The KEAP1-NRF2 System: a Thiol-Based Sensor-Effector Apparatus for Maintaining Redox Homeostasis.

The Kelch-like ECH-associated protein 1-NF-E2-related factor 2 (KEAP1-NRF2) system forms the major node of cellular and organismal defense against oxidative and electrophilic stresses of both exogenous and endogenous origins. KEAP1 acts as a cysteine thiol-rich sensor of redox insults, whereas NRF2 is a transcription factor that robustly transduces chemical signals to regulate a battery of cytoprotective genes. KEAP1 represses NRF2 activity under quiescent conditions, whereas NRF2 is liberated from KEAP1-mediated repression on exposure to stresses. The rapid inducibility of a response based on a derepression mechanism is an important feature of the KEAP1-NRF2 system. Recent studies have unveiled the complexities of the functional contributions of the KEAP1-NRF2 system and defined its broader involvement in biological processes, including cell proliferation and differentiation, as well as cytoprotection. In this review, we describe historical milestones in the initial characterization of the KEAP1-NRF2 system and provide a comprehensive overview of the molecular mechanisms governing the functions of KEAP1 and NRF2, as well as their roles in physiology and pathology. We also refer to the clinical significance of the KEAP1-NRF2 system as an important prophylactic and therapeutic target for various diseases, particularly aging-related disorders. We believe that controlled harnessing of the KEAP1-NRF2 system is a key to healthy aging and well-being in humans.

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.

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.

Novel NRF2-activated cancer treatments utilizing synthetic lethality.

The KEAP1-NRF2 pathway regulates the main inducible cellular response to oxidative and electrophilic stresses. Activating mutations in the KEAP1-NRF2 pathway occur commonly in human cancer, where they contribute to the formation of aggressive tumours that are associated with a poor prognosis for patients. An important clinical feature of these tumours is their defiance to all current anti-cancer treatment regimens, highlighting the need for the development of new therapeutic strategies to target NRF2-activated cancers. In this review, we discuss the mechanisms through which acquired NRF2 hyperactivation can result in resistance of tumours to immune checkpoint inhibitor therapies in addition to classical chemotherapeutics, and propose with examples that using a synthetic lethal strategy mediated by NRF2-target gene-dependent bioactivation of prodrugs represents a promising strategy to specifically enhance toxicity to heretofore untreatable NRF2-hyperactivated human tumours.

Adolescent and early adulthood inflammation-associated dietary patterns in relation to premenopausal mammographic density.

BACKGROUND: Adolescence and early adulthood has been identified as a critical time window for establishing breast cancer risk. Mammographic density is an independent risk factor for breast cancer that may be influenced by diet, but there has been limited research conducted on the impact of diet on mammographic density. Thus, we sought to examine the association between adolescent and early adulthood inflammatory dietary patterns, which have previously been associated with breast cancer risk, and premenopausal mammographic density among women in the Nurses' Health Study II (NHSII). METHODS: This study included control participants with premenopausal mammograms from an existing breast cancer case-control study nested within the NHSII who completed a Food Frequency Questionnaire in 1998 about their diet during high school (HS-FFQ) (n = 685) and/or a Food Frequency Questionnaire in 1991 (Adult-FFQ) when they were 27-44 years old (n = 1068). Digitized analog film mammograms were used to calculate the percent density, absolute dense, and non-dense areas. Generalized linear models were fit to evaluate the associations of a pro-inflammatory dietary pattern and the Alternative Healthy Eating Index (AHEI, an anti-inflammatory dietary pattern) with each breast density measure. RESULTS: Significant associations were observed between an adolescent pro-inflammatory dietary pattern and mammographic density in some age-adjusted models; however, these associations did not remain after adjustment for BMI and other breast cancer risk factors. No associations were observed with the pro-inflammatory pattern or with the AHEI pattern in adolescence or early adulthood in fully adjusted models. CONCLUSIONS: To our knowledge, this is the first study to evaluate the dietary patterns during adolescence and early adulthood in relation to mammographic density phenotypes. Our findings do not support an association between adolescent and early adulthood diet and breast density in mid-adulthood that is independent of BMI or other breast cancer risk factors.

Genetic or pharmacologic Nrf2 activation increases proteinuria in chronic kidney disease in mice.

