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Sample records for nrf2 translocation undermines

  1. Frequency Modulated Translocational Oscillations of Nrf2 Mediate the Antioxidant Response Element Cytoprotective Transcriptional Response

    PubMed Central

    Xue, Mingzhan; Momiji, Hiroshi; Rabbani, Naila; Barker, Guy; Bretschneider, Till; Shmygol, Anatoly; Rand, David A.

    2015-01-01

    Abstract Aims: Stress responsive signaling coordinated by nuclear factor erythroid 2-related factor 2 (Nrf2) provides an adaptive response for protection of cells against toxic insults, oxidative stress and metabolic dysfunction. Nrf2 regulates a battery of protective genes by binding to regulatory antioxidant response elements (AREs). The aim of this study was to examine how Nrf2 signals cell stress status and regulates transcription to maintain homeostasis. Results: In live cell microscopy we observed that Nrf2 undergoes autonomous translocational frequency-modulated oscillations between cytoplasm and nucleus. Oscillations occurred in quiescence and when cells were stimulated at physiological levels of activators, they decrease in period and amplitude and then evoke a cytoprotective transcriptional response. We propose a mechanism whereby oscillations are produced by negative feedback involving successive de-phosphorylation and phosphorylation steps. Nrf2 was inactivated in the nucleus and reactivated on return to the cytoplasm. Increased frequency of Nrf2 on return to the cytoplasm with increased reactivation or refresh-rate under stress conditions activated the transcriptional response mediating cytoprotective effects. The serine/threonine-protein phosphatase PGAM5, member of the Nrf2 interactome, was a key regulatory component. Innovation: We found that Nrf2 is activated in cells without change in total cellular Nrf2 protein concentration. Regulation of ARE-linked protective gene transcription occurs rather through translocational oscillations of Nrf2. We discovered cytoplasmic refresh rate of Nrf2 is important in maintaining and regulating the transcriptional response and links stress challenge to increased cytoplasmic surveillance. We found silencing and inhibition of PGAM5 provides potent activation of Nrf2. Conclusion: Frequency modulated translocational oscillations of Nrf2 mediate the ARE-linked cytoprotective transcriptional response. Antioxid. Redox

  2. Activation of Nrf2 by H2O2: de novo synthesis versus nuclear translocation.

    PubMed

    Covas, Gonçalo; Marinho, H Susana; Cyrne, Luísa; Antunes, Fernando

    2013-01-01

    The most common mechanism described for the activation of the transcription factor Nrf2 is based on the inhibition of its degradation in the cytosol followed by its translocation to the nucleus. Recently, Nrf2 de novo synthesis was proposed as an additional mechanism for the rapid upregulation of Nrf2 by hydrogen peroxide (H2O2). Here, we describe a detailed protocol, including solutions, pilot experiments, and experimental setups, which allows exploring the role of H2O2, delivered either as a bolus or as a steady state, in endogenous Nrf2 translocation and synthesis. We also show experimental data, illustrating that H2O2 effects on Nrf2 activation in HeLa cells are strongly dependent both on the H2O2 concentration and on the method of H2O2 delivery. The de novo synthesis of Nrf2 is triggered within 5min of exposure to low concentrations of H2O2, preceding Nrf2 translocation to the nucleus which is slower. Evidence of de novo synthesis of Nrf2 is observed only for low H2O2 steady-state concentrations, a condition that is prevalent in vivo. This study illustrates the applicability of the steady-state delivery of H2O2 to uncover subtle regulatory effects elicited by H2O2 in narrow concentration and time ranges. Copyright © 2013 Elsevier Inc. All rights reserved.

  3. A novel mechanism for cytoprotection against hypoxic injury: δ–opioid receptor-mediated increase in Nrf2 translocation

    PubMed Central

    Cao, Shan; Chao, Dongman; Zhou, Honghao; Balboni, Gianfranco; Xia, Ying

    2015-01-01

    Background and Purpose Hypoxia/reoxygenation induces synthesis of reactive oxygen species (ROS) which can attack macromolecules and cause brain injury. The transcription factor, nuclear factor (erythroid-derived 2)-like 2, (Nrf2), ia potent activator of genes with an antioxidant responsive element and Nrf2 can counteract oxidative injury by increasing expression of several antioxidative genes in response to ROS stress. Here, we show that activation of the δ-opioid receptor (DOR) increasedNrf2 protein expression and translocation, thereby leading to cytoprotection. Experimental Approach We used HEK293t cells exposed to 0.5% O2 for 16 h and then reoxygenated for 4 h as a model of hypoxia-reperfusion (H/R) injury. Real time PCR, Western blotting, siRNA and immunohistochemical techniques were used to follow Nrf2 expression and activity. Cell viability and damage (as LDH leakage) were also measured. Key Results H/R injury triggered Nrf2 translocation into the nucleus and up-regulated expression of several downstream genes, relevant to antioxidation, such as NAD(P)H:quinone oxidoreductase (NQO1). Incubation with the DOR agonist UFP-512 enhanced Nrf2 protein expression and translocation and up-regulated its downstream genes in normoxia and further increased Nrf2 expression and translocation after H/R, protecting the cells against loss of viability and damage. The effect of UFP-512 on Nrf2 nuclear translocation was blocked by the DOR antagonist, naltrindole. Also, DOR–mediated cytoprotection was strongly inhibited after transfection of HEK293t cells with Nrf2 siRNA. Conclusions and Implications The DOR agonist UFP-512 was cytoprotective against H/R injury and this effect was partly dependent on DOR-mediated increase in Nrf2 function. PMID:25439010

  4. Cinnamaldehyde enhances Nrf2 nuclear translocation to upregulate phase II detoxifying enzyme expression in HepG2 cells.

    PubMed

    Huang, Tzou-Chi; Chung, Yu-Ling; Wu, Mei-Li; Chuang, Show-Mei

    2011-05-11

    Cinnamaldehyde has been demonstrated to stimulate glutathione production and the expression of phase II detoxifying enzymes in HepG2 cells. The mechanism underlying this cinnamaldehyde-mediated gene expression relies on Nrf2 transcriptional activity. Therefore, the molecular signaling events in cinnamaldehyde-mediated detoxifying enzyme expression were further investigated in this study. Cinnamaldehyde activated ERK1/2, Akt, and JNK signaling pathways, but not the p38 MAP kinase pathway, subsequently leading to Nrf2 nuclear translocation and eventually increasing phase II enzyme expression. In contrast, inhibition of ERK1/2, Akt, or JNK pathways attenuated Nrf2 nuclear translocation and phase II enzyme expression. Depletion of Nrf2 by small RNA interference (si-RNA) showed that the protein levels of phase II enzymes were no longer induced by cinnamaldehyde. A luciferase reporter assay and an electrophoretic mobility shift assay (EMSA) also demonstrated that cinnamaldehyde-activated signaling resulted in the increased transcriptional activity of Nrf2 through binding to the ARE4 enhancer sequence. Altogether, these data suggest that ERK1/2, Akt, and JNK pathways activated by cinnamaldehyde collectively control Nrf2 nuclear translocation and transcriptional activity, leading to the increase of phase II enzyme expression. Application of an appropriate chemopreventive agent such as cinnamaldehyde could potentially be an alternative strategy for cancer chemoprevention.

  5. Phosphorylation of Nrf2 in the transcription activation domain by casein kinase 2 (CK2) is critical for the nuclear translocation and transcription activation function of Nrf2 in IMR-32 neuroblastoma cells.

    PubMed

    Apopa, Patrick L; He, Xiaoqing; Ma, Qiang

    2008-02-01

    The antioxidant-activated transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) regulates the induction of cytoprotective genes against chemical toxicity and oxidative injuries. The role of phosphorylation in Nrf2 activation has been suggested but remains elusive. We report that phenolic antioxidant/pro-oxidant tert-butylhydroquinone (tBHQ) induced two forms of the Nrf2 protein in neuroblastoma cells (IMR-32), which migrated as distinctive bands on SDS-PAGE. In vitro treatment with lambda phosphatase eliminated the slower migrating form and increased the amount of the faster migrating form of Nrf2. In vivo (32)Pi-phosphorylation resulted in (32)Pi-labeling of the Nrf2 protein in the presence of tBHQ that can be dephosphorylated by lambda phosphotase, indicating that the slower migrating form is a phosphorylated Nrf2 protein and the faster form an unphosphorylated Nrf2. Unphosphorylated Nrf2 predominated in the cytoplasm, whereas the phosphorylated form preferentially localized in the nucleus. Nuclear Nrf2 can be dephosphorylated by lambda phosphotase in vitro and be converted to the faster migrating form, implicating phosphorylation of Nrf2 in the cytoplasmic-nuclear translocation of the protein. Deletional analyses from both the carboxyl- and amino-ends revealed the transcription activation (TA) domains Neh4 (Nrf2-ECH homology 4) and Neh5 (Nrf2-ECH homology 5) as a major region necessary for the phosphorylation. The TA domains are characterized by the presence of multiple phosphorylation sites of casein kinase 2 (CK2). Moreover, CK2 phosphorylated the TA domains in vitro. Treatment with CK2 inhibitor 2-dimethylamino-4,5,6,7,-tetrabromo-1H-benzimidazole (DMAT) blocked the induction of endogenous target genes of Nrf2 in cells and inhibited the TA activities of both the full length and the TA domains of Nrf2 to a large extent. Finally, phosphorylation of the TA domains correlated with the nuclear translocation of Nrf2 that was inhibited by DMAT in a

  6. The pesticide deltamethrin increases free radical production and promotes nuclear translocation of the stress response transcription factor Nrf2 in rat brain

    PubMed Central

    Li, HY; Wu, SY; Ma, Q; Shi, N

    2015-01-01

    The transcription factor NF-E2-related factor 2 (Nrf2) plays a critical role in the mammalian response to chemical and oxidative stress through induction of phase II detoxification enzymes and oxidative stress response proteins. We reported that Nrf2 expression was activated by deltamethrin (DM), a prototype of the widely used pyrithroid pesticides, in PC12 cells. However, no study has examined Nrf2 nuclear translocation and free radical production, two hallmarks of oxidative stress, in the mammalian brain in vivo. To this end, we examined translocation of Nrf2 and production of free radicals in rat brain exposed to DM. Indeed, DM initiated nuclear translocation of Nrf2 in a dose-dependent manner. Furthermore, Nrf2 translocation was accompanied by the expression of heme oxygenase-1 gene, an Nrf2-regulated gene linked to free radical production. Deltamethrin exposure promoted free radical formation in rat brain and reactive oxygen species generation in PC12 cells. Translocation of Nrf2 may be a response to DM-dependent induction of free radicals and DM may act as a mammalian neurotoxin by initiating oxidative stress. PMID:21398409

  7. Curcumin induces Nrf2 nuclear translocation and prevents glomerular hypertension, hyperfiltration, oxidant stress, and the decrease in antioxidant enzymes in 5/6 nephrectomized rats.

    PubMed

    Tapia, Edilia; Soto, Virgilia; Ortiz-Vega, Karla Mariana; Zarco-Márquez, Guillermo; Molina-Jijón, Eduardo; Cristóbal-García, Magdalena; Santamaría, José; García-Niño, Wylly Ramsés; Correa, Francisco; Zazueta, Cecilia; Pedraza-Chaverri, José

    2012-01-01

    Renal injury resulting from renal ablation induced by 5/6 nephrectomy (5/6NX) is associated with oxidant stress, glomerular hypertension, hyperfiltration, and impaired Nrf2-Keap1 pathway. The purpose of this work was to know if the bifunctional antioxidant curcumin may induce nuclear translocation of Nrf2 and prevents 5/6NX-induced oxidant stress, renal injury, decrease in antioxidant enzymes, and glomerular hypertension and hyperfiltration. Four groups of rats were studied: (1) control, (2) 5/6NX, (3) 5/6NX +CUR, and (4) CUR (n = 8-10). Curcumin was given by gavage to NX5/6 +CUR and CUR groups (60 mg/kg/day) starting seven days before surgery. Rats were studied 30 days after NX5/6 or sham surgery. Curcumin attenuated 5/6NX-induced proteinuria, systemic and glomerular hypertension, hyperfiltration, glomerular sclerosis, interstitial fibrosis, interstitial inflammation, and increase in plasma creatinine and blood urea nitrogen. This protective effect was associated with enhanced nuclear translocation of Nrf2 and with prevention of 5/6NX-induced oxidant stress and decrease in the activity of antioxidant enzymes. It is concluded that the protective effect of curcumin against 5/6NX-induced glomerular and systemic hypertension, hyperfiltration, renal dysfunction, and renal injury was associated with the nuclear translocation of Nrf2 and the prevention of both oxidant stress and the decrease of antioxidant enzymes.

  8. Nrf2 signaling and cell survival

    SciTech Connect

    Niture, Suryakant K.; Kaspar, James W.; Shen, Jun; Jaiswal, Anil K.

    2010-04-01

    Nrf2:INrf2 acts as a sensor for oxidative/electrophilic stress. INrf2 serves as an adaptor to link Nrf2 to the ubiquitin ligase Cul3-Rbx1 complex that ubiquitinate and degrade Nrf2. Under basal conditions, cytosolic INrf2/Cul3-Rbx1 is constantly degrading Nrf2. When a cell encounters stress Nrf2 dissociates from the INrf2 and translocates into the nucleus. Oxidative/electrophilic stress induced modification of INrf2Cysteine151 and/or protein kinase C (PKC)-mediated phosphorylation of Nrf2Serine40 controls Nrf2 release from INrf2 followed by stabilization and nuclear translocation of Nrf2. Nrf2 binds to the antioxidant response element (ARE) and activates a myriad of genes that protect cells against oxidative/electrophilic stress and neoplasia. A delayed response of oxidative/electrophilic stress activates GSK-3beta that phosphorylates Fyn at unknown threonine residue(s). Phosphorylated Fyn translocates to the nucleus and phosphorylates Nrf2Tyrosine568 that leads to nuclear export and degradation of Nrf2. Prothymosin-alpha mediated nuclear translocation of INrf2 also degrades nuclear Nrf2. The degradation of Nrf2 both in cytosol and nuclear compartments rapidly brings down its levels to normal resulting in suppression of Nrf2 downstream gene expression. An auto-regulatory loop between Nrf2 and INrf2 controls their cellular abundance. Nrf2 regulates INrf2 by controlling its transcription, and INrf2 controls Nrf2 by degrading it. In conclusion, switching on and off of Nrf2 combined with promoting an auto-regulatory loop between them regulates activation/deactivation of defensive genes leading to protection of cells against adverse effects of oxidative and electrophilic stress and promote cell survival.

  9. Glucagon-like peptide-1 improves beta-cell antioxidant capacity via extracellular regulated kinases pathway and Nrf2 translocation.

    PubMed

    Fernández-Millán, E; Martín, M A; Goya, L; Lizárraga-Mollinedo, E; Escrivá, F; Ramos, S; Álvarez, C

    2016-06-01

    Oxidative stress plays an important role in the development of beta-cell dysfunction and insulin resistance, two major pathophysiological abnormalities of type 2 diabetes. Expression levels of antioxidant enzymes in beta cells are very low, rendering them more susceptible to damage caused by reactive oxygen species (ROS). Although the antioxidant effects of glucagon-like peptide-1 (GLP-1) and its analogs have been previously reported, the exact mechanisms involved are still unclear. In this study, we demonstrated that GLP-1 was able to effectively inhibit oxidative stress and cell death of INS-1E beta cells induced by the pro-oxidant tert-butyl hydroperoxide (tert-BOOH). Incubation with GLP-1 enhanced cellular levels of glutathione and the activity of its related enzymes, glutathione-peroxidase (GPx) and -reductase (GR) in beta cells. However, inhibition of ERK, but not of the PI3K/AKT pathway abolished, at least in part, the antioxidant effect of GLP-1. Moreover, ERK activation seems to be protein kinase A (PKA)-dependent because inhibition of PKA with H-89 was sufficient to block the GLP-1-derived protective effect on beta cells. GLP-1 likewise increased the synthesis of GR and favored the translocation of the nuclear transcription factor erythroid 2p45-related factor (Nrf2), a transcription factor implicated in the expression of several antioxidant/detoxificant enzymes. Glucose-stimulated insulin secretion was also preserved in beta-cells challenged with tert-BOOH but pre-treated with GLP-1, probably through the down-regulation of the mitochondrial uncoupling-protein2 (UCP2). Thus, our results provide additional mechanisms of action of GLP-1 to prevent oxidative damage in beta cells through the modulation of signaling pathways involved in antioxidant enzyme regulation. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Thyroid Hormone-Induced Cytosol-to-Nuclear Translocation of Rat Liver Nrf2 Is Dependent on Kupffer Cell Functioning

    PubMed Central

    Videla, Luis A.; Cornejo, Pamela; Romanque, Pamela; Santibáñez, Catherine; Castillo, Iván; Vargas, Romina

    2012-01-01

    L-3,3′,5-triiodothyronine (T3) administration upregulates nuclear factor-E2-related factor 2 (Nrf2) in rat liver, which is redox-sensitive transcription factor mediating cytoprotection. In this work, we studied the role of Kupffer cell respiratory burst activity, a process related to reactive oxygen species generation and liver homeostasis, in Nrf2 activation using the macrophage inactivator gadolinium chloride (GdCl3; 10 mg/kg i.v. 72 h before T3 [0.1 mg/kg i.p.]) or NADPH oxidase inhibitor apocynin (1.5 mmol/L added to the drinking water for 7 days before T3), and determinations were performed 2 h after T3. T3 increased nuclear/cytosolic Nrf2 content ratio and levels of heme oxygenase 1 (HO-1), catalytic subunit of glutamate cysteine ligase, and thioredoxin (Western blot) over control values, proteins whose gene transcription is induced by Nrf2. These changes were suppressed by GdCl3 treatment prior to T3, an agent-eliciting Kupffer-cell depletion, inhibition of colloidal carbon phagocytosis, and the associated respiratory burst activity, with enhancement in nuclear inhibitor of Nrf2 kelch-like ECH-associated protein 1 (Keap1)/Nrf2 content ratios suggesting Nrf2 degradation. Under these conditions, T3-induced tumor necrosis factor-α (TNF-α) response was eliminated by previous GdCl3 administration. Similar to GdCl3, apocynin given before T3 significantly reduced liver Nrf2 activation and HO-1 expression, a NADPH oxidase inhibitor eliciting abolishment of colloidal carbon-induced respiratory burst activity without altering carbon phagocytosis. It is concluded that Kupffer cell functioning is essential for upregulation of liver Nrf2-signaling pathway by T3. This contention is supported by suppression of the respiratory burst activity of Kupffer cells and the associated reactive oxygen species production by GdCl3 or apocynin given prior to T3, thus hindering Nrf2 activation. PMID:22649286

  11. Ferulic acid (FA) abrogates γ-radiation induced oxidative stress and DNA damage by up-regulating nuclear translocation of Nrf2 and activation of NHEJ pathway.

    PubMed

    Das, Ujjal; Manna, Krishnendu; Khan, Amitava; Sinha, Mahuya; Biswas, Sushobhan; Sengupta, Aaveri; Chakraborty, Anindita; Dey, Sanjit

    2017-01-01

    The present study was aimed to evaluate the radioprotective effect of ferulic acid (FA), a naturally occurring plant flavonoid in terms of DNA damage and damage related alterations of repair pathways by gamma radiation. FA was administered at a dose of 50 mg/kg body weight for five consecutive days prior to exposing the swiss albino mice to a single dose of 10 Gy gamma radiation. Ionising radiation induces oxidative damage manifested by decreased expression of Cu, Zn-SOD (SOD stands for super oxide dismutase), Mn-SOD and catalase. Gamma radiation promulgated reactive oxygen species (ROS) mediated DNA damage and modified repair pathways. ROS enhanced nuclear translocation of p53, activated ATM (ataxia telangiectasia-mutated protein), increased expression of GADD45a (growth arrest and DNA-damage-inducible protein) gene and inactivated Non homologous end joining (NHEJ) repair pathway. The comet formation in irradiated mice peripheral blood mononuclear cells (PBMC) reiterated the DNA damage in IR exposed groups. FA pretreatment significantly prevented the comet formation and regulated the nuclear translocation of p53, inhibited ATM activation and expression of GADD45a gene. FA promoted the nuclear translocation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and activated NHEJ repair pathway to overcome ROS mediated oxidative stress and DNA damage. Therefore, the current study stated that FA can challenge the oxidative stress by (i) inducing nuclear translocation of Nrf2, (ii) scavenging ROS, and (iii) activating NHEJ DNA repair process.

  12. Expression of Nrf2 in Neurodegenerative Diseases

    PubMed Central

    Ramsey, Chenere P.; Glass, Charles A.; Montgomery, Marshall B.; Lindl, Kathryn A.; Ritson, Gillian P.; Chia, Luis A.; Hamilton, Ronald L.; Chu, Charleen T.; Jordan-Sciutto, Kelly L.

    2008-01-01

    In response to oxidative stress, the nuclear factor E2-related factor 2 (Nrf2) transcription factor translocates from the cytoplasm into the nucleus and transactivates expression of genes with antioxidant activity. Despite this cellular mechanism, oxidative damage is abundant in Alzheimer and Parkinson disease (AD and PD). To investigate mechanisms by which Nrf2 activity may be aberrant or insufficient in neurodegenerative conditions, we assessed Nrf2 localization in affected brain regions of AD, Lewy body variant of AD (LBVAD), and PD. By immunohistochemistry, Nrf2 is expressed in both the nucleus and the cytoplasm of neurons in normal hippocampi with predominant expression in the nucleus. In AD and LBVAD, Nrf2 was predominantly cytoplasmic in hippocampal neurons and was not a major component of beta amyloid plaques or neurofibrillary tangles. By immunoblotting, we observed a significant decrease in nuclear Nrf2 levels in AD cases. In contrast, Nrf2 was strongly nuclear in PD nigral neurons but cytoplasmic in substantia nigra of normal, AD, and LBVAD cases. These findings suggest that Nrf2-mediated transcription is not induced in neurons in AD despite the presence of oxidative stress. In PD, nuclear localization of Nrf2 is strongly induced, but this response may be insufficient to protect neurons from degeneration. PMID:17204939

  13. Pterostilbene-mediated Nrf2 activation: Mechanistic insights on Keap1:Nrf2 interface.

    PubMed

    Bhakkiyalakshmi, Elango; Dineshkumar, Kesavan; Karthik, Suresh; Sireesh, Dornadula; Hopper, Waheeta; Paulmurugan, Ramasamy; Ramkumar, Kunka Mohanram

    2016-08-15

    The discovery of Keap1-Nrf2 protein-protein interaction (PPI) inhibitors has become a promising strategy to develop novel lead molecules against variety of stress. Hence, Keap1-Nrf2 system plays an important role in oxidative/electrophilic stress associated disorders. Our earlier studies identified pterostilbene (PTS), a natural analogue of resveratrol, as a potent Nrf2 activator and Keap1-Nrf2 PPI inhibitor as assessed by luciferase complementation assay. In this study, we further identified the potential of PTS in Nrf2 activation and ARE-driven downstream target genes expression by nuclear translocation experiments and ARE-luciferase reporter assay, respectively. Further, the luciferase complementation assay identified that PTS inhibits Keap1-Nrf2 PPI in both dose and time-dependent manner. Computational studies using molecular docking and dynamic simulation revealed that PTS directly interacts with the basic amino acids of kelch domain of Keap1 and perturb Keap1-Nrf2 interaction pattern. This manuscript not only shows the binding determinants of Keap1-Nrf2 proteins but also provides mechanistic insights on Nrf2 activation potential of PTS.

  14. Hydroxytyrosol induces antioxidant/detoxificant enzymes and Nrf2 translocation via extracellular regulated kinases and phosphatidylinositol-3-kinase/protein kinase B pathways in HepG2 cells.

    PubMed

    Martín, María Angeles; Ramos, Sonia; Granado-Serrano, Ana Belén; Rodríguez-Ramiro, Ildefonso; Trujillo, Mariana; Bravo, Laura; Goya, Luis

    2010-07-01

    Hydroxytyrosol (HTy) is a natural polyphenol abundant in olive oil, which possesses multiple biological actions. Particularly, HTy has cytoprotective activity against oxidative-stress-induced cell damage, but the underlying mechanisms of action remain unclear. Here, we have investigated the molecular mechanism involved in the protection exerted by HTy on tert-butyl hydroperoxide-induced damage in human HepG2 liver cells. Treatment of HepG2 cells with HTy increased the expression and the activity of glutathione-related enzymes such as glutathione peroxidase, glutathione reductase and glutathione S-transferase. HTy also induced the nuclear transcription factor erythroid 2p45-related factor (Nrf2), a transcription factor implicated in the expression of several antioxidant/detoxificant enzymes. Moreover, two important signalling proteins involved in Nrf2 translocation, the protein kinase B and the extracellular regulated kinases, were also activated by HTy. Further studies with specific inhibitors confirmed that both molecular pathways are critical for the nuclear translocation of Nrf2, the increased enzyme expression and activity and the beneficial effect against oxidative stress induced by HTy. In conclusion, together with the inherent radical scavenging activity of HTy, our results provide an additional mechanism of action to prevent oxidative stress damage through the modulation of signalling pathways involved in antioxidant/detoxifying enzymes regulation.

  15. Restoration of Nrf2 Signaling Normalizes the Regenerative Niche

    PubMed Central

    Soares, Marc A.; Cohen, Oriana D.; Low, Yee Cheng; Sartor, Rita A.; Ellison, Trevor; Anil, Utkarsh; Anzai, Lavinia; Chang, Jessica B.; Saadeh, Pierre B.; Rabbani, Piul S.

    2016-01-01

    Chronic hyperglycemia impairs intracellular redox homeostasis and contributes to impaired diabetic tissue regeneration. The Keap1/Nrf2 pathway is a critical regulator of the endogenous antioxidant response system, and its dysfunction has been implicated in numerous pathologies. Here we characterize the effect of chronic hyperglycemia on Nrf2 signaling within a diabetic cutaneous regeneration model. We characterized the effects of chronic hyperglycemia on the Keap1/Nrf2 pathway within models of diabetic cutaneous wound regeneration. We assessed reactive oxygen species (ROS) production and antioxidant gene expression following alterations in the Nrf2 suppressor Keap1 and the subsequent changes in Nrf2 signaling. We also developed a topical small interfering RNA (siRNA)–based therapy to restore redox homeostasis within diabetic wounds. Western blotting demonstrated that chronic hyperglycemia–associated oxidative stress inhibits nuclear translocation of Nrf2 and impairs activation of antioxidant genes, thus contributing to ROS accumulation. Keap1 inhibition increased Nrf2 nuclear translocation, increased antioxidant gene expression, and reduced ROS production to normoglycemic levels, both in vitro and in vivo. Topical siKeap1 therapy resulted in improved regenerative capacity of diabetic wounds and accelerated closure. We report that chronic hyperglycemia weakens the endogenous antioxidant response, and the consequences of this defect are manifested by intracellular redox dysregulation, which can be restored by Keap1 inhibition. Targeted siRNA-based therapy represents a novel, efficacious strategy to reestablish redox homeostasis and accelerate diabetic cutaneous tissue regeneration. PMID:26647385

  16. A novel CXCR3-B chemokine receptor-induced growth-inhibitory signal in cancer cells is mediated through the regulation of Bach-1 protein and Nrf2 protein nuclear translocation.

    PubMed

    Balan, Murugabaskar; Pal, Soumitro

    2014-02-07

    Chemokines and their receptors play diverse roles in regulating cancer growth and progression. The receptor CXCR3 can have two splice variants with opposite functions. CXCR3-A promotes cell growth, whereas CXCR3-B mediates growth-inhibitory signals. However, the negative signals through CXCR3-B in cancer cells are not well characterized. In this study, we found that CXCR3-B-mediated signaling in MCF-7 and T47D breast cancer cells induced apoptotic cell death. Signals through CXCR3-B decreased the levels of the antiapoptotic proteins Bcl-2 and Bcl-xL and increased the expression of apoptotic cleaved poly(ADP-ribose) polymerase. Along with up-regulation in apoptosis, CXCR3-B signals were associated with a decrease in cellular autophagy with reduced levels of the autophagic markers Beclin-1 and LC3B. Notably, CXCR3-B down-regulated the expression of the cytoprotective and antiapoptotic molecule heme oxygenase-1 (HO-1) at the transcriptional level. There was an increased nuclear localization of Bach-1 and nuclear export of Nrf2, which are important negative and positive transcription factors, respectively, for HO-1 expression. We also observed that CXCR3-B promoted the activation of p38 MAPK and the inhibition of ERK-1/2. CXCR3-B could not induce cancer cell apoptosis at the optimal level when we either inhibited p38 activity or knocked down Bach-1. Further, CXCR3-B-induced apoptosis was down-regulated when we overexpressed HO-1. Together, our data suggest that CXCR3-B mediates a growth-inhibitory signal in breast cancer cells through the modulations of nuclear translocation of Bach-1 and Nrf2 and down-regulation of HO-1. We suggest that the induction of CXCR3-B-mediated signaling can serve as a novel therapeutic approach where the goal is to promote tumor cell apoptosis.

  17. Nrf2-mediated cardiac maladaptive remodeling and dysfunction in a setting of autophagy insufficiency

    PubMed Central

    Qin, Qingyun; Qu, Chen; Niu, Ting; Zang, Huimei; Qi, Lei; Lyu, Linmao; Wang, Xuejun; Nagarkatti, Mitzi; Nagarkatti, Prakash; Janicki, Joseph S; Wang, Xing Li; Cui, Taixing

    2016-01-01

    Nuclear factor erythroid-2 related factor 2 (Nrf2) appears to exert either a protective or detrimental effect on the heart; however, the underlying mechanism remains poorly understood. Herein, we uncovered a novel mechanism for turning off the Nrf2-mediated cardioprotection while switching on Nrf2-mediated cardiac dysfunction. In a murine model of pressure overload-induced cardiac remodeling and dysfunction via transverse aortic arch constriction (TAC), knockout of Nrf2 enhanced myocardial necrosis and death rate during an initial stage of cardiac adaptation when myocardial autophagy function is intact. However, knockout of Nrf2 turned out to be cardioprotective throughout the later stage of cardiac maladaptive remodeling when myocardial autophagy function became insufficient. TAC-induced activation of Nrf2 was dramatically enhanced in the heart with impaired autophagy which is induced by cardiomyocyte-specific knockout of autophagy related gene (Atg)5. Notably, Nrf2 activation coincided with upregulation of angiotensinogen (Agt) only in the autophagy impaired heart after TAC. Agt5 and Nrf2 gene loss of function approaches in combination with Jak2 and Fyn kinase inhibitors revealed that suppression of autophagy inactivated Jak2 and Fyn as well as nuclear translocation of Fyn while enhancing nuclear translocation of Nrf2 and Nrf2-driven Agt expression in cardiomyocytes. Taken together; these results indicate that the pathophysiological consequences of Nrf2 activation are closely linked with the functional integrity of myocardial autophagy during cardiac remodeling. When autophagy is intact, Nrf2 is required for cardiac adaptive responses; however, autophagy impairment most likely turns off Fyn-operated Nrf2 nuclear export thus activating Nrf2-driven Agt transcription, which exacerbates cardiac maladaptation leading to dysfunction. PMID:26573705

  18. NRF2 plays a protective role in diabetic retinopathy in mice

    PubMed Central

    Xu, Zhenhua; Wei, Yanhong; Gong, Junsong; Cho, Hongkwan; Park, James K.; Sung, Ee-Rah; Huang, Hu; Wu, Lijuan; Eberhart, Charles; Handa, James T.; Du, Yunpeng; Kern, Timothy S.; Thimmulappa, Rajesh; Barber, Alistair J.; Biswal, Shyam; Duh, Elia J.

