Nuclear factor erythroid 2–related factor 2 (Nrf2) is a transcription factor that is important in protection against oxidative stress. This study was designed to determine the role of Nrf2 signaling in transcriptional activation of detoxifying and antioxidant genes in an in vivo mouse fetal alcohol syndrome model. Maternal ethanol treatment was found to increase both Nrf2 protein levels and Nrf2-ARE binding in mouse embryos. It also resulted in a moderate increase in the mRNA expression of Nrf2 downstream target detoxifying and antioxidant genes as well as an increase in the expression of antioxidant proteins. Pretreatment with the Nrf2 inducer, 3H-1,2 dithiole-3-thione (D3T), significantly increased Nrf2 protein levels and Nrf2-ARE binding, and strongly induced the mRNA expression of Nrf2 downstream target genes. It also increased the expression of antioxidant proteins and the activities of the antioxidant enzymes. Additionally, D3T pretreatment resulted in a significant decrease in ethanol-induced reactive oxygen species generation and apoptosis in mouse embryos. These results demonstrate that Nrf2 signaling is involved in the induction of antioxidant response in ethanol-exposed embryos. In addition, the potency of D3T in inducing antioxidants as well as in diminishing ethanol-induced apoptosis suggests that further exploration of the antiteratogenic effect of this compound will be fruitful. Antioxid. Redox Signal. 11, 2023–2033.
Oxidative stress and inflammation are two critical factors that drive the formation of plaques in atherosclerosis. Nrf2 is a redox-sensitive transcription factor that upregulates a battery of antioxidative genes and cytoprotective enzymes that constitute the cellular response to oxidative stress. Our previous studies have shown that disruption of Nrf2 in mice (Nrf2−/−) causes increased susceptibility to pulmonary emphysema, asthma and sepsis due to increased oxidative stress and inflammation. Here we have tested the hypothesis that disruption of Nrf2 in mice causes increased atherosclerosis.
To investigate the role of Nrf2 in the development of atherosclerosis, we crossed Nrf2−/− mice with apoliporotein E-deficient (ApoE−/−) mice. ApoE−/− and ApoE−/− Nrf2−/− mice were fed an atherogenic diet for 20 weeks, and plaque area was assessed in the aortas. Surprisingly, ApoE−/− Nrf2−/− mice exhibited significantly smaller plaque area than ApoE−/− controls (11.5% vs 29.5%). This decrease in plaque area observed in ApoE−/− Nrf2−/− mice was associated with a significant decrease in uptake of modified low density lipoproteins (AcLDL) by isolated macrophages from ApoE−/− Nrf2−/− mice. Furthermore, atherosclerotic plaques and isolated macrophages from ApoE−/− Nrf2−/− mice exhibited decreased expression of the scavenger receptor CD36.
Nrf2 is pro-atherogenic in mice, despite its antioxidative function. The net pro-atherogenic effect of Nrf2 may be mediated via positive regulation of CD36. Our data demonstrates that the potential effects of Nrf2-targeted therapies on cardiovascular disease need to be investigated.
The apolipoprotein E (APOE) genotype is an important risk factor for ageing and age-related diseases. The APOE4 genotype (in contrast to APOE3) has been shown to be associated with oxidative stress and chronic inflammation. Metallothioneins (MT) exhibit antioxidant and anti-inflammatory activity, and MT overexpression has been shown to increase lifespan in mice. Interactions between APOE and MT, however, are largely unknown. Hence, we determined the effect of the APOE4 versus APOE3 genotype on MT levels in targeted gene replacement mice. APOE4 versus APOE3 mice exhibited significantly lower hepatic MT1 and MT2 mRNA as well as lower MT protein levels. The decrease in hepatic MT protein levels in APOE4 as compared to APOE3 mice was accompanied by lower nuclear Nrf1, a protein partly controlling MT gene expression. Cell culture experiments using hepatocytes identified allyl-isothiocyanate (AITC) as a potent MT inductor in vitro. Therefore, we supplemented APOE3 and APOE4 mice with AITC. However, AITC (15 mg/kg b.w.) could only partly correct for decreased MT1 and MT2 gene expression in APOE4 mice in vivo. Furthermore, cholesterol significantly decreased both Nrf1 and MT mRNA levels in Huh7 cells indicating that differences in MT gene expression between the two genotypes could be related to differences in hepatic cholesterol concentrations. Overall, present data suggest that the APOE genotype is an important determinant of tissue MT levels in mice and that MT gene expression may be impaired by the APOE4 genotype.
