Nrf2-null Mice Research Articles

Nrf2-Mediated Neuroprotection Against Recurrent Hypoglycemia Is Insufficient to Prevent Cognitive Impairment in a Rodent Model of Type 1 Diabetes.

It remains uncertain whether recurrent nonsevere hypoglycemia (Hypo) results in long-term cognitive impairment in type 1 diabetes (T1D). This study tested the hypothesis that specifically in the T1D state, Hypo leads to cognitive impairment via a pathological response to oxidative stress. Wild-type (Control) and nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) null mice were studied. Eight groups of mice (Control and Nrf2(-/-) ± T1D and ± Hypo) were subject to recurrent, twice-weekly, insulin or saline injections over 4 weeks, after which cognitive function was assessed and brain tissue analyzed. Recurrent moderate hypoglycemia in T1D, but not Control, mice significantly impaired cognitive performance, and this was associated with hippocampal oxidative damage and inflammation despite an enhanced expression of Nrf2 and its target genes Hmox1 and Nqo1 In Nrf2(-/-) mice, both T1D and Hypo independently resulted in impaired cognitive performance, and this was associated with oxidative cell damage and marked inflammation. Together, these data suggest that Hypo induces an Nrf2-dependent antioxidant response in the hippocampus, which counteracts oxidative damage. However, in T1D, this neuroprotective mechanism is insufficient to prevent neuronal oxidative damage, resulting in chronic deficits in working and long-term memory.

NF-E2-related factor 2 deletion facilitates hepatic fatty acids metabolism disorder induced by high-fat diet via regulating related genes in mice

There is increasing evidence that Nrf2 participates in hepatic fatty acid metabolism in non-alcoholic fatty liver disease; however, the mechanism remains unclear. We investigated the role of Nrf2 in hepatic fatty acid metabolism disorder induced by high-fat diet (HFD). Mice fed HFD developed hepatic steatosis and exhibited Nrf2 deficiency. Change of fatty acid composition mediated by Nrf2 deletion was observed predominantly in the liver and not the serum. HFD-induced variations in hepatic 18-carbon and 22-carbon fatty acids were enhanced by Nrf2 deficiency. In the HFD group, Nrf2 deficiency led to increases in the mRNA expression of PPARα, FXR, FAS, LXR and ACC-1, while levels of PGC-1α and Srebp-1c mRNA were decreased. Nrf2 mRNA expression was enhanced in the liver of HFD-induced wild type mice, whereas it was undetectable in Nrf2-null mice. These results suggest that Nrf2 deficiency induced by HFD promoted hepatic fatty acid metabolism disorder by altering 18-carbon and 22-carbon fatty acid composition. Changes in fatty acid content were also associated with alteration of the transcription of genes involved in hepatic fatty acid metabolism.

Distinct Nrf2 Signaling Mechanisms of Fumaric Acid Esters and Their Role in Neuroprotection against 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine-Induced Experimental Parkinson's-Like Disease.