The nuclear factor erythroid 2-related factor 2 (Nrf2) pathway upregulates key cellular defenses. Clinical trials are utilizing pharmacologic Nrf2 inducers such as bardoxolone methyl to treat chronic kidney disease, but Nrf2 activation has been linked to a paradoxical increase in proteinuria. To understand this effect, we examined genetically engineered mice with elevated Nrf2 signaling due to reduced expression of the Nrf2 inhibitor, Kelch-like ECH-associated protein 1 (Keap1). These Keap1FA/FA mice lacked baseline proteinuria but exhibited increased proteinuria in experimental models evoked by adriamycin, angiotensin II, or protein overload. After injury, Keap1FA/FA mice had increased glomerulosclerosis, nephrin disruption and shedding, podocyte injury, foot process effacement, and interstitial fibrosis. Keap1FA/FA mice also had higher daytime blood pressures and lower heart rates measured by radiotelemetry. Conversely, Nrf2 knockout mice were protected from proteinuria. We also examined the pharmacologic Nrf2 inducer CDDO-Im. Compared to angiotensin II alone, the combination of angiotensin II and CDDO-Im significantly increased proteinuria, a phenomenon not observed in Nrf2 knockout mice. This effect was not accompanied by additional increases in blood pressure. Finally, Nrf2 was found to be upregulated in the glomeruli of patients with focal segmental glomerulosclerosis, diabetic nephropathy, fibrillary glomerulonephritis, and membranous nephropathy. Thus, our studies demonstrate that Nrf2 induction in mice may exacerbate proteinuria in chronic kidney disease.

Biomonitoring of Ambient Outdoor Air Pollutant Exposure in Humans Using Targeted Serum Albumin Adductomics.

Outdoor air pollution, a spatially and temporally complex mixture, is a human carcinogen. However, ambient measurements may not reflect subject-level exposures, personal monitors do not assess internal dose, and spot assessments of urinary biomarkers may not recapitulate chronic exposures. Nucleophilic sites in serum albumin-particularly the free thiol at Cys34-form adducts with electrophiles. Due to the 4-week lifetime of albumin in circulation, accumulating adducts can serve as intermediate- to long-residence biomarkers of chronic exposure and implicate potential biological effects. Employing nanoflow liquid chromatography-high-resolution mass spectrometry (nLC-HRMS) and parallel reaction monitoring (PRM), we have developed and validated a novel targeted albumin adductomics platform capable of simultaneously monitoring dozens of Cys34 adducts per sample in only 2.5 µL of serum, with on-column limits of detection in the low-femtomolar range. Using this platform, we characterized the magnitude and impact of ambient outdoor air pollution exposures with three repeated measurements over 84 days in n = 26 nonsmoking women (n = 78 total samples) from Qidong, China, an area with a rising burden of lung cancer incidence. In concordance with seasonally rising ambient concentrations of NO2, SO2, and PM10 measured at stationary monitors, we observed elevations in concentrations of Cys34 adducts of benzoquinone (p < 0.05), benzene diol epoxide (BDE; p < 0.05), crotonaldehyde (p < 0.01), and oxidation (p < 0.001). Regression analysis revealed significant elevations in oxidation and BDE adduct concentrations of 300% to nearly 700% per doubling of ambient airborne pollutant levels (p < 0.05). Notably, the ratio of irreversibly oxidized to reduced Cys34 rose more than 3-fold during the 84-day period, revealing a dramatic perturbation of serum redox balance and potentially serving as a portent of increased pollution-related mortality risk. Our targeted albumin adductomics assay represents a novel and flexible approach for sensitive and multiplexed internal dosimetry of environmental exposures, providing a new strategy for personalized biomonitoring and prevention.

Serum miR-182 is a predictive biomarker for dichotomization of risk of hepatocellular carcinoma in rats.

