

NRF2 modulates aryl hydrocarbon receptor signaling: influence on adipogenesis.

The NF-E2 p45-related factor 2 (NRF2) and the aryl hydrocarbon receptor (AHR) are transcription factors controlling pathways modulating xenobiotic metabolism. AHR has recently been shown to affect Nrf2 expression. Conversely, this study demonstrates that NRF2 regulates expression of Ahr and subsequently modulates several downstream events of the AHR signaling cascade, including (i) transcriptional control of the xenobiotic metabolism genes Cyp1a1 and Cyp1b1 and (ii) inhibition of adipogenesis in mouse embryonic fibroblasts (MEFs). Constitutive expression of AHR was affected by Nrf2 genotype. Moreover, a pharmacological activator of NRF2 signaling, CDDO-IM {1-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole}, induced Ahr, Cyp1a1, and Cyp1b1 transcription in Nrf2+/+ MEFs but not in Nrf2-/- MEFs. Reporter analysis and chromatin immunoprecipitation assay revealed that NRF2 directly binds to one antioxidant response element (ARE) found in the -230-bp region of the promoter of Ahr. Since AHR negatively controls adipocyte differentiation, we postulated that NRF2 would inhibit adipogenesis through the interaction with the AHR pathway. Nrf2-/- MEFs showed markedly accelerated adipogenesis upon stimulation, while Keap1-/- MEFs (which exhibit higher NRF2 signaling) differentiated slowly compared to their congenic wild-type MEFs. Ectopic expression of Ahr and dominant-positive Nrf2 in Nrf2-/- MEFs also substantially delayed differentiation. Thus, NRF2 directly modulates AHR signaling, highlighting bidirectional interactions of these pathways.

Nrf2 prevents Notch-induced insulin resistance and tumorigenesis in mice.

Insulin resistance is associated with increased incidence and enhanced progression of cancers. However, little is known about strategies that can effectively ameliorate insulin resistance and consequently halt cancer progression. Herein, we propose that the transcription factor Nrf2 (also known as Nfe2l2) may be such a target, given its central role in disease prevention. To this end, we developed a mouse that overexpresses the Notch intracellular domain in adipocytes (AdNICD), leading to lipodystrophy-induced severe insulin resistance and subsequent development of sarcomas, as a model reflecting that Notch signaling is deregulated in cancers and shows positive associations with insulin resistance and fatty liver disease in humans. Nrf2 pathway activation was achieved by knocking down Keap1, a repressor of Nrf2, in the AdNICD background. Constitutively enhanced Nrf2 signaling in this setting led to prevention of hepatic steatosis, dyslipidemia, and insulin resistance by repressing hepatic lipogenic pathways and restoration of the hepatic fatty acid profile to control levels. This protective effect of Nrf2 against diabetes extended to significant reduction and delay in sarcoma incidence and latency. Our study highlights that the Nrf2 pathway, which has been induced by small molecules in clinical trials, is a potential therapeutic target against insulin resistance and subsequent risk of cancer.

Antioxidants enhance mammalian proteasome expression through the Keap1-Nrf2 signaling pathway.

Proteasomes degrade damaged proteins formed during oxidative stress, thereby promoting cell survival. Neurodegenerative and other age-related disorders are associated with reduced proteasome activity. We show herein that expression of most subunits of 20S and 19S proteasomes, which collectively assemble the 26S proteasome, was enhanced up to threefold in livers of mice following treatment with dithiolethiones, which act as indirect antioxidants. Subunit protein levels and proteasome activity were coordinately increased. No induction was seen in mice where the transcription factor Nrf2 was disrupted. Promoter activity of the PSMB5 subunit of the 20S proteasome increased with either Nrf2 overexpression or treatment with antioxidants in mouse embryonic fibroblasts. Tandem antioxidant response elements in the proximal promoter of PSMB5 that controlled these responses were identified. We propose that induction of the 26S proteasome through the Nrf2 pathway represents an important indirect action of these antioxidants that can contribute to their protective effects against chronic diseases.