    2014-01-01

    Aims/hypothesis Although much is known about the pathophysiological processes contributing to diabetic retinopathy (DR), the role of protective pathways has received less attention. The transcription factor nuclear factor erythroid-2-related factor 2 (also known as NFE2L2 or NRF2) is an important regulator of oxidative stress and also has anti-inflammatory effects. The objective of this study was to explore the potential role of NRF2 as a protective mechanism in DR. Methods Retinal expression of NRF2 was investigated in human donor and mouse eyes by immunohistochemistry. The effect of NRF2 modulation on oxidative stress was studied in the human Müller cell line MIO-M1. Non-diabetic and streptozotocin-induced diabetic wild-type and Nrf2 knockout mice were evaluated for multiple DR endpoints. Results NRF2 was expressed prominently in Müller glial cells and astrocytes in both human and mouse retinas. In cultured MIO-M1 cells, NRF2 inhibition significantly decreased antioxidant gene expression and exacerbated tert-butyl hydroperoxide- and hydrogen peroxide-induced oxidative stress. NRF2 activation strongly increased NRF2 target gene expression and suppressed oxidant-induced reactive oxygen species. Diabetic mice exhibited retinal NRF2 activation, indicated by nuclear translocation. Superoxide levels were significantly increased by diabetes in Nrf2 knockout mice as compared with wild-type mice. Diabetic Nrf2 knockout mice exhibited a reduction in retinal glutathione and an increase in TNF-α protein compared with wild-type mice. Nrf2 knockout mice exhibited early onset of blood–retina barrier dysfunction and exacerbation of neuronal dysfunction in diabetes. Conclusions/interpretation These results indicate that NRF2 is an important protective mechanism regulating the progression of DR and suggest enhancement of the NRF2 pathway as a potential therapeutic strategy. PMID:24186494

  19. Enhancement of the Effect of Methyl Pyropheophorbide-a-Mediated Photodynamic Therapy was Achieved by Increasing ROS via Inhibition of Nrf2-HO-1 or Nrf2-ABCG2 Signaling.

    PubMed

    Tian, Si; Yong, Min; Zhu, Jiang; Zhang, Li; Pan, Li; Chen, Qing; Li, Kai-Ting; Kong, Yu-Han; Jiang, Yuan; Yu, Ting-He; Yu, Le-Hua; Bai, Ding-Qun

    2017-03-27

    Emerging evidence indicates that the transcription factor nuclear factor-E2-related factor 2 (-NRF2) plays an essential role in cellular defense against oxidative stress; its activation has been related to cytoprotection. Here, we investigated the role of Nrf2 in improving the efficacy of methyl pyropheophorbide-a-mediated photodynamic therapy (Mppa-PDT) via the downregulation of Nrf2 in human ovarian cancer A2780 cells and SKOV3 cells. We found that Nrf2 translocated from the cytoplasm to the nucleus in vitro and in vivo, and the expression of Nrf2 and P-Nrf2 increased through a possible mechanism regulated by mitogen-activated protein kinase (MAPK) after Mppa-PDT treatment. Furthermore, cytotoxicity and apoptosis induced by Mppa-PDT increased after Nrf2down-regulation. Nrf2 down -regulation increased reactive oxygen species (ROS) levels by attenuating antioxidants or pumping Mppa out of cells, which resulted from the inhibition of Nrf2-HO-1 or Nrf2-ABCG2 signaling. In addition, SKOV3 cells exhibited increased resistance to Mppa-PDT, and the expression levels of P-Nrf2 and ABCG2 were higher in SKOV3 cells than in A2780 cells, suggesting that Nrf2-ABCG2 signaling might be involved in the intrinsic resistanceto Mppa-PDT. Taken together, these results provided evidence that Nrf2 down-regulation can enhance the effect of Mppa-PDT.

  20. Multiple nuclear localization signals function in the nuclear import of the transcription factor Nrf2.

    PubMed

    Theodore, Melanie; Kawai, Yumiko; Yang, Jianqi; Kleshchenko, Yuliya; Reddy, Sekhar P; Villalta, Fernando; Arinze, Ifeanyi J

    2008-04-04

    Nuclear factor erythroid 2-related factor 2 (Nrf2) mediates the transcriptional response of cells to oxidative stress and is translocated into the nucleus following, or concomitant with, its activation by electrophiles or reactive oxygen species. The mechanism of its translocation into the nucleus is not entirely elucidated. Here we have identified two novel nuclear localization signal (NLS) motifs in murine Nrf2, one located near the N-terminal region (amino acid residues 42-53) and the other (residues 587-593) located near the C-terminal region. Imaging of green fluorescent protein (GFP)-tagged Nrf2 revealed that mutation(s) in any of these sequences resulted in decreased nuclear fluorescence intensity compared with the wild-type Nrf2 when Nrf2 activation was induced with the electrophile tert-butylhydroquinone. The mutations also impaired Nrf2-induced transactivation of antioxidant response element-driven reporter gene expression to the same extent as the Nrf2 construct bearing mutation in a previously identified bipartite NLS that maps at residues 494-511. When linked to GFP or to GFP-PEPCK-C each of the novel NLS motifs was sufficient to drive nuclear translocation of the fusion proteins. Co-immunoprecipitation assays demonstrated that importins alpha5 and beta1 associate with Nrf2, an interaction that was blocked by the nuclear import inhibitor SN50. SN50 also blocked tert-butylhydroquinone-induced nuclear fluorescence of GFP-Nrf2 in cells transfected with wild-type GFP-Nrf2. Overall these results reveal that multiple NLS motifs in Nrf2 function in its nuclear translocation in response to pro-oxidant stimuli and that the importin alpha-beta heterodimer nuclear import receptor system plays a critical role in the import process.

  1. NRF2-regulation in brain health and disease: implication of cerebral inflammation

    PubMed Central

    Sandberg, Mats; Patil, Jaspal; D’Angelo, Barbara; Weber, Stephen G; Mallard, Carina

    2014-01-01

    The nuclear factor erythroid 2 related factor 2 (NRF2) is a key regulator of endogenous inducible defense systems in the body. Under physiological conditions NRF2 is mainly located in the cytoplasm. However, in response to oxidative stress, NRF2 translocates to the nucleus and binds to specific DNA sites termed “anti-oxidant response elements” or “electrophile response elements” to initiate transcription of cytoprotective genes. Acute oxidative stress to the brain, such as stroke and traumatic brain injury is increased in animals that are deficient in NRF2. Insufficient NRF2 activation in humans has been linked to chronic diseases such as Parkinson’s disease, Alzheimer’s disease and amyotrophic lateral sclerosis. New findings have also linked activation of the NRF2 system to anti-inflammatory effects via interactions with NF-κB. Here we review literature on cellular mechanisms of NRF2 regulation, how to maintain and restore NRF2 function and the relationship between NRF2 regulation and brain damage. We bring forward the hypothesis that inflammation via prolonged activation of key kinases (p38 and GSK-3β) and activation of histone deacetylases gives rise to dysregulation of the NRF2 system in the brain, which contributes to oxidative stress and injury. PMID:24262633

  2. DNA Demethylation Upregulated Nrf2 Expression in Alzheimer’s Disease Cellular Model

    PubMed Central

    Cao, Huimin; Wang, Li; Chen, Beibei; Zheng, Peng; He, Yi; Ding, Yubin; Deng, Yushuang; Lu, Xi; Guo, Xiuming; Zhang, Yuping; Li, Yu; Yu, Gang

    2016-01-01

    Nuclear factor erythroid 2-related factor 2 (Nrf2) is an important transcription factor in the defense against oxidative stress. Cumulative evidence has shown that oxidative stress plays a key role in the pathogenesis of Alzheimer’s disease (AD). Previous animal and clinical studies had observed decreased expression of Nrf2 in AD. However, the underlying regulation mechanisms of Nrf2 in AD remain unclear. Here, we used the DNA methyltransferases (Dnmts) inhibitor 5-aza-2′-deoxycytidine (5-Aza) to test whether Nrf2 expression was regulated by methylation in N2a cells characterizing by expressing human Swedish mutant amyloid precursor protein (N2a/APPswe). We found 5-Aza treatment increased Nrf2 at both messenger RNA and protein levels via downregulating the expression of Dnmts and DNA demethylation. In addition, 5-Aza-mediated upregulation of Nrf2 expression was concomitant with increased nuclear translocation of Nrf2 and higher expression of Nrf2 downstream target gene NAD(P)H:quinone oxidoreductas (NQO1). Our study showed that DNA demethylation promoted the Nrf2 cell signaling pathway, which may enhance the antioxidant system against AD development. PMID:26779013

  3. Salidroside Suppresses HUVECs Cell Injury Induced by Oxidative Stress through Activating the Nrf2 Signaling Pathway.

    PubMed

    Zhu, Yao; Zhang, Ya-Jie; Liu, Wei-Wei; Shi, Ai-Wu; Gu, Ning

    2016-08-09

    Oxidative stress plays an important role in the pathogenesis of cardiovascular diseases. Salidroside (SAL), one of the main effective constituents of Rhodiola rosea, has been reported to suppress oxidative stress-induced cardiomyocyte injury and necrosis by promoting transcription of nuclear factor E2-related factor 2 (Nrf2)-regulated genes such as heme oxygenase-1 (HO-1) and NAD(P)H dehydrogenase (quinone1) (NQO1). However, it has not been indicated whether SAL might ameliorate endothelial injury induced by oxidative stress. Here, our study demonstrated that SAL might suppress HUVEC cell injury induced by oxidative stress through activating the Nrf2 signaling pathway. The results of our study indicated that SAL decreased the levels of intercellular reactive oxygen species (ROS) and malondialdehyde (MDA), and improved the activities of superoxide dismutase (SOD) and catalase (CAT), resulting in protective effects against oxidative stress-induced cell damage in HUVECs. It suppressed oxidative stress damage by inducing Nrf2 nuclear translocation and activating the expression of Nrf2-regulated antioxidant enzyme genes such as HO-1 and NQO1 in HUVECs. Knockdown of Nrf2 with siRNA abolished the cytoprotective effects against oxidative stress, decreased the expression of Nrf2, HO-1, and NQO1, and inhibited the nucleus translocation of Nrf2 in HUVECs. This study is the first to demonstrate that SAL suppresses HUVECs cell injury induced by oxidative stress through activating the Nrf2 signaling pathway.

  4. Activation of Nrf2 in defense against cadmium-induced oxidative stress.

    PubMed

    He, Xiaoqing; Chen, Michael G; Ma, Qiang

    2008-07-01

    Exposure to cadmium (Cd) elicits a range of adverse responses including oxidative damage and cancer. The molecular targets of Cd remain largely unidentified. Here, we analyzed the function and signal transduction of transcription factor Nrf2 in protection against Cd-induced oxidative stress. Wild-type (Nrf2 (+/+)) mouse embryonic fibroblasts (MEF) produced reactive oxygen species (ROS) at a low level, whereas treatment with Cd significantly increased the ROS production. On the other hand, Nrf2 knockout (Nrf2 (-/-)) MEF cells exhibited an elevated level of ROS under a basal condition, and Cd dramatically increased the ROS production at concentrations as low as 2 microM, resulting in increased sensitivity to Cd-induced cell death. Cd induced the basal and inducible expression of cytoprotective enzymes NQO1 and HO1 in WT MEF cells, but induction was lost in Nrf2 (-/-) MEF cells. Induction of the genes required antioxidant response elements (ARE) as Cd drove ARE-dependent reporter expression and Cd-activated Nrf2 bound to endogenous AREs in mouse hepa1c1c7 cells. Activation of Nrf2 by Cd involved stabilization of the Nrf2 protein, increased formation of Nrf2/Keap1 complex in the cytoplasm, translocation of the complex into the nucleus, and subsequently disruption of the complex. Lastly, Nrf2 was found ubiquitinated in the cytoplasm but deubiquitinated in the nucleus. The study provided a mechanistic transcriptional model in which Cd activates Nrf2 through a metal-activated signaling pathway involving a dynamic interplay between ubiquitination/deubiquitination and complex formation/dissociation of Nrf2 and Keap1.

  5. NRF2, cancer and calorie restriction

    PubMed Central

    Martín-Montalvo, A; Villalba, JM; Navas, P; de Cabo, R

    2015-01-01

    The transcription factor NF-E2-related factor (NRF2) is a key regulator of several enzymatic pathways, including cytoprotective enzymes in highly metabolic organs. In this review, we summarize the ongoing research related to NRF2 activity in cancer development, focusing on in vivo studies using NRF2 knockout (KO) mice, which have helped in defining the crucial role of NRF2 in chemoprevention. The lower cancer protection observed in NRF2 KO mice under calorie restriction (CR) suggests that most of the beneficial effects of CR on the carcinogenesis process are likely mediated by NRF2. We propose that future interventions in cancer treatment would be carried out through the activation of NRF2 in somatic cells, which will lead to a delay or prevention of the onset of some forms of human cancers, and subsequently an extension of health- and lifespan. PMID:21057541

  6. Targeting Nrf2 Signaling to Combat Chemoresistance

    PubMed Central

    No, Jae Hong; Kim, Yong-Beom; Song, Yong Sang

    2014-01-01

    Nuclear factor E2-related factor 2 (Nrf2) is a transcription factor that upregulates expression of a battery of genes to combat oxidative and electrophilic stress. Modification of Kelch-like ECH-associated protein 1 (Keap1) by reactive oxygen species stabilizes Nrf2 by escaping from degradation. Nrf2 then binds to antioxidant response elements (AREs) on the promoter region of various genes. Activation of the Keap1-Nrf2-ARE pathway plays critical roles in the chemopreventive effect of various phytochemicals. However, Nrf2 can protect cancer cells from oxidative stress and promote cell proliferation. Moreover, recent studies reveal that activation of the Nrf2 pathway is critical for resistance to chemotherapeutic agents. The aim of this review is to provide a molecular basis for the use of Nrf2 inhibitors in overcoming chemoresistance. PMID:25337579

  7. Coordinated induction of Nrf2 target genes protects against iron nitrilotriacetate (FeNTA)-induced nephrotoxicity

    SciTech Connect

    Tanaka, Yuji; Aleksunes, Lauren M. |; Goedken, Michael J.; Chen, Chuan; Reisman, Scott A.; Manautou, Jose E.; Klaassen, Curtis D.

    2008-09-15

    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 6h 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.

  8. Neuroprotective effect of fermented papaya preparation by activation of Nrf2 pathway in astrocytes.

    PubMed

    Murakami, Shinki; Miyazaki, Ikuko; Asanuma, Masato

    2016-11-14

    Nuclear factor erythroid 2-related factor (Nrf2) in astrocyte plays important roles in brain homeostasis. Fermented papaya preparation (FPP) has anti-oxidative, anti-inflammatory, immunoregulatory properties. The present study investigated the effects of FPP on activation of Nrf2 and release of Nrf2-regulated neuroprotective antioxidants and detoxifying molecules. Primary cultured astrocytes from rat embryos were treated with FPP for 6 or 24 hours. The expression levels of nuclear Nrf2 and cytoplasmic Nrf2-regulated molecules were determined by western blot analysis and immunohistochemistry. Glutathione levels were measured in cells and medium. Dopaminergic neurons were exposed 6-hydroxydopamine (6-OHDA) with/without pre-treatment with FPP astrocytes. Mice were treated orally with FPP for 2 weeks. FPP increased nuclear translocation of Nrf2 in striatal astrocytes, induced up-regulation of NAD(P)H quinine oxidoreductase-1, glutathione-S transferase and hemeoxygenase-1, and increased glutathione level and the percentage of metallothionein-expressing astrocytes. Moreover, FPP suppressed 6-OHDA-induced dopaminergic neuronal loss in not only neuron-astrocyte mixed culture, but also neuron-rich cultures pre-treated with glial conditioned medium. Two-week oral treatment of mice with FPP resulted in Nrf2 activation and increase in glutathione level in striatum. The results indicated that FPP enhances the anti-oxidative capacity through activation of Nrf2 in astrocytes, suggesting it may provide neuroprotection in oxidative stress-related neurodegenerative diseases.

  9. Hsp90 Interaction with INrf2(Keap1) Mediates Stress-induced Nrf2 Activation*

    PubMed Central

    Niture, Suryakant K.; Jaiswal, Anil K.

    2010-01-01

    INrf2(Keap1) functions as an adapter for Cul3/Rbx1-mediated degradation of Nrf2. In response to stress, Nrf2 is released from INrf2 and translocates inside the nucleus leading to activation of cytoprotective proteins critical in protection against adverse effects including cancer. We demonstrate here a novel role of heat shock protein 90 (Hsp90) in control of the INrf2 and Nrf2 activation. Hsp90 interacted with INrf2 that leds to stabilization of INrf2 during heat shock stress. Domain mapping showed the requirement of INrf2-NTR and the Hsp90-CLD region for interaction of Hsp90 with INrf2. Heat shock and antioxidants induced Hsp90, and casein kinase 2 (CK2) phosphorylated INrf2Thr55. This led to increased Hsp90-INrf2 interaction, dissociation of the Rbx1/Cul3·INrf2·Nrf2 complex, and activation of Nrf2. Inhibitors of CK2 and Hsp90, and mutation of INrf2Thr55 abolished the Hsp90-INrf2 interaction and downstream signaling. INrf2 is released from Hsp90 once the heat shock or antioxidant stress subsidized, thereby allowing INrf2 to interact with Nrf2 and facilitate Nrf2 ubiquitination and degradation. The results together demonstrate a novel role for the stress-induced Hsp90-INrf2 interaction in regulation of Nrf2 activation and induction of cytoprotective proteins. PMID:20864537

  10. Nrf2 activity as a potential biomarker for the pan-epigenetic anticancer agent, RRx-001

    PubMed Central

    Ning, Shoucheng; Sekar, Thillai Veerapazham; Scicinski, Jan; Oronsky, Bryan; Peehl, Donna M.; Knox, Susan J.; Paulmurugan, Ramasamy

    2015-01-01

    Nuclear factor erythroid 2-related factor 2 (Nrf2) is a master regulatory transcription factor that plays an important role in the antioxidant response pathway against anticancer drug-induced cytotoxic effects. RRx-001 is a new anticancer agent that generates reactive oxygen and nitrogen species, and leads to epigenetic alterations in cancer cells. Here we report the RRx-001 mediated nuclear translocation of Nrf2 and the activation of expression of its downstream enzymes HO-1 and NQO1 in tumor cells. Inhibition of intrinsic Nrf2 expression by Nrf2-specific siRNA increased cell sensitivity to RRx-001. Molecular imaging of tumor cells co-expressing pARE-Firefly luciferase and pCMV-Renilla luciferase-mRFP in vitro and in vivo in mice revealed that RRx-001 significantly increased ARE-FLUC signal in cells in a dose- and time-dependent manner, suggesting that RRx-001 is an effective activator of the Nrf2-ARE signaling pathway. The pre-treatment level of ARE-FLUC signal in cells, reflecting basal activity of Nrf2, negatively correlated with the tumor response to RRx-001. The results support the concept that RRx-001 activates Nrf2-ARE antioxidant signaling pathways in tumor cells. Hence measurement of Nrf2-mediated activation of downstream target genes through ARE signaling may constitute a useful molecular biomarker for the early prediction of response to RRx-001 treatment, and thereby guide therapeutic decision-making. PMID:26280276

  11. Glycosylation enables aesculin to activate Nrf2

    PubMed Central

    Kim, Kyun Ha; Park, Hyunsu; Park, Hee Jin; Choi, Kyoung-Hwa; Sadikot, Ruxana T.; Cha, Jaeho; Joo, Myungsoo

    2016-01-01

    Since aesculin, 6,7-dihydroxycoumarin-6-O-β-glucopyranoside, suppresses inflammation, we asked whether its anti-inflammatory activity is associated with the activation of nuclear factor-E2-related factor 2 (Nrf2), a key anti-inflammatory factor. Our results, however, show that aesculin marginally activated Nrf2. Since glycosylation can enhance the function of a compound, we then asked whether adding a glucose makes aesculin activate Nrf2. Our results show that the glycosylated aesculin, 3-O-β-d-glycosyl aesculin, robustly activated Nrf2, inducing the expression of Nrf2-dependent genes, such as heme oxygenase-1, glutamate-cysteine ligase catalytic subunit, and NAD(P)H quinone oxidoreductase 1 in macrophages. Mechanistically, 3-O-β-d-glycosyl aesculin suppressed ubiquitination of Nrf2, retarding degradation of Nrf2. Unlike aesculin, 3-O-β-d-glycosyl aesculin significantly suppressed neutrophilic lung inflammation, a hallmark of acute lung injury (ALI), in mice, which was not recapitulated in Nrf2 knockout mice, suggesting that the anti-inflammatory function of the compound largely acts through Nrf2. In a mouse model of sepsis, a major cause of ALI, 3-O-β-d-glycosyl aesculin significantly enhanced the survival of mice, compared with aesculin. Together, these results show that glycosylation could confer the ability to activate Nrf2 on aesculin, enhancing the anti-inflammatory function of aesculin. These results suggest that glycosylation can be a way to improve or alter the function of aesculin. PMID:27417293

  12. Nrf2 protects against airway disorders

    SciTech Connect

    Cho, Hye-Youn; Kleeberger, Steven R.

    2010-04-01

    Nuclear factor-erythroid 2 related factor 2 (Nrf2) is a ubiquitous master transcription factor that regulates antioxidant response elements (AREs)-mediated expression of antioxidant enzyme and cytoprotective proteins. In the unstressed condition, Kelch-like ECH-associated protein 1 (Keap1) suppresses cellular Nrf2 in cytoplasm and drives its proteasomal degradation. Nrf2 can be activated by diverse stimuli including oxidants, pro-oxidants, antioxidants, and chemopreventive agents. Nrf2 induces cellular rescue pathways against oxidative injury, abnormal inflammatory and immune responses, apoptosis, and carcinogenesis. Application of Nrf2 germ-line mutant mice has identified an extensive range of protective roles for Nrf2 in experimental models of human disorders in the liver, gastrointestinal tract, airway, kidney, brain, circulation, and immune or nerve system. In the lung, lack of Nrf2 exacerbated toxicity caused by multiple oxidative insults including supplemental respiratory therapy (e.g., hyperoxia, mechanical ventilation), cigarette smoke, allergen, virus, bacterial endotoxin and other inflammatory agents (e.g., carrageenin), environmental pollution (e.g., particles), and a fibrotic agent bleomycin. Microarray analyses and bioinformatic studies elucidated functional AREs and Nrf2-directed genes that are critical components of signaling mechanisms in pulmonary protection by Nrf2. Association of loss of function with promoter polymorphisms in NRF2 or somatic and epigenetic mutations in KEAP1 and NRF2 has been found in cohorts of patients with acute lung injury/acute respiratory distress syndrome or lung cancer, which further supports the role for NRF2 in these lung diseases. In the current review, we address the role of Nrf2 in airways based on emerging evidence from experimental oxidative disease models and human studies.

  13. Nimesulide-induced electrophile stress activates Nrf2 in human hepatocytes and mice but is not sufficient to induce hepatotoxicity in Nrf2-deficient mice.

    PubMed

    Kale, Vijay M; Hsiao, Chin-ju J; Boelsterli, Urs A

    2010-05-17

    Nimesulide is a widely prescribed nitroaromatic sulfoanilide-type nonsteroidal anti-inflammatory drug that, despite its favorable safety profile, has been associated with rare cases of idiosyncratic drug-induced liver injury (DILI). Because reactive metabolites have been implicated in DILI, we aimed at investigating whether hepatic bioactivation of nimesulide produces a protein-reactive intermediate in hepatocytes. Also, we explored whether nimesulide can activate the transcription factor Nrf2 that would protect from drug-induced hepatocyte injury. We found that [(14)C]-nimesulide covalently bound to human liver microsomes (<50 pmol/mg under standard conditions) or immortalized human hepatocytes in a sulfaphenazole-sensitive, rifampicin-inducible manner; yet the overall extent of binding was modest. Although exposure of hepatocytes to nimesulide was not associated with increased net levels of superoxide anion, nimesulide (100 microM, 24 h) caused nuclear translocation of Nrf2 in a sulfaphenazole-sensitive manner, indicating a role of electrophilic metabolites. However, knockdown of Nrf2 with siRNA did not make the cells more sensitive to nimesulide-induced cell injury. Similarly, exposure of wild-type C57BL/6x129 Sv mice to nimesulide (100 mg/kg/day, po, for 5 days) was associated with nuclear translocation of immunoreactive Nrf2 in a small number of hepatocytes and induced >2-fold the expression levels of the Nrf2-target gene Nqo1 in wild-type but not Nrf2-null mice. Nimesulide administered to Nrf2(-/-) knockout mice did not cause increases in serum ALT activity or any apparent histopathological signs of liver injury. In conclusion, these data indicate that nimesulide is bioactivated by CYP2C to a protein-reactive electrophilic intermediate that activates the Nrf2 pathway even at nontoxic exposure levels.

  14. Keap1-Nrf2 Activation in the Presence and Absence of DJ-1

    PubMed Central

    Gan, Li; Johnson, Delinda A.; Johnson, Jeffrey A.

    2012-01-01

    The molecular mechanisms leading to neurodegeneration in Parkinson’s disease remain elusive. Deletion and mutations of DJ-1 (PARK7) have been reported to cause autosomal recessive familial Parkinson’s disease. Wildtype DJ-1 scavenges H2O2 by cysteine oxidation in response to oxidative stress, and thus confers neuroprotection. Activation of the transcription factor NF-E2 related factor-2 (Nrf2) has also been shown to be important for protection against oxidative stress in many models of neurodegenerative diseases. Previous data indicate that DJ-1 affects the transcriptional functions and stability of Nrf2. However, this observation has not been confirmed. In the current study, the role of DJ-1 in the regulation of Nrf2 is examined in primary cultured neurons, astrocytes and in vivo. The prototypical Nrf2 activator, tBHQ, protected primary cortical neurons derived from DJ-1 knockout (KO) as well as DJ-1 wildtype mice by activation of Nrf2-ARE pathway. Nrf2 nuclear translocation, robust increases of canonical Nrf2-driven genes and proteins, and dramatic activation of the ARE reporter gene, hPAP, were observed after tBHQ treatment. These results were further confirmed by siRNA mediated DJ-1 knockdown in primary cortical astrocytes from ARE-hPAP mice and tBHQ administration into the striatum of mouse brain. In addition, over-expression of Nrf2 with adenovirus preferentially in astrocytes from DJ-1 KO mice enhanced survival of neurons under oxidative insults. These findings indicate that activation of the Nrf2-ARE pathway is independent of DJ-1, and Nrf2 activation is a potential therapeutic target to prevent neurodegeneration in sporadic and DJ-1 familial Parkinson’s disease. PMID:20377612

  15. CDDO-Im protects from acetaminophen hepatotoxicity through induction of Nrf2-dependent genes

    SciTech Connect

    Reisman, Scott A.; Buckley, David B.; Tanaka, Yuji; Klaassen, Curtis D.

    2009-04-01

    CDDO-Im is a synthetic triterpenoid recently shown to induce cytoprotective genes through the Nrf2-Keap1 pathway, an important mechanism for the induction of cytoprotective genes in response to oxidative stress. Upon oxidative or electrophilic insult, the transcription factor Nrf2 translocates to the nucleus, heterodimerizes with small Maf proteins, and binds to antioxidant response elements (AREs) in the upstream promoter regions of various cytoprotective genes. To further elucidate the hepatoprotective effects of CDDO-Im, wild-type and Nrf2-null mice were pretreated with CDDO-Im (1 mg/kg, i.p.) or vehicle (DMSO), and then administered acetaminophen (500 mg/kg, i.p.). Pretreatment of wild-type mice with CDDO-Im reduced liver injury caused by acetaminophen. In contrast, hepatoprotection by CDDO-Im was not observed in Nrf2-null mice. CDDO-Im increased Nrf2 protein expression and Nrf2-ARE binding in wild-type, but not Nrf2-null mice. Furthermore, CDDO-Im increased the mRNA expression of the Nrf2 target genes NAD(P)H: quinone oxidoreductase-1 (Nqo1); glutamate-cysteine ligase, catalytic subunit (Gclc); and heme-oxygenase-1 (Ho-1), in both a dose- and time-dependent manner. Conversely, CDDO-Im did not induce Nqo1, Gclc, and Ho-1 mRNA expression in Nrf2-null mice. Collectively, the present study shows that CDDO-Im pretreatment induces Nrf2-dependent cytoprotective genes and protects the liver from acetaminophen-induced hepatic injury.