Exploration of animal models leads to discoveries that can reveal candidate biomarkers for translation to human populations. Herein, a model of hepatocarcinogenesis and protection was used in which rats treated with aflatoxin (AFB1 ) daily for 28 days (200 µg/kg BW) developed tumors compared with rats completely protected from tumors by concurrent administration of the chemoprotective agent, 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im). Differential expression of miRNAs in tumors (AFB1 ) and nontumor (AFB1 + CDDO-Im) bearing livers and their levels in sera over the life-course of the animals was determined. miRNA transcriptome analysis identified 17 miRNAs significantly upregulated at greater than five-fold in the tumors. The ten most dysregulated miRNAs judged by fold-change and biological significance were selected for further study, including liver-specific miR-122-5p. Validation of sequencing results by real-time PCR confirmed the upregulation of the majority of these miRNAs in tumors, including miR-182, as well as miR-224-5p as the most dysregulated of these miRNAs (over 400-fold). The longitudinal analysis of levels of miR-182 in sera demonstrated significant and persistent increases (5.13-fold; 95% CI: 4.59-5.70). The increase in miR-182 was detected months before any clinical symptoms were present in the animals. By the terminal time point of the study, in addition to elevated levels of serum miR-182, serum miR-122-5p was also found to be increased (>1.5-fold) in animals that developed hepatocarcinomas. Thus, using the data from an unbiased discovery approach of the tissue findings, serum miR-182 was found to track across the complex, multistage process of hepatocarcinogenesis opening an opportunity for translation to human populations.

Broccoli or Sulforaphane: Is It the Source or Dose That Matters?

There is robust epidemiological evidence for the beneficial effects of broccoli consumption on health, many of them clearly mediated by the isothiocyanate sulforaphane. Present in the plant as its precursor, glucoraphanin, sulforaphane is formed through the actions of myrosinase, a ß-thioglucosidase present in either the plant tissue or the mammalian microbiome. Since first isolated from broccoli and demonstrated to have cancer chemoprotective properties in rats in the early 1990s, over 3000 publications have described its efficacy in rodent disease models, underlying mechanisms of action or, to date, over 50 clinical trials examining pharmacokinetics, pharmacodynamics and disease mitigation. This review evaluates the current state of knowledge regarding the relationships between formulation (e.g., plants, sprouts, beverages, supplements), bioavailability and efficacy, and the doses of glucoraphanin and/or sulforaphane that have been used in pre-clinical and clinical studies. We pay special attention to the challenges for better integration of animal model and clinical studies, particularly with regard to selection of dose and route of administration. More effort is required to elucidate underlying mechanisms of action and to develop and validate biomarkers of pharmacodynamic action in humans. A sobering lesson is that changes in approach will be required to implement a public health paradigm for dispensing benefit across all spectrums of the global population.

Pharmacogenomics of Chemically Distinct Classes of Keap1-Nrf2 Activators Identify Common and Unique Gene, Protein, and Pathway Responses In Vivo.

The Kelch-like erythroid-associated protein 1 (Keap1)-NF-E2-related factor 2 (Nrf2) signaling pathway is the subject of several clinical trials evaluating the effects of Nrf2 activation on the prevention of cancer and diabetes and the treatment of chronic kidney disease and multiple sclerosis. 3H-1,2-dithiole-3-thione (D3T) and 1-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im) are representative members of two distinct series of Nrf2 chemical activators. Previous reports have described activator-specific effects on Nrf2-dependent gene regulation and physiologic outcomes. Here we used a robust chemical genomics approach to characterize expression profiles between D3T and CDDO-Im in livers from wild-type and Nrf2-null mice. At equally efficacious doses in wild-type mice, 406 genes show common RNA responses to both treatments. These genes enriched the Nrf2-regulated pathways of antioxidant defense and xenobiotic metabolism. In addition, 197 and 745 genes were regulated uniquely in response to either D3T or CDDO-Im, respectively. Functional analysis of the D3T-regulated set showed a significant enrichment of Nrf2-regulated enzymes involved in cholesterol biosynthesis. This result was supported by Nrf2-dependent increases in lanosterol synthase and CYP51 protein expression. CDDO-Im had no effect on cholesterol biosynthesis regardless of the dose tested. However, unlike D3T, CDDO-Im resulted in Nrf2-dependent elevation of peroxisome proliferator α and Kruppel-like factor 13, as well as the coactivator peroxisome proliferator γ coactivator 1ß, together indicating regulation of ß-oxidation and lipid metabolic pathways. These findings provide novel insights into the pharmacodynamic action of these two activators of Keap1-Nrf2 signaling. Although both compounds modify Keap1 to affect canonical cytoprotective gene expression, additional unique sets of Nrf2-dependent genes were regulated by each agent with enrichment of selective metabolic pathways.