A promising approach to neurotherapeutics involves activating the nuclear-factor-E2-related factor 2 (Nrf2)/antioxidant response element signaling, which regulates expression of antioxidant, anti-inflammatory, and cytoprotective genes. Tecfidera, a putative Nrf2 activator, is an oral formulation of dimethylfumarate (DMF) used to treat multiple sclerosis. We compared the effects of DMF and its bioactive metabolite monomethylfumarate (MMF) on Nrf2 signaling and their ability to block 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced experimental Parkinson's disease (PD). We show that in vitro DMF and MMF activate the Nrf2 pathway via S-alkylation of the Nrf2 inhibitor Keap1 and by causing nuclear exit of the Nrf2 repressor Bach1. Nrf2 activation by DMF but not MMF was associated with depletion of glutathione, decreased cell viability, and inhibition of mitochondrial oxygen consumption and glycolysis rates in a dose-dependent manner, whereas MMF increased these activities in vitro However, both DMF and MMF upregulated mitochondrial biogenesis in vitro in an Nrf2-dependent manner. Despite the in vitro differences, both DMF and MMF exerted similar neuroprotective effects and blocked MPTP neurotoxicity in wild-type but not in Nrf2 null mice. Our data suggest that DMF and MMF exhibit neuroprotective effects against MPTP neurotoxicity because of their distinct Nrf2-mediated antioxidant, anti-inflammatory, and mitochondrial functional/biogenetic effects, but MMF does so without depleting glutathione and inhibiting mitochondrial and glycolytic functions. Given that oxidative damage, neuroinflammation, and mitochondrial dysfunction are all implicated in PD pathogenesis, our results provide preclinical evidence for the development of MMF rather than DMF as a novel PD therapeutic. Almost two centuries since its first description by James Parkinson, Parkinson's disease (PD) remains an incurable disease with limited symptomatic treatment. The current study provides preclinical evidence that a Food and Drug Administration-approved drug, dimethylfumarate (DMF), and its metabolite monomethylfumarate (MMF) can block nigrostriatal dopaminergic neurodegeneration in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of PD. We elucidated mechanisms by which DMF and its active metabolite MMF activates the redox-sensitive transcription factor nuclear-factor-E2-related factor 2 (Nrf2) to upregulate antioxidant, anti-inflammatory, mitochondrial biosynthetic and cytoprotective genes to render neuroprotection via distinct S-alkylating properties and depletion of glutathione. Our data suggest that targeting Nrf2-mediated gene transcription using MMF rather than DMF is a promising approach to block oxidative stress, neuroinflammation, and mitochondrial dysfunction for therapeutic intervention in PD while minimizing side effects.

Nuclear Factor Erythroid 2-Related Factor 2 Deficiency Results in Amplification of the Liver Fat-Lowering Effect of Estrogen.

Transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) regulates multiple biologic processes, including hepatic lipid metabolism. Estrogen exerts actions affecting energy homeostasis, including a liver fat-lowering effect. Increasing evidence indicates the crosstalk between these two molecules. The aim of this study was to evaluate whether Nrf2 modulates estrogen signaling in hepatic lipid metabolism. Nonalcoholic fatty liver disease (NAFLD) was induced in wild-type and Nrf2-null mice fed a high-fat diet and the liver fat-lowering effect of exogenous estrogen was subsequently assessed. We found that exogenous estrogen eliminated 49% and 90% of hepatic triglycerides in wild-type and Nrf2-null mice with NAFLD, respectively. This observation demonstrates that Nrf2 signaling is antagonistic to estrogen signaling in hepatic fat metabolism; thus, Nrf2 absence results in striking amplification of the liver fat-lowering effect of estrogen. In addition, we found the association of trefoil factor 3 and fatty acid binding protein 5 with the liver fat-lowering effect of estrogen. In summary, we identified Nrf2 as a novel and potent inhibitor of estrogen signaling in hepatic lipid metabolism. Our finding may provide a potential strategy to treat NAFLD by dually targeting Nrf2 and estrogen signaling.

Nrf2 activation skews murine and human CD4 T cell differentiation towards a Th2 phenotype

Abstract CD4 T cell differentiation is a critical part of the adaptive immune response during which CD4 T cells adapt a response to a specific pathogen. Nuclear factor erythroid-derived 2 like-2 (Nrf2) is a cell-stress responsive transcription factor that regulates a battery of antioxidant, detoxification, and cytoprotective genes. Nrf2 is activated by stressors such as reactive oxygen species and electrophilic xenobiotics, including tert-butylhydroquinone (tBHQ), a commonly used food preservative. Nrf2 has been shown to be immunomodulatory: Nrf2 null mice are more susceptible to inflammatory stimuli such as sepsis, lung injury, and experimental autoimmune encephalomyelitis. In addition, Nrf2 has been shown to modulate dendritic cell and macrophage function, however, the role of Nrf2 in CD4 T cells remains only partially characterized. Thus, the goal of the present studies is to determine the role of Nrf2 in CD4 T cell differentiation in primary human and mouse CD4 T cells. Treatment with the Nrf2 activator tBHQ increased production of the Th2 cytokines IL-4, IL-5, and IL-13 in CD4 T cells isolated from wild-type, but not Nrf2-null mice, and decreased production of the Th1 cytokine IFNγ. tBHQ increased GATA-3 and decreased T-bet DNA binding in wild type but not Nrf2-null mice. In primary human CD4 T cells, tBHQ treatment increased production of the cytokines IL-4, IL-5, and IL-13, and had no effect on IFNγ production upon restimulation. Collectively, these data suggest that Nrf2 activation promotes Th2 differentiation of primary human and mouse CD4 T cells. (Funded by NIH grants ES018885 and ES007255).