  16. t-BHQ Provides Protection against Lead Neurotoxicity via Nrf2/HO-1 Pathway

    PubMed Central

    Ye, Fang; Li, Xiaoyi; Li, Lili; Yuan, Jing; Chen, Jun

    2016-01-01

    The neurotoxicity of lead has been well established, and oxidative stress is strongly associated with lead-induced neurotoxicity. Nrf2 is important for protection against oxidative stress in many disease models. We applied t-BHQ, which is an Nrf2 activator, to investigate the possible role of Nrf2 in the protection against lead neurotoxicity. t-BHQ significantly attenuated the oxidative stress in developmental rats by decreasing MDA level, as well as by increasing SOD activity and GSH content, in the hippocampus and frontal cortex. Furthermore, neuronal apoptosis was detected by Nissl staining, and Bax expression was inhibited in the t-BHQ-treated group. Results showed that t-BHQ suppressed ROS production and caspase 3/7 activity but increased intracellular GSH content, in SH-SY5Y cells under lead exposure. Moreover, in vivo and in vitro, t-BHQ enhanced the nuclear translocation of Nrf2 and binding to ARE areas but did not induce Nrf2 transcription. These phenomena were confirmed using RT-PCR, EMSA, Western blot, and immunofluorescence analyses. Subsequent upregulation of the expression of HO-1, NQO1, and GCLC was observed. However, knockdown of Nrf2 or HO-1 adversely affected the protective effects of t-BHQ against lead toxicity in SH-SY5Y cells. Thus, t-BHQ can protect against lead neurotoxicity, depending on the Nrf2/HO-1 pathway. PMID:26798413

  17. NRF2/miR-140 signaling confers radioprotection to human lung fibroblasts

    PubMed Central

    Duru, Nadire; Gernapudi, Ramkishore; Zhang, Yongshu; Yao, Yuan; Lo, Pang-Kuo; Wolfson, Benjamin; Zhou, Qun

    2016-01-01

    Breast and lung cancer patients who are treated with radiotherapy often have severe side effects, including radiation-induced lung damage and secondary cancers. Activation of the NRF2 pathway is a well-known mechanism that protects cells against radiation induced oxidative stress, but its role in radiation-induced lung damage is not well understood. Using human lung fibroblasts (HLFs) we found that ionizing radiation (IR) leads to BRCA1-dependent activation of NRF2 through the inhibition of KEAP1 function, promoting the nuclear accumulation of NRF2, and activating critical radioprotective mechanisms. We discovered that NRF2 directly binds to the miR-140 promoter and increases its expression in response to IR treatment. Gain and loss of function studies further showed the ability of miR-140 to regulate lung fibroblast self-renewal upon irradiation, a potential mechanism to contribute to the regulation of DNA repair. We verified our in vitro findings using primary lung fibroblast cultures from wild type and Nrf2 (KO) mice. Using these models we showed that IR induces overexpression of Brca1, Nrf2 and miR-140 in lung tissue after irradiation. These data reveal a novel radioprotective mechanism in which IR promotes NRF2 nuclear translocation and subsequent activation of miR-140 transcription in HLFs. PMID:26300493

  18. Phosphorylation and dephosphorylation of tyrosine 141 regulate stability and degradation of INrf2: a novel mechanism in Nrf2 activation.

    PubMed

    Jain, Abhinav K; Mahajan, Shilpi; Jaiswal, Anil K

    2008-06-20

    INrf2-Nrf2 proteins are sensors of chemical/radiation stress. Nrf2, in response to stresses, is released from INrf2. Nrf2 is translocated into the nucleus where it binds to the antioxidant response element and coordinately activates the expression of a battery of genes that protect cells against oxidative and electrophilic stress. An autoregulatory loop between INrf2 and Nrf2 regulates their cellular abundance. Nrf2 activates INrf2 gene expression, and INrf2 serves as an adapter for degradation of Nrf2. In this report, we demonstrate that mutation of tyrosine 141 in bric-a-bric, tramtrack, broad complex domain to alanine rendered INrf2 unstable and nonfunctional. INrf2Y141A mutant degraded rapidly as compared with wild type INrf2, although it could dimerize and bind Nrf2. De novo synthesized INrf2 protein was phosphorylated at tyrosine 141. Tyrosine 141-phosphorylated INrf2 was highly stable. Treatment with hydrogen peroxide, which is an oxidizing agent, led to dephosphorylation of INrf2Y141, resulting in rapid degradation of INrf2. This resulted in stabilization of Nrf2 and activation of ARE-mediated gene expression. These results demonstrate that stress-induced dephosphorylation of tyrosine 141 is a novel mechanism in Nrf2 activation and cellular protection.

  19. Targeting NRF2 signaling for cancer chemoprevention

    SciTech Connect

    Kwak, Mi-Kyoung; Kensler, Thomas W.

    2010-04-01

    Modulation of the metabolism and disposition of carcinogens through induction of cytoprotective enzymes is one of several promising strategies to prevent cancer. Chemopreventive efficacies of inducers such as dithiolethiones and sulforaphane have been extensively studied in animals as well as in humans. The KEAP1-NRF2 system is a key, but not unilateral, molecular target for these chemopreventive agents. The transcription factor NRF2 (NF-E2-related factor 2) is a master regulator of the expression of a subset of genes, which produce proteins responsible for the detoxication of electrophiles and reactive oxygen species as well as the removal or repair of some of their damage products. It is believed that chemopreventive enzyme inducers affect the interaction between KEAP1 and NRF2 through either mediating conformational changes of the KEAP1 protein or activating phosphorylation cascades targeting the KEAP1-NRF2 complex. These events in turn affect NRF2 stability and trafficking. Recent advances elucidating the underlying structural biology of KEAP1-NRF2 signaling and identification of the gene clusters under the transcriptional control of NRF2 are facilitating understanding of the potential pleiotropic effects of NRF2 activators and discovery of novel classes of potent chemopreventive agents such as the triterpenoids. Although there is appropriately a concern regarding a deleterious role of the KEAP1-NRF2 system in cancer cell biology, especially as the pathway affects cell survival and drug resistance, the development and the use of NRF2 activators as chemopreventive agents still holds a great promise for protection of normal cells from a diversity of environmental stresses that contribute to the burden of cancer and other chronic, degenerative diseases.

  20. Nrf2 protects photoreceptor cells from photo-oxidative stress induced by blue light.

    PubMed

    Chen, Wan-Ju; Wu, Caiying; Xu, Zhenhua; Kuse, Yoshiki; Hara, Hideaki; Duh, Elia J

    2017-01-01

    Oxidative stress plays a key role in age-related macular degeneration and hereditary retinal degenerations. Light damage in rodents has been used extensively to model oxidative stress-induced photoreceptor degeneration, and photo-oxidative injury from blue light is particularly damaging to photoreceptors. The endogenous factors protecting photoreceptors from oxidative stress, including photo-oxidative stress, are continuing to be elucidated. In this study, we evaluated the effect of blue light exposure on photoreceptors and its relationship to Nrf2 using cultured murine photoreceptor (661W) cells. 661W cells were exposed to blue light at 2500 lux. Exposure to blue light for 6-24 h resulted in a significant increase in intracellular reactive oxygen species (ROS) and death of 661W cells in a time-dependent fashion. Blue light exposure resulted in activation of Nrf2, as indicated by an increase in nuclear translocation of Nrf2. This was associated with a significant induction of expression of Nrf2 as well as an array of Nrf2 target genes, including antioxidant genes, as indicated by quantitative reverse transcription PCR (qRT-PCR). In order to determine the functional role of Nrf2, siRNA-mediated knockdown studies were performed. Nrf2-knockdown in 661W cells resulted in significant exacerbation of blue light-induced reactive oxygen species levels as well as cell death. Taken together, these findings indicate that Nrf2 is an important endogenous protective factor against oxidative stress in photoreceptor cells. This suggests that drugs targeting Nrf2 could be considered as a neuroprotective strategy for photoreceptors in AMD and other retinal conditions. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. The chalcone compound isosalipurposide (ISPP) exerts a cytoprotective effect against oxidative injury via Nrf2 activation

    SciTech Connect

    Han, Jae Yun; Cho, Seung Sik; Yang, Ji Hye; Kim, Kyu Min; Jang, Chang Ho; Park, Da Eon; Bang, Joon Seok; Jung, Young Suk; Ki, Sung Hwan

    2015-08-15

    The chalcone compound isosalipurposide (ISPP) has been successfully isolated from the native Korean plant species Corylopsis coreana Uyeki (Korean winter hazel). However, the therapeutic efficacy of ISPP remains poorly understood. This study investigated whether ISPP has the capacity to activate NF-E2-related factor (Nrf2)-antioxidant response element (ARE) signaling and induce its target gene expression, and to determined the protective role of ISPP against oxidative injury of hepatocytes. In HepG2 cells, nuclear translocation of Nrf2 is augmented by ISPP treatment. Consistently, ISPP increased ARE reporter gene activity and the protein levels of glutamate cysteine ligase (GCL) and hemeoxygenase (HO-1), resulting in increased intracellular glutathione levels. Cells pretreated with ISPP were rescued from tert-butylhydroperoxide-induced reactive oxygen species (ROS) production and glutathione depletion and consequently, apoptotic cell death. Moreover, ISPP ameliorated the mitochondrial dysfunction and apoptosis induced by rotenone which is an inhibitor of complex 1 of the mitochondrial respiratory chain. The specific role of Nrf2 activation by ISPP was demonstrated using an ARE-deletion mutant plasmid and Nrf2-knockout cells. Finally, we observed that extracellular signal-regulated kinase (ERK) and AMP-activated protein kinase (AMPK), but not protein kinase C (PKC)-δ or other mitogen-activated protein kinases (MAPKs), are involved in the activation of Nrf2 by ISPP. Taken together, our results demonstrate that ISPP has a cytoprotective effect against oxidative damage mediated through Nrf2 activation and induction of its target gene expression in hepatocytes. - Highlights: • We investigated the effect of ISPP on Nrf2 activation. • ISPP increased Nrf2 activity and its target gene expression. • ISPP inhibited the mitochondrial dysfunction and ROS production. • Nrf2 activation by ISPP is dependent on ERK1/2 and AMPK phosphorylation. • ISPP may be a promising

  2. Nrf2 activation attenuates both orthodontic tooth movement and relapse.

    PubMed

    Kanzaki, H; Shinohara, F; Itohiya-Kasuya, K; Ishikawa, M; Nakamura, Y

    2015-06-01

    During orthodontic tooth movement, osteoclasts resorb the alveolar bone at the compress side of periodontium. Reactive oxygen species (ROS) works as intracellular signaling molecules of RANKL during osteoclastogenesis, although ROS has cytotoxicity against cells such as lipid oxidation. To deal with oxidative stress, cells have a defense system that is scavenging ROS by augmented antioxidative stress enzymes via transcriptional regulation with nuclear factor E2-related factor 2 (Nrf2). Previously, we reported that augmented antioxidative stress enzymes by Nrf2-gene transfer inhibited bone destruction. In the present study, we examined the effects of Nrf2 activation on osteoclastogenesis and, thereby, orthodontic tooth movement and orthodontic relapse. Mouse macrophage cell line RAW264.7 cells were used as osteoclast progenitor cells and stimulated with recombinant RANKL (100 ng/mL) with or without Nrf2 activator sulforaphane (SFN) and epigallocatechin gallate (EGCG) or ROS scavenger catechin. Osteoclastogenesis, resorption activity, and osteoclast marker gene expression were examined. Intracellular ROS was analyzed by flow cytometry. Maxillary first molars of C57BL6 male mice were moved palatally with 0.012-inch NiTi wire (100-mN force); SFN or EGCG was injected into the palatal gingiva once a week; and phosphate buffered saline was injected on the contralateral side. Tooth movement was monitored using a stone model with precise impression, and the amount of the tooth movement was compared among groups. SFN and EGCG significantly, but catechin weakly, inhibited RANKL-mediated osteoclastogenesis in vitro. Western blot analysis revealed that SFN and EGCG augmented the nuclear translocation of Nrf2 and the expression of anti-oxidative stress enzymes such as HO-1, although catechin did not. SFN and EGCG significantly, but catechin weakly, attenuated the intracellular ROS. Finally, animal experiment revealed that both SFN and EGCG successfully inhibited the orthodontic

  3. MDA-7/IL-24 inhibits Nrf2-mediated antioxidant response through activation of p38 pathway and inhibition of ERK pathway involved in cancer cell apoptosis.

    PubMed

    Tian, H; Zhang, D; Gao, Z; Li, H; Zhang, B; Zhang, Q; Li, L; Cheng, Q; Pei, D; Zheng, J

    2014-10-01

    Reactive oxygen species (ROS) have a crucial role in melanoma differentiation-associated gene-7 (MDA-7)/interleukin-24 (IL-24)-induced cancer cell apoptosis. However, cancer cell has a series of protective mechanisms to resist ROS damage. Nuclear factor erythroid 2-related factor 2 (Nrf2) activates antioxidant response element (ARE)-mediated gene expression involved in cellular protection against oxidative stress. As the Nrf2 repressor, Kelch-like ECH-associated protein-1 (Keap1) sequesters Nrf2 in cytoplasm to block Nrf2 nuclear translocation. In the present study, administration of MDA-7/IL-24 by means of tumor-selective replicating adenovirus (ZD55-IL-24) was used to investigate whether ZD55-IL-24 could attenuate Nrf2-mediated oxidative stress response in cancer cell. We found that ZD55-IL-24 effectively strengthened the association between Nrf2 and Keap1 to restrict Nrf2 nuclear translocation, thereby inhibiting ARE-dependent transcriptional response. To evaluate the detailed mechanism underlying the suppression of ZD55-IL-24 on Nrf2-mediated oxidative stress response, we further tested three different mitogen-activated protein kinase (MAPK) signaling pathways in A549 and HeLa cells transfected by ZD55-IL-24. Our data showed that ZD55-IL-24 inhibited extracellular signal-regulated kinase (ERK) signal pathway but activated p38 and c-Jun-NH2-kinase (JNK) signal pathways to exert the tumor-specific apoptosis. Moreover, ERK pathway inhibitor U0126 prevented Nrf2 phosphorylation at Ser40 to retard Nrf2 nuclear translocation, thus decreasing antioxidant gene transcription. In contrast, p38 pathway inhibitor SB203580 obviously promoted the dissociation of Nrf2 from Keap1 to promote antioxidant gene transcription. However, JNK pathway had no effect on Nrf2 subcellular localization or the association of Nrf2 with Keap1. Conclusively, our results indicate that ZD55-IL-24 inhibits Nrf2-mediated oxidative stress response not only by activating p38 signal pathway to

  4. The mtDNA NARP mutation activates the actin-Nrf2 signaling of antioxidant defenses

    SciTech Connect

    Dassa, Emmanuel Philippe; Paupe, Vincent; Goncalves, Sergio; Rustin, Pierre

    2008-04-11

    An efficient handling of superoxides by antioxidant defenses is a crucial issue for cells with respiratory chain deficient mitochondria. We used human cultured skin fibroblasts to delineate the mechanism controlling the expression of antioxidant defenses in the case of a severe ATPase deficiency resulting from an 8993T>G mutation in the mitochondrial ATPase6 gene. We observed the nuclear translocation of the transcription factor Nrf2 associated with thinning of the actin stress fibers. The mobilization of the Nrf2 signaling pathway could be mimicked by a chemical blockade of the ATPase with a specific inhibitor, oligomycin. Interestingly enough, Nrf2 nuclear translocation was not observed in the case of a severe cytochrome oxidase deficiency, indicating that studying the status of this signaling pathway could throw some light on the importance of the oxidative insult associated with different respiratory chain defects.

  5. Ursolic Acid Ameliorates Early Brain Injury After Experimental Traumatic Brain Injury in Mice by Activating the Nrf2 Pathway.

    PubMed

    Ding, Hui; Wang, Handong; Zhu, Lin; Wei, Wuting

    2017-02-01

    Previous studies have indicated oxidative stress and inflammatory injury as significant contributors to the secondary damage associated with traumatic brain injury (TBI). Ursolic acid (UA) has been demonstrated to exert anti-oxidative and anti-inflammatory effects on cerebral ischemia by activating the nuclear factor-erythroid 2-related factor 2 (Nrf2) pathway. However, the effects of UA on TBI remain unclear. The aim of this study is to evaluate the potential roles of UA in the activation of the Nrf2 pathway using an experimental TBI model and the underlying mechanism. Wild-type (WT) and Nrf2((-/-)) mice were divided into eight groups: (1) sham; (2) TBI; (3) TBI + vehicle; (4) TBI + 50 mg/kg UA; (5) TBI + 100 mg/kg UA; (6) TBI + 150 mg/kg UA; (7) TBI + Nrf2((-/-)) + vehicle; (8) TBI + Nrf2((-/-)) + UA. All mice underwent the TBI with the exception of the sham group. The neurologic outcomes of the mice were evaluated at 24 h after TBI, as well as the expression of Nrf2, NQO1, HO1,SOD, GPx, and MDA. Treatment of UA significantly ameliorated brain edema and the neurological insufficiencies after TBI. In addition, UA treatment markedly strengthened the nuclear translocation of Nrf2 protein and increased the expression of NQO1 and HO1. Moreover, UA significantly increased the expression of AKT, an Nrf2 upstream factor, suggesting that UA play a neuroprotective role through the activation of the Nrf2-ARE signal pathway. On the contrary, UA showed no neuroprotective effect on the Nrf2((-/-)) mice. These data indicated that UA increases the activity of antioxidant enzymes and attenuated brain injury via Nrf2 factor.

  6. Escin activates AKT-Nrf2 signaling to protect retinal pigment epithelium cells from oxidative stress.

    PubMed

    Wang, Kaijun; Jiang, Yiqian; Wang, Wei; Ma, Jian; Chen, Min

    2015-12-25

    Here we explored the anti-oxidative and cytoprotective potentials of escin, a natural triterpene-saponin, against hydrogen peroxide (H2O2) in retinal pigment epithelium (RPE) cells. We showed that escin remarkably attenuated H2O2-induced death and apoptosis of established (ARPE-19) and primary murine RPE cells. Meanwhile, ROS production and lipid peroxidation by H2O2 were remarkably inhibited by escin. Escin treatment in RPE cells resulted in NF-E2-related factor 2 (Nrf2) signaling activation, evidenced by transcription of anti-oxidant-responsive element (ARE)-regulated genes, including HO-1, NQO-1 and SRXN-1. Knockdown of Nrf2 through targeted shRNAs/siRNAs alleviated escin-mediated ARE gene transcription, and almost abolished escin-mediated anti-oxidant activity and RPE cytoprotection against H2O2. Reversely, escin was more potent against H2O2 damages in Nrf2-over-expressed ARPE-19 cells. Further studies showed that escin-induced Nrf2 activation in RPE cells required AKT signaling. AKT inhibitors (LY294002 and perifosine) blocked escin-induced AKT activation, and dramatically inhibited Nrf2 phosphorylation, its cytosol accumulation and nuclear translocation in RPE cells. Escin-induced RPE cytoprotection against H2O2 was also alleviated by the AKT inhibitors. Together, these results demonstrate that escin protects RPE cells from oxidative stress possibly through activating AKT-Nrf2 signaling. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. Induction of human fetal hemoglobin via the NRF2 antioxidant response signaling pathway

    PubMed Central

    Macari, Elizabeth R.

    2011-01-01

    Although hematopoietic stem cell transplantation and gene therapy have the potential to cure β-thalassemia and sickle cell disease, they are not currently available to most people with these diseases. In the near term, pharmacologic induction of fetal hemoglobin (HbF) may offer the best possibility for safe, effective, and widely available therapy. In an effort to define new pathways for targeted drug development for HbF induction, we evaluated the nuclear factor erythroid 2–related factor 2 (NRF2) antioxidant response element signaling pathway. We found that 3 well-known activators of this pathway increased γ-globin mRNA at nontoxic doses in K562 cells. Tert-butylhydroquinone (tBHQ), the most active of these compounds, increased cellular levels and nuclear translocation of NRF2 and binding of NRF2 to the γ-globin promoter. siRNA knockdown of NRF2 inhibited γ-globin induction by tBHQ. When tested in human primary erythroid cells, tBHQ induced NRF2 binding to the γ-globin promoter, increased γ-globin mRNA and HbF, and suppressed β-globin mRNA and HbA, resulting in a > 3-fold increase in the percentage of HbF. These results suggest that drugs that activate the NRF2/antioxidant response element signaling pathway have the potential to induce therapeutic levels of HbF in people with β-hemoglobinopathies. PMID:21464371

  8. Activation of the Nrf2 Pathway by Inorganic Arsenic in Human Hepatocytes and the Role of Transcriptional Repressor Bach1

    PubMed Central

    Liu, Dan; Duan, Xiaoxu; Dong, Dandan; Bai, Caijun; Li, Xin; Sun, Guifan; Li, Bing

    2013-01-01

    Previous studies have proved that the environmental toxicant, inorganic arsenic, activates nuclear factor erythroid 2-related factor 2 (Nrf2) pathway in many different cell types. This study tried to explore the hepatic Nrf2 pathway upon arsenic treatment comprehensively, since liver is one of the major target organs of arsenical toxicity. Our results showed that inorganic arsenic significantly induced Nrf2 protein and mRNA expression in Chang human hepatocytes. We also observed a dose-dependent increase of antioxidant response element- (ARE-) luciferase activity. Both the mRNA and protein levels of NAD(P)H:quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1) were all upregulated dramatically. On the other hand, entry and accumulation of Nrf2 protein in the nucleus, while exportting the transcriptional repressor BTB and CNC homology 1 (Bach1) from nucleus to cytoplasm, were also confirmed by western blot and immunofluorescence assay. Our results therefore confirmed the arsenic-induced Nrf2 pathway activation in hepatocytes and also suggested that the translocation of Bach1 was associated with the regulation of Nrf2 pathway by arsenic. Hepatic Nrf2 pathway plays indispensable roles for cellular defenses against arsenic hepatotoxicity, and the interplay of Bach1 and Nrf2 may be helpful to understand the self-defensive responses and the diverse biological effects of arsenicals. PMID:23738048

  9. Photoprotection by dietary phenolics against melanogenesis induced by UVA through Nrf2-dependent antioxidant responses

    PubMed Central

    Chaiprasongsuk, Anyamanee; Onkoksoong, Tasanee; Pluemsamran, Thanyawan; Limsaengurai, Saowalak; Panich, Uraiwan

    2015-01-01

    Dietary phenolics may play a protective role in UV-mediated skin pigmentation through their antioxidant and UV-absorbing actions. In this study, we investigated whether genetic silencing of Nrf2, regulating the transcription of antioxidant genes, affected melanogenesis in primary human epidermal melanocytes (HEMn) and B16F10 melanoma cells subjected to UVA (8 J/cm2) exposure. Then, we explored the antimelanogenic actions of phenolics; caffeic acid (CA) and ferulic acid (FA) providing partial UVA protection; quercetin (QU) and rutin (RU) providing strong UVA protection and; avobenzone (AV), an efficient UVA filter, in association with modulation of Nrf2-mediated antioxidant defenses in response to UVA insults in B16F10 cells. Upon oxidative insults, Nrf2 silencing promoted melanogenesis in both HEMn and B16F10 cells irradiated with UVA. Stimulation of melanogenesis by UVA correlated with increased ROS and oxidative DNA damage (8-OHdG), GSH depletion as well as a transient downregulation of Nrf2 nuclear translocation and of Nrf2-ARE signaling in B16F10 cells. All test compounds exerted antimelanogenic effects with respect to their abilities to reverse UVA-mediated oxidative damage as well as downregulation of Nrf2 activity and its target antioxidants (GCLC, GST and NQO1) in B16F10 cells. In conclusion, defective Nrf2 may promote melanogenesis under UVA irradiation through oxidative stress mechanisms. Compounds with antioxidant and/or UVA absorption properties could protect against UVA-induced melanogenesis through indirect regulatory effect on Nrf2-ARE pathway. PMID:26765101

  10. Photoprotection by dietary phenolics against melanogenesis induced by UVA through Nrf2-dependent antioxidant responses.

    PubMed

    Chaiprasongsuk, Anyamanee; Onkoksoong, Tasanee; Pluemsamran, Thanyawan; Limsaengurai, Saowalak; Panich, Uraiwan

    2016-08-01

    Dietary phenolics may play a protective role in UV-mediated skin pigmentation through their antioxidant and UV-absorbing actions. In this study, we investigated whether genetic silencing of Nrf2, regulating the transcription of antioxidant genes, affected melanogenesis in primary human epidermal melanocytes (HEMn) and B16F10 melanoma cells subjected to UVA (8J/cm(2)) exposure. Then, we explored the antimelanogenic actions of phenolics; caffeic acid (CA) and ferulic acid (FA) providing partial UVA protection; quercetin (QU) and rutin (RU) providing strong UVA protection and; avobenzone (AV), an efficient UVA filter, in association with modulation of Nrf2-mediated antioxidant defenses in response to UVA insults in B16F10 cells. Upon oxidative insults, Nrf2 silencing promoted melanogenesis in both HEMn and B16F10 cells irradiated with UVA. Stimulation of melanogenesis by UVA correlated with increased ROS and oxidative DNA damage (8-OHdG), GSH depletion as well as a transient downregulation of Nrf2 nuclear translocation and of Nrf2-ARE signaling in B16F10 cells. All test compounds exerted antimelanogenic effects with respect to their abilities to reverse UVA-mediated oxidative damage as well as downregulation of Nrf2 activity and its target antioxidants (GCLC, GST and NQO1) in B16F10 cells. In conclusion, defective Nrf2 may promote melanogenesis under UVA irradiation through oxidative stress mechanisms. Compounds with antioxidant and/or UVA absorption properties could protect against UVA-induced melanogenesis through indirect regulatory effect on Nrf2-ARE pathway.

  11. Myopathic Lamin Mutations Cause Reductive Stress and Activate the Nrf2/Keap-1 Pathway

    PubMed Central

    Dialynas, George; Shrestha, Om K.; Ponce, Jessica M.; Zwerger, Monika; Thiemann, Dylan A.; Young, Grant H.; Moore, Steven A.; Yu, Liping; Lammerding, Jan; Wallrath, Lori L.

    2015-01-01

    Mutations in the human LMNA gene cause muscular dystrophy by mechanisms that are incompletely understood. The LMNA gene encodes A-type lamins, intermediate filaments that form a network underlying the inner nuclear membrane, providing structural support for the nucleus and organizing the genome. To better understand the pathogenesis caused by mutant lamins, we performed a structural and functional analysis on LMNA missense mutations identified in muscular dystrophy patients. These mutations perturb the tertiary structure of the conserved A-type lamin Ig-fold domain. To identify the effects of these structural perturbations on lamin function, we modeled these mutations in Drosophila Lamin C and expressed the mutant lamins in muscle. We found that the structural perturbations had minimal dominant effects on nuclear stiffness, suggesting that the muscle pathology was not accompanied by major structural disruption of the peripheral nuclear lamina. However, subtle alterations in the lamina network and subnuclear reorganization of lamins remain possible. Affected muscles had cytoplasmic aggregation of lamins and additional nuclear envelope proteins. Transcription profiling revealed upregulation of many Nrf2 target genes. Nrf2 is normally sequestered in the cytoplasm by Keap-1. Under oxidative stress Nrf2 dissociates from Keap-1, translocates into the nucleus, and activates gene expression. Unexpectedly, biochemical analyses revealed high levels of reducing agents, indicative of reductive stress. The accumulation of cytoplasmic lamin aggregates correlated with elevated levels of the autophagy adaptor p62/SQSTM1, which also binds Keap-1, abrogating Nrf2 cytoplasmic sequestration, allowing Nrf2 nuclear translocation and target gene activation. Elevated p62/SQSTM1 and nuclear enrichment of Nrf2 were identified in muscle biopsies from the corresponding muscular dystrophy patients, validating the disease relevance of our Drosophila model. Thus, novel connections were made

  12. Notch-Nrf2 Axis: Regulation of Nrf2 Gene Expression and Cytoprotection by Notch Signaling

    PubMed Central

    Skoko, John J.; Chartoumpekis, Dionysios V.; Kimura, Shoko; Slocum, Stephen L.; Noda, Kentaro; Palliyaguru, Dushani L.; Fujimuro, Masahiro; Boley, Patricia A.; Tanaka, Yugo; Shigemura, Norihisa; Biswal, Shyam; Yamamoto, Masayuki; Kensler, Thomas W.

    2014-01-01

    The Notch signaling pathway enables regulation and control of development, differentiation, and homeostasis through cell-cell communication. Our investigation shows that Notch signaling directly activates the Nrf2 stress adaptive response pathway through recruitment of the Notch intracellular domain (NICD) transcriptosome to a conserved Rbpjκ site in the promoter of Nrf2. Stimulation of Notch signaling through Notch ligand expression in cells and by overexpression of the NICD in RosaNICD/−::AlbCre mice in vivo induces expression of Nrf2 and its target genes. Continuous and transient NICD expression in the liver produces a Notch-dependent cytoprotective response through direct transcriptional activation of Nrf2 signaling to rescue mice from acute acetaminophen toxicity. This response can be reversed upon genetic disruption of Nrf2. Morphological studies showed that the characteristic phenotype of high-density intrahepatic bile ducts and enlarged liver in RosaNICD/−::AlbCre mice could be at least partially reversed after Nrf2 disruption. Furthermore, the liver and bile duct phenotypes could be recapitulated with constitutive activation of Nrf2 signaling in Keap1F/F::AlbCre mice. It appears that Notch-to-Nrf2 signaling is another important determinant in liver development and function and promotes cell-cell cytoprotective signaling responses. PMID:24298019

  13. DJ-1 Protects Breast Cancer Cells Against 2'-Benzoyloxycinnamaldehyde-induced Oxidative Stress Independent of Nrf2.

    PubMed

    Ismail, Ismail Ahmed; Abdel Shakor, Abo Bakr; Hong, Su-Hyung

    2015-09-01

    2'-Benzoyloxycinnamaldehyde (BCA) is a promising antitumor agent. BCA effectively inhibited proliferation of MDA-MB-435 more than in MCF-7 breast cancer cells. Our recent findings showed that DJ-1 protects MCF7 cells from BCA-induced oxidative stress via its mitochondrial translocation and inhibition of the mitochondrial perturbation (Ismail et al., 2012). In this study, we addressed the question of whether Nrf2 works downstream to DJ-1 in mediating differential antiproliferation effects in MCF-7 and MDAMB-435 breast cancer cells induced by BCA treatment. BCA upregulated the expression and induced nuclear translocalization of DJ-1 and Nrf2 in only MCF-7 cells. However, in MDA-MB-435, BCA increased only Nrf2 expression without inducing DJ-1 and/or Nrf2 protein translocalization to the nucleus. Furthermore, DJ-1 knockdown decreased DJ-1 expression in both cells without affecting Nrf2 and its downstream target γ-GCS, suggesting that DJ-1-induced cell protection and works independent of Nrf2 signaling pathway.

  14. Impaired Nrf2 regulation of mitochondrial biogenesis in rostral ventrolateral medulla on hypertension induced by systemic inflammation.