APOE; Metallothionein; Nrf1; Ageing; Mice
The iron chelate, ferric nitrilotriacetate (FeNTA), induces acute proximal tubular necrosis as a consequence of lipid peroxidation and oxidative tissue damage. Chronic exposure of FeNTA leads to a high incidence of renal adenocarcinomas in rodents. NF-e2-related factor 2 (Nrf2) is a transcription factor that is activated by oxidative stress and electrophiles, and regulates the basal and inducible expression of numerous detoxifying and antioxidant genes. To determine the roles of Nrf2 in regulating renal gene expression and protecting against oxidative stress-induced kidney damage, wild-type and Nrf2-null mice were administered FeNTA. Renal Nrf2 protein translocated to the nucleus at 6 h after FeNTA treatment. FeNTA increased mRNA levels of Nrf2 target genes, including NQO1, GCLC, GSTpi1/2, Mrp1, 2, and 4 in kidneys from wild-type mice, but not Nrf2-null mice. Protein expression of NQO1, a prototypical Nrf2 target gene, was increased in wild-type mice, with no change in Nrf2-null mice. FeNTA produced more nephrotoxicity in Nrf2-null mice than wild-type mice as indicated by higher serum urea nitrogen and creatinine levels, as more urinary NAG, stronger 4-hydroxynonenal protein adduct staining, and more extensive proximal tubule damage. Furthermore, pretreatment with CDDO-Im, a potent small molecule Nrf2 activator, protected mice against FeNTA-induced renal toxicity. Collectively, these results suggest that activation of Nrf2 protects mouse kidneys from FeNTA-induced oxidative stress damage by coordinately up-regulating the expression of cytoprotective genes.
Nrf2; FeNTA; NQO1; oxidative stress; Mrp; kidney
Pyrazole can induce CYP2E1 and 2A5, which produce reactive oxygen species (ROS). Nuclear factor erythroid 2-related factor 2 (Nrf2) regulates important antioxidant enzymes to remove ROS. In this study, we applied Nrf2 knockout mice to test the hypothesis that pyrazole will cause hepatotoxicity and elevate oxidative stress to a greater extent in Nrf2 knockout compared to wild type mice. Pyrazole induced severe oxidative liver damage in Nrf2 knockout mice but not in wild type mice. Activities and levels of CYP2E1 and 2A5 were elevated by pyrazole in the wild type mice but not in the Nrf2 knockout mice. However, expression or activity of Nrf2-regulated antioxidant enzymes, such as γ-glutamylcysteine synthetase (GCS), heme oxygenase-1(HO-1) and glutathione S-transferase (GST), were upregulated in the pyrazole-treated wild type mice, but to a lesser extent or not at all in the pyrazole-treated Nrf2 knockout mice. Treatment with antioxidants such as vitamin C or S-Adenosyl-L-methionine (SAM) or an inhibitor of iNOS prevented the pyrazole-induced oxidative liver damage, thus validating the role of oxidative/nitrosative stress in the pyrazole-induced liver injury to the Nrf2 knockout mice. In summary, even though ROS-producing CYP2E1/2A5 were not elevated by pyrazole, impaired antioxidant capacity resulting from Nrf2 deficiency appear to be sufficient to promote pyrazole-induced oxidative liver injury.
Nuclear factor-erythroid 2-related factor 2 (Nrf2); pyrazole; CYP2E1; CYP2A5; oxidative/nitrosative stress; free radicals
Activation of the transcription factor NF-E2 related factor 2 (Nrf2) by oxidative stress induces the expression of a variety of antioxidant and anti-inflammatory genes. Yet, genetic ablation of Nrf2 was shown to protect mice from high-fat diet (HFD)-induced obesity and insulin resistance. The mechanisms that underlay this seemingly paradoxical finding remain largely unexplored.
Here we examined whether Nrf2 deficiency in myeloid cells contributes to protection against HFD-induced metabolic changes by decreasing adipose tissue inflammation. In vitro, induction of IL-1β by inflammatory stimuli was significantly reduced in Nrf2-deficient macrophages. While, inflammatory gene expression in the stromal vascular fraction was reduced in both global and chimeric Nrf2 KO mice, only global Nrf2-deficient but not bone marrow-transplanted Nrf2 chimeric mice were protected against HFD-induced adipose tissue inflammation. While global Nrf2 deficiency resulted in significantly decreased expression of inflammatory genes and PPARγ2, there was no difference when Nrf2 was absent only in myeloid cells. In vitro co-culture with adipocytes demonstrated that macrophage Nrf2 regulated inflammatory gene expression in macrophages, however, was not required to induce inflammatory gene expression in adipocytes. Finally, in contrast to global Nrf2 knock-out, Nrf2 deficiency in myeloid cells did not protect against HFD-induced insulin resistance.
Together, our data demonstrate a dominant role of nonmyeloid Nrf2 in controlling HFD-induced adipose tissue inflammation and the development of insulin resistance.