The Nrf2 activator, tBHQ, inhibits IL-2 production by human T cells in a largely Nrf2-independent manner

Abstract Nrf2 is a transcription factor that is activated by cell stress, such as oxidative insult and exposure to xenobiotics. Upon activation, Nrf2 translocates to the nucleus, where it upregulates its target genes, many of which arecytoprotective in function. Nrf2 has been shown to modulate immune function; Nrf2-null mice are more sensitive to many models of inflammatory disease, including experimental autoimmune encephalitis and sepsis induced by cecal ligation and puncture. We have previously shown that activation of Nrf2 in primary mouse CD4 T cells promotes Th2 differentiation, which may have implications on the development of allergy and asthma. However, the role of Nrf2 in human CD4 T cell function remains uncharacterized. Thus, the purpose of the present studies was to determine the role of Nrf2 in the early events following CD4 T cell activation. Primary human CD4 T cells from peripheral blood were treated with varying concentrations of tBHQ (a known Nrf2 activator) prior to activation with anti-CD3/anti-CD28. Although treatment of human T cells with tBHQ suppressed the production of early cytokines, such as IL-2, the model fell short in determining whether these effects were Nrf2-dependent. Toward that end, Nrf2-null T cells were generated by implementation of CRISPR-CAS9 gene editing, in human Jurkat T cells. Treatment of Nrf2-null and Wild-type Jurkat T cells demonstrated that the effects of tBHQ upon cytokine production are largely independent of Nrf2. In contrast, suppression of CD25 expression by tBHQ is partially Nrf2-dependent. The current studies demonstrate that many of the effects of tBHQ on early events following CD4 T cell activation are independent of Nrf2. (This work was funded by NIH grants ES018885 and GM092715)

Green tea extract provides extensive Nrf2-independent protection against lipid accumulation and NFκB pro- inflammatory responses during nonalcoholic steatohepatitis in mice fed a high-fat diet.

Green tea extract (GTE) reduces liver steatosis and inflammation during nonalcoholic steatohepatitis (NASH). We hypothesized GTE would mitigate NASH in a nuclear factor erythroid-2-related-factor-2 (Nrf2)-dependent manner in a high fat (HF) induced model. Nrf2-null and wild-type (WT) mice were fed an HF diet containing 0 or 2% GTE for eight weeks prior to assessing parameters of NASH. Compared to WT mice, Nrf2-null mice had increased serum alanine aminotransferase, hepatic triglyceride, expression of free fatty acid uptake and lipogenic genes, malondialdehyde and NFκB phosphorylation and expression of pro-inflammatory genes. In WT mice, GTE increased Nrf2 and NADPH:quinone oxidoreductase-1 mRNA, and lowered hepatic steatosis, lipid uptake and lipogenic gene expression, malondialdehyde, and NFκB-dependent inflammation. In Nrf2-null mice, GTE lowered NFκB phosphorylation and TNF-α and MCP1 mRNA to levels observed in WT mice fed GTE whereas hepatic triglyceride and lipogenic genes were lowered only to those of WT mice fed no GTE. Malondialdehyde was lowered in Nrf2-null mice fed GTE, but not to levels of WT mice, and without improving the hepatic antioxidants α-tocopherol, ascorbic acid and uric acid. Nrf2 deficiency exacerbates NASH whereas anti-inflammatory and hypolipidemic activities of GTE likely occur largely independent of Nrf2 signaling.

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.

Nrf2 Deficiency Promotes Melanoma Growth and Lung Metastasis.

The role of Nrf2, a key regulator of antioxidant and cytoprotective genes, in tumorigenesis remains controversial. Here we showed that Nrf2 deficiency led to increased local tumor growth in mice following subcutaneous injection of B16-F10 melanoma cells, as indicated by increased proportion of animals with locally palpable tumor mass and time-dependent increases in tumor volume at the injection site. In vivo bioluminescence imaging also revealed increased growth of melanoma in Nrf2-null mice as compared with wild-type mice. By using a highly sensitive bioluminometric assay, we further found that Nrf2 deficiency resulted in a remarkable increase in lung metastasis of B16-F10 melanoma cells as compared with wild-type mice. Taken together, the results of this short communication for the first time demonstrated that Nrf2 deficiency promoted melanoma growth and lung metastasis following subcutaneous inoculation of B16-F10 cells in mice.