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.

Profound changes in miRNA expression during cancer initiation by aflatoxin B1 and their abrogation by the chemopreventive triterpenoid CDDO-Im.

Aflatoxin B1 (AFB1 ) is a potent human and animal hepatocarcinogen. To investigate the effects of aflatoxin on miRNA expression during the initiation phase of carcinogenesis, next-generation sequencing was used to analyze liver tissues from F344 rats exposed to 200 µg/kg per day AFB1 for 4 week. A panel of miRNAs was identified that was upregulated with AFB1 treatment compared to controls: rno-miR-434-3p, rno-miR-411-5p, rno-miR-221-3p, rno-miR-127-3p, rno-miR-205, rno-miR-429, rno-miR-34a-5p, rno-miR-181c-3p, rno-miR-200b-3p, and rno-miR-541-5p. Analysis of rat livers exposed to AFB1 plus the chemopreventive triterpenoid CDDO-Im revealed a striking abrogation of this upregulation. These changes were validated by real-time PCR. We also explored the temporal variation in expression of the candidate miRNAs during the 4-week dosing period. Most of the candidate miRNAs were upregulated at week 1 and increased for the duration of AFB1 dosing over the 4-week period. Treatment with CDDO-Im ameliorated these effects at all time points. All candidate miRNAs were detectable in serum from aflatoxin treated animals; however, there was no significant difference in expression for 7 of the 11 miRNAs examined. Exposure to AFB1 upregulated miR-122-5p (fivefold), 34a-5p (13-fold), and 181c-3p (170-fold) compared with controls. The findings from this study give insight into epigenetic changes induced by aflatoxin taking place during the initial step of carcinogenesis.

Disruption of nuclear factor (erythroid-derived-2)-like 2 antioxidant signaling: a mechanism for impaired activation of stem cells and delayed regeneration of skeletal muscle.

Recently we have reported that age-dependent decline in antioxidant levels accelerated apoptosis and skeletal muscle degeneration. Here, we demonstrate genetic ablation of the master cytoprotective transcription factor, nuclear factor (erythroid-derived-2)-like 2 (Nrf2), aggravates cardiotoxin (CTX)-induced tibialis anterior (TA) muscle damage. Disruption of Nrf2 signaling sustained the CTX-induced burden of reactive oxygen species together with compromised expression of antioxidant genes and proteins. Transcript/protein expression of phenotypic markers of muscle differentiation, namely paired box 7 (satellite cell) and early myogenic differentiation and terminal differentiation (myogenin and myosin heavy chain 2) were increased on d 2 and 4 postinjury but later returned to baseline levels on d 8 and 15 in wild-type (WT) mice. In contrast, these responses were persistently augmented in Nrf2-null mice suggesting that regulation of the regeneration-related signaling mechanisms require Nrf2 for normal functioning. Furthermore, Nrf2-null mice displayed slower regeneration marked by dysregulation of embryonic myosin heavy chain temporal expression. Histologic observations illustrated that Nrf2-null mice displayed smaller, immature TA muscle fibers compared with WT counterparts on d 15 after CTX injury. Improvement in TA muscle morphology and gain in muscle mass evident in the WT mice was not noticeable in the Nrf2-null animals. Taken together these data show that the satellite cell activation, proliferation, and differentiation requires a functional Nrf2 system for effective healing following injury.-Shelar, S. B., Narasimhan, M., Shanmugam, G., Litovsky, S. H., Gounder, S. S., Karan, G., Arulvasu, C., Kensler, T. W., Hoidal, J. R., Darley-Usmar, V. M., Rajasekaran, N. S. Disruption of nuclear factor (erythroid-derived-2)-like 2 antioxidant signaling: a mechanism for impaired activation of stem cells and delayed regeneration of skeletal muscle.

KEAP1 and Done? Targeting the NRF2 Pathway with Sulforaphane.