Nrf2; inflammation; insulin resistance; bone marrow transplantation; stromal vascular fraction and adipocyte/macrophage co-culture
Oxidative stress occurs when generation of reactive oxygen species (ROS) overwhelms antioxidant defenses. Oxidative stress has been associated with male infertility. The transcription factor Nuclear Factor-Erythroid 2-Related Factor 2 (NRF2) regulates basal and inducible transcription of genes encoding enzymes important for protection against ROS. We hypothesized that deletion of the Nrf2 gene causes testicular and epididymal oxidative stress, which disrupts spermatogenesis. Our results show that male Nrf2−/− mice have decreased fertility compared to wild type and heterozygous littermates, due to accumulating seminiferous tubule damage with increasing age. Testicular sperm head counts, epididymal sperm counts, and epididymal sperm motility in 2 month old Nrf2−/− males did not differ from wild type littermates; however, by age 6 months, Nrf2−/− males had 44% lower testicular sperm head counts, 65% lower epididymal sperm counts, and 66% lower epididymal sperm motility than wild type males. Two to 4 month old Nrf2−/− males had elevated levels of testicular and epididymal lipid peroxidation and testicular germ cell apoptosis, and decreased levels of antioxidants compared to wild type males. These results provide evidence that oxidative stress has deleterious effects on the testis and epididymis and demonstrate a critical role for the transcription factor NRF2 in preventing oxidative disruption of spermatogenesis.
oxidative stress; spermatogenesis; NRF2; testis; reproduction
Introduction. The transcription factor NFE2-related factor 2 (Nrf2) is a central regulator of antioxidant and detoxification gene expression in response to electrophilic or oxidative stress. Nrf2 has recently been shown to cross-talk with metabolic pathways, and its gene deletion protected mice from high-fat-diet-(HFD-) induced obesity and insulin resistance. This study aimed to identify potential Nrf2-regulated genes of metabolic interest by comparing gene expression profiles of livers of wild-type (WT) versus Nrf2 knockout (Nrf2-KO) mice after a long-term HFD. Methods. WT and Nrf2-KO mice were fed an HFD for 180 days; total RNA was prepared from liver and used for microarray analysis and quantitative real-time RT-PCR (qRT-PCR). Results. The microarray analysis identified 601 genes that were differentially expressed between WT and Nrf2-KO mice after long-term HFD. Selected genes, including ones known to be involved in metabolic regulation, were prioritized for verification by qRT-PCR: Cyp7a1 and Fabp5 were significantly overexpressed in Nrf2-KO mice; in contrast, Car, Cyp2b10, Lipocalin 13, Aquaporin 8, Cbr3, Me1, and Nqo1 were significantly underexpressed in Nrf2-KO mice. Conclusion. Transcriptome profiling after HFD-induced obesity confirms that Nrf2 is implicated in liver metabolic gene networks. The specific genes identified here may provide insights into Nrf2-dependent mechanisms of metabolic regulation.
Nrf2:INrf2(Keap1) are cellular sensors of chemical and radiation induced oxidative and electrophilic stress. Nrf2 is a nuclear transcription factor that controls the expression and coordinated induction of a battery of defensive genes encoding detoxifying enzymes and antioxidant proteins. This is a mechanism of critical importance for cellular protection and cell survival. Nrf2 is retained in the cytoplasm by an inhibitor INrf2. INrf2 functions as an adapter for Cul3/Rbx1 mediated degradation of Nrf2. In response to oxidative/electrophilic stress, Nrf2 is switched on and then off by distinct early and delayed mechanisms. Oxidative/electrophilic modification of INrf2cysteine151 and/or PKC phosphorylation of Nrf2serine40 results in the escape or release of Nrf2 from INrf2. Nrf2 is stabilized and translocates to the nucleus, forms heterodimers with unknown proteins, and binds antioxidant response element (ARE) that leads to coordinated activation of gene expression. It takes less than fifteen minutes from the time of exposure to switch on nuclear import of Nrf2. This is followed by activation of a delayed mechanism that controls switching off of Nrf2 activation of gene expression. GSK3β phosphorylates Fyn at unknown threonine residue(s) leading to nuclear localization of Fyn. Fyn phosphorylates Nrf2tyrosine568 resulting in nuclear export of Nrf2, binding with INrf2 and degradation of Nrf2. The switching on and off of Nrf2 protect cells against free radical damage, prevents apoptosis and promotes cell survival.
Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that binds to the antioxidant response element, a cis-acting regulatory element that increases expression of detoxifying enzymes and antioxidant proteins. Kelch-like ECH associating protein 1 (Keap1) protein is a negative regulator of Nrf2. Previous work has shown that genetic overexpression of Nrf2 is protective in vitro and in vivo. To modulate the Nrf2-ARE system without overexpressing Nrf2, we used short interfering RNA (siRNA) directed against Keap1. Keap1 siRNA administration in primary astrocytes increased the levels of Nrf2-ARE driven genes and protected against oxidative stress. Moreover, Keap1 siRNA resulted in a persistent upregulation of the Nrf2-ARE pathway and protection against oxidative stress in primary astrocytes. Keap1 siRNA injected into the striatum was also modestly protective against MPTP-induced dopaminergic terminal damage. These data indicate that activation of endogenous intracellular levels of Nrf2 is sufficient to protect in models of oxidative stress and Parkinson's disease.