Metallothionein plays a prominent role in the prevention of diabetic nephropathy by sulforaphane via up-regulation of Nrf2

Sulforaphane (SFN) prevents diabetic nephropathy (DN) in type 1 diabetes via up-regulation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2). However, it has not been addressed whether SFN also prevents DN from type 2 diabetes or which Nrf2 downstream gene(s) play(s) the key role in SFN renal protection. Here we investigated whether Nrf2 is required for SFN protection against type 2 diabetes-induced DN and whether metallothionein (MT) is an Nrf2 downstream antioxidant using Nrf2 knockout (Nrf2-null) mice. In addition, MT knockout mice were used to further verify if MT is indispensable for SFN protection against DN. Diabetes-increased albuminuria, renal fibrosis, and inflammation were significantly prevented by SFN, and Nrf2 and MT expression was increased. However, SFN renal protection was completely lost in Nrf2-null diabetic mice, confirming the pivotal role of Nrf2 in SFN protection from type 2 diabetes-induced DN. Moreover, SFN failed to up-regulate MT in the absence of Nrf2, suggesting that MT is an Nrf2 downstream antioxidant. MT deletion resulted in a partial, but significant attenuation of SFN renal protection from type 2 diabetes, demonstrating a partial requirement for MT for SFN renal protection. Therefore, the present study demonstrates for the first time that as an Nrf2 downstream antioxidant, MT plays an important, though partial, role in mediating SFN renal protection from type 2 diabetes.

Nrf2 is essential for timely M phase entry of replicating hepatocytes during liver regeneration.

The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) regulates various cellular activities, including redox balance, detoxification, metabolism, autophagy, proliferation, and apoptosis. Several studies have demonstrated that Nrf2 regulates hepatocyte proliferation during liver regeneration. The aim of this study was to investigate how Nrf2 modulates the cell cycle of replicating hepatocytes in regenerating livers. Wild-type and Nrf2 null mice were subjected to 2/3 partial hepatectomy (PH) and killed at multiple time points for various analyses. Nrf2 null mice exhibited delayed liver regrowth, although the lost liver mass was eventually restored 7 days after PH. Nrf2 deficiency did not affect the number of hepatocytes entering the cell cycle but did delay hepatocyte mitosis. Mechanistically, the lack of Nrf2 resulted in increased mRNA and protein levels of hepatic cyclin A2 when the remaining hepatocytes were replicating in response to PH. Moreover, Nrf2 deficiency in regenerating livers caused dysregulation of Wee1, Cdc2, and cyclin B1 mRNA and protein expression, leading to decreased Cdc2 activity. Thus, Nrf2 is required for timely M phase entry of replicating hepatocytes by ensuring proper regulation of cyclin A2 and the Wee1/Cdc2/cyclin B1 pathway during liver regeneration.

Protection against phalloidin-induced liver injury by oleanolic acid involves Nrf2 activation and suppression of Oatp1b2

This study utilized pharmacological activation of Nrf2 with oleanolic acid (OA, 22.5mg/kg, sc for 4 days) and the genetic alteration of Nrf2 (Nrf2-null, wild-type, and Keap1-HKO mice) to examine the role of Nrf2 in protection against phalloidin hepatotoxicity. Mice were given phalloidin (1.5mg/kg, ip for 8h) to examine liver injury and the expression of toxicity-related genes. Phalloidin increased serum enzyme activities and caused extensive hepatic hemorrhage and necrosis in Nrf2-null and wild-type mice, but less injury was seen in Keap1-HKO mice and OA-pretreated mice. Phalloidin increased the expression of neutrophil-specific chemokine mKC and MIP-2 in Nrf2-null and WT mice, but such increases were attenuated in Keap1-HKO and OA-pretreated mice. Phalloidin increased, while Nrf2 activation attenuated, the expression of genes involved in acute-phase response (Ho-1) and DNA-damage response genes (Gadd45 and Chop10). Phalloidin is taken up by hepatocytes through Oatp1b2, but there was no difference in basal and phalloidin-induced Oatp1b2 expression among Nrf2-null, wild-type, and Keap1-HKO mice. In contrast, OA decreased phalloidin-induced Oatp1b2. Phalloidin activated MAPK signaling (p-JNK), which was attenuated by activation of Nrf2. In conclusion, this study demonstrates that protection against phalloidin hepatotoxicity by OA involves activation of Nrf2 and suppression of Oatp1b2.