    PubMed

    Wu, Kay L H; Wu, Chih-Wei; Chao, Yung-Mei; Hung, Chun-Ying; Chan, Julie Y H

    2016-08-01

    Oxidative stress in rostral ventrolateral medulla (RVLM), where sympathetic premotor neurons reside, is involved in the development of hypertension under systemic inflammation. Mitochondrial dysfunction contributes to tissue oxidative stress. In this study, we sought to investigate whether hypertension developed under systemic inflammation is attributable to impaired mitochondrial biogenesis in RVLM. In normotensive Sprague-Dawley rats, intraperitoneal infusion of a low dose Escherichia coli lipopolysaccharide (LPS) for 7 days promoted a pressor response, alongside a decrease in mitochondrial DNA (mtDNA) copy number, reductions in protein expression of nuclear DNA-encoded transcription factors for mitochondrial biogenesis, including mitochondrial transcription factor A (TFAM) and nuclear factor erythroid-derived 2-like 2 (Nrf2), and suppression of nuclear translocation of the phosphorylated Nrf2 (p-Nrf2) in RVLM neurons; all of which were abrogated by treatment with intracisternal infusion of an interleukin-1β (IL-1β) blocker, IL-1Ra, or a mobile mitochondrial electron carrier, coenzyme Q10 (CoQ10). Microinjection into RVLM of IL-1β suppressed the expressions of p-Nrf2 and TFAM, and evoked a pressor response; conversely, the Nrf2 inducer, tert-butylhydroquinone, lessened the LPS-induced suppression of TFAM expression and pressor response. At cellular level, exposure of neuronal N2a cells to IL-1β decreased mtDNA copy number, increased protein interaction of Nrf2 to its negative regulator, kelch-like ECH-associated protein 1 (Keap1), and reduced DNA binding activity of p-Nrf2 to Tfam gene. Together these results indicate that defect mitochondrial biogenesis in RVLM neurons entailing redox-sensitive and IL-1β-dependent suppression of TFAM because of the increase in the formation of Keap1/Nrf2 complex, reductions in nuclear translocation of the activated Nrf2 and its binding to the Tfam gene promoter may underlie hypertension developed under the LPS

  15. Sinomenine activation of Nrf2 signaling prevents hyperactive inflammation and kidney injury in a mouse model of obstructive nephropathy.

    PubMed

    Qin, Tian; Du, Ronghui; Huang, Fengjie; Yin, Shasha; Yang, Jun; Qin, Siyuan; Cao, Wangsen

    2016-03-01

    Sinomenine is originally derived from medicinal herb and used preferentially in treatment of rheumatoid diseases in Far East regions. SIN has strong anti-inflammatory and immune-regulatory properties, acting mainly through inhibiting NF-kB signaling. Although the upstream target through which SIN affects NF-kB activity is unknown, evidence suggests that SIN might regulate inflammation through Nrf2 signaling. In this study we explored the role of Nrf2 in mediating SIN's anti-inflammation and kidney protection in a mouse model of obstructive nephropathy. We found that SIN is an activator of Nrf2 signaling. It markedly increased Nrf2 protein level, Nrf2 nuclear translocation, Nef2 transcription capacity, and the downstream protein expression. We further demonstrated that SIN activation of Nrf2 is likely due to its repression of the Nrf2 inhibitor Keap1 since it drastically reduced Keap1 protein through the PKC-sensitive ubiquitination-proteasomal degradation. SIN treatment of nephropathy mice effectively reduced the kidney damage and inflammatory responses, balanced renal oxidative stress, and improved the pathological protein expression in an Nrf2 dependent manner. In addition, SIN also Nrf2-dependently modulated macrophage M1/M2 polarization and inhibited the IkBα phosphorylation and NF-kB nuclear translocation, hence revealing an important upstream event that contributed to its anti-inflammation and tissue protection. Taken together our study has identified a novel pathway through which SIN exerts its anti-inflammation and renal protective functions, and provided a molecular basis for SIN potential applications in the treatment of kidney and other inflammatory disorders.

  16. Isorhamnetin protects against oxidative stress by activating Nrf2 and inducing the expression of its target genes.

    PubMed

    Yang, Ji Hye; Shin, Bo Yeon; Han, Jae Yun; Kim, Mi Gwang; Wi, Ji Eun; Kim, Young Woo; Cho, Il Je; Kim, Sang Chan; Shin, Sang Mi; Ki, Sung Hwan

    2014-01-15

    Isorhamentin is a 3'-O-methylated metabolite of quercetin, and has been reported to have anti-inflammatory and anti-proliferative effects. However, the effects of isorhamnetin on Nrf2 activation and on the expressions of its downstream genes in hepatocytes have not been elucidated. Here, we investigated whether isorhamnetin has the ability to activate Nrf2 and induce phase II antioxidant enzyme expression, and to determine the protective role of isorhamnetin on oxidative injury in hepatocytes. In HepG2 cells, isorhamnetin increased the nuclear translocation of Nrf2 in a dose- and time-dependent manner, and consistently, increased antioxidant response element (ARE) reporter gene activity and the protein levels of hemeoxygenase (HO-1) and of glutamate cysteine ligase (GCL), which resulted in intracellular GSH level increases. The specific role of Nrf2 in isorhamnetin-induced Nrf2 target gene expression was verified using an ARE-deletion mutant plasmid and Nrf2-knockout MEF cells. Deletion of the ARE in the promoter region of the sestrin2 gene, which is recently identified as the Nrf2 target gene by us, abolished the ability of isorhamnetin to increase luciferase activity. In addition, Nrf2 deficiency completely blocked the ability of isorhamnetin to induce HO-1 and GCL. Furthermore, isorhamnetin pretreatment blocked t-BHP-induced ROS production and reversed GSH depletion by t-BHP and consequently, due to reduced ROS levels, decreased t-BHP-induced cell death. In addition isorhamnetin increased ERK1/2, PKCδ and AMPK phosphorylation. Finally, we showed that Nrf2 deficiency blocked the ability of isorhamnetin to protect cells from injury induced by t-BHP. Taken together, our results demonstrate that isorhamnetin is efficacious in protecting hepatocytes against oxidative stress by Nrf2 activation and in inducing the expressions of its downstream genes.

  17. Acupuncture ameliorates cognitive impairment and hippocampus neuronal loss in experimental vascular dementia through Nrf2-mediated antioxidant response.

    PubMed

    Wang, Xue-Rui; Shi, Guang-Xia; Yang, Jing-Wen; Yan, Chao-Qun; Lin, Li-Ting; Du, Si-Qi; Zhu, Wen; He, Tian; Zeng, Xiang-Hong; Xu, Qian; Liu, Cun-Zhi

    2015-12-01

    Emerging evidence suggests acupuncture could exert neuroprotection in the vascular dementia via anti-oxidative effects. However, the involvement of Nrf2, a master regulator of antioxidant defense, in acupuncture-induced neuroprotection in vascular dementia remains undetermined. The goal of our study was to investigate the contribution of Nrf2 in acupuncture and its effects on vascular dementia. Morris water maze and Nissl staining were used to assess the effect of acupuncture on cognitive function and hippocampal neurodegeneration in experimental vascular dementia. The distribution of Nrf2 in neurons in hippocampus, the protein expression of Nrf2 in both cytosol and nucleus, and the protein and mRNA levels of its downstream target genes NQO1 and HO-1 were detected by double immunofluorescent staining, Western blotting and realtime PCR analysis respectively. Cognitive function and microglia activation were measured in both wild-type and Nrf2 gene knockout mice after acupuncture treatment. We found that acupuncture could remarkably reverse the cognitive deficits, neuron cell loss, reactive oxygen species production, and decreased cerebral blood flow. It was notable that acupuncture enhanced nuclear translocation of Nrf2 in neurons and up-regulate the protein and mRNA levels of Nrf2 and its target genes HO-1 and NQO1. Moreover, acupuncture could significantly down-regulated the over-activation of microglia after common carotid artery occlusion surgery. However, the reversed cognitive deficits, neuron cell loss and microglia activation by acupuncture were abolished in Nrf2 gene knockout mice. In conclusion, these findings provide evidence that the neuroprotection of acupuncture in models of vascular dementia was via the Nrf2 activation and Nrf2-dependent microglia activation. Copyright © 2015. Published by Elsevier Inc.

  18. NRF2-ome: an integrated web resource to discover protein interaction and regulatory networks of NRF2.

    PubMed

    Türei, Dénes; Papp, Diána; Fazekas, Dávid; Földvári-Nagy, László; Módos, Dezső; Lenti, Katalin; Csermely, Péter; Korcsmáros, Tamás

    2013-01-01

    NRF2 is the master transcriptional regulator of oxidative and xenobiotic stress responses. NRF2 has important roles in carcinogenesis, inflammation, and neurodegenerative diseases. We developed an online resource, NRF2-ome, to provide an integrated and systems-level database for NRF2. The database contains manually curated and predicted interactions of NRF2 as well as data from external interaction databases. We integrated NRF2 interactome with NRF2 target genes, NRF2 regulating TFs, and miRNAs. We connected NRF2-ome to signaling pathways to allow mapping upstream NRF2 regulatory components that could directly or indirectly influence NRF2 activity totaling 35,967 protein-protein and signaling interactions. The user-friendly website allows researchers without computational background to search, browse, and download the database. The database can be downloaded in SQL, CSV, BioPAX, SBML, PSI-MI, and in a Cytoscape CYS file formats. We illustrated the applicability of the website by suggesting a posttranscriptional negative feedback of NRF2 by MAFG protein and raised the possibility of a connection between NRF2 and the JAK/STAT pathway through STAT1 and STAT3. NRF2-ome can also be used as an evaluation tool to help researchers and drug developers to understand the hidden regulatory mechanisms in the complex network of NRF2.

  19. Nrf2: a modulator of Parkinson's disease?

    PubMed

    Todorovic, Michael; Wood, Stephen A; Mellick, George D

    2016-06-01

    Parkinson's disease (PD) is a complex multifactorial disorder that has been associated with the processes of oxidative stress. In the absence of curative therapies, modification of the neurodegenerative process-including the manipulation of endogenous antioxidant pathways-is the focus of intensive research. Recently, genetic and pharmacological accretion of the transcription factor, and phase II antioxidant 'master regulator' Nrf2, has shown to demonstrably mitigate the toxic neuronal effects of parkinsonian agents such as MPP(+), rotenone, and hydrogen peroxide in vitro and in vivo. Furthermore, baseline genetic variability in Nrf2-dependant pathways may promote neuronal susceptibility to exogenous agents and correlate with PD onset within certain populations. While contemporary evidence directly implicating Nrf2 in the pathogenesis of PD is not conclusive and likely contingent upon the evaluation of complex interacting factors-including genetic variation and a history of environmental exposures-it remains a promising target for therapeutic benefit in the modulation of oxidative stress.

  20. The Nrf2 activator, tBHQ, differentially affects early events following stimulation of Jurkat cells.

    PubMed

    Zagorski, Joseph W; Turley, Alexandra E; Dover, Heather E; VanDenBerg, Kelly R; Compton, Jacob R; Rockwell, Cheryl E

    2013-11-01

    Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that is activated by cellular stresses, such as oxidative compounds. After activation, Nrf2 induces transcription of its target genes, many of which have cytoprotective functions. Previously, we have shown that activation of Nrf2 by tert-butylhydroquinone (tBHQ) skews murine CD4⁺ T-cell differentiation. Although the role of Nrf2 in murine T cells is somewhat characterized, it is largely uncharacterized in human T cells. Therefore, the aim of the current studies was to characterize the effects of the Nrf2 activator, tBHQ, on the early events of human CD4⁺ T-cell activation. Pretreatment of Jurkat T cells with tBHQ, prior to activation with anti-CD3/anti-CD28, diminished the production of interleukin-2 (IL-2) at both the transcript and protein levels. Similarly, the expression of CD25 also diminished, albeit to a lesser degree than IL-2, after pretreatment with tBHQ. The decrease in IL-2 production was not due to decreased nuclear translocation of c-fos or c-jun. Although tBHQ caused both a delay and a decrease in Ca²⁺ influx in activated Jurkat cells, no decrease in nuclear factor of activated T cells (NFAT) DNA binding or transcriptional activity was observed. In contrast to NFAT, tBHQ significantly decreased NFκB transcriptional activity. Collectively, our studies show that the Nrf2 activator, tBHQ, inhibits IL-2 and CD25 expression, which correlates with decreased NFκB transcriptional activity in activated Jurkat cells. Overall, our studies suggest that Nrf2 represents a novel mechanism for the regulation of both human and mouse T cell function.

  1. The Nrf2 Activator, tBHQ, Differentially Affects Early Events Following Stimulation of Jurkat Cells

    PubMed Central

    Rockwell, Cheryl E.

    2013-01-01

    Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that is activated by cellular stresses, such as oxidative compounds. After activation, Nrf2 induces transcription of its target genes, many of which have cytoprotective functions. Previously, we have shown that activation of Nrf2 by tert-butylhydroquinone (tBHQ) skews murine CD4+ T-cell differentiation. Although the role of Nrf2 in murine T cells is somewhat characterized, it is largely uncharacterized in human T cells. Therefore, the aim of the current studies was to characterize the effects of the Nrf2 activator, tBHQ, on the early events of human CD4+ T-cell activation. Pretreatment of Jurkat T cells with tBHQ, prior to activation with anti-CD3/anti-CD28, diminished the production of interleukin-2 (IL-2) at both the transcript and protein levels. Similarly, the expression of CD25 also diminished, albeit to a lesser degree than IL-2, after pretreatment with tBHQ. The decrease in IL-2 production was not due to decreased nuclear translocation of c-fos or c-jun. Although tBHQ caused both a delay and a decrease in Ca2+ influx in activated Jurkat cells, no decrease in nuclear factor of activated T cells (NFAT) DNA binding or transcriptional activity was observed. In contrast to NFAT, tBHQ significantly decreased NFκB transcriptional activity. Collectively, our studies show that the Nrf2 activator, tBHQ, inhibits IL-2 and CD25 expression, which correlates with decreased NFκB transcriptional activity in activated Jurkat cells. Overall, our studies suggest that Nrf2 represents a novel mechanism for the regulation of both human and mouse T cell function. PMID:23945499

  2. Activation of the transcription factor Nrf2 in macrophages, Caco-2 cells and intact human gut tissue by Maillard reaction products and coffee.

    PubMed

    Sauer, Tanja; Raithel, Martin; Kressel, Jürgen; Münch, Gerald; Pischetsrieder, Monika

    2013-06-01

    In addition to direct antioxidative effects, Maillard reaction products (MRPs) could increase the antioxidative capacity of cells through the induction of cytoprotective enzymes. Since many of those enzymes are regulated by the transcription factor Nrf2, the effect of MRPs on nuclear translocation of Nrf2 in macrophages and Caco-2 cells was investigated. Stimulation of both cell types by MRPs showed a concentration-dependent significant increase in nuclear translocation of Nrf2 up to fivefold after short-term (2 h) and up to 50-fold after long-term treatment (24 h). In intact human gut tissue, nuclear translocation of Nrf2 was significantly twofold increased after short-term incubation. To study the activation mechanisms, macrophages and Caco-2 cells were stimulated with MRPs in the presence of catalase, which significantly suppressed Nrf2 activation. Thus, activation was related to extracellular H2O2 continuously formed from MRPs. Short-term incubation with coffee, a MRP-rich beverage, led to a trend towards Nrf2 activation in macrophages, but not in Caco-2 cells or intact human gut tissue. Long-term incubation with coffee (1-4 mg/mL) significantly increased nuclear Nrf2 up to 17-fold. Since raw coffee was inactive under the tested conditions, the effect was related to roasting products. Coffee-induced Nrf2 translocation was, however, only slightly reversed by catalase. Therefore, the Nrf2 activity of coffee can only partially be explained by MRP-induced, H2O2-dependent mechanisms. Thus, it can be concluded that MRPs may increase the antioxidative capacity inside the cell by inducing Nrf2-regulated signalling pathways not only in different cell types, but also in intact gut tissue.

  3. Neuroprotective Effects of the Triterpenoid, CDDO Methyl Amide, a Potent Inducer of Nrf2-Mediated Transcription

    PubMed Central

    Yang, Lichuan; Calingasan, Noel Y.; Thomas, Bobby; Chaturvedi, Rajnish K.; Kiaei, Mahmoud; Wille, Elizabeth J.; Liby, Karen T.; Williams, Charlotte; Royce, Darlene; Risingsong, Renee; Musiek, Eric S.; Morrow, Jason D.; Sporn, Michael; Beal, M. Flint

    2009-01-01

    The NF-E2-related factor-2 (Nrf2)/antioxidant response element (ARE) signaling pathway regulates phase 2 detoxification genes, including a variety of antioxidative enzymes. We tested neuroprotective effects of the synthetic triterpenoid CDDO-MA, a potent activator of the Nrf2/ARE signaling. CDDO-MA treatment of neuroblastoma SH-SY5Y cells resulted in Nrf2 upregulation and translocation from cytosol to nucleus and subsequent activation of ARE pathway genes. CDDO-MA blocked t-butylhydroperoxide-induced production of reactive oxygen species (ROS) by activation of ARE genes only in wild type, but not Nrf2 knockout mouse embryonic fibroblasts. Oral administration of CDDO-MA resulted in significant protection against MPTP-induced nigrostriatal dopaminergic neurodegeneration, pathological alpha-synuclein accumulation and oxidative damage in mice. Additionally, CDDO-MA treatment in rats produced significant rescue against striatal lesions caused by the neurotoxin 3-NP, and associated increases in the oxidative damage markers malondialdehyde, F2-Isoprostanes, 8-hydroxy-2-deoxyguanosine, 3-nitrotyrosine, and impaired glutathione homeostasis. Our results indicate that the CDDO-MA renders its neuroprotective effects through its potent activation of the Nrf2/ARE pathway, and suggest that triterpenoids may be beneficial for the treatment of neurodegenerative diseases like Parkinson's disease and Huntington's disease. PMID:19484125

  4. Hepatoprotective effect of 7-Hydroxycoumarin against Methyl glyoxal toxicity via activation of Nrf2.

    PubMed

    Li, Dan; Wang, Na; Zhang, Jingdong; Ma, Shuren; Zhao, Zhuangzhuang; Ellis, Elizabeth M

    2017-03-02

    Methyl glyoxal (MG), a major precursor of advanced glycation end-products, has been identified as significant in the progression of several diseases including aging, diabetes and neurodegenerative diseases as well as causing hepatic damages. 7-hydroxycoumarin (7-HC), a natural-occurring derivative of coumarin from fruits and plants, has been reported to exert antioxidant and free radical-scavenging properties, protecting cells from aldehydes and oxidants. In this study, the ability of 7-HC to protect human HepG2 cells against MG-induced toxicity and oxidative stress was investigated. Results show that 7-HC pretreatment significantly attenuates MG-induced cytotoxicity, apoptotic changes and ROS accumulation and that this protection is shown to be associated with the induction of the nuclear factor erythroid 2-related factor 2 (NRF2) and its downstream detoxifying enzymes. In response to 7-HC, NRF2 protein translocates from cytosol to the nuclei. In addition, depletion of NRF2 by siRNA significantly reduces the protective effect of 7-HC against MG, suggesting that NRF2 plays an important role in the protective function of 7-HC. These findings highlight the potential for the interventional activation of the NRF2 induction via the non-toxic natural phytochemical 7-HC as a novel therapeutic approach towards the detoxification of MG, with the aim of halting the progression of diseases in which MG has been implicated.

  5. Astrocyte NMDA receptors' activity sustains neuronal survival through a Cdk5–Nrf2 pathway

    PubMed Central

    Jimenez-Blasco, D; Santofimia-Castaño, P; Gonzalez, A; Almeida, A; Bolaños, J P

    2015-01-01

    Neurotransmission unavoidably increases mitochondrial reactive oxygen species. However, the intrinsic antioxidant defense of neurons is weak and hence the mechanism whereby these cells are physiologically protected against oxidative damage is unknown. Here we found that the antioxidant defense of neurons is repressed owing to the continuous protein destabilization of the master antioxidant transcriptional activator, nuclear factor-erythroid 2-related factor-2 (Nrf2). By contrast, Nrf2 is highly stable in neighbor astrocytes explaining their robust antioxidant defense and resistance against oxidative stress. We also show that subtle and persistent stimulation of N-methyl-d-aspartate receptors (NMDAR) in astrocytes, through a mechanism not requiring extracellular Ca2+ influx, upregulates a signal transduction pathway involving phospholipase C-mediated endoplasmic reticulum release of Ca2+ and protein kinase Cδ activation. Active protein kinase Cδ promotes, by phosphorylation, the stabilization of p35, a cyclin-dependent kinase-5 (Cdk5) cofactor. Active p35/Cdk5 complex in the cytosol phosphorylates Nrf2 at Thr395, Ser433 and Thr439 that is sufficient to promote Nrf2 translocation to the nucleus and induce the expression of antioxidant genes. Furthermore, this Cdk5–Nrf2 transduction pathway boosts glutathione metabolism in astrocytes efficiently protecting closely spaced neurons against oxidative damage. Thus, intercellular communication through NMDAR couples neurotransmission with neuronal survival. PMID:25909891

  6. Brg1-mediated Nrf2/HO-1 pathway activation alleviates hepatic ischemia-reperfusion injury.

    PubMed

    Ge, Mian; Yao, Weifeng; Yuan, Dongdong; Zhou, Shaoli; Chen, Xi; Zhang, Yihan; Li, Haobo; Xia, Zhengyuan; Hei, Ziqing

    2017-06-01

    Cytoprotective gene heme oxygenase 1 (HO-1) could be induced by nuclear factor E2-related factor 2 (Nrf2) nuclear translocation. The purpose of this study was to determine the role of Brahma-related gene 1 (Brg1), a catalytic subunit of SWI2/SNF2-like chromatin remodeling complexes, in Nrf2/HO-1 pathway activation during hepatic ischemia-reperfusion (HIR). Our results showed that hepatic Brg1 was inhibited during early HIR while Brg1 overexpression reduced oxidative injury in CMV-Brg1 mice subjected to HIR. Moreover, promoter-driven luciferase assay showed that overexpression of Brg1 by adenovirus transfection in AML12 cells selectively enhanced HO-1 gene expression after hypoxia/reoxygenation (H/R) treatment but did not affect the other Nrf2 target gene NQO1. Furthermore, inhibition of HO-1 by the selective HO-1 inhibitor zinc protoporphyria could partly reverse the hepatic protective effects of Brg1 overexpression while HO-1-Adv attenuated AML12 cells H/R damage. Further, chromatin immunoprecipitation analysis revealed that Brg1 overexpression, which could significantly increase the recruitment of Brg1 protein to HO-1 but not NQO1 promoter, was recruited by Nrf2 to the HO-1 regulatory regions in AML12 hepatocytes subjected to H/R. In conclusion, our results demonstrated that restoration of Brg1 during reperfusion could enhance Nrf2-mediated inducible expression of HO-1 during HIR to effectively increase antioxidant ability to combat against hepatocytes damage.

  7. Increased Glutathione Synthesis Following Nrf2 Activation by Vanadyl Sulfate in Human Chang Liver Cells

    PubMed Central

    Kim, Areum Daseul; Zhang, Rui; Kang, Kyoung Ah; You, Ho Jin; Hyun, Jin Won

    2011-01-01

    Jeju ground water, containing vanadium compounds, was shown to increase glutathione (GSH) levels as determined by a colorimetric assay and confocal microscopy. To investigate whether the effects of Jeju ground water on GSH were specifically mediated by vanadium compounds, human Chang liver cells were incubated for 10 passages in media containing deionized distilled water (DDW), Jeju ground water (S1 and S3), and vanadyl sulfate (VOSO4). Vanadyl sulfate scavenged superoxide anion, hydroxyl radical and intracellular reactive oxygen species. Vanadyl sulfate effectively increased cellular GSH level and up-regulated mRNA and protein expression of a catalytic subunit of glutamate cysteine ligase (GCLC), which is involved in GSH synthesis. The induction of GCLC expression by vanadyl sulfate was found to be mediated by transcription factor erythroid transcription factor NF-E2 (Nrf2), which critically regulates GCLC by binding to the antioxidant response elements (AREs). Vanadyl sulfate treatment increased the nuclear translocation of Nrf2 and the accumulation of phosphorylated Nrf2. Extracellular regulated kinase (ERK) contributed to ARE-driven GCLC expression via Nrf2 activation. Vanadyl sulfate induced the expression of the active phospho form of ERK. Taken together, these results suggest that the increase in GSH level by Jeju ground water is, at least in part, due to the effects of vanadyl sulfate via the Nrf2-mediated induction of GCLC. PMID:22272109

  8. Proteasome inhibition induces both antioxidant and hb f responses in sickle cell disease via the nrf2 pathway.

    PubMed

    Pullarkat, Vinod; Meng, Zhuo; Tahara, Stanley M; Johnson, Cage S; Kalra, Vijay K

    2014-01-01

    Oxidant stress is implicated in the manifestations of sickle cell disease including hemolysis and vascular occlusion. Strategies to induce antioxidant response as well as Hb F (α2γ2) have the potential to ameliorate the severity of sickle cell disease. Nuclear factor (erythroid-derived 2)-like 2 (NFE2L2 or Nrf2) is a transcription factor that regulates antioxidant enzymes as well as γ-globin transcription. The Nrf2 in the cytoplasm is bound to its adapter protein Keap-1 that targets Nrf2 for proteasomal degradation, thereby preventing its nuclear translocation. We examined whether inhibiting the 26S proteasome using the clinically applicable proteasome inhibitors bortezomib and MLN 9708 would promote nuclear translocation of Nrf2, and thereby induce an antioxidant response and as well as Hb F in sickle cell disease. Proteasome inhibitors induced reactive oxygen species (ROS) and thereby increased Nrf2-dependent antioxidant enzyme transcripts, elevated cellular glutathione (GSH) levels and γ-globin transcripts as well as Hb F levels in the K562 cell line and also in erythroid burst forming units (BFU-E) generated from peripheral blood mononuclear cells of sickle cell disease patients. These responses were abolished by siRNA-mediated knockdown of Nrf2. Proteasome inhibitors, especially newer oral agents such as MLN9708 have the potential to be readily translated to clinical trials in sickle cell disease with the dual end points of antioxidant response and Hb F induction.

  9. Natural Nrf2 activators in diabetes.

    PubMed

    Jiménez-Osorio, Angélica Saraí; González-Reyes, Susana; Pedraza-Chaverri, José

    2015-08-25

    Prediabetes and diabetes are rising worldwide. Control of blood glucose is crucial to prevent or delay diabetic complications that frequently result in increased morbidity and mortality. Most strategies include medical treatment and changes in lifestyle and diet. Some nutraceutical compounds have been recognized as adjuvants in diabetes control. Many of them can activate the nuclear factor (erythroid-derived 2)-like 2 (Nrf2), which has been recognized as a master regulator of the antioxidant response. Recent studies have described the role of Nrf2 in obesity, metabolic syndrome, nephropathy, retinopathy and neuropathy, where its activation prevents the development of diabetes and its complications. It has been demonstrated that natural compounds derived from plants, vegetables, fungi and micronutrients (such as curcumin, sulforaphane, resveratrol and vitamin D among others) can activate Nrf2 and, thus, promote antioxidant pathways to mitigate oxidative stress and hyperglycemic damage. The role of some natural Nrf2 activators and its effect in diabetes is discussed. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Andrographolide Activates Keap1/Nrf2/ARE/HO-1 Pathway in HT22 Cells and Suppresses Microglial Activation by Aβ42 through Nrf2-Related Inflammatory Response

    PubMed Central

    Seo, Ji Yeon; Pyo, Euisun; An, Jin-Pyo; Kim, Jinwoong; Sung, Sang Hyun

    2017-01-01

    Therapeutic approach of Alzheimer's disease (AD) has been gradually diversified. We examined the therapeutic and preventive potential of andrographolide, which is a lactone diterpenoid from Andrographis paniculata, and focused on the Kelch-like ECH-associated protein 1 (Keap1)/nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-mediated heme oxygenase (HO)-1-inducing effects and the inhibitory activity of amyloid beta (Aβ)42-induced microglial activation related to Nrf2 and nuclear factor κB (NF-κB)-mediated inflammatory responses. Andrographolide induced the expression and translocation of Nrf2 from the cytoplasm to the nucleus, thereby activating antioxidant response element (ARE) gene transcription and HO-1 expression in murine hippocampal HT22 cells. Andrographolide eliminated intracellular Aβ42 in BV-2 cells and decreased the production of interleukin (IL)-6, IL-1β, prostaglandin (PG)E2, and nitric oxide (NO) because of artificial phagocytic Aβ42. It decreased pNF-κB accumulation in the nucleus and the expression of inducible nitric oxide synthase (i-NOS) and cyclooxygenase II (COX-II) in the microglial BV-2 cell line. In summary, andrographolide activates Nrf2-mediated HO-1 expression and inhibits Aβ42-overexpressed microglial BV-2 cell activation. These results suggested that andrographolide might have the potential for further examination of the therapeutics of AD. PMID:28373747

  11. Early modulation of the transcription factor Nrf2 in rodent striatal slices by quinolinic acid, a toxic metabolite of the kynurenine pathway.

    PubMed

    Colín-González, A L; Luna-López, A; Königsberg, M; Ali, S F; Pedraza-Chaverrí, J; Santamaría, A

    2014-02-28

    Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a transcription factor involved in the orchestration of antioxidant responses. Although its pharmacological activation has been largely hypothesized as a promising tool to ameliorate the progression of neurodegenerative events, the actual knowledge about its modulation in neurotoxic paradigms remains scarce. In this study, we investigated the early profile of Nrf2 modulation in striatal slices of rodents incubated in the presence of the toxic kynurenine pathway metabolite, quinolinic acid (QUIN). Tissue slices from rats and mice were obtained and used throughout the experiments in order to compare inter-species responses. Nuclear Nrf2 protein levels and oxidative damage to lipids were compared. Time- and concentration-response curves of all markers were explored. Nrf2 nuclear activation was corroborated through phase 2 antioxidant protein expression. The effects of QUIN on Nrf2 modulation and oxidative stress were also compared between slices of wild-type (Nrf2(+/+)) and Nrf2 knock-out (Nrf2(-/-)) mice. The possible involvement of the N-methyl-d-aspartate receptor (NMDAr) in the Nrf2 modulation and lipid peroxidation was further explored in mice striatal slices. In rat striatal slices, QUIN stimulated the Nrf2 nuclear translocation. This effect was accompanied by augmented lipid peroxidation. In the mouse striatum, QUIN per se exerted an induction of Nrf2 factor only at 1h of incubation, and a concentration-response effect on lipid peroxidation after 3h of incubation. QUIN stimulated the striatal content of phase 2 enzymes. Nrf2(-/-) mice were slightly more responsive than Nrf2(+/+) mice to the QUIN-induced oxidative damage, and completely unresponsive to the NMDAr antagonist MK-801 when tested against QUIN. Findings of this study indicate that: (1) Nrf2 is modulated in rodent striatal tissue in response to QUIN; (2) Nrf2(-/-) striatal tissue was moderately more vulnerable to oxidative damage than the Wt

  12. Synergistic Interaction Between Heme Oxygenase (HO) and Nuclear-Factor E2- Related Factor-2 (Nrf2) against Oxidative Stress in Cardiovascular Related Diseases.