Rationale: The NF-E2 related factor 2 (Nrf2)–antioxidant response element (ARE) pathway is essential for protection against oxidative injury and inflammation including hyperoxia-induced acute lung injury. Microarray expression profiling revealed that lung peroxisome proliferator activated receptor γ (PPARγ) induction is suppressed in hyperoxia-susceptible Nrf2-deficient (Nrf2−/−) mice compared with wild-type (Nrf2+/+) mice. PPARγ has pleiotropic beneficial effects including antiinflammation in multiple tissues.
Objectives: We tested the hypothesis that PPARγ is an important determinant of pulmonary responsivity to hyperoxia regulated by Nrf2.
Methods: A computational bioinformatic method was applied to screen potential AREs in the Pparg promoter for Nrf2 binding. The functional role of a potential ARE was investigated by in vitro promoter analysis. A role for PPARγ in hyperoxia-induced acute lung injury was determined by temporal silencing of PPARγ via intranasal delivery of PPARγ-specific interference RNA and by administration of a PPARγ ligand 15-deoxy-Δ12,14-prostaglandin J2 in mice.
Measurements and Main Results: Deletion or site-directed mutagenesis of a potential ARE spanning -784/-764 sequence significantly attenuated hyperoxia-increased Pparg promoter activity in airway epithelial cells overexpressing Nrf2, indicating that the -784/-764 ARE is critical for Nrf2-regulated PPARγ expression. Mice with decreased lung PPARγ by specific interference RNA treatment had significantly augmented hyperoxia-induced pulmonary inflammation and injury. 15 Deoxy-Δ12,14-prostaglandin J2 administration significantly reduced hyperoxia-induced lung inflammation and edema in Nrf2+/+, but not in Nrf2−/− mice.
Conclusions: Results indicate for the first time that Nrf2-driven PPARγ induction has an essential protective role in pulmonary oxidant injury. Our observations provide new insights into the therapeutic potential of PPARγ in airway oxidative inflammatory disorders.
antioxidant response element; hyperoxia; inflammation; siRNA; 15d-PGJ2
Oxidative stress-mediated destruction of normal parenchymal cells during hepatic inflammatory responses contributes to the pathogenesis of immune-mediated hepatitis and is implicated in the progression of acute inflammatory liver injury to chronic inflammatory liver disease. The transcription factor NF-E2-related factor 2 (Nrf2) regulates the expression of a battery of antioxidative enzymes and Nrf2 signaling can be activated by small-molecule drugs that disrupt Keap1-mediated repression of Nrf2 signaling. Therefore, genetic and pharmacologic approaches were used to activate Nrf2 signaling to assess protection against inflammatory liver injury. Profound increases in ind of cell death were observed in both Nrf2 wild-type (Nrf2-WT) mice and Nrf2-disrupted (Nrf2-KO) mice 24-hr following intravenous injection of concanavalin A (12.5 mg/kg, ConA), a model for T cell-mediated acute inflammatory liver injury. However, hepatocyte-specific conditional Keap1 null (Alb-Cre:Keap1flox/−, cKeap1-KO) mice with constitutively enhanced expression of Nrf2-regulated antioxidative genes as well as Nrf2-WT mice but not Nrf2-KO mice pretreated with three daily doses of a triterpenoid that potently activates Nrf2 (30 µmole/kg, CDDO-Im) were highly resistant to ConA-mediated inflammatory liver injury. CDDO-Im pretreatment of both Nrf2-WT and Nrf2-KO mice resulted in equivalent suppression of serum pro-inflammatory soluble proteins suggesting that the hepatoprotection afforded by CDDO-Im pretreatment of Nrf2-WT mice but not Nrf2-KO mice was not due to suppression of systemic pro-inflammatory signaling, but instead was due to activation of Nrf2 signaling in the liver. Enhanced hepatic expression of Nrf2-regulated antioxidative genes inhibited inflammation-mediated oxidative stress, thereby preventing hepatocyte necrosis. Attenuation of hepatocyte death in cKeap1-KO mice and CDDO-Im pretreated Nrf2-WT mice resulted in decreased late-phase pro-inflammatory gene expression in the liver thereby diminishing the sustained influx of inflammatory cells initially stimulated by the ConA challenge. Taken together, these results clearly illustrate that targeted cytoprotection of hepatocytes through Nrf2 signaling during inflammation prevents the amplification of inflammatory responses in the liver.