Nrf2 is involved in maintaining hepatocyte identity during liver regeneration.

Nrf2, a central regulator of the cellular defense against oxidative stress and inflammation, participates in modulating hepatocyte proliferation during liver regeneration. It is not clear, however, whether Nrf2 regulates hepatocyte growth, an important cellular mechanism to regain the lost liver mass after partial hepatectomy (PH). To determine this, various analyses were performed in wild-type and Nrf2-null mice following PH. We found that, at 60 h post-PH, the vast majority of hepatocytes lacking Nrf2 reduced their sizes, activated hepatic progenitor markers (CD133, TWEAK receptor, and trefoil factor family 3), depleted HNF4α protein, and downregulated the expression of a group of genes critical for their functions. Thus, the identity of hepatocytes deficient in Nrf2 was transiently but massively impaired in response to liver mass loss. This event was associated with the coupling of protein depletion of hepatic HNF4α, a master regulator of hepatocyte differentiation, and concomitant inactivation of hepatic Akt1 and p70S6K, critical hepatocyte growth signaling molecules. We conclude that Nrf2 participates in maintaining newly regenerated hepatocytes in a fully differentiated state by ensuring proper regulation of HNF4α, Akt1, and p70S6K during liver regeneration.

Susceptibility of Nrf2-null mice to steatohepatitis and cirrhosis upon consumption of a high-fat diet is associated with oxidative stress, perturbation of the unfolded protein response, and disturbance in the expression of metabolic enzymes but not with insulin resistance.

Mice lacking the transcription factor NF-E2 p45-related factor 2 (Nrf2) develop more severe nonalcoholic steatohepatitis (NASH), with cirrhosis, than wild-type (Nrf2+/+) mice when fed a high-fat (HF) diet for 24 weeks. Although NASH is usually associated with insulin resistance, HF-fed Nrf2−/− mice exhibited better insulin sensitivity than HF-fed Nrf2+/+ mice. In livers of HF-fed mice, loss of Nrf2 resulted in greater induction of lipogenic genes, lower expression of β-oxidation genes, greater reduction in AMP-activated protein kinase (AMPK) levels, and diminished acetyl coenzyme A (CoA) carboxylase phosphorylation than in the wild-type livers, which is consistent with greater fatty acid (FA) synthesis in Nrf2−/− livers. Moreover, primary Nrf2−/− hepatocytes displayed lower glucose and FA oxidation than Nrf2+/+ hepatocytes, with FA oxidation partially rescued by treatment with AMPK activators. The unfolded protein response (UPR) was perturbed in control regular-chow (RC)-fed Nrf2−/− mouse livers, and this was associated with constitutive activation of NF-κB and JNK, along with upregulation of inflammatory genes. The HF diet elicited an antioxidant response in Nrf2+/+ livers, and as this was compromised in Nrf2−/− livers, they suffered oxidative stress. Therefore, Nrf2 protects against NASH by suppressing lipogenesis, supporting mitochondrial function, increasing the threshold for the UPR and inflammation, and enabling adaptation to HF-diet-induced oxidative stress.

Nrf2 upregulates ATP binding cassette transporter expression and activity at the blood-brain and blood-spinal cord barriers.