    PubMed

    Ndisang, Joseph Fomusi

    2017-01-01

    Nuclear factor-erythroid related factor-2 (Nrf2) is a master regulator of transcriptional activation of anti-oxidants in cells. Similarly, heme oxygenase (HO) is a cytoprotective protein with anti-oxidant effects. This review article will shed more light on the interaction between Nrf2 and HO. A PubMed search was done for recent articles on Nrf2 and HO. These studies suggested that under normal physiological conditions, Nrf2 is bound within the cytoplasm to its repressor, Kelch-like ECHassociated protein (Keap1), an oxidative stress sensor. Upon activation, Nrf2 translocates to the nucleus and binds to the antioxidant-response-element located at the promoter region of some anti-oxidants including the cytoprotective protein HO. Since the HO-1 gene harbors binding site for Nrf2, mutual stimulatory and regulatory effects between Nrf2 and HO-1 have been reported. Accordingly, the interaction between Nrf2 and HO-1 has been implicated in the regulation of many physiological anti-oxidants including superoxide dismutases, catalase, glutathione S-transferase, peroxidase, NAD(P)H quinone oxidoreductase, and thioredoxin. Although an overwhelming body of evidence has underscored unique anti-oxidant attributes of HO- 1 and Nrf2, emerging evidence suggests that the cytoprotective activities of Nrf2 and HO-1 may be attributed, at least in part, to the potentiation of different anti-oxidants in physiological mileu. Since Nrf2 binds to the antioxidant responsive element of HO-1, the coordinated regulation of Nrf2 and keap1 by the HO-system may constitute the basis of many physiological effects of HO-1 including its effects against oxidative stress and inflammation in a wide spectrum of cardiovascular, cardio-metabolic and other related diseases. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  13. Toxico-pharmacological perspective of the Nrf2-Keap1 defense system against oxidative stress in kidney diseases.

    PubMed

    Saito, Hideyuki

    2013-04-01

    Oxidative stress, including the generation of reactive oxygen species (ROS), appears to be responsible for the high incidence of cardiovascular events in patients with chronic kidney disease (CKD), and for the progression of CKD to end-stage renal disease. The processes for oxidative stress include increased generation and decreased elimination of ROS that could be caused by an impaired antioxidant defense system. Nuclear factor-erythroid-2-related factor 2 (Nrf2) helps protect the kidney against oxidative stress by playing a pivotal role in the cooperative induction of genes that encode antioxidant and detoxifying enzymes. Nrf2 is confined to the cytoplasm as an inactive complex bound to a repressor Kelch-like ECH-associated protein 1 (Keap1), which facilitates ubiquitination of Nrf2. Studies using CKD model animals showed that despite stimulated oxidative stress the nuclear Nrf2 level was suppressed, which led to downregulation of the antioxidant enzymes. Hence, deterioration in Nrf2-Keap1 signaling could contribute to the severity of oxidative stress and the progression of CKD. By contrast, acute kidney injury (AKI) induces activation of renal Nrf2. Nrf2 activators or its proteasomal degradation inhibitors enhance nuclear Nrf2 translocation, inducing potential renoprotective actions against CKD and AKI. In both chronic and acute kidney diseases, sulfate-conjugated uremic toxins appear to enhance ROS production when accumulated in renal cells. An intestinal indole adsorbent ameliorates the progression of CKD by decreasing accumulation of indoxyl sulfate. Therapeutic approaches to prevent oxidative stress via activation of the Nrf2-Keap1 signaling and/or suppression of uremic toxin-induced ROS production could be effective strategies for maintaining kidney function.

  14. Apigenin reactivates Nrf2 anti-oxidative stress signaling in mouse skin epidermal JB6 P + cells through epigenetics modifications.

    PubMed

    Paredes-Gonzalez, Ximena; Fuentes, Francisco; Su, Zheng-Yuan; Kong, Ah-Ng Tony

    2014-07-01

    Nrf2 is a crucial transcription factor that controls a critical anti-oxidative stress defense system and is implicated in skin homeostasis. Apigenin (API), a potent cancer chemopreventive agent, protects against skin carcinogenesis and elicits multiple molecular signaling pathways. However, the potential epigenetic effect of API in skin cancer chemoprotection is not known. In this study, bisulfite genomic DNA sequencing and methylated DNA immunoprecipitation were utilized to investigate the demethylation effect of API at 15 CpG sites in the Nrf2 promoter in mouse skin epidermal JB6 P + cells. In addition, qPCR and Western blot analyses were performed to evaluate the mRNA and protein expression of Nrf2 and the Nrf2 ARE downstream gene, NQO1. Finally, the protein expression levels of DNA methyltransferases (DNMTs) and histone deacetylases (HDACs) were evaluated using API and the DNMT/HDAC inhibitor 5-aza/ trichostatin A. Our results showed that API effectively reversed the hypermethylated status of the 15 CpG sites in the Nrf2 promoter in a dose-dependent manner. API enhanced the nuclear translocation of Nrf2 and increased the mRNA and protein expression of Nrf2 and the Nrf2 downstream target gene, NQO1. Furthermore, API reduced the expression of the DNMT1, DNMT3a, and DNMT3b epigenetic proteins as well as the expression of some HDACs (1-8). Taken together, our results showed that API can restore the silenced status of Nrf2 in skin epidermal JB6 P + cells by CpG demethylation coupled with attenuated DNMT and HDAC activity. These results may provide new therapeutic insights into the prevention of skin cancer by dietary phytochemicals.

  15. The Involvement of NRF2 in Lung Cancer

    PubMed Central

    Bauer, Alison K.; Hill, Thomas

    2013-01-01

    Nuclear factor, erythroid-derived 2, like 2 (NRF2) is a key regulator of antioxidants and cellular stress responses. The role of NRF2 in pulmonary neoplasia, a diverse disease for which few biomarkers exist, is complicated and appears to depend on several main factors including the existence of activating mutations in NRF2 and/or loss of function mutations in KEAP1 and the stage of carcinogenesis studied, particularly in the mouse models tested. Therapeutic strategies for lung cancer targeting NRF2 have observed mixed results, both anti- and protumorigenic effects; however, these differences seem to reflect the mutation status of NRF2 or KEAP1. In this paper, we will discuss the studies on human NRF2 and the mechanisms proposed, several mouse models using various mice deficient in NRF2, as well as xenograft models, and the chemotherapeutic strategies using the NRF2 pathway. PMID:23577226

  16. Anti-Inflammatory Therapy Modulates Nrf2-Keap1 in Kidney from Rats with Diabetes

    PubMed Central

    Arellano-Buendía, Abraham Said; Tostado-González, Montserrat; García-Arroyo, Fernando Enrique; Cristóbal-García, Magdalena; Loredo-Mendoza, María Lilia; Tapia, Edilia; Sánchez-Lozada, Laura-Gabriela; Osorio-Alonso, Horacio

    2016-01-01

    This study addressed the relationship of proinflammatory cytokines and Nrf2-Keap1 system in diabetic nephropathy. The experimental groups were control, diabetic, and diabetic treated with mycophenolate mofetil (MMF). The renal function, proinflammatory and profibrotic cytokines, oxidative stress, morphology, and nephrin expression were assessed. Diabetic group showed impaired renal function in association with oxidative stress and decreased Nrf2 nuclear translocation. These results were associated with increased mesangial matrix index, interstitial fibrosis, and increased nephrin expression in cortex and urine excretion. Additionally, interleukin-1β, IL-6, and transforming growth factor-β1 were increased in plasma and kidney. MMF treatment conserved renal function, prevented renal structural alterations, and partially prevented the proinflammatory and profibrotic cytokines overexpression. Despite that MMF treatment induced nephrin overexpression in renal tissue, preventing its urinary loss. MMF salutary effects were associated with a partial prevention of oxidative stress, increased Nrf2 nuclear translocation, and conservation of antioxidant enzymes in renal tissue. In conclusion, our results confirm that inflammation is a key factor in the progression of diabetic nephropathy and suggest that treatment with MMF protects the kidney by an antioxidant mechanism, possibly regulated at least in part by the Nrf2/Keap1 system, in addition to its well-known anti-inflammatory effects. PMID:26955430

  17. Anti-Inflammatory Therapy Modulates Nrf2-Keap1 in Kidney from Rats with Diabetes.

    PubMed

    Arellano-Buendía, Abraham Said; Tostado-González, Montserrat; García-Arroyo, Fernando Enrique; Cristóbal-García, Magdalena; Loredo-Mendoza, María Lilia; Tapia, Edilia; Sánchez-Lozada, Laura-Gabriela; Osorio-Alonso, Horacio

    2016-01-01

    This study addressed the relationship of proinflammatory cytokines and Nrf2-Keap1 system in diabetic nephropathy. The experimental groups were control, diabetic, and diabetic treated with mycophenolate mofetil (MMF). The renal function, proinflammatory and profibrotic cytokines, oxidative stress, morphology, and nephrin expression were assessed. Diabetic group showed impaired renal function in association with oxidative stress and decreased Nrf2 nuclear translocation. These results were associated with increased mesangial matrix index, interstitial fibrosis, and increased nephrin expression in cortex and urine excretion. Additionally, interleukin-1β, IL-6, and transforming growth factor-β1 were increased in plasma and kidney. MMF treatment conserved renal function, prevented renal structural alterations, and partially prevented the proinflammatory and profibrotic cytokines overexpression. Despite that MMF treatment induced nephrin overexpression in renal tissue, preventing its urinary loss. MMF salutary effects were associated with a partial prevention of oxidative stress, increased Nrf2 nuclear translocation, and conservation of antioxidant enzymes in renal tissue. In conclusion, our results confirm that inflammation is a key factor in the progression of diabetic nephropathy and suggest that treatment with MMF protects the kidney by an antioxidant mechanism, possibly regulated at least in part by the Nrf2/Keap1 system, in addition to its well-known anti-inflammatory effects.

  18. Nrf2 deficiency impairs fracture healing in mice.

    PubMed

    Lippross, Sebastian; Beckmann, Rainer; Streubesand, Nadine; Ayub, Ferda; Tohidnezhad, Mersedeh; Campbell, Graeme; Kan, Yuet Wai; Horst, Fischer; Sönmez, Tolga Taha; Varoga, Deike; Lichte, Philipp; Jahr, Holger; Pufe, Thomas; Wruck, Christoph Jan

    2014-10-01

    Oxidative stress plays an important role in wound healing but data relating oxidative stress to fracture healing are scarce. Nuclear factor erythroid 2-related factor 2 (Nrf2) is the major transcription factor that controls the cellular defence essential to combat oxidative stress by regulating the expression of antioxidative enzymes. This study examined the impact of Nrf2 on fracture healing using a standard closed femoral shaft fracture model in wild-type (WT) and Nrf2-knockout (Nrf2-KO)-mice. Healing was evaluated by histology, real-time RT-PCR, µCT and biomechanical measurements. We showed that Nrf2 expression is activated during fracture healing. Bone healing and remodelling were retarded in the Nrf2-KO compared to the WT-mice. Nrf2-KO-mice developed significantly less callus tissue compared to WT-mice. In addition, biomechanical testing demonstrated lower strength against shear stress in the Nrf2-KO-group compared to WT. The expression of vascular endothelial growth factor (VEGF) and osteocalcin is reduced during fracture healing in Nrf2-KO-mice. Taken together, our results demonstrate that Nrf2 deficiency in mice results in impaired fracture healing suggesting that Nrf2 plays an essential role in bone regeneration. Pharmacological activation of Nrf2 may have therapeutic potential for the enhancement of fracture healing.

  19. Nrf2 protects against furosemide-induced hepatotoxicity.

    PubMed

    Qu, Qiang; Liu, Jie; Zhou, Hong-Hao; Klaassen, Curtis D

    2014-10-03

    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.

  20. Isorhamnetin protects against oxidative stress by activating Nrf2 and inducing the expression of its target genes

    SciTech Connect

    Yang, Ji Hye; Shin, Bo Yeon; Han, Jae Yun; Kim, Mi Gwang; Wi, Ji Eun; Kim, Young Woo; Cho, Il Je; Kim, Sang Chan; Shin, Sang Mi; Ki, Sung Hwan

    2014-01-15

    Isorhamentin is a 3′-O-methylated metabolite of quercetin, and has been reported to have anti-inflammatory and anti-proliferative effects. However, the effects of isorhamnetin on Nrf2 activation and on the expressions of its downstream genes in hepatocytes have not been elucidated. Here, we investigated whether isorhamnetin has the ability to activate Nrf2 and induce phase II antioxidant enzyme expression, and to determine the protective role of isorhamnetin on oxidative injury in hepatocytes. In HepG2 cells, isorhamnetin increased the nuclear translocation of Nrf2 in a dose- and time-dependent manner, and consistently, increased antioxidant response element (ARE) reporter gene activity and the protein levels of hemeoxygenase (HO-1) and of glutamate cysteine ligase (GCL), which resulted in intracellular GSH level increases. The specific role of Nrf2 in isorhamnetin-induced Nrf2 target gene expression was verified using an ARE-deletion mutant plasmid and Nrf2-knockout MEF cells. Deletion of the ARE in the promoter region of the sestrin2 gene, which is recently identified as the Nrf2 target gene by us, abolished the ability of isorhamnetin to increase luciferase activity. In addition, Nrf2 deficiency completely blocked the ability of isorhamnetin to induce HO-1 and GCL. Furthermore, isorhamnetin pretreatment blocked t-BHP-induced ROS production and reversed GSH depletion by t-BHP and consequently, due to reduced ROS levels, decreased t-BHP-induced cell death. In addition isorhamnetin increased ERK1/2, PKCδ and AMPK phosphorylation. Finally, we showed that Nrf2 deficiency blocked the ability of isorhamnetin to protect cells from injury induced by t-BHP. Taken together, our results demonstrate that isorhamnetin is efficacious in protecting hepatocytes against oxidative stress by Nrf2 activation and in inducing the expressions of its downstream genes. - Highlights: • We investigated the effect of isorhamnetin on Nrf2 activation. • Isorhamnetin increased Nrf2

  1. Copper diethyldithiocarbamate as an activator of Nrf2 in cultured vascular endothelial cells.

    PubMed

    Fujie, Tomoya; Murakami, Masaki; Yoshida, Eiko; Tachinami, Tadashi; Shinkai, Yasuhiro; Fujiwara, Yasuyuki; Yamamoto, Chika; Kumagai, Yoshito; Naka, Hiroshi; Kaji, Toshiyuki

    2016-04-01

    The interest in organic-inorganic hybrid molecules as molecular probes for biological systems has been growing rapidly. Such hybrid molecules exhibit unique biological activities. Herein, copper(II) bis(diethyldithiocarbamate) (Cu10) was found to activate the transcription factor NF-E2-related factor 2 (Nrf2), which is responsible for regulating antioxidant and phase II xenobiotic enzymes, in vascular endothelial cells. The copper complex rapidly accumulated within cells and induced nuclear translocation of Nrf2, leading to upregulation of the expression of downstream proteins without cytotoxic effects. However, while copper bis(2-hydroxyethyl)dithiocarbamate activated Nrf2, copper ion, diethyldithiocarbamate ligand with or without zinc or iron failed to exhibit this activity. Intracellular accumulation of Cu10 was higher than that of Cu(II) and Cu(I). While the accumulation of copper(II) bis(dimethyldithiocarbamate) was reduced by small interfering RNA (siRNA)-mediated knockdown of the copper transporter CTR1, the knockdown did not affect Cu10 accumulation, indicating that Cu10 rapidly enters vascular endothelial cells via CTR1-independent mechanisms. In addition, copper and iron complexes with other ligands tested could not activate Nrf2, suggesting that the intramolecular interaction between copper and dithiocarbamate ligand is important for the activation of the transcription factor. Cu10 induced the expression of heme oxygenase-1, NAD(P)H quinone oxidoreductase 1, and γ-glutamylcysteine synthetase, downstream proteins of Nrf2. It was suggested that Cu10-induced activation of Nrf2 was due to proteasome inhibition as well as binding to Kelch-like ECH-associated protein 1. Since the effects of Cu10 on vascular endothelial cells are unique and diverse, the copper complex may be a good molecular probe to analyze the functions of the cells.

  2. Escin activates AKT-Nrf2 signaling to protect retinal pigment epithelium cells from oxidative stress

    SciTech Connect

    Wang, Kaijun; Jiang, Yiqian; Wang, Wei; Ma, Jian; Chen, Min

    2015-12-25

    Here we explored the anti-oxidative and cytoprotective potentials of escin, a natural triterpene-saponin, against hydrogen peroxide (H{sub 2}O{sub 2}) in retinal pigment epithelium (RPE) cells. We showed that escin remarkably attenuated H{sub 2}O{sub 2}-induced death and apoptosis of established (ARPE-19) and primary murine RPE cells. Meanwhile, ROS production and lipid peroxidation by H{sub 2}O{sub 2} were remarkably inhibited by escin. Escin treatment in RPE cells resulted in NF-E2-related factor 2 (Nrf2) signaling activation, evidenced by transcription of anti-oxidant-responsive element (ARE)-regulated genes, including HO-1, NQO-1 and SRXN-1. Knockdown of Nrf2 through targeted shRNAs/siRNAs alleviated escin-mediated ARE gene transcription, and almost abolished escin-mediated anti-oxidant activity and RPE cytoprotection against H{sub 2}O{sub 2}. Reversely, escin was more potent against H{sub 2}O{sub 2} damages in Nrf2-over-expressed ARPE-19 cells. Further studies showed that escin-induced Nrf2 activation in RPE cells required AKT signaling. AKT inhibitors (LY294002 and perifosine) blocked escin-induced AKT activation, and dramatically inhibited Nrf2 phosphorylation, its cytosol accumulation and nuclear translocation in RPE cells. Escin-induced RPE cytoprotection against H{sub 2}O{sub 2} was also alleviated by the AKT inhibitors. Together, these results demonstrate that escin protects RPE cells from oxidative stress possibly through activating AKT-Nrf2 signaling.

  3. Nrf2 promotes survival following exposure to ionizing radiation.

    PubMed

    Sekhar, Konjeti R; Freeman, Michael L

    2015-11-01

    Nrf2 is a transcription factor that promotes antioxidant and drug-metabolizing gene expression. It also regulates the transcription of genes involved in carbohydrate and lipid metabolism, NADPH regeneration, and heme and iron metabolism, as well as proteasome metabolism. Emerging research has identified Nrf2 as a critical factor for promoting survival of mammalian cells subjected to ionizing radiation. At a mechanistic level, Nrf2 promotes the repair of DNA damage and drives detoxification of superoxide that is generated hours to days after irradiation. This review summarizes research in these areas and discusses targeting of Nrf2 in radiation-resistant cancer and Nrf2׳s role in mitigating acute radiation syndrome.

  4. Nrf2: friend or foe for chemoprevention?

    PubMed Central

    Kensler, Thomas W.; Wakabayashi, Nobunao

    2010-01-01

    Health reflects the ability of an organism to adapt to stress. Stresses—metabolic, proteotoxic, mitotic, oxidative and DNA-damage stresses—not only contribute to the etiology of cancer and other chronic degenerative diseases but are also hallmarks of the cancer phenotype. Activation of the Kelch-like ECH-associated protein 1 (KEAP1)–NF-E2-related factor 2 (NRF2)-signaling pathway is an adaptive response to environmental and endogenous stresses and serves to render animals resistant to chemical carcinogenesis and other forms of toxicity, whilst disruption of the pathway exacerbates these outcomes. This pathway can be induced by thiol-reactive small molecules that demonstrate protective efficacy in preclinical chemoprevention models and in clinical trials. However, mutations and epigenetic modifications affecting the regulation and fate of NRF2 can lead to constitutive dominant hyperactivation of signaling that preserves rather than attenuates cancer phenotypes by providing selective resistance to stresses. This review provides a synopsis of KEAP1–NRF2 signaling, compares the impact of genetic versus pharmacologic activation and considers both the attributes and concerns of targeting the pathway in chemoprevention. PMID:19793802

  5. Activation of Keap1/Nrf2 signaling pathway by nuclear epidermal growth factor receptor in cancer cells

    PubMed Central

    Huo, Longfei; Li, Chia-Wei; Huang, Tzu-Hsuan; Lam, Yung Carmen; Xia, Weiya; Tu, Chun; Chang, Wei-Chao; Hsu, Jennifer L; Lee, Dung-Fang; Nie, Lei; Yamaguchi, Hirohito; Wang, Yan; Lang, Jingyu; Li, Long-Yuan; Chen, Chung-Hsuan; Mishra, Lopa; Hung, Mien-Chie

    2014-01-01

    Nuclear translocation of EGFR has been shown to be important for tumor cell growth, survival, and therapeutic resistance. Previously, we detected the association of EGFR with Keap1 in the nucleus. Keap1 is a Kelch-like ECH-associated protein, which plays an important role in cellular response to chemical and oxidative stress by regulating Nrf2 protein stability and nuclear translocation. In this study, we investigate the role of EGFR in regulating Keap1/Nrf2 cascade in the nucleus and provide evidence to show that nuclear EGFR interacts with and phosphorylates nuclear Keap1 to reduce its nuclear protein level. The reduction of nuclear Keap1 consequently stabilizes nuclear Nrf2 and increases its transcriptional activity in cancer cells, which contributes to tumor cell resistance to chemotherapy. PMID:25628777

  6. Activation of the Nrf2 response by intrinsic hepatotoxic drugs correlates with suppression of NF-κB activation and sensitizes toward TNFα-induced cytotoxicity.

    PubMed

    Herpers, Bram; Wink, Steven; Fredriksson, Lisa; Di, Zi; Hendriks, Giel; Vrieling, Harry; de Bont, Hans; van de Water, Bob

    2016-05-01

    Drug-induced liver injury (DILI) is an important problem both in the clinic and in the development of new safer medicines. Two pivotal adaptation and survival responses to adverse drug reactions are oxidative stress and cytokine signaling based on the activation of the transcription factors Nrf2 and NF-κB, respectively. Here, we systematically investigated Nrf2 and NF-κB signaling upon DILI-related drug exposure. Transcriptomics analyses of 90 DILI compounds in primary human hepatocytes revealed that a strong Nrf2 activation is associated with a suppression of endogenous NF-κB activity. These responses were translated into quantitative high-content live-cell imaging of induction of a selective Nrf2 target, GFP-tagged Srxn1, and the altered nuclear translocation dynamics of a subunit of NF-κB, GFP-tagged p65, upon TNFR signaling induced by TNFα using HepG2 cells. Strong activation of GFP-Srxn1 expression by DILI compounds typically correlated with suppression of NF-κB nuclear translocation, yet reversely, activation of NF-κB by TNFα did not affect the Nrf2 response. DILI compounds that provided strong Nrf2 activation, including diclofenac, carbamazepine and ketoconazole, sensitized toward TNFα-mediated cytotoxicity. This was related to an adaptive primary protective response of Nrf2, since loss of Nrf2 enhanced this cytotoxic synergy with TNFα, while KEAP1 downregulation was cytoprotective. These data indicate that both Nrf2 and NF-κB signaling may be pivotal in the regulation of DILI. We propose that the NF-κB-inhibiting effects that coincide with a strong Nrf2 stress response likely sensitize liver cells to pro-apoptotic signaling cascades induced by intrinsic cytotoxic pro-inflammatory cytokines.

  7. Nrf2 Weaves an Elaborate Network of Neuroprotection Against Stroke.

    PubMed

    Jiang, Shuai; Deng, Chao; Lv, Jianjun; Fan, Chongxi; Hu, Wei; Di, Shouyin; Yan, Xiaolong; Ma, Zhiqiang; Liang, Zhenxing; Yang, Yang

    2017-03-01

    Nuclear factor erythroid 2-related factor 2 (Nrf2) is a neuroprotective transcription factor that has recently attracted increased attention. Stroke, a common and serious neurological disease, is currently a leading cause of death in the USA so far. It is therefore of vital importance to explore how Nrf2 behaves in stroke. In this review, we first introduce the structural features of Nrf2 and Kelch-like ECH-associated protein 1 (Keap1) and briefly depict the activation, inactivation, and regulation processes of the Nrf2 pathway. Next, we discuss the physiopathological mechanisms, upstream modulators, and downstream targets of the Nrf2 pathway. Following this background, we expand our discussion to the roles of Nrf2 in ischemic and hemorrhagic stroke and provide several potential future directions. The information presented here may be useful in the design of future experimental research and increase the likelihood of using Nrf2 as a therapeutic target for stroke in the future.

  8. Protective effects of diallyl disulfide on carbon tetrachloride-induced hepatotoxicity through activation of Nrf2.

    PubMed

    Lee, In-Chul; Kim, Sung-Hwan; Baek, Hyung-Seon; Moon, Changjong; Kim, Sung-Ho; Kim, Yun-Bae; Yun, Won-Kee; Kim, Hyoung-Chin; Kim, Jong-Choon

    2015-05-01

    This study was conducted to investigate the potential effects of diallyl disulfide (DADS) on carbon tetrachloride (CCl4 )-induced acute hepatotoxicity and to determine the molecular mechanisms of protection offered by DADS in rats. DADS was administered orally at 50 and 100 mg/kg/day once daily for 5 consecutive days prior to CCl4 administration. The single oral dose of CCl4 (2 mL/kg) caused a significant elevation in serum aspartate and alanine aminotransferase activities, which decreased upon pretreatment with DADS. Histopathological examinations showed extensive liver injury, characterized by extensive hepatocellular degeneration/necrosis, fatty changes, inflammatory cell infiltration, and congestion, which were reversed following pretreatment with DADS. The effects of DADS on cytochrome P450 2E1 (CYP2E1), the major isozyme involved in CCl4 bioactivation, were also investigated. DADS pretreatment resulted in a significant decrease in CYP2E1 protein levels in dose-dependent manner. In addition, CCl4 caused a decrease in protein level of cytoplasmic nuclear factor E2-related factor 2 (Nrf2) and suppression of nuclear translocation of Nrf2 concurrent with downregulation of detoxifying phase II enzymes and a decrease in antioxidant enzyme activities. In contrast, DADS prevented the depletion of cytoplasmic Nrf2 and enhanced nuclear translocation of Nrf2, which, in turn, upregulated antioxidant and/or phase II enzymes. These results indicate that the protective effects of DADS against CCl4 -induced hepatotoxicity possibly involve mechanisms related to its ability to induce antioxidant or detoxifying enzymes by activating Nrf2 and block metabolic activation of CCl4 by suppressing CYP2E1. © 2013 Wiley Periodicals, Inc.

  9. Nrf2 activation prevents cadmium-induced acute liver injury

    SciTech Connect

    Wu, Kai C.; Liu, Jie J.; Klaassen, Curtis D.

    2012-08-15

    Oxidative stress plays an important role in cadmium-induced liver injury. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that up-regulates cytoprotective genes in response to oxidative stress. To investigate the role of Nrf2 in cadmium-induced hepatotoxicity, Nrf2-null mice, wild-type mice, kelch-like ECH-associated protein 1-knockdown (Keap1-KD) mice with enhanced Nrf2, and Keap1-hepatocyte knockout (Keap1-HKO) mice with maximum Nrf2 activation were treated with cadmium chloride (3.5 mg Cd/kg, i.p.). Blood and liver samples were collected 8 h thereafter. Cadmium increased serum alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) activities, and caused extensive hepatic hemorrhage and necrosis in the Nrf2-null mice. In contrast, Nrf2-enhanced mice had lower serum ALT and LDH activities and less morphological alternations in the livers than wild-type mice. H{sub 2}DCFDA (2′,7′-dichlorodihydrofluoresein diacetate) staining of primary hepatocytes isolated from the four genotypes of mice indicated that oxidative stress was higher in Nrf2-null cells, and lower in Nrf2-enhanced cells than in wild-type cells. To further investigate the mechanism of the protective effect of Nrf2, mRNA of metallothionein (MT) and other cytoprotective genes were determined. Cadmium markedly induced MT-1 and MT-2 in livers of all four genotypes of mice. In contrast, genes involved in glutathione synthesis and reducing reactive oxygen species, including glutamate-cysteine ligase (Gclc), glutathione peroxidase-2 (Gpx2), and sulfiredoxin-1 (Srxn-1) were only induced in Nrf2-enhanced mice, but not in Nrf2-null mice. In conclusion, the present study shows that Nrf2 activation prevents cadmium-induced oxidative stress and liver injury through induction of genes involved in antioxidant defense rather than genes that scavenge Cd. -- Highlights: ► Cadmium caused extensive hepatic hemorrhage and necrosis in Nrf2-null mice. ► Keap1-KD and Keap1-HKO mice

  10. l-carnitine protects human hepatocytes from oxidative stress-induced toxicity through Akt-mediated activation of Nrf2 signaling pathway.

    PubMed

    Li, Jinlian; Zhang, Yanli; Luan, Haiyun; Chen, Xuehong; Han, Yantao; Wang, Chunbo

    2016-05-01

    In our previous study, l-carnitine was shown to have cytoprotective effect against hydrogen peroxide (H2O2)-induced injury in human normal HL7702 hepatocytes. The aim of this study was to investigate whether the protective effect of l-carnitine was associated with the nuclear factor erythroid 2 (NFE2)-related factor 2 (Nrf2) pathway. Our results showed that pretreatment with l-carnitine augmented Nrf2 nuclear translocation, DNA binding activity and heme oxygenase-1 (HO-1) expression in H2O2-treated HL7702 cells, although l-carnitine treatment alone had no effect on them. Analysis using Nrf2 siRNA demonstrated that Nrf2 activation was involved in l-carnitine-induced HO-1 expression. In addition, l-carnitine-mediated protection against H2O2 toxicity was abrogated by Nrf2 siRNA, indicating the important role of Nrf2 in l-carnitine-induced cytoprotection. Further experiments revealed that l-carnitine pretreatment enhanced the phosphorylation of Akt in H2O2-treated cells. Blocking Akt pathway with inhibitor partly abrogated the protective effect of l-carnitine. Moreover, our finding demonstrated that the induction of Nrf2 translocation and HO-1 expression by l-carnitine directly correlated with the Akt pathway because Akt inhibitor showed inhibitory effects on the Nrf2 translocation and HO-1 expression. Altogether, these results demonstrate that l-carnitine protects HL7702 cells against H2O2-induced cell damage through Akt-mediated activation of Nrf2 signaling pathway.

  11. Role of pterostilbene in attenuating immune mediated devastation of pancreatic beta cells via Nrf2 signaling cascade.