Liver inflammation; Nrf2; Keap1; antioxidative enzymes; cytoprotection; triterpenoid
Oleanolic acid is a plant-derived triterpenoid, which protects against various hepatotoxicants in rodents. In order to determine whether oleanolic acid activates nuclear factor erythroid-2 related factor 2 (Nrf2), a transcription factor known to induce various antioxidant and cytoprotective genes, wild-type and Nrf2-null mice were treated with oleanolic acid (90 mg/kg, i.p.) once daily for three days. Oleanolic acid increased nuclear accumulation of Nrf2 in wild-type but not Nrf2-null mice, as determined by Western blot and immunofluorescence. Oleanolic acid-treated wild-type mice had increased hepatic mRNA expression of the Nrf2 target genes NAD(P)H:quinone oxidoreductase 1 (Nqo1); glutamate-cysteine ligase, catalytic subunit (Gclc); heme oxygenase-1 (Ho-1); as well as Nrf2 itself. In addition, oleanolic acid increased protein expression and enzyme activity of the prototypical Nrf2 target gene, Nqo1, in wild-type, but not in Nrf2-null mice. Oleanolic acid protected against acetaminophen hepatotoxicity in wild-type mice but to a lesser extent in Nrf2-null mice. Oleanolic acid-mediated Nrf2-independent protection from acetaminophen is, in part, due to induction of Nrf2-independent cytoprotective genes, such as metallothionein. Collectively, the present study demonstrates that oleanolic acid facilitates Nrf2 nuclear accumulation, causing induction of Nrf2-dependent genes, which contributes to protection from acetaminophen hepatotoxicity.
Nrf2; oleanolic acid; hepatoprotection; oxidative stress; acetaminophen
Nuclear factor erythroid 2–related factor 2 (Nrf2) is a transcription factor that promotes the transcription of cytoprotective genes in response to oxidative and electrophilic stresses. Most functions of Nrf2 were identified by studying biological models with Nrf2 deficiency, however, little is known about the effects of graded Nrf2 activation. In the present study, genomic gene expression profiles by microarray analysis were characterized with a “gene dose-response” model in livers of Nrf2-null mice, wild-type mice, Kelch-like ECH associating protein 1 (Keap1)-knockdown (Keap1-KD) mice with enhanced Nrf2 activation, and Keap1-hepatocyte knockout (Keap1-HKO) mice with maximum hepatic Nrf2 activation. Hepatic nuclear Nrf2 protein, glutathione concentrations, and known Nrf2 target genes were increased in a dose-dependent manner. In total, 115 genes were identified to be constitutively induced and 80 genes suppressed with graded Nrf2 activation. Messenger RNA of genes encoding enzymes in the pentose phosphate pathway and enzyme were low with Nrf2 deficiency and high with Nrf2 activation, indicating that Nrf2 is important for NADPH production. NADPH is the major reducing resource to scavenge oxidative stress, including regenerating glutathione and thioredoxin and is also used for anabolic pathways including lipid synthesis. High performance liquid chromatography-ultraviolet absorbance analysis confirmed that hepatic NADPH concentration was lowest in Nrf2-null mice and highest in Keap1-HKO mice. In addition, genes involved in fatty acid synthesis and desaturation were downregulated with graded Nrf2 activation. In conclusion, the present study suggests that Nrf2 protects against environmental insults by promoting the generation of NADPH, which is preferentially consumed by aiding scavenging of oxidative stress rather than fatty acid synthesis and desaturation.
Nrf2; microarray; liver
Apolipoprotein E (apoE) plays a key role in lipoprotein metabolism and may have other important biological functions. In humans, there are three common, naturally occurring isoforms of apoE that are associated with differences in lipid levels and atherosclerosis. However, the direct in vivo effects of the apoE isoforms on lipoprotein metabolism and atherosclerosis are not yet fully understood. To investigate the effect of the apoE isoforms in vivo, we constructed second-generation recombinant adenoviruses encoding each of the apoE isoforms. These recombinant adenoviruses were injected intravenously into apoE-deficient mice fed a Western diet (mean baseline cholesterol level 1401 mg/dl) in order to study their effects in the absence of endogenous mouse apoE. Hepatic expression of apoE3 and apoE4 completely normalized the lipoprotein profile; 3 d after injection, mean plasma cholesterol levels were 194 and 217 mg/ dl, respectively, and this effect was maintained for at least 6 wk. Expression of apoE2 had much less effect on lipoprotein levels (mean cholesterol level 752 mg/dl 3 d after injection), despite much higher plasma levels of apoE2 compared with apoE3 and apoE4; by 6 wk after injection the cholesterol levels had returned to baseline levels in the apoE2-expressing mice. Expression of all three isoforms significantly increased HDL cholesterol levels by approximately threefold and was independent of the cholesterol-lowering effect. ApoE transgene expression was substantially prolonged compared with that achieved using a first generation adenovirus and apoE was readily detected in plasma 3 mo after virus injection. These studies demonstrate: (a) prolonged in vivo expression of human apoE isoforms in apoE deficient mice after second-generation recombinant adenovirus-mediated somatic gene transfer; and (b) significantly impaired ability of apoE2 in vivo to mediate clearance of remnant lipoproteins in apoE-deficient mice fed a Western diet compared with apoE3 and apoE4.