Activation of nuclear factor E2-related factor-2 (Nrf2), a sensor of oxidative stress, is neuroprotective in animal models of cerebral ischemia, traumatic brain injury, subarachnoid hemorrhage, and spinal cord injury. We show here that Nrf2 activation with sulforaphane (SFN) in vivo or in vitro increases expression and transport activity of three ATP-driven drug efflux pumps at the blood-brain barrier [P-glycoprotein, ATP binding cassette b1 (Abcb1); multidrug resistance-associated protein-2 (Mrp2), Abcc2; and breast cancer resistance protein (Bcrp), Abcg2]. Dosing rats with SFN increased protein expression of all three transporters in brain capillaries and decreased by 50% brain accumulation of the P-glycoprotein substrate verapamil. Exposing rat or mouse brain capillaries to SFN increased P-glycoprotein, Bcrp, and Mrp2 transport activity and protein expression; SFN increased P-glycoprotein activity in mouse spinal cord capillaries. Inhibiting transcription or translation abolished upregulation of P-glycoprotein activity. No such effects were seen in brain capillaries from Nrf2-null mice, indicating Nrf2 dependence. Nrf2 signaled indirectly to increase transporter activity/expression. The p53 inhibitor pifithrin abolished the SFN-induced increase in transporter activity/expression, and the p53-activator nutlin-3 increased P-glycoprotein activity. SFN did not alter P-glycoprotein transport activity in brain and spinal cord capillaries from p53-null mice. Inhibitors of p38 MAPK and nuclear factor κB (NF-κB) blocked the effects of SFN and nutlin-3 on P-glycoprotein activity. These results implicate Nrf2, p53, and NF-κB in the upregulation of P-glycoprotein, Bcrp, and Mrp2 at blood-CNS barriers. They imply that the barriers are tightened selectively (efflux transporter upregulation) by oxidative stress, providing increased neuroprotection, but also reduced penetration of many therapeutic drugs.

Identification and quantification of the basal and inducible Nrf2-dependent proteomes in mouse liver: biochemical, pharmacological and toxicological implications.

The transcription factor Nrf2 is a master regulator of cellular defence: Nrf2 null mice (Nrf2(−/−)) are highly susceptible to chemically induced toxicities. We report a comparative iTRAQ-based study in Nrf2(−/−) mice treated with a potent inducer, methyl-2-cyano-3,12-dioxooleana-1,9(11)dien-28-oate (CDDO-me; bardoxolone -methyl), to define both the Nrf2-dependent basal and inducible hepatoproteomes. One thousand five hundred twenty-one proteins were fully quantified (FDR < 1%). One hundred sixty-one were significantly different (P < 0.05) between WT and Nrf2(−/−) mice, confirming extensive constitutive regulation by Nrf2. Treatment with CDDO-me (3 mg/kg; i.p.) resulted in significantly altered expression of 43 proteins at 24 h in WT animals. Six proteins were regulated at both basal and inducible levels exhibiting the largest dynamic range of Nrf2 regulation: cytochrome P4502A5 (CYP2A5; 17.2-fold), glutathione-S-transferase-Mu 3 (GSTM3; 6.4-fold), glutathione-S-transferase Mu 1 (GSTM1; 5.9-fold), ectonucleoside-triphosphate diphosphohydrolase (ENTPD5; 4.6-fold), UDP-glucose-6-dehydrogenase (UDPGDH; 4.1-fold) and epoxide hydrolase (EPHX1; 3.0-fold). These proteins, or their products, thus provide a potential source of biomarkers for Nrf2 activity. ENTPD5 is of interest due to its emerging role in AKT signalling and, to our knowledge, this protein has not been previously shown to be Nrf2-dependent. Only two proteins altered by CDDO-me in WT animals were similarly affected in Nrf2(−/−) mice, demonstrating the high degree of selectivity of CDDO-me for the Nrf2:Keap1 signalling pathway.Biological significanceThe Nrf2:Keap1 signalling pathway is attracting considerable interest as a therapeutic target for different disease conditions. For example, CDDO-me (bardoxolone methyl) was investigated in clinical trials for the treatment of acute kidney disease, and dimethyl fumarate, recently approved for reducing relapse rate in multiple sclerosis, is a potent Nrf2 inducer. Such compounds have been suggested to act through multiple mechanisms; therefore, it is important to define the selectivity of Nrf2 inducers to assess the potential for off-target effects that may lead to adverse drug reactions, and to provide biomarkers with which to assess therapeutic efficacy. Whilst there is considerable information on the global action of such inducers at the mRNA level, this is the first study to catalogue the hepatic protein expression profile following acute exposure to CDDO-me in mice. At a dose shown to evoke maximal Nrf2 induction in the liver, CDDO-me appeared highly selective for known Nrf2-regulated proteins. Using the transgenic Nrf2(−/−) mouse model, it could be shown that 97% of proteins induced in wild type mice were associated with a functioning Nrf2 signalling pathway. This analysis allowed us to identify a panel of proteins that were regulated both basally and following Nrf2 induction. Identification of these proteins, which display a large magnitude of variation in their expression, provides a rich source of potential biomarkers for Nrf2 activity for use in experimental animals, and which may be translatable to man to define individual susceptibility to chemical stress, including that associated with drugs, and also to monitor the pharmacological response to Nrf2 inducers.