    PubMed

    Sireesh, Dornadula; Ganesh, Munuswamy-Ramanujam; Dhamodharan, Umapathy; Sakthivadivel, Murugesan; Sivasubramanian, Srinivasan; Gunasekaran, Palani; Ramkumar, Kunka Mohanram

    2017-03-06

    Nrf2 (nuclear factor erythroid 2-related factor-2) is a transcription factor that regulates oxidative/xenobiotic stress response and also suppress inflammation. Nrf2 signaling is associated with an increased susceptibility to various kinds of stress. Nrf2 has been shown as a promising therapeutic target in various human diseases including diabetes. Our earlier studies showed Pterostilbene (PTS) as a potent Nrf2 activator, and it protects the pancreatic β-cells against oxidative stress. In this study, we investigated PTS confer protection against cytokine-induced β-cell apoptosis and its role on insulin secretion in streptozotocin (STZ)-induced diabetic mice. The Nrf2 activation potential of PTS was assessed by dissociation of the Nrf2-Keap1 complex and by expression of ARE-driven downstream target genes in MIN6 cells. Further, the nuclear Nrf2 translocation and blockage of apoptotic signaling as demonstrated by the reduction of BAX/Bcl-2 ratio, Annexin-V positive cells and caspase-3 activity conferred the cyto-protection of PTS against cytokine-induced cellular damage. In addition, PTS treatment markedly improved glucose homeostasis and abated inflammatory response evidenced by the reduction of proinflammatory cytokines in diabetic mice. The inhibition of β-cell apoptosis by PTS as assessed by BAX/Bcl-2 ratio and caspase-3 activity in the pancreas was associated with the activation of Nrf2 and the expression of its downstream target genes. PTS also inhibited the activation of iNOS and decreased nitric oxide (NO) formation in the pancreas of diabetic animals. The results obtained from both in vitro and in vivo experiments showed that PTS improves β-cell function and survival against cytokine stress and also prevents STZ-induced diabetes.

  12. Antimalarial Drug Artemether Inhibits Neuroinflammation in BV2 Microglia Through Nrf2-Dependent Mechanisms.

    PubMed

    Okorji, Uchechukwu P; Velagapudi, Ravikanth; El-Bakoush, Abdelmeneim; Fiebich, Bernd L; Olajide, Olumayokun A

    2016-11-01

    Nrf2 activity by increasing nuclear translocation of Nrf2 and its binding to antioxidant response elements in BV2 cells. Transfection of BV2 microglia with Nrf2 siRNA resulted in the loss of both anti-inflammatory and neuroprotective activities of artemether. We conclude that artemether induces Nrf2 expression and suggest that Nrf2 mediates the anti-inflammatory effect of artemether in BV2 microglia. Our results suggest that this drug has a therapeutic potential in neurodegenerative disorders.

  13. Berberine, a natural antidiabetes drug, attenuates glucose neurotoxicity and promotes Nrf2-related neurite outgrowth

    SciTech Connect

    Hsu, Ya-Yun; Tseng, Yu-Ting; Lo, Yi-Ching

    2013-11-01

    production and neuronal cell death. • BBR activates IGF-1/Akt/GSK-3β signaling under normal and high glucose conditions. • BBR enhances HO-1 and NGF expression through stimulating Nrf2 translocation. • BBR promotes neurite outgrowth through Nrf2-dependent pathway.

  14. Activation of Nrf2 by the dengue virus causes an increase in CLEC5A, which enhances TNF-α production by mononuclear phagocytes.

    PubMed

    Cheng, Yi-Lin; Lin, Yee-Shin; Chen, Chia-Ling; Tsai, Tsung-Ting; Tsai, Cheng-Chieh; Wu, Yan-Wei; Ou, Yi-Dan; Chu, Yu-Yi; Wang, Ju-Ming; Yu, Chia-Yi; Lin, Chiou-Feng

    2016-08-26

    Infection by the dengue virus (DENV) threatens global public health due to its high prevalence and the lack of effective treatments. Host factors may contribute to the pathogenesis of DENV; herein, we investigated the role of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), which is activated by DENV in mononuclear phagocytes. DENV infection selectively activates Nrf2 following nuclear translocation. Following endoplasmic reticular (ER) stress, protein kinase R-like ER kinase (PERK) facilitated Nrf2-mediated transcriptional activation of C-type lectin domain family 5, member A (CLEC5A) to increase CLEC5A expression. Signaling downstream of the Nrf2-CLEC5A interaction enhances Toll-like receptor 3 (TLR3)-independent tumor necrosis factor (TNF)-α production following DENV infection. Forced expression of the NS2B3 viral protein induces Nrf2 nuclear translocation/activation and CLEC5A expression which increases DENV-induced TNF-α production. Animal studies confirmed Nrf2-induced CLEC5A and TNF-α in brains of DENV-infected mice. These results demonstrate that DENV infection causes Nrf2-regulated TNF-α production by increasing levels of CLEC5A.

  15. Oxyresveratrol abrogates oxidative stress by activating ERK-Nrf2 pathway in the liver.

    PubMed

    Choi, Hee Yoon; Lee, Ju-Hee; Jegal, Kyung Hwan; Cho, Il Je; Kim, Young Woo; Kim, Sang Chan

    2016-02-05

    Oxyresveratrol is a polyphenolic phytoalexin produced by plants as an antioxidant. This study investigated the hepatoprotective effects of oxyresveratrol as well as its underlying mechanism of action. Here, we evaluated the protective effects of oxyresveratrol against tert-butyl hydroperoxide (tBHP)-induced severe oxidative stress in HepG2 cells as well as acute liver injury caused by carbon tetrachloride (CCl4) in mice. tBHP-induced reactive oxygen species production and cell death in hepatocytes were blocked by oxyresveratrol, as indicated by MTT, TUNEL, and FACS analyses. Moreover, pretreatment with oxyresveratrol increased nuclear translocation and transactivation of NF-E2-related factor 2 (Nrf2), as assessed by antioxidant response element reporter gene expression and immunofluorescence staining, and transactivated expression of both hemeoxygenase-1 and glutamate-cysteine ligase catalytic subunit. More importantly, oxyresveratrol induced phosphorylation of Nrf2 mediated through activation of extracellular signal-regulated kinase 1/2 (ERK1/2). Further, ERK inhibitors such as PD98059 and U0126 blocked phosphorylation of Nrf2 as well as the protective effect of oxyresveratrol in mitochondria. In mice, oral administration of oxyresveratrol significantly prevented hepatocyte degeneration, inflammatory cell infiltration, as well as elevation of plasma markers such as ALT and AST induced by CCl4 injection. In conclusion, this study confirmed that oxyresveratrol protected hepatocytes against oxidative stress and mitochondrial dysfunction, which might be associated with activation of Nrf2. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  16. Catalase prevents maternal diabetes-induced perinatal programming via the Nrf2-HO-1 defense system.

    PubMed

    Chang, Shiao-Ying; Chen, Yun-Wen; Zhao, Xin-Ping; Chenier, Isabelle; Tran, Stella; Sauvé, Alexandre; Ingelfinger, Julie R; Zhang, Shao-Ling

    2012-10-01

    We investigated whether overexpression of catalase (CAT) in renal proximal tubular cells (RPTCs) could prevent the programming of hypertension and kidney disease in the offspring of dams with maternal diabetes. Male offspring of nondiabetic and diabetic dams from two transgenic (Tg) lines (Hoxb7-green fluorescent protein [GFP]-Tg [controls] and Hoxb7/CAT-GFP-Tg, which overexpress CAT in RPTCs) were studied from the prenatal period into adulthood. Nephrogenesis, systolic blood pressure, renal hyperfiltration, kidney injury, and reactive oxygen species (ROS) generation were assessed. Gene expression of transforming growth factor-β1 (TGF-β1), nuclear factor erythroid 2p45-related factor-2 (Nrf2), and heme oxygenase-1 (HO-1) was tested in both in vitro and in vivo studies. Renal dysmorphogenesis was observed in offspring of Hoxb7-GFP-Tg dams with severe maternal diabetes; the affected male offspring displayed higher renal ROS generation and developed hypertension and renal hyperfiltration as well as renal injury with heightened TGF-β1 expression in adulthood. These changes were ameliorated in male offspring of diabetic Hoxb7/CAT-GFP-Tg dams via the Nrf2-HO-1 defense system. CAT promoted Nrf2 nuclear translocation and HO-1 gene expression, seen in both in vitro and in vivo studies. In conclusion, CAT overexpression in the RPTCs ameliorated maternal diabetes-induced perinatal programming, mediated, at least in part, by triggering the Nrf2-HO-1 defense system.

  17. Opposing effects of Nrf2 and Nrf2-activating compounds on the NLRP3 inflammasome independent of Nrf2-mediated gene expression.

    PubMed

    Garstkiewicz, Martha; Strittmatter, Gerhard E; Grossi, Serena; Sand, Jennifer; Fenini, Gabriele; Werner, Sabine; French, Lars E; Beer, Hans-Dietmar

    2017-03-01

    The transcription factor Nrf2 regulates the expression of genes required for protection from xenobiotic and oxidative stress. Under normal conditions Nrf2 is constantly degraded upon ubiquitination, mediated by the Nrf2 inhibitor Keap1. Inflammasomes represent stress-induced protein complexes. They are critically involved in acute and chronic inflammation through caspase-1-mediated activation of pro-inflammatory cytokines. Here, we demonstrate that Nrf2 is as a positive regulator of the NLRP3 inflammasome. In contrast, Nrf2-activating compounds, including the anti-inflammatory drug dimethyl fumarate (DMF), inhibit inflammasome activation. Both effects are independent of the transcriptional activity of Nrf2 and, at least in part, not interdependent. On the other hand, NLRP3 inflammasome activation induces a rapid and partly caspase-1- and Keap1-independent degradation of Nrf2. These data argue against a simultaneous activation of both stress-related pathways. Finally, we provide evidence that the cross-regulation of both pathways is controlled by a physical interaction between the Nrf2/Keap1 and NLRP3 complexes. This article is protected by copyright. All rights reserved.

  18. Diphenyl diselenide improves the antioxidant response via activation of the Nrf-2 pathway in macrophage cells.

    PubMed

    Mancini, Gianni; Raniel Straliotto, Marcos; da Rocha, João Batista Texeira; de Bem, Andreza Fabro

    2014-10-01

    Diphenyl diselenide [(PhSe)2] is an organoselenium compound that can mimic endogenous antioxidant enzymes, such as glutathione peroxidase (GPx), or be metabolized by thioredoxin reductase to form selenol intermediate, which can copy the function of the antioxidant selenoenzymes. This compound has shown potential role in preventing atherosclerosis and other oxidative stress-related diseases. The understanding of the underlying mechanism by which (PhSe)2 modulates the glutathione-related antioxidant defenses is a relevant question. Therefore, we tested its ability to promote the nuclear translocation of the nuclear factor (erythroid 2-like)-related factor 2 (Nrf-2), increasing the expression of enzymes related to the antioxidant system, such as heme oxygenase 1 (HO-1) and peroxiredoxin 1 (Prx-1), in addition to the main enzyme in the glutathione synthesis - gamma glutamylcysteine synthetase (?-GCS) - in murine J774 macrophage cells. (PhSe)2 (1µM) was able to promote nuclear translocation and increased the expression of the Nrf-2 factor in the nucleus in a time-dependent manner (1-24hours). In addition, this compound significantly increased the expression of HO-1 and Prx-1 at 24hours and GPx-1 after the first hour. Furthermore, (PhSe)2 was able to enhance GSH levels in a time-dependent manner, as well as GPx and GGCS activities. The increase in GPx and GGCS activities was dependent on the activation of PI3K, JNK, and p38MAPKs signaling pathways that may activate the Nrf2 factor. Altogether, these results show that (PhSe)2 improved the antioxidant defense by increasing the expression of HO-1 and Prx-1 and the synthesis of GSH as a consequence of the activation and nuclear translocation of Nrf-2 factor. Copyright © 2014. Published by Elsevier Inc.

  19. Nrf2b, Novel Zebrafish Paralog of Oxidant-responsive Transcription Factor NF-E2-related Factor 2 (NRF2)*

    PubMed Central

    Timme-Laragy, Alicia R.; Karchner, Sibel I.; Franks, Diana G.; Jenny, Matthew J.; Harbeitner, Rachel C.; Goldstone, Jared V.; McArthur, Andrew G.; Hahn, Mark E.

    2012-01-01

    NF-E2-related factor 2 (NRF2; also called NFE2L2) and related NRF family members regulate antioxidant defenses by activating gene expression via antioxidant response elements (AREs), but their roles in embryonic development are not well understood. We report here that zebrafish (Danio rerio), an important developmental model species, possesses six nrf genes, including duplicated nrf1 and nrf2 genes. We cloned a novel zebrafish nrf2 paralog, nrf2b. The predicted Nrf2b protein sequence shares several domains with the original Nrf2 (now Nrf2a) but lacks the Neh4 transactivation domain. Zebrafish-human comparisons demonstrate conserved synteny involving nrf2 and hox genes, indicating that nrf2a and nrf2b are co-orthologs of human NRF2. nrf2a and nrf2b displayed distinct patterns of expression during embryonic development; nrf2b was more highly expressed at all stages. Embryos in which Nrf2a expression had been knocked down with morpholino oligonucleotides were more sensitive to tert-butylhydroperoxide but not tert-butylhydroquinone, whereas knockdown of Nrf2b did not affect sensitivity of embryos to either chemical. Gene expression profiling by microarray identified a specific role for Nrf2b as a negative regulator of several genes, including p53, cyclin G1, and heme oxygenase 1, in embryos. Nrf2a and Nrf2b exhibited different mechanisms of cross-talk with the Ahr2 signaling pathway. Together, these results demonstrate distinct roles for nrf2a and nrf2b, consistent with subfunction partitioning, and identify a novel negative regulatory role for Nrf2b during development. The identification of zebrafish nrf2 co-orthologs will facilitate new understanding of the multiple roles of NRF2 in protecting vertebrate embryos from oxidative damage. PMID:22174413

  20. Structural basis of Keap1 interactions with Nrf2.

    PubMed

    Canning, Peter; Sorrell, Fiona J; Bullock, Alex N

    2015-11-01

    Keap1 is a highly redox-sensitive member of the BTB-Kelch family that assembles with the Cul3 protein to form a Cullin-RING E3 ligase complex for the degradation of Nrf2. Oxidative stress disables Keap1, allowing Nrf2 protein levels to accumulate for the transactivation of critical stress response genes. Consequently, the Keap1-Nrf2 system is extensively pursued for the development of protein-protein interaction inhibitors that will stabilize Nrf2 for therapeutic effect in conditions of neurodegeneration, inflammation, and cancer. Here we review current progress toward the structure determination of Keap1 and its protein complexes with Cul3, Nrf2 substrate, and small-molecule antagonists. Together the available structures establish a rational three-dimensional model to explain the two-site binding of Nrf2 as well as its efficient ubiquitination.

  1. Structural basis of Keap1 interactions with Nrf2

    PubMed Central

    Canning, Peter; Sorrell, Fiona J.; Bullock, Alex N.

    2015-01-01

    Keap1 is a highly redox-sensitive member of the BTB-Kelch family that assembles with the Cul3 protein to form a Cullin–RING E3 ligase complex for the degradation of Nrf2. Oxidative stress disables Keap1, allowing Nrf2 protein levels to accumulate for the transactivation of critical stress response genes. Consequently, the Keap1–Nrf2 system is extensively pursued for the development of protein–protein interaction inhibitors that will stabilize Nrf2 for therapeutic effect in conditions of neurodegeneration, inflammation, and cancer. Here we review current progress toward the structure determination of Keap1 and its protein complexes with Cul3, Nrf2 substrate, and small-molecule antagonists. Together the available structures establish a rational three-dimensional model to explain the two-site binding of Nrf2 as well as its efficient ubiquitination. PMID:26057936

  2. Resveratrol preconditioning protects against cerebral ischemic injury via Nrf2

    PubMed Central

    Narayanan, Srinivasan V.; Dave, Kunjan R.; Saul, Isa; Perez-Pinzon, Miguel A.

    2015-01-01

    Background and Purpose Nuclear erythroid 2 related factor 2 (Nrf2) is an astrocyte-enriched transcription factor that has previously been shown to upregulate cellular antioxidant systems in response to ischemia. While resveratrol preconditioning (RPC) has emerged as a potential neuroprotective therapy, the involvement of Nrf2 in RPC-induced neuroprotection and mitochondrial reactive oxygen species (ROS) production following cerebral ischemia remains unclear. The goal of our study was to study the contribution of Nrf2 to RPC and its effects on mitochondrial function. Methods We used rodent astrocyte cultures and an in vivo stroke model with RPC. An Nrf2 DNA-binding ELISA and protein analysis via Western blotting of downstream Nrf2 targets were performed to determine RPC-induced activation of Nrf2 in rat and mouse astrocytes. Following RPC, mitochondrial function was determined by measuring ROS production and mitochondrial respiration in both wild-type (WT) and Nrf2−/− mice. Infarct volume was measured to determine neuroprotection, while protein levels were measured by immunoblotting. Results We report that Nrf2 is activated by RPC in rodent astrocyte cultures, and that loss of Nrf2 reduced RPC-mediated neuroprotection in a mouse model of focal cerebral ischemia. In addition, we observed that wild-type and Nrf2−/− cortical mitochondria exhibited increased uncoupling and ROS production following RPC treatments, Finally, Nrf2−/− astrocytes exhibited decreased mitochondrial antioxidant expression and were unable to upregulate cellular antioxidants following RPC treatment. Conclusion Nrf2 contributes to RPC-induced neuroprotection through maintaining mitochondrial coupling and antioxidant protein expression. PMID:25908459

  3. When NRF2 Talks, Who's Listening?

    PubMed Central

    Wakabayashi, Nobunao; Slocum, Stephen L.; Skoko, John J.; Shin, Soona

    2010-01-01

    Abstract Activation of the KEAP1-NRF2 signaling pathway is an adaptive response to environmental and endogenous stresses and serves to render animals resistant to chemical carcinogenesis and other forms of toxicity, whereas disruption of the pathway exacerbates these outcomes. This pathway, which can be activated by sulfhydryl-reactive, small-molecule pharmacologic agents, regulates the inducible expression of an extended battery of cytoprotective genes, often by direct binding of the transcription factor to antioxidant response elements in the promoter regions of target genes. However, it is becoming evident that some of the protective effects may be mediated indirectly through cross talk with additional pathways affecting cell survival and other aspects of cell fate. These interactions provide a multi-tiered, integrated response to chemical stresses. This review highlights recent observations on the molecular interactions and their functional consequences between NRF2 and the arylhydrocarbon receptor (AhR), NF-κB, p53, and Notch1 signaling pathways. Antioxid. Redox Signal. 13, 1649–1663. PMID:20367496

  4. 4-Acetoxyphenol Prevents RPE Oxidative Stress–Induced Necrosis by Functioning as an NRF2 Stabilizer

    PubMed Central

    Hanus, Jakub; Kolkin, Alexander; Chimienti, Julia; Botsay, Sara; Wang, Shusheng

    2015-01-01

    Purpose Oxidative stress has been suggested to be a major risk factor for the pathogenesis of AMD. Retinal pigment epithelial (RPE) cells are essential for maintaining the homeostasis of the retina, and RPE cell death and the resultant photoreceptor apoptosis have been observed in dry AMD, especially in geographic atrophy. The purpose of this article was to identify and repurpose the Food and Drug Administration–approved natural compound 4-Acetoxyphenol (4-AC), and to evaluate its effect and mechanism in protecting against oxidative stress–induced RPE necrosis. Methods We exposed ARPE-19 cells to tert-Butyl hydroperoxide (tBHP) after pretreatment with 4-AC, and measured cell viability by MTT assay. Aggregation of RIPK3 and HMGB1 nuclear release were analyzed by transfected reporter genes. Reactive oxygen species (ROS) were measured using a commercially available ROS detection system. The importance of the NRF2/NQO1/HO-1 pathway in mediating 4-AC function was corroborated by siRNA studies, qRT-PCR, and immunostaining. Results We have identified a natural antioxidant, 4-AC, which demonstrates strong abilities to protect RPE cells from oxidative stress–induced necrosis. Mechanistically, 4-AC blocked the increase of cellular ROS induced by oxidative stress, and upregulated NQO1 and HO-1 genes by stabilizing and inducing the nuclear translocation of NRF2 transcription factor. The NQO1, HO-1, and NRF2 were further shown to be required for 4-AC protection of RPE cells from death induced by tBHP. The tBHQ, an NRF2 stabilizer, consistently mimicked the protective effect of 4-AC against tBHP-induced RPE death. Conclusions The compound 4-AC protects ARPE-19 cells from oxidative stress–induced necrosis through upregulation of NQO1 and HO-1 genes by stabilization of NRF2. PMID:26241392

  5. Nrf2 Protects Against TWEAK-mediated Skeletal Muscle Wasting

    NASA Astrophysics Data System (ADS)

    Al-Sawaf, Othman; Fragoulis, Athanassios; Rosen, Christian; Kan, Yuet Wai; Sönmez, Tolga Taha; Pufe, Thomas; Wruck, Christoph Jan

    2014-01-01

    Skeletal muscle (SM) regeneration after injury is impaired by excessive inflammation. Particularly, the inflammatory cytokine tumour necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) is a potent inducer of skeletal muscle wasting and fibrosis. In this study we investigated the role of Nrf2, a major regulator of oxidative stress defence, in SM ischemia/reperfusion (I/R) injury and TWEAK induced atrophy. We explored the time-dependent expression of TWEAK after I/R in SM of Nrf2-wildtype (WT) and knockout (KO) mice. Nrf2-KO mice expressed significant higher levels of TWEAK as compared to WT mice. Consequently, Nrf2-KO mice present an insufficient regeneration as compared to Nrf2-WT mice. Moreover, TWEAK stimulation activates Nrf2 in the mouse myoblast cell line C2C12. This Nrf2 activation inhibits TWEAK induced atrophy in C2C12 differentiated myotubes. In summary, we show that Nrf2 protects SM from TWEAK-induced cell death in vitro and that Nrf2-deficient mice therefore have poorer muscle regeneration.

  6. Nrf2: a potential therapeutic target for diabetic neuropathy.

    PubMed

    Kumar, Anil; Mittal, Ruchika

    2017-08-01

    Different aspects involved in pathophysiology of diabetic neuropathy are related to inflammatory and apoptotic pathways. This article summarizes evidence that Nrf2 acts as a bridging link in various inflammatory and apoptotic pathways impacting progression of diabetic neuropathy. Nrf2 is involved in expression of various antioxidant proteins (such as detoxifying enzymes) via antioxidant response element (ARE) binding site. Under normal conditions, Nrf2 is inactive and remains in the cytosol. Hyperglycemia is a strong stimulus for oxidative stress and inflammation that downregulates the activity of Nrf2 through various neuroinflammatory pathways. Acute hyperglycemia increases the expression of Nrf2, but persistent hyperglycemia decreases its expression. This downregulation of Nrf2 causes various microvascular changes, which result in diabetic neuropathy. The key contribution of Nrf2 in progression of diabetic neuropathy has been summarized in the article. Despite involvement of Nrf2 in progression of diabetic neuropathy, targeting Nrf2 activators as a therapeutic potential will provide important new insights into the ways that influence treatment of diabetic neuropathy.

  7. Oxidative Stress and Cancer: The role of Nrf2.

    PubMed

    Sajadimajd, Soraya; Khazaei, Mozafar

    2017-10-02

    Oxidative stress due to imbalance between ROS production and detoxification plays the pivotal role in determining cell fate. In response to excessive ROS, apoptotic signaling pathway is activated to promote normal cell death. However, through deregulation of biomolecules, high amount of ROS promotes carcinogenesis in cells with defective signaling factors. In this line, NRF2 appears to be as the master regulator to protect cells from oxidative and electrophilic stress. Nrf2 is an intracellular transcription factor that regulates the expression of a number of genes to encode anti-oxidative enzymes, detoxifying factors, anti-apoptotic proteins and drug transporters. Under normal condition, Nrf2 is commonly degraded in cytoplasm by interaction with Keap1 inhibitor as an adaptor for ubiquitination factors. However, high amount of ROS activates tyrosine kinases to dissociate Nrf2:Keap1complex, nuclear import of Nrf2 and coordinated activation of cytoprotective gene expression. Nevertheless, deregulation of Nrf2 and/or Keap1 due to mutation and activated upstream oncogenes is associated with nuclear accumulation and constitutive activation of Nrf2 to protect cells from apoptosis and induce proliferation, metastasis and chemoresistance. Owning to the interplay of ROS and Nrf2 signaling pathways with carcinogenesis, Nrf2 modulation seems to be important in personalization of cancer therapy. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  8. Nrf2 Is an Attractive Therapeutic Target for Retinal Diseases.

    PubMed

    Nakagami, Yasuhiro

    2016-01-01

    Nuclear factor erythroid 2-related factor 2 (Nrf2) is a redox-sensitive transcription factor that binds to antioxidant response elements located in the promoter region of genes encoding many antioxidant enzymes and phase II detoxifying enzymes. Activation of Nrf2 functions is one of the critical defensive mechanisms against oxidative stress in many species. The retina is constantly exposed to reactive oxygen species, and oxidative stress is a major contributor to age-related macular diseases. Moreover, the resulting inflammation and neuronal degeneration are also related to other retinal diseases. The well-known Nrf2 activators, bardoxolone methyl and its derivatives, have been the subject of a number of clinical trials, including those aimed at treating chronic kidney disease, pulmonary arterial hypertension, and mitochondrial myopathies. Recent studies suggest that Nrf2 activation protects the retina from retinal diseases. In particular, this is supported by the finding that Nrf2 knockout mice display age-related retinal degeneration. Moreover, the concept has been validated by the efficacy of Nrf2 activators in a number of retinal pathological models. We have also recently succeeded in generating a novel Nrf2 activator, RS9, using a biotransformation technique. This review discusses current links between retinal diseases and Nrf2 and the possibility of treating retinal diseases by activating the Nrf2 signaling pathway.

  9. Nrf2 Is an Attractive Therapeutic Target for Retinal Diseases

    PubMed Central

    2016-01-01

    Nuclear factor erythroid 2-related factor 2 (Nrf2) is a redox-sensitive transcription factor that binds to antioxidant response elements located in the promoter region of genes encoding many antioxidant enzymes and phase II detoxifying enzymes. Activation of Nrf2 functions is one of the critical defensive mechanisms against oxidative stress in many species. The retina is constantly exposed to reactive oxygen species, and oxidative stress is a major contributor to age-related macular diseases. Moreover, the resulting inflammation and neuronal degeneration are also related to other retinal diseases. The well-known Nrf2 activators, bardoxolone methyl and its derivatives, have been the subject of a number of clinical trials, including those aimed at treating chronic kidney disease, pulmonary arterial hypertension, and mitochondrial myopathies. Recent studies suggest that Nrf2 activation protects the retina from retinal diseases. In particular, this is supported by the finding that Nrf2 knockout mice display age-related retinal degeneration. Moreover, the concept has been validated by the efficacy of Nrf2 activators in a number of retinal pathological models. We have also recently succeeded in generating a novel Nrf2 activator, RS9, using a biotransformation technique. This review discusses current links between retinal diseases and Nrf2 and the possibility of treating retinal diseases by activating the Nrf2 signaling pathway. PMID:27818722

  10. Translational control of Nrf2 within the open reading frame

    SciTech Connect

    Perez-Leal, Oscar Barrero, Carlos A.; Merali, Salim

    2013-07-19

    Highlights: •Identification of a novel Nrf2 translational repression mechanism. •The repressor is within the 3′ portion of the Nrf2 ORF. •The translation of Nrf2 or eGFP is reduced by the regulatory element. •The translational repression can be reversed with synonymous codon substitutions. •The molecular mechanism requires the mRNA sequence, but not the encoded amino acids. -- Abstract: Nuclear Factor Erythroid 2-Related Factor 2 (Nrf2) is a transcription factor that is essential for the regulation of an effective antioxidant and detoxifying response. The regulation of its activity can occur at transcription, translation and post-translational levels. Evidence suggests that under environmental stress conditions, new synthesis of Nrf2 is required – a process that is regulated by translational control and is not fully understood. Here we described the identification of a novel molecular process that under basal conditions strongly represses the translation of Nrf2 within the open reading frame (ORF). This mechanism is dependent on the mRNA sequence within the 3′ portion of the ORF of Nrf2 but not in the encoded amino acid sequence. The Nrf2 translational repression can be reversed with the use of synonymous codon substitutions. This discovery suggests an additional layer of control to explain the reason for the low Nrf2 concentration under quiescent state.

  11. Retinoic acid synergizes ATO-mediated cytotoxicity by precluding Nrf2 activity in AML cells

    PubMed Central

    Valenzuela, M; Glorieux, C; Stockis, J; Sid, B; Sandoval, J M; Felipe, K B; Kviecinski, M R; Verrax, J; Calderon, P Buc

    2014-01-01

    Background: Standard therapy for acute promyelocytic leukaemia (APL) includes retinoic acid (all-trans retinoic acid (ATRA)), which promotes differentiation of promyelocytic blasts. Although co-administration of arsenic trioxide (ATO) with ATRA has emerged as an effective option to treat APL, the molecular basis of this effect remains unclear. Methods: Four leukaemia cancer human models (HL60, THP-1, NBR4 and NBR4-R2 cells) were treated either with ATO alone or ATO plus ATRA. Cancer cell survival was monitored by trypan blue exclusion and DEVDase activity assays. Gene and protein expression changes were assessed by RT-PCR and western blot. Results: ATO induced an antioxidant response characterised by Nrf2 nuclear translocation and enhanced transcription of downstream target genes (that is, HO-1, NQO1, GCLM, ferritin). In cells exposed to ATO plus ATRA, the Nrf2 nuclear translocation was prevented and cytotoxicity was enhanced. HO-1 overexpression reversed partially the cytotoxicity by ATRA-ATO in HL60 cells. The inhibitory effects of ATRA on ATO-mediated responses were not observed in either the ATRA-resistant NB4-R2 cells or in NB4 cells pre-incubated with the RARα antagonist Ro-41-52-53. Conclusions: The augmented cytotoxicity observed in leukaemia cells following combined ATO-ATRA treatment is likely due to inhibition of Nrf2 activity, thus explaining the efficacy of combined ATO-ATRA treatment in the APL therapy. PMID:25003661

  12. Cerium oxide nanoparticles alleviate oxidative stress and decreases Nrf-2/HO-1 in D-GALN/LPS induced hepatotoxicity.

    PubMed

    Hashem, Reem M; Rashd, Laila A; Hashem, Khalid S; Soliman, Hatem M

    2015-07-01

    Translocation of the master regulator of antioxidant-response element-driven antioxidant gene, nuclear factor erythroid 2 (Nrf-2) from the cytoplasm into the nucleus and triggering the transcription of hemoxygenase-1 (HO-1) to counteract the oxidative stress is a key feature in D-galactoseamine and lipopolysaccharide (D-GALN/LPS) induced hepatotoxicity. We mainly aimed to study the effect of cerium oxide (CeO2) nanoparticles on Nrf-2/HO-1 pathway whereas; it has previously shown to have an antioxidant effect in liver models. Administration of CeO2 nanoparticles significantly decreased the translocation of the cytoplasmic Nrf-2 with a concomitant decrement in the gene expression of HO-1 as it reveals a powerful antioxidative effect as indicated by the significant increase in the levels of glutathione (GSH), glutathione peroxidase (GPX1), glutathione reductase (GR), superoxide dismutase (SOD) and catalase. In synchronization, a substantial decrement in the levels of inducible nitric oxide synthase (iNOS), TBARS and percentage of DNA fragmentation was established. These results were confirmed by histopathology examination which showed a severe degeneration, haemorrhages, widened sinusoids and focal leukocyte infiltration in D-GALN/LPS treatment and these features were alleviated with CeO2 administration. In conclusion, CeO2 is a potential antioxidant that can effectively decrease the translocation of the cytoplasmic Nrf-2 into the nucleus and decrease HO-1 in D-GALN/LPS induced hepatotoxicity.