In response to oxidative stress, the transcription factor NF-E2-related factor 2 (Nrf2) controls the fate of cells through transcriptional upregulation of antioxidant response element (ARE)-bearing genes, including those encoding endogenous antioxidants, phase II detoxifying enzymes, and transporters. Expression of the Nrf2-dependent proteins is critical for ameliorating or eliminating toxicants/carcinogens to maintain cellular redox homeostasis. As a result, activation of the Nrf2 pathway, by naturally-occurring compounds or synthetic chemicals at sub-toxic doses, confers protection against subsequent toxic/carcinogenic exposure. Thus, the use of dietary compounds or synthetic chemicals to boost the Nrf2-dependent adaptive response to counteract environmental insults has emerged to be a promising strategy for cancer prevention. Interestingly, recent emerging data has revealed the “dark” side of Nrf2. Nrf2 and its downstream genes are overexpressed in many cancer cell lines and human cancer tissues, giving cancer cells an advantage for survival and growth. Furthermore, Nrf2 is upregulated in resistant cancer cells and is thought to be responsible for acquired chemoresistance. Therefore, it may be necessary to inhibit the Nrf2 pathway during chemotherapy. This review is primarily focused on the role of Nrf2 in cancer, with emphasis on the recent findings indicating the cancer promoting function of Nrf2 and its role in acquired chemoresistance.
The cardiovascular system is susceptible to a group of diseases that are responsible for a larger proportion of morbidity and mortality than any other disease. Many cardiovascular diseases are associated with a failure of defenses against oxidative stress-induced cellular damage and/or death, leading to organ dysfunction. The pleiotropic transcription factor, nuclear factor-erythroid (NF-E) 2-related factor 2 (Nrf2), regulates the expression of antioxidant enzymes and proteins through the antioxidant response element. Nrf2 is an important component in antioxidant defenses in cardiovascular diseases such as atherosclerosis, hypertension, and heart failure. Nrf2 is also involved in protection against oxidant stress during the processes of ischemia-reperfusion injury and aging. However, evidence suggests that Nrf2 activity does not always lead to a positive outcome and may accelerate the pathogenesis of some cardiovascular diseases (e.g., atherosclerosis). The precise conditions under which Nrf2 acts to attenuate or stimulate cardiovascular disease processes are unclear. Further studies on the cellular environments related to cardiovascular diseases that influence Nrf2 pathways are required before Nrf2 can be considered a therapeutic target for the treatment of cardiovascular diseases.
Lycopene can be cleaved by carotene 9′,10′-oxygenase at its 9′,10′ double bond to form apo-10′-lycopenoids, including apo-10′-lycopenal, -lycopenol and -lycopenoic acid. The latter has been recently shown to inhibit lung carcinogenesis both in vivo and in vitro, however, the mechanism(s) underlying this protection is not well defined. In the present study, we report that treatment with apo-10′-lycopenoic acid, in a time- and dose-dependent manner, results in the nuclear accumulation of transcription factor Nrf2 (nuclear factor E2-related factor 2) protein in BEAS-2B human bronchial epithelial cells. The activation of Nrf2 by apo-10′-lycopenoic acid is associated with the induction of phase II detoxifying/antioxidant enzymes including heme oxygenase-1, NAD(P)H:quinone oxidoreductase 1, glutathione S-transferases, and glutamate–cysteine ligases in BEAS-2B cells. Furthermore, apo-10′-lycopenoic acid treatment increased total intracellular glutathione levels and suppressed both endogenous reactive oxygen species generation and H2O2-induced oxidative damage in BEAS-2B cells. In addition, both apo-10′-lycopenol and apo-10′-lycopenal induced heme oxygenase-1 gene expression in BEAS-2B cells. These data strongly suggest that the anti-carcinogenic and antioxidant functions of lycopene may be mediated by apo-10′-lycopenoids via activating Nrf2 and inducing phase II detoxifying/antioxidant enzymes.
lycopene; apo-10′-lycopenoic acid; phase II enzymes; Nrf2; GSH; oxidative damage
NF-E2-related factor 2 (NRF2) regulates a battery of antioxidative and phase II drug metabolizing/detoxifying genes through binding to the antioxidant response elements (ARE). NRF2-ARE signaling plays a central role in protecting cells from a wide spectrum of reactive toxic species including reactive oxygen/nitrogen species (RONS). 4-hydroxylnonenal (4-HNE) is a major end product from lipid peroxidation of omega-6 polyunsaturated fatty acids (PUFA) induced by oxidative stress, and it is highly reactive to nucleophilic sites in DNA and proteins, causing cytotoxicity and genotoxicity. In this study, we examined the role of NRF2 in regulating the 4-HNE induced gene expression of antioxidant and detoxifying enzymes.