Nrf2 protects against furosemide-induced hepatotoxicity.

Furosemide is a diuretic drug, but its reactive intermediates lead to acute liver injury in mice. Given the essential role of Nrf2 as a cellular defense regulator, we investigated whether Nrf2 would protect against furosemide-induced liver injury using the Nrf2 "gene-dose response" mouse model (Nrf2-null with Nrf2 knock-out, wild-type with normal expression of Nrf2, Keap1-KD with enhanced Nrf2 activation and Keap1-HKO mice with maximum Nrf2 activation). Twenty-four hours after furosemide administration (250mg/kg, i.p.), serum ALT activities and histopathological analysis indicated severe hepatotoxicity in Nrf2-null and WT mice, but significantly less in the Nrf2-overexpressing Keap1-KD and Keap1-HKO mice. Furosemide increased the mRNA of genes involved in the acute phase response (hemeoxygenase-1 and metallothionein-1), ER stress (C/Ebp-homologous protein and Growth arrest and DNA-damage-inducible protein), inflammatory cytokine (interleukin 1 beta), chemokines (macrophage inflammatory protein 2 and mouse keratinocyte-derived chemokine), as well as apoptosis (early growth response factor and BCL2-associated X protein) in livers of Nrf2-null and wild-type mice, but these genes increased less in mice with more Nrf2. The two genotypes of over-expressed Nrf2 mice had increased expression of the Nrf2 target genes Gclm, Gclc and Nqo1 prior to furosemide administration, and the expressions of these genes were increased further after furosemide administration. Thus, our findings provide strong evidence that over-expression of Nrf2 in Keap1-KD and Keap1-HKO mice and the increases in mRNA of a number of genes involved in anti-oxidative stress, anti-inflammation, anti-ER stress and anti-apoptosis protect against furosemide-induced hepatotoxicity.

Overexpression of Nrf2 Protects against Microcystin-Induced Hepatotoxicity in Mice

Oxidative stress and glutathione (GSH) depletion are implicated in mycocystin hepatotoxicity. To investigate the role of nuclear factor erythroid 2-related factor 2 (Nrf2) in microcystin-induced liver injury, Nrf2-null, wild-type, and Keap1-hepatocyte knockout (Keap1-HKO) mice were treated with microcystin (50 μg/kg, i.p.). Blood and liver samples were collected 8 h thereafter. Microcystin increased serum alanine aminotransferase and aspartate aminotransferase activities, and caused extensive inflammation and necrosis in Nrf2-null and wild-type mice, but not in Keap1-HKO mice. Oxidative stress and inflammation are implicated in microcystin-induced hepatotoxicity, as evidenced by increased lipid peroxidation and increased expression of pro-inflammatory genes, such as neutrophil-specific chemokines mKC and MIP-2, and pro-inflammatory cytokines IL-1β and IL-6. The increased expression of these pro-inflammatory genes was attenuated in Keap1-HKO mice. Nrf2 and Nqo1 mRNA and protein were higher in Keap1-HKO mice at constitutive levels and after microcystin. To further investigate the mechanism of the protection, hepatic GSH and the mRNA of GSH-related enzymes were determined. Microcystin markedly depleted liver GSH by 60–70% in Nrf2 and WT mice but only 35% in Keap1-HKO mice. The mRNAs of GSH conjugation and peroxide reduction enzymes, such as Gstα1, Gstα4, Gstμ, and Gpx2 were higher in livers of Keap1-HKO mice, together with higher expression of the rate-limiting enzyme for GSH synthesis (Gclc). Organic anion transport polypeptides were increased by microcystin with the most increase in Keap1-HKO mice. In conclusion, this study demonstrates that higher basal levels of Nrf2 and GSH-related genes in Keap1-HKO mice prevented microcystin-induced oxidative stress and liver injury.