  13. Nrf2-inducing anti-oxidation stress response in the rat liver--new beneficial effect of lansoprazole.

    PubMed

    Yamashita, Yasunobu; Ueyama, Takashi; Nishi, Toshio; Yamamoto, Yuta; Kawakoshi, Akatsuki; Sunami, Shogo; Iguchi, Mikitaka; Tamai, Hideyuki; Ueda, Kazuki; Ito, Takao; Tsuruo, Yoshihiro; Ichinose, Masao

    2014-01-01

    Lansoprazole is a potent anti-gastric ulcer drug that inhibits gastric proton pump activity. We identified a novel function for lansoprazole, as an inducer of anti-oxidative stress responses in the liver. Gastric administration of lansoprazole (10-100 mg/kg) to male Wistar rats produced a dose-dependent increase in hepatic mRNA levels of nuclear factor, erythroid-derived 2, -like 2 (Nrf2), a redox-sensitive transcription factor, at 3 h and Nrf2 immunoreactivity (IR) in whole hepatic lysates at 6 h. Conversely, the levels of Kelch-like ECH-associated protein (Keap1), which sequesters Nrf2 in the cytoplasm under un-stimulated conditions, were unchanged. Translocation of Nrf2 into the nuclei of hepatocytes was observed using western blotting and immunohistochemistry. Expression of mRNAs for Nrf2-dependent antioxidant and phase II enzymes, such as heme oxygenase 1 (HO-1), NAD (P) H dehydrogenase, quinone 1 (Nqo1), glutathione S-transferase A2 (Gsta2), UDP glucuronosyltransferase 1 family polypeptide A6 (Ugt1a6), were dose-dependently up-regulated at 3 h. Furthermore, the levels of HO-1 IR were dose-dependently increased in hepatocytes at 6 h. Subcutaneous administration of lansoprazole (30 mg/kg/day) for 7 successive days resulted in up-regulation and nuclear translocation of Nrf2 IR in hepatocytes and up-regulation of HO-1 IR in the liver. Pretreatment with lansoprazole attenuated thioacetamide (500 mg/kg)-induced acute hepatic damage via both HO-1-dependent and -independent pathways. Up-stream networks related to Nrf2 expression were investigated using microarray analysis, followed by data mining with Ingenuity Pathway Analysis. Up-regulation of the aryl hydrocarbon receptor (AhR)-cytochrome P450, family 1, subfamily a, polypeptide 1 (Cyp1a1) pathway was associated with up-regulation of Nrf2 mRNA. In conclusion, lansoprazole might have an alternative indication in the prevention and treatment of oxidative hepatic damage through the induction of both phase I and phase

  14. Nrf2-Inducing Anti-Oxidation Stress Response in the Rat Liver - New Beneficial Effect of Lansoprazole

    PubMed Central

    Yamashita, Yasunobu; Ueyama, Takashi; Nishi, Toshio; Yamamoto, Yuta; Kawakoshi, Akatsuki; Sunami, Shogo; Iguchi, Mikitaka; Tamai, Hideyuki; Ueda, Kazuki; Ito, Takao; Tsuruo, Yoshihiro; Ichinose, Masao

    2014-01-01

    Lansoprazole is a potent anti-gastric ulcer drug that inhibits gastric proton pump activity. We identified a novel function for lansoprazole, as an inducer of anti-oxidative stress responses in the liver. Gastric administration of lansoprazole (10–100 mg/kg) to male Wistar rats produced a dose-dependent increase in hepatic mRNA levels of nuclear factor, erythroid-derived 2, -like 2 (Nrf2), a redox-sensitive transcription factor, at 3 h and Nrf2 immunoreactivity (IR) in whole hepatic lysates at 6 h. Conversely, the levels of Kelch-like ECH-associated protein (Keap1), which sequesters Nrf2 in the cytoplasm under un-stimulated conditions, were unchanged. Translocation of Nrf2 into the nuclei of hepatocytes was observed using western blotting and immunohistochemistry. Expression of mRNAs for Nrf2-dependent antioxidant and phase II enzymes, such as heme oxygenase 1 (HO-1), NAD (P) H dehydrogenase, quinone 1 (Nqo1), glutathione S-transferase A2 (Gsta2), UDP glucuronosyltransferase 1 family polypeptide A6 (Ugt1a6), were dose-dependently up-regulated at 3 h. Furthermore, the levels of HO-1 IR were dose-dependently increased in hepatocytes at 6 h. Subcutaneous administration of lansoprazole (30 mg/kg/day) for 7 successive days resulted in up-regulation and nuclear translocation of Nrf2 IR in hepatocytes and up-regulation of HO-1 IR in the liver. Pretreatment with lansoprazole attenuated thioacetamide (500 mg/kg)-induced acute hepatic damage via both HO-1-dependent and -independent pathways. Up-stream networks related to Nrf2 expression were investigated using microarray analysis, followed by data mining with Ingenuity Pathway Analysis. Up-regulation of the aryl hydrocarbon receptor (AhR)-cytochrome P450, family 1, subfamily a, polypeptide 1 (Cyp1a1) pathway was associated with up-regulation of Nrf2 mRNA. In conclusion, lansoprazole might have an alternative indication in the prevention and treatment of oxidative hepatic damage through the induction of both phase I and

  15. Nrf2 deficiency prevents reductive stress-induced hypertrophic cardiomyopathy

    PubMed Central

    Kannan, Sankaranarayanan; Muthusamy, Vasanthi R.; Whitehead, Kevin J.; Wang, Li; Gomes, Aldrin V.; Litwin, Sheldon E.; Kensler, Thomas W.; Abel, E. Dale; Hoidal, John R.; Rajasekaran, Namakkal S.

    2013-01-01

    Aims Mutant protein aggregation (PA) cardiomyopathy (MPAC) is characterized by reductive stress (RS), PA (of chaperones and cytoskeletal components), and ventricular dysfunction in transgenic mice expressing human mutant CryAB (hmCryAB). Sustained activation of nuclear erythroid-2 like factor-2 (Nrf2) causes RS, which contributes to proteotoxic cardiac disease. The goals of this pre-clinical study were to (i) investigate whether disrupting Nrf2-antioxidant signalling prevents RS and rescues redox homeostasis in hearts expressing the mutant chaperone and (ii) elucidate mechanisms that could delay proteotoxic cardiac disease. Methods and results Non-transgenic (NTG), transgenic (TG) with MPAC and MPAC-TG:Nrf2-deficient (Nrf2-def) mice were used in this study. The effects of Nrf2 diminution (Nrf2±) on RS mediated MPAC in TG mice were assessed at 6–7 and 10 months of age. The diminution of Nrf2 prevented RS and prolonged the survival of TG mice (∼50 weeks) by an additional 20–25 weeks. The TG:Nrf2-def mice did not exhibit cardiac hypertrophy at even 60 weeks, while the MPAC-TG mice developed pathological hypertrophy and heart failure starting at 24–28 weeks of age. Aggregation of cardiac proteins was significantly reduced in TG:Nrf2-def when compared with TG mice at 7 months. Preventing RS and maintaining redox homeostasis in the TG:Nrf2-def mice ameliorated PA, leading to decreased ubiquitination of proteins. Conclusion Nrf2 deficiency rescues redox homeostasis, which reduces aggregation of mutant proteins, thereby delaying the proteotoxic pathological cardiac remodelling caused by RS and toxic protein aggregates. PMID:23761402

  16. Quinone Induced Activation of Keap1/Nrf2 Signaling by Aspirin Prodrugs Masquerading as Nitric Oxide

    PubMed Central

    Dunlap, Tareisha; Piyankarage, Sujeewa C.; Wijewickrama, Gihani T.; Abdul-Hay, Samer; Vanni, Michael; Litosh, Vladislav; Luo, Jia; Thatcher, Gregory R. J.

    2013-01-01

    The promising therapeutic potential of the NO-donating hybrid aspirin prodrugs (NO-ASA), includes induction of chemopreventive mechanisms, and has been reported in almost 100 publications. One example, NCX-4040 (pNO-ASA), is bioactivated by esterase to a quinone methide (QM) electrophile. In cell cultures, pNO-ASA and QM-donating X-ASA prodrugs that cannot release NO rapidly depleted intracellular GSH and caused DNA damage; however, induction of Nrf2 signaling elicited cellular defense mechanisms including upregulation of NAD(P)H:quinone oxidoreductase-1 (NQO1) and glutamate-cysteine ligase (GCL). In HepG2 cells, the “NO-specific” 4,5-diaminofluorescein reporter, DAF-DA, responded to NO-ASA and X-ASA, with QM-induced oxidative stress masquerading as NO. LC-MS/MS analysis demonstrated efficient alkylation of Cys residues of proteins including glutathione-S-transferase-P1 (GST-P1) and Kelch-like ECH-associated protein 1 (Keap1). Evidence was obtained for alkylation of Keap1 Cys residues associated with Nrf2 translocation to the nucleus, nuclear translocation of Nrf2, activation of antioxidant response element (ARE), and upregulation of cytoprotective target genes. At least in cell culture, pNO-ASA acts as a QM-donor, bioactivated by cellular esterase activity to release salicylates, NO3−, and an electrophilic QM. Finally, two novel aspirin prodrugs were synthesized, both potent activators of ARE, designed to release only the QM and salicylates on bioactivation. Current interest in electrophilic drugs acting via Nrf2 signaling suggests that QM-donating hybrid drugs can be designed as informative chemical probes in drug discovery. PMID:23035985

  17. MHY1485 ameliorates UV-induced skin cell damages via activating mTOR-Nrf2 signaling.

    PubMed

    Yang, Bo; Xu, Qiu-Yun; Guo, Chun-Yan; Huang, Jin-Wen; Wang, Shu-Mei; Li, Yong-Mei; Tu, Ying; He, Li; Bi, Zhi-Gang; Ji, Chao; Cheng, Bo

    2017-02-21

    Ultra Violet (UV)-caused skin cell damage is a main cause of skin cancer. Here, we studied the activity of MHY1485, a mTOR activator, in UV-treated skin cells. In primary human skin keratinocytes, HaCaT keratinocytes and human skin fibroblasts, MHY1485 ameliorated UV-induced cell death and apoptosis. mTOR activation is required for MHY1485-induced above cytoprotective actions. mTOR kinase inhibitors (OSI-027, AZD-8055 and AZD-2014) or mTOR shRNA knockdown almost abolished MHY1485-induced cytoprotection. Further, MHY1485 treatment in skin cells activated mTOR downstream NF-E2-related factor 2 (Nrf2) signaling, causing Nrf2 Ser-40 phosphorylation, stabilization/upregulation and nuclear translocation, as well as mRNA expression of Nrf2-dictated genes. Contrarily, Nrf2 knockdown or S40T mutation almost nullified MHY1485-induced cytoprotection. MHY1485 suppressed UV-induced reactive oxygen species production and DNA single strand breaks in skin keratinocytes and fibroblasts. Together, we conclude that MHY1485 inhibits UV-induced skin cell damages via activating mTOR-Nrf2 signaling.

  18. The NRF2 transcriptional target, OSGIN1, contributes to monomethyl fumarate-mediated cytoprotection in human astrocytes

    PubMed Central

    Brennan, Melanie S.; Matos, Maria F.; Richter, Karl E.; Li, Bing; Scannevin, Robert H.

    2017-01-01

    Dimethyl fumarate (DMF) is indicated for the treatment of relapsing multiple sclerosis and may exert therapeutic effects via activation of the nuclear factor (erythroid-derived 2)-like 2 (NRF2) pathway. Following oral DMF administration, central nervous system (CNS) tissue is predominantly exposed to monomethyl fumarate (MMF), the bioactive metabolite of DMF, which can stabilize NRF2 and induce antioxidant gene expression; however, the detailed NRF2-dependent mechanisms modulated by MMF that lead to cytoprotection are unknown. Our data identify a mechanism for MMF-mediated cytoprotection in human astrocytes that functions in an OSGIN1-dependent manner, specifically via upregulation of the OSGIN1-61 kDa isoform. NRF2-dependent OSGIN1 expression induced P53 nuclear translocation following MMF administration, leading to cell-cycle inhibition and cell protection against oxidative challenge. This study provides mechanistic insight into MMF-mediated cytoprotection via NRF2, OSGIN1, and P53 in human CNS-derived cells and contributes to our understanding of how DMF may act clinically to ameliorate pathological processes in neurodegenerative disease. PMID:28181536

  19. Apigenin exhibits protective effects in a mouse model of d-galactose-induced aging via activating the Nrf2 pathway.

    PubMed

    Sang, Ying; Zhang, Fan; Wang, Heng; Yao, Jianqiao; Chen, Ruichuan; Zhou, Zhengdao; Yang, Kun; Xie, Yan; Wan, Tianfeng; Ding, Hong

    2017-06-21

    The aim of the present research was to study the protective effects and underlying mechanisms of apigenin on d-galactose-induced aging mice. Firstly, apigenin exhibited a potent antioxidant activity in vitro. Secondly, d-galactose was administered by subcutaneous injection once daily for 8 weeks to establish an aging mouse model to investigate the protective effect of apigenin. We found that apigenin supplementation significantly ameliorated aging-related changes such as behavioral impairment, decreased organic index, histopathological injury, increased senescence-associated β-galactosidase (SAβ-gal) activity and advanced glycation end product (AGE) level. Further data showed that apigenin facilitated Nrf2 nuclear translocation both in aging mice and normal young mice, and the Nrf2 expression of normal young mice was higher than that of natural senile mice. In addition, the expressions of Nrf2 downstream gene targets, including HO-1 and NQO1, were also promoted by apigenin administration. Moreover, apigenin also decreased the MDA level and elevated SOD and CAT activities. In conclusion, focusing on the Nrf2 pathway is a suitable strategy to delay the aging process, and apigenin may exert an anti-senescent effect process via activating the Nrf2 pathway.

  20. Mining a human transcriptome database for Nrf2 modulators

    EPA Science Inventory

    Nuclear factor erythroid-2 related factor 2 (Nrf2) is a key transcription factor important in the protection against oxidative stress. We developed computational procedures to enable the identification of chemical, genetic and environmental modulators of Nrf2 in a large database ...

  1. Nrf2 links epidermal barrier function with antioxidant defense

    PubMed Central

    Schäfer, Matthias; Farwanah, Hany; Willrodt, Ann-Helen; Huebner, Aaron J; Sandhoff, Konrad; Roop, Dennis; Hohl, Daniel; Bloch, Wilhelm; Werner, Sabine

    2012-01-01

    The skin provides an efficient permeability barrier and protects from microbial invasion and oxidative stress. Here, we show that these essential functions are linked through the Nrf2 transcription factor. To test the hypothesis that activation of Nrf2 provides skin protection under stress conditions, we determined the consequences of pharmacological or genetic activation of Nrf2 in keratinocytes. Surprisingly, mice with enhanced Nrf2 activity in keratinocytes developed epidermal thickening, hyperkeratosis and inflammation resembling lamellar ichthyosis. This resulted from upregulation of the cornified envelope proteins small proline-rich proteins (Sprr) 2d and 2h and of secretory leukocyte peptidase inhibitor (Slpi), which we identified as novel Nrf2 targets in keratinocytes. Since Sprrs are potent scavengers of reactive oxygen species and since Slpi has antimicrobial activities, their upregulation contributes to Nrf2's protective function. However, it also caused corneocyte fragility and impaired desquamation, followed by alterations in the epidermal lipid barrier, inflammation and overexpression of mitogens that induced keratinocyte hyperproliferation. These results identify an unexpected role of Nrf2 in epidermal barrier function, which needs to be considered for pharmacological use of Nrf2 activators. PMID:22383093

  2. The Keap1-Nrf2 system and diabetes mellitus.

    PubMed

    Uruno, Akira; Yagishita, Yoko; Yamamoto, Masayuki

    2015-01-15

    Nrf2 (NF-E2-related factor 2) plays a key role in the protection of vertebrates against environmental stress by contributing to the inducible expression of detoxification and antioxidant enzymes. Keap1 (Kelch-like ECH-associated protein 1) is a sensor for oxidative and electrophilic stresses. Keap1 also acts as an E3 ubiquitin ligase substrate-recognition subunit that specifically targets Nrf2. Keap1 causes Nrf2 to be degraded through the ubiquitin-proteasome pathway and thus ensures that Nrf2 is constitutively suppressed under unstressed conditions. Upon exposure to oxidative or electrophilic stress, Keap1 loses its ability to ubiquitinate Nrf2. Many lines of evidence have recently clarified that the Keap1-Nrf2 system also plays critical roles in the maintenance of cellular homeostasis. One of the most salient examples is the contribution of Keap1-Nrf2 to metabolic and energy-balance regulation. In particular, how the Keap1-Nrf2 system protects the body against diabetes mellitus and how perturbations in this system provoke the disease condition are now under intense investigation. This review will summarize the recent progress made in this area.

  3. Mining a human transcriptome database for Nrf2 modulators

    EPA Science Inventory

    Nuclear factor erythroid-2 related factor 2 (Nrf2) is a key transcription factor important in the protection against oxidative stress. We developed computational procedures to enable the identification of chemical, genetic and environmental modulators of Nrf2 in a large database ...

  4. An Essential Role of NRF2 in Diabetic Wound Healing

    PubMed Central

    Long, Min; Rojo de la Vega, Montserrat; Wen, Qing; Bharara, Manish; Jiang, Tao; Zhang, Rui; Zhou, Shiwen; Wong, Pak K.

    2016-01-01

    The high mortality and disability of diabetic nonhealing skin ulcers create an urgent need for the development of more efficacious strategies targeting diabetic wound healing. In the current study, using human clinical specimens, we show that perilesional skin tissues from patients with diabetes are under more severe oxidative stress and display higher activation of the nuclear factor-E2–related factor 2 (NRF2)–mediated antioxidant response than perilesional skin tissues from normoglycemic patients. In a streptozotocin-induced diabetes mouse model, Nrf2−/− mice have delayed wound closure rates compared with Nrf2+/+ mice, which is, at least partially, due to greater oxidative DNA damage, low transforming growth factor-β1 (TGF-β1) and high matrix metalloproteinase 9 (MMP9) expression, and increased apoptosis. More importantly, pharmacological activation of the NRF2 pathway significantly improves diabetic wound healing. In vitro experiments in human immortalized keratinocyte cells confirm that NRF2 contributes to wound healing by alleviating oxidative stress, increasing proliferation and migration, decreasing apoptosis, and increasing the expression of TGF-β1 and lowering MMP9 under high-glucose conditions. This study indicates an essential role for NRF2 in diabetic wound healing and the therapeutic benefits of activating NRF2 in this disease, laying the foundation for future clinical trials using NRF2 activators in treating diabetic skin ulcers. PMID:26718502

  5. Agrimonolide and Desmethylagrimonolide Induced HO-1 Expression in HepG2 Cells through Nrf2-Transduction and p38 Inactivation

    PubMed Central

    Chen, Lei; Teng, Hui; Zhang, Kalin Yanbo; Skalicka-Woźniak, Krystyna; Georgiev, Milen I.; Xiao, Jianbo

    2017-01-01

    Agrimonolide and desmethylagrimonolide are the main bioactive polyphenols in agrimony with well-documented antioxidant, anti-diabetic, and anti-inflammatory potential. We report here for the first time that agrimonolide and desmethylagrimonolide stimulate the expression of phase II detoxifying enzymes through the Nrf2-dependent signaling pathway. Agrimonolide and desmethylagrimonolide also possess considerable protective activity from oxidative DNA damage. In order to explore the cytoprotective potential of agrimonolide and desmethylagrimonolide on oxidative stress in liver, we developed an oxidative stress model in HepG2 cells, and check the hypothesis whether Nrf2 pathway is involved. Western blotting and luciferase assay revealed that exposure of HepG2 cells to agrimonolide or desmethylagrimonolide leads to increased heme oxygenase-1 (HO-1) expression by activating ARE through induction of Nrf2 and suppression of Kelch-like ECH-associated protein 1 (Keap1). Moreover, agrimonolide and desmethylagrimonolide also activated ERK signaling pathways and significantly attenuated individual p38 MAPK expression, subsequently leading to Nrf2 nuclear translocation. In conclusion, our results indicated that transcriptional activation of Nrf2/ARE is critical in agrimonolide and desmethylagrimonolide-mediated HO-1 induction, which can be regulated partially by the blockade of p38 MAPK signaling pathway and inhibiting nuclear translocation of Nrf2. PMID:28119605

  6. The protective role of Nrf2-Gadd45b against antimony-induced oxidative stress and apoptosis in HEK293 cells.

    PubMed

    Jiang, Xingkang; An, Zesheng; Lu, Chao; Chen, Yue; Du, E; Qi, Shiyong; Yang, Kuo; Zhang, Zhihong; Xu, Yong

    2016-08-10

    Antimony (Sb) is one of the most prevalent heavy metals and frequently causes biological toxicity. However, the specific mechanisms by which Sb elicits its toxic effects remains to be fully elucidated. In this study, we found antimony trioxide (Sb2O3) caused a dose-dependent cytotoxicity against HEK293 cells, and Sb2O3-induced excessive reactive oxygen species (ROS) was closely correlated with increased cell apoptosis. Mechanistic investigation manifested that nuclear factor NF-E2-related factor 2 (Nrf2) expression and nuclear translocation were significantly induced under Sb2O3 treatment in HEK293 cells, and Nrf2 knockdown aggregated Sb2O3-induced cell apoptosis. Moreover, elevated Gadd45b expression actives the phosphorylation of MAPKs upon Sb2O3 exposure, whereas Gadd45b knockdown diminished Sb2O3-induced activation of MAPKs and promoted cell apoptosis. In the meantime, however, the antioxidant N-acetylcysteine (NAC) was found to ameliorate Nrf2 expression and nuclear translocation as well as Gadd45b expression and MAPKs activation by repressing Sb2O3-induced ROS production. More importantly, we found Gadd45b was transcriptionally enhanced by Nrf2 through binding to three canonical antioxidant response elements (AREs) within its promoter region. Either Sb2O3 or TBHQ (a selective Nrf2 activator) treatment, Gadd45b expression was significantly increased by luciferase assay. Nrf2 inhibition greatly diminished Gadd45b expression due to reduced binding of Nrf2 in Gadd45b promoter under Sb2O3 treatment. To summarize, this study demonstrated the Nrf2-Gadd45b signaling axis exhibited a protective role in Sb-induced cell apoptosis.

  7. Effects of mutant TDP-43 on the Nrf2/ARE pathway and protein expression of MafK and JDP2 in NSC-34 cells.

    PubMed

    Tian, Y P; Che, F Y; Su, Q P; Lu, Y C; You, C P; Huang, L M; Wang, S G; Wang, L; Yu, J X

    2017-05-10

    Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that affects motor neurons and lacks an effective treatment. The disease pathogenesis has not been clarified at present. Pathological transactive response DNA-binding protein 43 (TDP-43) plays an important role in the pathogenesis of ALS. Nuclear translocation of nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2) is found in a mutant TDP-43 transgenic cell model, but its downstream antioxidant enzyme expression is decreased. To elucidate the specific mechanism of Nrf2/ARE (antioxidant responsive element) signaling dysfunction, we constructed an ALS cell model with human mutant TDP-43 using the NSC-34 cell line to evaluate the impact of the TDP-43 mutation on the Nrf2/ARE pathway. We found the nuclear translocation of Nrf2, but the expression of total Nrf2, cytoplasmic Nrf2, and downstream phase II detoxifying enzyme (NQO1) was decreased in NSC-34 cells transfected with the TDP-43-M337V plasmid. Besides, TDP-43-M337V plasmid-transfected NSC-34 cells were rounded with reduced neurites, shortened axons, increased levels of intracellular lipid peroxidation products, and decreased viability, which suggests that the TDP-43-M337V plasmid weakened the antioxidant capacity of NSC-34 cells and increased their susceptibility to oxidative damage. We further showed that expression of the MafK protein and the Jun dimerization protein 2 (JDP2) was reduced in TDP-43-M337V plasmid-transfected NSC-34 cells, which might cause accumulation of Nrf2 in nuclei but a decrease in NQO1 expression. Taken together, our results confirmed that TDP-43-M337V impaired the Nrf2/ARE pathway by reducing the expression of MafK and JDP2 proteins, and provided information for further research on the molecular mechanisms of TDP-43-M337V in ALS.

  8. tBHQ-induced HO-1 expression is mediated by calcium through regulation of Nrf2 binding to enhancer and polymerase II to promoter region of HO-1.

    PubMed

    Cheung, Ka Lung; Yu, Siwang; Pan, Zui; Ma, Jianjie; Wu, Tien Yuan; Kong, Ah-Ng Tony

    2011-05-16

    Induction of Nrf2-mediated detoxifying/antioxidant enzymes is an effective strategy for cancer chemoprevention. The goal of this study was to examine the role of calcium [Ca(2+)] in regulating a well-known phenolic chemopreventive compound tertiary-butylhydroquinone (tBHQ) activation of Nrf2 and induction of Nrf2 downstream target gene heme-oxygenase (HO-1). tBHQ alone caused Nrf2 nuclear localization and induced HO-1 mRNA and protein expression in a dose-dependent manner. Using RT-PCR and Western blotting, we showed that tBHQ-induced transcription of HO-1 is Ca(2+)-dependent. Chelation of [Ca(2+)](ext) or [Ca(2+)](intra) by EGTA or BAPTA attenuated tBHQ-induced HO-1. Cotreatment of tBHQ with inhibitors of [Ca(2+)]-sensitive protein kinase C and camodulin kinase did not attenuate HO-1 induction. Nuclear translocation of Nrf2 induced by tBHQ was also not affected by treatment of EGTA or BAPTA. Additionally, EGTA and BAPTA treatments decreased basal nuclear phosphorylation of CREB and decreased tBHQ-induced Nrf2-CBP binding and Nrf2 binding to enhancer as well as polymerase II binding to the promoter of HO-1 gene. Furthermore, tBHQ in combination with higher [Ca(2+)](ext) augmented HO-1 induction both in vitro and in vivo, indicating that the modulation of [Ca(2+)](int) could be used as an adjuvant to increase the efficacy of chemopreventive agents. Taken together, our results indicated that in addition to tBHQ-induced oxidative stress-mediated Nrf2 translocation, HO-1 induction by tBHQ also appears to be dependent on a series of Ca(2+)-regulated mechanisms.

  9. Ginger Compound [6]-Shogaol and Its Cysteine-Conjugated Metabolite (M2) Activate Nrf2 in Colon Epithelial Cells in Vitro and in Vivo

    PubMed Central

    2015-01-01

    In this study, we identified Nrf2 as a molecular target of [6]-shogaol (6S), a bioactive compound isolated from ginger, in colon epithelial cells in vitro and in vivo. Following 6S treatment of HCT-116 cells, the intracellular GSH/GSSG ratio was initially diminished but was then elevated above the basal level. Intracellular reactive oxygen species (ROS) correlated inversely with the GSH/GSSG ratio. Further analysis using gene microarray showed that 6S upregulated the expression of Nrf2 target genes (AKR1B10, FTL, GGTLA4, and HMOX1) in HCT-116 cells. Western blotting confirmed upregulation, phosphorylation, and nuclear translocation of Nrf2 protein followed by Keap1 decrease and upregulation of Nrf2 target genes (AKR1B10, FTL, GGTLA4, HMOX1, and MT1) and glutathione synthesis genes (GCLC and GCLM). Pretreatment of cells with a specific inhibitor of p38 (SB202190), PI3K (LY294002), or MEK1 (PD098059) attenuated these effects of 6S. Using ultra-high-performance liquid chromatography–tandem mass spectrometry, we found that 6S modified multiple cysteine residues of Keap1 protein. In vivo 6S treatment induced Nrf2 nuclear translocation and significantly upregulated the expression of MT1, HMOX1, and GCLC in the colon of wild-type mice but not Nrf2–/– mice. Similar to 6S, a cysteine-conjugated metabolite of 6S (M2), which was previously found to be a carrier of 6S in vitro and in vivo, also activated Nrf2. Our data demonstrated that 6S and its cysteine-conjugated metabolite M2 activate Nrf2 in colon epithelial cells in vitro and in vivo through Keap1-dependent and -independent mechanisms. PMID:25148906

  10. Aspirin induces Nrf2-mediated transcriptional activation of haem oxygenase-1 in protection of human melanocytes from H2 O2 -induced oxidative stress.