When HeLa cells were treated with 4-HNE, NRF2 rapidly transloated into the nucleus, as determined by the distribution of NRF2 tagged with the enhanced green fluorescent protein (EGFP) and increased NRF2 protein in the nuclear fraction. Transcriptional activity of ARE-luciferase was significantly induced by 0.01-10 μM of 4-HNE in a dose-dependent manner, and the induction could be blocked by pretreatment with glutathione (GSH). 4-HNE induced transcriptional expression of glutathione S-transferase (GST) A4, aldoketone reductase (AKR) 1C1 and heme oxygenase-1 (HO-1), and the induction was attenuated by knocking down NRF2 using small interfering RNA.
NRF2 is critical in mediating 4-HNE induced expression of antioxidant and detoxifying genes. This may account for one of the major cellular defense mechanisms against reactive metabolites of lipids peroxidation induced by oxidative stress and protect cells from cytotoxicity.
NRF2; 4-hydroxynonenal (4-HNE); Antioxidant response element (ARE); Oxidative stress
Acute fasting causes elevated oxidative stress. The current study investigated the effects of the nuclear factor erythoid 2-related factor 2 (Nrf2), the sensor of oxidative stress in cells, on energy homeostasis and liver pathophysiology during fasting. Feed was removed from mice possessing none (Nrf2-null), normal (wild-type, WT), enhanced (Keap1-knockdown, K1-KD), and maximum (hepatocyte-specific Keap1-knockout, K1-HKO) Nrf2 activity in liver for 24 h. Body weight, blood glucose, and blood lipid profiles were similar among mice with graded Nrf2 activity under either fed or fasted conditions. Fasting reduced liver size in mice expressing Nrf2, but not in Nrf2-null mice. Nrf2-null mice accumulated more non-esterified free fatty acids and triglycerides in liver after fasting than the other genotypes of mice. Fatty acids are mainly catabolized in mitochondria, and Nrf2-null mice had lower mitochondrial content in liver under control feeding conditions, which was further reduced by fasting. In contrast, mitochondrial contents in mice with enhanced Nrf2 activity were not affected by fasting. Oxidative stress, determined by staining of free radicals and quantification of malondialdehyde equivalents, was highest in Nrf2-null and lowest in K1-HKO mice after fasting. The exacerbated oxidative stress in livers of Nrf2-null mice is predicted to lead to damages to mitochondria, and therefore diminished oxidation and increased accumulation of lipids in livers of Nrf2-null mice. In summary, the Nrf2-regulated signaling pathway is critical in protecting mitochondria from oxidative stress during feed deprivation, which ensures efficient utilization of fatty acids in livers of mice.
Oxidative stress has been postulated to play an important role in the pathogenesis of asthma; although a defect in antioxidant responses has been speculated to exacerbate asthma severity, this has been difficult to demonstrate with certainty. Nuclear erythroid 2 p45-related factor 2 (Nrf2) is a redox-sensitive basic leucine zipper transcription factor that is involved in the transcriptional regulation of many antioxidant genes. We show that disruption of the Nrf2 gene leads to severe allergen-driven airway inflammation and hyperresponsiveness in mice. Enhanced asthmatic response as a result of ovalbumin sensitization and challenge in Nrf2-disrupted mice was associated with more pronounced mucus cell hyperplasia and infiltration of eosinophils into the lungs than seen in wild-type littermates. Nrf2 disruption resulted in an increased expression of the T helper type 2 cytokines interleukin (IL)-4 and IL-13 in bronchoalveolar lavage fluid and in splenocytes after allergen challenge. The enhanced severity of the asthmatic response from disruption of the Nrf2 pathway was a result of a lowered antioxidant status of the lungs caused by lower basal expression, as well as marked attenuation, of the transcriptional induction of multiple antioxidant genes. Our studies suggest that the responsiveness of Nrf2-directed antioxidant pathways may act as a major determinant of susceptibility to allergen-mediated asthma.
The Keap1-Nrf2 system protects animals from oxidative and electrophilic stresses. Nrf2 is a transcription factor that induces the expression of genes essential for detoxifying reactive oxygen species (ROS) and cytotoxic electrophiles. Keap1 is a stress sensor protein that binds to and ubiquitinates Nrf2 under unstressed conditions, leading to the rapid proteasomal degradation of Nrf2. Upon exposure to stress, Keap1 is modified and inactivated, which allows Nrf2 to accumulate and activate the transcription of a battery of cytoprotective genes. Antioxidative and detoxification activities are important for many types of cells to avoid DNA damage and cell death. Accumulating lines of recent evidence suggest that Nrf2 is also required for the primary functions of myeloid cells, which include phagocytosis, inflammation regulation, and ROS generation for bactericidal activities. In fact, results from several mouse models have shown that Nrf2 expression in myeloid cells is required for the proper regulation of inflammation, antitumor immunity, and atherosclerosis. Moreover, several molecules generated upon inflammation activate Nrf2. Although ROS detoxification mediated by Nrf2 is assumed to be required for anti-inflammation, the entire picture of the Nrf2-mediated regulation of myeloid cell primary functions has yet to be elucidated. In this review, we describe the Nrf2 inducers characteristic of myeloid cells and the contributions of Nrf2 to diseases.