Nrf2 deficiency promotes apoptosis and impairs PAX7/MyoD expression in aging skeletal muscle cells

Skeletal muscle redox homeostasis is transcriptionally regulated by nuclear erythroid-2-p45-related factor-2 (Nrf2). We recently demonstrated that age-associated stress impairs Nrf2–ARE (antioxidant-response element) transcriptional signaling. Here, we hypothesize that age-dependent decline or genetic ablation of Nrf2 leads to accelerated apoptosis and skeletal muscle degeneration. Under basal-physiological conditions, disruption of Nrf2 significantly downregulates antioxidants and causes oxidative stress. Surprisingly, Nrf2-null mice had enhanced antioxidant capacity identical to wild-type (WT) upon acute endurance exercise stress (AEES), suggesting activation of Nrf2-independent mechanisms (i.e., PGC1α) against oxidative stress. Analysis of prosurvival pathways in the basal state reveals decreased AKT levels, whereas p-p53, a repressor of AKT, was increased in Nrf2-null vs WT mice. Upon AEES, AKT and p-AKT levels were significantly (p < 0.001) increased (>10-fold) along with profound downregulation of p-p53 (p < 0.01) in Nrf2-null vs WT skeletal muscle, indicating the onset of prosurvival mechanisms to compensate for the loss of Nrf2 signaling. However, we found a decreased stem cell population (PAX7) and MyoD expression (differentiation) along with profound activation of ubiquitin and apoptotic pathways in Nrf2-null vs WT mice upon AEES, suggesting that compensatory prosurvival mechanisms failed to overcome the programmed cell death and degeneration in skeletal muscle. Further, the impaired regeneration was sustained in Nrf2-null vs WT mice after 1 week of post-AEES recovery. In an age-associated oxidative stress condition, ablation of Nrf2 results in induction of apoptosis and impaired muscle regeneration.

Keap1 Cysteine 288 as a Potential Target for Diallyl Trisulfide-Induced Nrf2 Activation

Diallyl sulfide, diallyl disulfide, and daillyl trisulfide (DATS) are major volatile components of garlic oil. In this study, we assessed their relative potency in inducing antioxidant enzyme expression. Among the three organosulfur compounds, DATS was found to be most potent in inducing heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase-1 (NQO1) in human gastric epithelial (AGS) cells. Furthermore, DATS administration by gavage increased the expression of HO-1 and NQO1 in C57BL/6 mouse stomach. Treatment with DATS increased the accumulation of nuclear factor-erythroid-2-related factor-2 (Nrf2) in the nucleus of cultured AGS cells and in mouse stomach in vivo. The DATS-induced expression of HO-1 and NQO1 was abrogated in the cells transiently transfected with Nrf2-siRNA or in the embryonic fibroblasts from Nrf2-null mice, indicating that Nrf2 is a key mediator of the cytoprotective effects of DATS. Pretreatment of AGS cells with N-acetylcysteine or dithiothreitol attenuated DATS-induced nuclear localization of Nrf2 and the expression of HO-1 and NQO1. Cysteine-151, -273 and -288 of Kelch-like ECH-associated protein-1 (Keap1), a cytosolic repressor of Nrf2, have been considered to act as a redox sensor and play a role in Nrf2 activation. To determine whether DATS could inactivate Keap1 through thiol modification, we established cell lines constitutively expressing wild type-Keap1 or three different mutant constructs in which cysteine-151, -273, or -288 of Keap1 was replaced with serine by retroviral gene transfer. DATS failed to activate Nrf2, and to induce expression of HO-1 and NQO1 only in Keap1-C288S mutant cells. LC-ESI-MS/MS analysis of recombinant Keap1 treated with DATS revealed that the peptide fragment containing Cys288 gained a molecular mass of 72.1 Da equivalent to the molecular weight of mono-allyl mono-sulfide. Taken together, these findings suggest that DATS may directly interact with the Cys288 residue of Keap1, which partly accounts for its ability to induce Nrf2 activation and upregulate defensive gene expression.