BACKGROUND: Since the re-discovery of sulforaphane in 1992 and the recognition of the bioactivity of this phytochemical, many studies have examined its mode of action in cells, animals and humans. Broccoli, especially as young sprouts, is a rich source of sulforaphane and broccoli-based preparations are now used in clinical studies probing efficacy in health preservation and disease mitigation. Many putative cellular targets are affected by sulforaphane although only one, KEAP1-NRF2 signaling, can be considered a validated target at this time. The transcription factor NRF2 is a master regulator of cell survival responses to endogenous and exogenous stressors. SCOPE AND APPROACH: This review summarizes the chemical biology of sulforaphane as an inducer of NRF2 signaling and efficacy as an inhibitor of carcinogenesis. It also provides a summary of the current findings from clinical trials using a suite of broccoli sprout preparations on a series of short-term endpoints reflecting a diversity of molecular actions. KEY FINDINGS AND CONCLUSIONS: Sulforaphane, as a pure chemical, protects against chemical-induced skin, oral, stomach, colon, lung and bladder carcinogenesis and in genetic models of colon and prostate carcinogenesis. In many of these settings the antitumorigenic efficacy of sulforaphane is dampened in Nrf2-disrupted animals. Broccoli preparations rich in glucoraphanin or sulforaphane exert demonstrable pharmacodynamic action in over a score of clinical trials. Measures of NRF2 pathway response and function are serving as guideposts for the optimization of dose, schedule and formulation as clinical trials with broccoli-based preparations become more commonplace and more rigorous in design and implementation.

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.

NRF2/long noncoding RNA ROR signaling regulates mammary stem cell expansion and protects against estrogen genotoxicity.

Long noncoding RNAs (lncRNAs) have emerged as key regulators of gene expression in embryonic stem cell (ESC) self-renewal and differentiation. In ESCs, lncRNAs are regulated at the genetic level via transcription factor binding to lncRNA gene promoters. Here we demonstrate that the key cytoprotective transcription factor NRF2 controls lncRNA expression in mammary stem cells. By profiling lncRNAs in wild-type and NRF2 knockdown mammary stem cells, we demonstrate that the lncRNA ROR, a regulator of embryonic stem cell pluripotency, is overexpressed upon NRF2 knockdown. We performed promoter analyses and examined predicted NRF2 binding elements in the ROR promoter using luciferase reporter constructs of a ROR promoter deletion series. Our studies revealed that NRF2 binds to two specific NRF2 response elements flanking the ROR promoter and that these two NRF2 response elements are equally important to suppress ROR transcription. In addition, we identified associated H3K27me3 chromatin modification and EZH2 binding at the ROR promoter that was dependent on NRF2 binding. We observed that NRF2 knockdown or ROR overexpression leads to increased stem cell self-renewal in mammary stem cells. Furthermore, we demonstrate Nrf2 regulation of the mammary stem cell population in vivo. These observations provide further evidence for the critical role of NRF2 in maintaining normal stem cell subpopulations in mammary epithelium.

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.

The Nrf2 triterpenoid activator, CDDO-imidazolide, protects kidneys from ischemia-reperfusion injury in mice.

Acute kidney injury (AKI) caused by ischemia-reperfusion is a major clinical problem in both native and transplanted kidneys. We had previously shown that deficiency of Nrf2, a potent bZIP transcription factor that binds to the antioxidant response element, enhances susceptibility to experimental ischemic AKI. Here we further explored the role of Nrf2 in AKI by amplifying Nrf2 activation in vivo and in vitro with the synthetic triterpenoid CDDO-imidazolide. Mice treated with CDDO-imidazolide and undergoing experimental bilateral ischemic AKI had improved survival and renal function. Treated mice had improved renal histology with a decrease in tubular injury, as well as a decrease in proinflammatory cytokine and chemokine production compared with vehicle-treated mice. In an exploration of protective mechanisms, we found an upregulation of Nrf2 target antioxidant genes in CDDO-imidazolide-treated mouse kidneys. Furthermore, Nrf2-deficient mice treated with CDDO-imidazolide had no significant improvement in mortality, renal function or histology, proinflammatory cytokine gene expression, and no significant increase in antioxidant gene expression. In vitro studies demonstrated that the renal epithelial cells were likely an important target of CDDO-imidazolide. Thus, activation of Nrf2 signaling with CDDO-imidazolide confers protection from AKI, and presents a new therapeutic opportunity for this common and serious condition.