To evaluate the effects of a genetic reduction of Lias gene expression on atherosclerosis development.
Methods and Results
Heterozygous knockout mice for the lipoid acid synthase gene (Lias+/−) were crossed with apolipoprotein E–deficient (ApoE−/−) mice, and the plaque size in aortic sinuses of Lias+/− ApoE−/− mice was evaluated at 6 months of age. Lesions at the aortic sinus in Lias+/− ApoE−/− males were significantly larger (1.5X) than those in Lias+/+ ApoE−/− littermate males. The lesion size was inversely correlated with an increased erythrocyte reduced glutathione/ oxidized glutathione (GSH/GSSH) ratio, an systemic index of body redox balance. Lias+/− ApoE−/− males also had significantly increased plasma cholesterol and reduced pyruvate dehydrogenase complex activity in the liver. Significant reductions in the expression of genes for antioxidant enzymes, including superoxide dismutase 1 (SOD1) and SOD2, were observed in aortas of Lias+/− ApoE−/− males. Female Lias+/− ApoE−/− also exhibited changes in these parameters, parallel to those observed in males. However, the Lias gene effects for the majority of these factors, including atherosclerotic lesion size, were not significant in females.
Our data provide evidence that Lias deficiency enhances atherosclerosis in male mice, at least in part due to reduce antioxidant capacity. The notable absence of such effects in females leaves open the possibility of a gender-specific protection mechanism.
antioxidant; lipoic acid; Lias mouse model; atherosclerosis; apolipoprotein E null mice
Nuclear factor erythroid 2–related factor 2 (Nrf2) is a transcription factor critical for protection against electrophilic and oxidative stress. In a recently engineered mouse with knockdown of kelch-like ECH associated protein 1 (Keap1-kd mice), the cytosolic repressor of Nrf2, there is a 55% decrease in Keap1 mRNA and a 200% increase in Nrf2 protein in liver. Experiments with Nrf2-null mice have demonstrated the effects of a lack of Nrf2. However, little is known about the biological effects of more Nrf2 activation. Accordingly, the hepatic phenotype of Keap1-kd mice, as well as the hepatic mRNA expression of cytoprotective genes were compared among wild-type, Nrf2-null, and Keap1-kd mice. Three distinct patterns of hepatic gene expression were identified among wild-type, Nrf2-null, and Keap1-kd mice. The first pattern encompassed genes that were lower in Nrf2-null mice and considerably higher in Keap1-kd mice than wild-type mice, which included genes mainly responsible for the detoxification and elimination of electrophiles, such as NAD(P)H:quinone oxidoreductase 1 and glutathione-S-transferases (Gst), and multidrug resistance–associated proteins. The second pattern encompassed genes that were lower in Nrf2-null mice but not increased in Keap1-kd mice, and included genes, such as epoxide hydrolase-1, UDP-glucuronosyltransferases, aldehyde dehydrogenases, as well as genes important in the detoxification of reactive oxygen species, such as superoxide dismutase 1 and 2, catalase, and peroxiredoxin 1. The third pattern encompassed genes that were not different among wild-type, Nrf2-null, and Keap1-kd mice and included genes such as glutathione peroxidase, microsomal Gsts, and uptake transporters. In conclusion, the present study suggests that increased activation of hepatic Nrf2 is more important for the detoxification and elimination of electrophiles than reactive oxygen species.
Nrf2; electrophilic stress; oxidative stress; cytoprotection
Oxidative stress plays an important part in the pathogenesis of a variety of diseases. The ability to mount an efficient response against the continuous threat posed by exogenous and endogenous oxidants is essential for cellular homeostasis and survival. Oxidative stress activates transcription of a variety of antioxidant genes through cis-acting sequence known as antioxidant response element (ARE). Members of the Cap-N-Collar family of transcription factors, including Nrf1 and Nrf2, have been identified that bind ARE. Nrf1 and Nrf2 are expressed in a wide range of tissues and cell types, and both bind the ARE as heterodimers with small-Maf proteins. Numerous studies indicate a pivotal role of Nrf2 in ARE function. Herein, we review data derived from cell-based studies and knockout mice in an attempt to define the role and regulation of Nrf1 in oxidative stress response and other functions.