    PubMed

    Jian, Zhe; Tang, Lingzhen; Yi, Xiuli; Liu, Bangmin; Zhang, Qian; Zhu, Guannan; Wang, Gang; Gao, Tianwen; Li, Chunying

    2016-07-01

    The removal of hydrogen peroxide (H2 O2 ) by antioxidants has been proven to be beneficial to patients with vitiligo. Aspirin (acetylsalicylic acid, ASA) has antioxidant activity and has great preventive and therapeutical effect in many oxidative stress-relevant diseases. Whether ASA can protect human melanocytes against oxidative stress needs to be further studied. Here, we investigated the potential protective effect and mechanisms of ASA against H2 O2 -induced oxidative injury in human melanocytes. Human melanocytes were pre-treated with different concentrations of ASA, followed by exposure to 1.0 mM H2 O2 . Cell apoptosis, intracellular reactive oxygen species (ROS) levels were evaluated by flow cytometry, and cell viability was determined by an Cell Counting Kit-8 assay. Total and phosphorylated NRF2 expression, NRF2 nuclear translocation and antioxidant response element (ARE) transcriptional activity were assayed with or without Nrf2-siRNA transfection to investigate the possible molecular mechanisms. Concomitant with an increase in viability, pre-treatment of 10-90 μmol/l ASA resulted in decreased rate of apoptotic cells, lactate dehydrogenase release and intracellular ROS levels in primary human melanocytes. Furthermore, we found ASA dramatically induced NRF2 nuclear translocation, enhanced ARE-luciferase activity, increased both p- NRF2 and total NRF2 levels, and induced the expression of haem oxygenase-1 (HO-1) in human melanocytes. In addition, knockdown of Nrf2 expression or pharmacological inhibition of HO-1 abrogated the protective action of ASA on melanocytes against H2 O2 -induced cytotoxicity and apoptosis. These results suggest that ASA protects human melanocytes against H2 O2 -induced oxidative stress via Nrf2-driven transcriptional activation of HO-1. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  11. Mechanisms underlying the perifocal neuroprotective effect of the Nrf2–ARE signaling pathway after intracranial hemorrhage

    PubMed Central

    Yin, Xiao-ping; Chen, Zhi-ying; Zhou, Jun; Wu, Dan; Bao, Bing

    2015-01-01

    Background It has been found that nuclear factor erythroid 2-related factor 2/antioxidant response element (Nrf2–ARE) signaling pathway plays a role in antioxidative response, anti-inflammatory response, and neuron-protection in intracerebral hemorrhage (ICH). The aim of this study is to explore mechanisms underlying the perifocal neuroprotective effect of the Nrf2–ARE signaling pathway after ICH. Methods There were a total of 90 rats with basal ganglia hemorrhage, which were randomly divided into the following four groups: ICH (Sprague–Dawley rats with autologous femoral arterial blood injection into the basal ganglia), sulforaphane (SFN) (SFN was intraperitoneally administered into rats), retinoic acid (RA) (RA was intraperitoneally administered into rats), and dimethyl sulfoxide (the rats were treated with dimethyl sulfoxide). We observed the neurological score of the rats in the different groups, and collected brain tissues for immunofluorescence, Western blot, and reverse transcription polymerase chain reaction to detect expression of Nrf2, heme oxygenase (HO-1), nuclear factor-κB (NF-κB), and tumor necrosis factor-α (TNF-α). Results The results indicated that neurological dysfunction of rats was significantly improved in the SFN group, and the expressions of Nrf2 and HO-1 in tissues surrounding the hemorrhage were increased. Also, the level of NF-κB and TNF-α were reduced compared to the ICH group. The RA group exhibited more severe neurological dysfunction and lower levels of Nrf2 and HO-1 than the SFN and ICH groups. Compared to the ICH group, the NF-κB and TNF-α expression in the RA groups was increased. In conclusion, RA inhibits Nrf2 dissociation and translocation into nucleus, thereby suppressing the anti-inflammatory effect of Nrf2–ARE signaling pathway. The activation of Nrf2–ARE signaling pathway by SFN can elevate expression of antioxidant enzyme HO-1, reduce perifocal inflammatory response after ICH, and thus may play a

  12. Antioxidant axis Nrf2-keap1-ARE in inhibition of alcoholic liver fibrosis by IL-22

    PubMed Central

    Ni, Ya-Hui; Huo, Li-Juan; Li, Ting-Ting

    2017-01-01

    level was attenuated but the GSH level was further increased. These changes were dose-dependent. CONCLUSION IL-22 inhibits acetaldehyde-induced HSC activation and proliferation, which may be related to nuclear translocation of Nrf2 and increased activity of the antioxidant axis Nrf2-keap1-ARE. PMID:28373766

  13. Tim-3 inhibits macrophage control of Listeria monocytogenes by inhibiting Nrf2

    PubMed Central

    Wang, Zhiding; Sun, Dejun; Chen, Guojiang; Li, Ge; Dou, Shuaijie; Wang, Renxi; Xiao, He; Hou, Chunmei; Li, Yan; Feng, Jiannan; Shen, Beifen; Han, Gencheng

    2017-01-01

    T cell immunoglobulin mucin-3 (Tim-3) is an immune checkpoint inhibitor and its dysregulation has been related to T cell tolerance and many immune disorders, such as tumors and infection tolerance. However, the physiopathology roles of Tim-3 in innate immunity remain elusive. Here, we demonstrate that Tim-3 inhibits macrophage phagocytosis of L. monocytogenes by inhibiting the nuclear erythroid 2-related factor 2 (Nrf2) signaling pathway and increases bacterial burden. Tim-3 signaling promotes Nrf2 degradation by increasing its ubiquitination and, as a result, decreasing its nuclear translocation. CD36 and heme oxygenase-1 (HO-1), two downstream molecules in the Tim-3-Nrf2 signaling axis, are involved in the Tim-3- mediated immune evasion of L. monocytogenes both in vitro and in vivo. We here identified new mechanisms by which Tim-3 induces infection tolerance. By modulating the Tim-3 pathway, we demonstrate the feasibility of manipulating macrophage function as a potent tool for treating infectious diseases, such as Listeria infection. PMID:28205579

  14. Tim-3 inhibits macrophage control of Listeria monocytogenes by inhibiting Nrf2.

    PubMed

    Wang, Zhiding; Sun, Dejun; Chen, Guojiang; Li, Ge; Dou, Shuaijie; Wang, Renxi; Xiao, He; Hou, Chunmei; Li, Yan; Feng, Jiannan; Shen, Beifen; Han, Gencheng

    2017-02-16

    T cell immunoglobulin mucin-3 (Tim-3) is an immune checkpoint inhibitor and its dysregulation has been related to T cell tolerance and many immune disorders, such as tumors and infection tolerance. However, the physiopathology roles of Tim-3 in innate immunity remain elusive. Here, we demonstrate that Tim-3 inhibits macrophage phagocytosis of L. monocytogenes by inhibiting the nuclear erythroid 2-related factor 2 (Nrf2) signaling pathway and increases bacterial burden. Tim-3 signaling promotes Nrf2 degradation by increasing its ubiquitination and, as a result, decreasing its nuclear translocation. CD36 and heme oxygenase-1 (HO-1), two downstream molecules in the Tim-3-Nrf2 signaling axis, are involved in the Tim-3- mediated immune evasion of L. monocytogenes both in vitro and in vivo. We here identified new mechanisms by which Tim-3 induces infection tolerance. By modulating the Tim-3 pathway, we demonstrate the feasibility of manipulating macrophage function as a potent tool for treating infectious diseases, such as Listeria infection.

  15. The PTEN/NRF2 axis promotes human carcinogenesis.

    PubMed

    Rojo, Ana I; Rada, Patricia; Mendiola, Marta; Ortega-Molina, Ana; Wojdyla, Katarzyna; Rogowska-Wrzesinska, Adelina; Hardisson, David; Serrano, Manuel; Cuadrado, Antonio

    2014-12-20

    A recent study conducted in mice reported that liver-specific knockout of tumor suppressor Pten augments nuclear factor (erythroid-derived 2)-like 2 (NRF2) transcriptional activity. Here, we further investigated how phosphatase and tensin homolog deleted on chromosome 10 (PTEN) controls NRF2 and the relevance of this pathway in human carcin ogenesis. Drug and genetic targeting to PTEN and phosphoproteomics approaches indicated that PTEN leads to glycogen synthase kinase-3 (GSK-3)-mediated phosphorylation of NRF2 at residues Ser(335) and Ser(338) and subsequent beta-transducin repeat containing protein (β-TrCP)-dependent but Kelch-like ECH-associated protein 1 (KEAP1)-independent degradation. Rescue experiments in PTEN-deficient cells and xerographs in athymic mice indicated that loss of PTEN leads to increased NRF2 signature which provides a proliferating and tumorigenic advantage. Tissue microarrays from endometrioid carcinomas showed that 80% of PTEN-negative tumors expressed high levels of NRF2 or its target heme oxygenase-1 (HO-1). These results uncover a new mechanism of oncogenic activation of NRF2 by loss of its negative regulation by PTEN/GSK-3/β-TrCP that may be relevant to a large number of tumors, including endometrioid carcinomas. Increased activity of NRF2 due to loss of PTEN is instrumental in human carcinogenesis and represents a novel therapeutic target.

  16. Rising levels of atmospheric oxygen and evolution of Nrf2.

    PubMed

    Gacesa, Ranko; Dunlap, Walter C; Barlow, David J; Laskowski, Roman A; Long, Paul F

    2016-06-14

    In mammals, the master transcription regulator of antioxidant defences is provided by the Nrf2 protein. Phylogenetic analyses of Nrf2 sequences are used here to derive a molecular clock that manifests persuasive evidence that Nrf2 orthologues emerged, and then diverged, at two time points that correlate with well-established geochemical and palaeobiological chronologies during progression of the 'Great Oxygenation Event'. We demonstrate that orthologues of Nrf2 first appeared in fungi around 1.5 Ga during the Paleoproterozoic when photosynthetic oxygen was being absorbed into the oceans. A subsequent significant divergence in Nrf2 is seen during the split between fungi and the Metazoa approximately 1.0-1.2 Ga, at a time when oceanic ventilation released free oxygen to the atmosphere, but with most being absorbed by methane oxidation and oxidative weathering of land surfaces until approximately 800 Ma. Atmospheric oxygen levels thereafter accumulated giving rise to metazoan success known as the Cambrian explosion commencing at ~541 Ma. Atmospheric O2 levels then rose in the mid Paleozoic (359-252 Ma), and Nrf2 diverged once again at the division between mammals and non-mammalian vertebrates during the Permian-Triassic boundary (~252 Ma). Understanding Nrf2 evolution as an effective antioxidant response may have repercussions for improved human health.

  17. Mechanisms and functions of Nrf2 signaling in Drosophila.

    PubMed

    Pitoniak, Andrew; Bohmann, Dirk

    2015-11-01

    The Nrf2 transcription factor belongs to the Cap'n'collar family, named after the founding member of this group, the product of the Drosophila Cap'n'collar gene. The encoded protein, Cap'n'collar, abbreviated Cnc, offers a convenient and accessible model to study the structure, function, and biology of Nrf2 transcription factors at the organismic, tissular, cellular, and molecular levels, using the powerful genetic, genomic, and biochemical tools available in Drosophila. In this review we provide an account of the original identification of Cnc as a regulator of embryonic development. We then describe the discovery of Nrf2-like functions of Cnc and its role in acute stress signaling and aging. The establishment of Drosophila as a model organism in which the mechanisms and functions of Nrf2 signaling can be studied has led to several discoveries: the regulation of stem cell activity by an Nrf2-mediated redox mechanism, the interaction of Nrf2 with p62 and Myc in the control of tissue growth and the unfolded protein response, and more. Several of these more recent lines of investigation are highlighted. Model organisms such as the fly and the worm remain powerful experimental platforms that can help to unravel the many remaining puzzles regarding the role of Nrf2 and its relatives in controlling the physiology and maintaining the health of multicellular organisms. Copyright © 2015. Published by Elsevier Inc.

  18. WDR23 regulates NRF2 independently of KEAP1

    PubMed Central

    Lo, Jacqueline Y.; Spatola, Brett N.

    2017-01-01

    Cellular adaptation to stress is essential to ensure organismal survival. NRF2/NFE2L2 is a key determinant of xenobiotic stress responses, and loss of negative regulation by the KEAP1-CUL3 proteasome system is implicated in several chemo- and radiation-resistant cancers. Advantageously using C. elegans alongside human cell culture models, we establish a new WDR23-DDB1-CUL4 regulatory axis for NRF2 activity that operates independently of the canonical KEAP1-CUL3 system. WDR23 binds the DIDLID sequence within the Neh2 domain of NRF2 to regulate its stability; this regulation is not dependent on the KEAP1-binding DLG or ETGE motifs. The C-terminal domain of WDR23 is highly conserved and involved in regulation of NRF2 by the DDB1-CUL4 complex. The addition of WDR23 increases cellular sensitivity to cytotoxic chemotherapeutic drugs and suppresses NRF2 in KEAP1-negative cancer cell lines. Together, our results identify WDR23 as an alternative regulator of NRF2 proteostasis and uncover a cellular pathway that regulates NRF2 activity and capacity for cytoprotection independently of KEAP1. PMID:28453520

  19. Rising levels of atmospheric oxygen and evolution of Nrf2

    PubMed Central

    Gacesa, Ranko; Dunlap, Walter C.; Barlow, David J.; Laskowski, Roman A.; Long, Paul F.

    2016-01-01

    In mammals, the master transcription regulator of antioxidant defences is provided by the Nrf2 protein. Phylogenetic analyses of Nrf2 sequences are used here to derive a molecular clock that manifests persuasive evidence that Nrf2 orthologues emerged, and then diverged, at two time points that correlate with well-established geochemical and palaeobiological chronologies during progression of the ‘Great Oxygenation Event’. We demonstrate that orthologues of Nrf2 first appeared in fungi around 1.5 Ga during the Paleoproterozoic when photosynthetic oxygen was being absorbed into the oceans. A subsequent significant divergence in Nrf2 is seen during the split between fungi and the Metazoa approximately 1.0–1.2 Ga, at a time when oceanic ventilation released free oxygen to the atmosphere, but with most being absorbed by methane oxidation and oxidative weathering of land surfaces until approximately 800 Ma. Atmospheric oxygen levels thereafter accumulated giving rise to metazoan success known as the Cambrian explosion commencing at ~541 Ma. Atmospheric O2 levels then rose in the mid Paleozoic (359–252 Ma), and Nrf2 diverged once again at the division between mammals and non-mammalian vertebrates during the Permian-Triassic boundary (~252 Ma). Understanding Nrf2 evolution as an effective antioxidant response may have repercussions for improved human health. PMID:27297177

  20. NRF2 activation by antioxidant antidiabetic agents accelerates tumor metastasis.

    PubMed

    Wang, Hui; Liu, Xiufei; Long, Min; Huang, Yi; Zhang, Linlin; Zhang, Rui; Zheng, Yi; Liao, Xiaoyu; Wang, Yuren; Liao, Qian; Li, Wenjie; Tang, Zili; Tong, Qiang; Wang, Xiaocui; Fang, Fang; Rojo de la Vega, Montserrat; Ouyang, Qin; Zhang, Donna D; Yu, Shicang; Zheng, Hongting

    2016-04-13

    Cancer is a common comorbidity of diabetic patients; however, little is known about the effects that antidiabetic drugs have on tumors. We discovered that common classes of drugs used in type 2 diabetes mellitus, the hypoglycemic dipeptidyl peptidase-4 inhibitors (DPP-4i) saxagliptin and sitagliptin, as well as the antineuropathic α-lipoic acid (ALA), do not increase tumor incidence but increase the risk of metastasis of existing tumors. Specifically, these drugs induce prolonged activation of the nuclear factor E2-related factor 2 (NRF2)-mediated antioxidant response through inhibition of KEAP1-C151-dependent ubiquitination and subsequent degradation of NRF2, resulting in up-regulated expression of metastasis-associated proteins, increased cancer cell migration, and promotion of metastasis in xenograft mouse models. Accordingly, knockdown of NRF2 attenuated naturally occurring and DPP-4i-induced tumor metastasis, whereas NRF2 activation accelerated metastasis. Furthermore, in human liver cancer tissue samples, increased NRF2 expression correlated with metastasis. Our findings suggest that antioxidants that activate NRF2 signaling may need to be administered with caution in cancer patients, such as diabetic patients with cancer. Moreover, NRF2 may be a potential biomarker and therapeutic target for tumor metastasis.

  1. The PTEN/NRF2 Axis Promotes Human Carcinogenesis

    PubMed Central

    Rada, Patricia; Mendiola, Marta; Ortega-Molina, Ana; Wojdyla, Katarzyna; Rogowska-Wrzesinska, Adelina; Hardisson, David; Serrano, Manuel

    2014-01-01

    Abstract Aims: A recent study conducted in mice reported that liver-specific knockout of tumor suppressor Pten augments nuclear factor (erythroid-derived 2)-like 2 (NRF2) transcriptional activity. Here, we further investigated how phosphatase and tensin homolog deleted on chromosome 10 (PTEN) controls NRF2 and the relevance of this pathway in human carcin ogenesis. Results: Drug and genetic targeting to PTEN and phosphoproteomics approaches indicated that PTEN leads to glycogen synthase kinase-3 (GSK-3)-mediated phosphorylation of NRF2 at residues Ser335 and Ser338 and subsequent beta-transducin repeat containing protein (β-TrCP)-dependent but Kelch-like ECH-associated protein 1 (KEAP1)-independent degradation. Rescue experiments in PTEN-deficient cells and xerographs in athymic mice indicated that loss of PTEN leads to increased NRF2 signature which provides a proliferating and tumorigenic advantage. Tissue microarrays from endometrioid carcinomas showed that 80% of PTEN-negative tumors expressed high levels of NRF2 or its target heme oxygenase-1 (HO-1). Innovation: These results uncover a new mechanism of oncogenic activation of NRF2 by loss of its negative regulation by PTEN/GSK-3/β-TrCP that may be relevant to a large number of tumors, including endometrioid carcinomas. Conclusion: Increased activity of NRF2 due to loss of PTEN is instrumental in human carcinogenesis and represents a novel therapeutic target. Antioxid. Redox Signal. 21, 2498–2514. PMID:24892215

  2. Beyond antioxidant genes in the ancient NRF2 regulatory network

    PubMed Central

    Lacher, Sarah E.; Lee, Joslynn S.; Wang, Xuting; Campbell, Michelle R.; Bell, Douglas A.; Slattery, Matthew

    2016-01-01

    NRF2, a basic leucine zipper transcription factor encoded by the gene NFE2L2, is a master regulator of the transcriptional response to oxidative stress. NRF2 is structurally and functionally conserved from insects to humans, and it heterodimerizes with the small MAF transcription factors to bind a consensus DNA sequence (the antioxidant response element, or ARE) and regulate gene expression. We have used genome-wide chromatin immunoprecipitation (ChIP-seq) and gene expression data to identify direct NRF2 target genes in Drosophila and humans. These data have allowed us to construct the deeply conserved ancient NRF2 regulatory network – target genes that are conserved from Drosophila to human. The ancient network consists of canonical antioxidant genes, as well as genes related to proteasomal pathways, metabolism, and a number of less expected genes. We have also used enhancer reporter assays and electrophoretic mobility shift assays to confirm NRF2-mediated regulation of ARE (antioxidant response element) activity at a number of these novel target genes. Interestingly, the ancient network also highlights a prominent negative feedback loop; this, combined with the finding that and NRF2-mediated regulatory output is tightly linked to the quality of the ARE it is targeting, suggests that precise regulation of nuclear NRF2 concentration is necessary to achieve proper quantitative regulation of distinct gene sets. Together, these findings highlight the importance of balance in the NRF2-ARE pathway, and indicate that NRF2-mediated regulation of xenobiotic metabolism, glucose metabolism, and proteostasis have been central to this pathway since its inception. PMID:26163000

  3. Nrf2-Mediated Regulation of Skeletal Muscle Glycogen Metabolism

    PubMed Central

    Yagishita, Yoko; Katsuoka, Fumiki; Kitajima, Yasuo; Nunomiya, Aki; Nagatomi, Ryoichi; Pi, Jingbo; Biswal, Shyam S.

    2016-01-01

    Nrf2 (NF-E2-related factor 2) contributes to the maintenance of glucose homeostasis in vivo. Nrf2 suppresses blood glucose levels by protecting pancreatic β cells from oxidative stress and improving peripheral tissue glucose utilization. To elucidate the molecular mechanisms by which Nrf2 contributes to the maintenance of glucose homeostasis, we generated skeletal muscle (SkM)-specific Keap1 knockout (Keap1MuKO) mice that express abundant Nrf2 in their SkM and then examined Nrf2 target gene expression in that tissue. In Keap1MuKO mice, blood glucose levels were significantly downregulated and the levels of the glycogen branching enzyme (Gbe1) and muscle-type PhKα subunit (Phka1) mRNAs, along with those of the glycogen branching enzyme (GBE) and the phosphorylase b kinase α subunit (PhKα) protein, were significantly upregulated in mouse SkM. Consistent with this result, chemical Nrf2 inducers promoted Gbe1 and Phka1 mRNA expression in both mouse SkM and C2C12 myotubes. Chromatin immunoprecipitation analysis demonstrated that Nrf2 binds the Gbe1 and Phka1 upstream promoter regions. In Keap1MuKO mice, muscle glycogen content was strongly reduced and forced GBE expression in C2C12 myotubes promoted glucose uptake. Therefore, our results demonstrate that Nrf2 induction in SkM increases GBE and PhKα expression and reduces muscle glycogen content, resulting in improved glucose tolerance. Our results also indicate that Nrf2 differentially regulates glycogen metabolism in SkM and the liver. PMID:27044864

  4. Nrf2 expression in endometrial serous carcinomas and its precancers.

    PubMed

    Chen, Ning; Yi, Xiaofang; Abushahin, Nisreen; Pang, Shujie; Zhang, Donna; Kong, Beihua; Zheng, Wenxin

    2010-12-24

    Endometrial serous carcinoma (ESC) is the most aggressive subtype of endometrial cancer. Its aggressive behavior and poor clinical outcome may be partially attributed to lack of early diagnostic markers and unclear patho-genesis. The transcription factor Erythroid-E2-related factor 2 (Nrf2) is a recently identified protein marker, which plays a role in carcinogenesis as well as responsible for poor prognosis of many human cancers. The aim of this study is to determine the Nrf2 expression in benign endometrium (n=28), endometrial cancers (n=122) as well as their precursor lesions (n=81) trying to see whether Nrf2 has any diagnostic usage and is potentially involved in endometrial carcinogenesis. The level of Nrf2 was evaluated by immunohistochemical (IHC) and verified by using Western blots. Among the malignant cases, Nrf2 was positive in 28 (68%) of 50 ESCs, which was significantly more than in 3 (6%) of 50 endometrioid carcinomas (p < 0.001) and 2 (13%) of 15 clear cell carcinomas (p = 0.001) and other histologic types of endometrial cancers. Among endometrial precursor lesions, both serous endometrial glandular dysplasia (EmGD, 40%) and serous endometrial intraepithelial carcinoma (EIC, 44%) showed a significantly higher Nrf2 expression than that in atypical endometrial hyperplasia or endometrial intraepithelial neoplasia (0%), clear cell EmGD (10%), and clear cell EIC (25%), respectively. We conclude that Nrf2 overexpression is closely associated with endometrial neoplasms with serous differentiation. Alteration of Nrf2 expression may represent one of the early molecular events in ESC carcinogenesis and overexpression of Nrf2 may used as a diagnostic marker in surgical pathology.

  5. Zeaxanthin induces Nrf2-mediated phase II enzymes in protection of cell death

    PubMed Central

    Zou, X; Gao, J; Zheng, Y; Wang, X; Chen, C; Cao, K; Xu, J; Li, Y; Lu, W; Liu, J; Feng, Z

    2014-01-01

    Zeaxanthin (Zea) is a major carotenoid pigment contained in human retina, and its daily supplementation associated with lower risk of age-related macular degeneration. Despite known property of Zea as an antioxidant, its underlying molecular mechanisms of action remain poorly understood. In this study, we aim to study the regulation mechanism of Zea on phase II detoxification enzymes. In normal human retinal pigment epithelium cells, Zea promoted the nuclear translocation of NF-E2-related factor 2 (Nrf2) and induced mRNA and protein expression of phase II enzymes, the induction was suppressed by specific knockdown of Nrf2. Zea also effectively protected against tert-butyl hydroperoxide-induced mitochondrial dysfunction and apoptosis. Glutathione (GSH) as the most important antioxidant was also induced by Zea through Nrf2 activation in a time- and dose-dependent manner, whereas the protective effects of Zea were decimated by inhibition of GSH synthesis. Finally, Zea activated the PI3K/Akt and MAPK/ERK pathway, whereas only PI3K/Akt activation correlated with phase II enzymes induction and Zea protection. In further in vivo analyses, Zea showed effects of inducing phase II enzymes and increased GSH content, which contributed to the reduced lipid and protein peroxidation in the retina as well as the liver, heart, and serum of the Sprague–Dawley rats. For the first time, Zea is presented as a phase II enzymes inducer instead of being an antioxidant. By activating Nrf2-mediated phase II enzymes, Zea could enhance anti-oxidative capacity and prevent cell death both in vivo and in vitro. PMID:24810054

  6. Mangiferin Upregulates Glyoxalase 1 Through Activation of Nrf2/ARE Signaling in Central Neurons Cultured with High Glucose.

    PubMed

    Liu, Yao-Wu; Cheng, Ya-Qin; Liu, Xiao-Li; Hao, Yun-Chao; Li, Yu; Zhu, Xia; Zhang, Fan; Yin, Xiao-Xing

    2017-08-01

    Mangiferin, a natural C-glucoside xanthone, has anti-inflammatory, anti-oxidative, neuroprotective actions. Our previous study showed that mangiferin could attenuate diabetes-associated cognitive impairment of rats by enhancing the function of glyoxalase 1 (Glo-1) in brain. The aim of this study was to investigate whether Glo-1 upregulation by mangiferin in central neurons exposed to chronic high glucose may be related to activation of Nrf2/ARE pathway. Compared with normal glucose (25 mmol/L) culture, Glo-1 protein, mRNA, and activity levels were markedly decreased in primary hippocampal and cerebral cortical neurons cultured with high glucose (50 mmol/L) for 72 h, accompanied by the declined Nrf2 nuclear translocation and protein expression of Nrf2 in cell nucleus, as well as protein expression and mRNA level of γ-glutamylcysteine synthetase (γ-GCS) and superoxide dismutase activity, target genes of Nrf2/ARE signaling. Nonetheless, high glucose cotreating with mangiferin or sulforaphane, a typical inducer of Nrf2 activation, attenuated the above changes in both central neurons. In addition, mangiferin and sulforaphane significantly prevented the formation of advanced glycation end-products (AGEs) reflecting Glo-1 activity, while elevated the level of glutathione, a cofactor of Glo-1 activity and production of γ-GCS, in high glucose cultured central neurons. These findings demonstrated that Glo-1 was greatly downregulated in central neurons exposed to chronic high glucose, which is expected to lead the formation of AGEs and oxidative stress damages. We also proved that mangiferin enhanced the function of Glo-1 under high glucose condition by inducing activation of Nrf2/ARE signaling pathway.

  7. Nrf2 and Redox Status in Prediabetic and Diabetic Patients

    PubMed Central

    Jiménez-Osorio, Angélica S.; Picazo, Alejandra; González-Reyes, Susana; Barrera-Oviedo, Diana; Rodríguez-Arellano, Martha E.; Pedraza-Chaverri, José

    2014-01-01

    The redox status associated with nuclear factor erythroid 2-related factor-2 (Nrf2) was evaluated in prediabetic and diabetic subjects. Total antioxidant status (TAS) in plasma and erythrocytes, glutathione (GSH) and malondialdehyde (MDA) content and activity of antioxidant enzymes were measured as redox status markers in 259 controls, 111 prediabetics and 186 diabetic type 2 subjects. Nrf2 was measured in nuclear extract fractions from peripheral blood mononuclear cells (PBMC). Nrf2 levels were lower in prediabetic and diabetic patients. TAS, GSH and activity of glutamate cysteine ligase were lower in diabetic subjects. An increase of MDA and superoxide dismutase activity was found in diabetic subjects. These results suggest that low levels of Nrf2 are involved in the development of oxidative stress and redox status disbalance in diabetic patients. PMID:25383674

  8. Oxidative stress response and Nrf2 signaling in aging

    PubMed Central

    Zhang, Hongqiao; Davies, Kelvin J. A.; Forman, Henry Jay

    2015-01-01

    Increasing oxidative stress, a major characteristic of aging, has been implicated in variety of age-related pathologies. In aging, oxidant production from several sources is increased while antioxidant enzymes, the primary lines of defense, are decreased. Repair systems, including the proteasomal degradation of damaged proteins also declines. Importantly, the adaptive response to oxidative stress declines with aging. Nrf2/EpRE signaling regulates the basal and inducible expression of many antioxidant enzymes and the proteasome. Nrf2/EpRE activity is regulated at several levels including transcription, post-translation, and interaction with other proteins. This review summarizes current studies on age-related impairment of Nrf2/EpRE function and discusses the change of Nrf2 regulatory mechanisms with aging. PMID:26066302

  9. Effect of coffee combining green coffee bean constituents with typical roasting products on the Nrf2/ARE pathway in vitro and in vivo.

    PubMed

    Volz, Nadine; Boettler, Ute; Winkler, Swantje; Teller, Nicole; Schwarz, Christoph; Bakuradze, Tamara; Eisenbrand, Gerhard; Haupt, Larissa; Griffiths, Lyn R; Stiebitz, Herbert; Bytof, Gerhard; Lantz, Ingo; Lang, Roman; Hofmann, Thomas; Somoza, Veronika; Marko, Doris

    2012-09-26

    This study investigated Nrf2-activating properties of a coffee blend combining raw coffee bean constituents with 5-O-caffeoylquinic acid (CGA) as a lead component with typical roasting products such as N-methylpyridinium (NMP). In cell culture (HT29) the respective coffee extract (CN-CE) increased nuclear Nrf2 translocation and enhanced the transcription of ARE-dependent genes as exemplified for NAD(P)H:quinone oxidoreductase and glutathione-S-transferase (GST)A1, reflected in the protein level by an increase in GST enzyme activity. In a pilot human intervention study (29 healthy volunteers), daily consumption of 750 mL of CN-coffee for 4 weeks increased Nrf2 transcription in peripheral blood lymphocytes on average. However, the transcriptional response pattern of Nrf2/ARE-dependent genes showed substantial interindividual variations. The presence of SNPs in the Nrf2-promoter, reported recently, as well as the detection of GSTT1*0 (null) genotypes in the study collective strengthens the hypothesis that coffee acts as a modulator of Nrf2-dependent gene response in humans, but genetic polymorphisms play an important role in the individual response pattern.

  10. miRNA-141 attenuates UV-induced oxidative stress via activating Keap1-Nrf2 signaling in human retinal pigment epithelium cells and retinal ganglion cells.

    PubMed

    Cheng, Li-Bo; Li, Ke-Ran; Yi, Nan; Li, Xiu-Miao; Wang, Feng; Xue, Bo; Pan, Ying-Shun; Yao, Jin; Jiang, Qin; Wu, Zhi-Feng

    2017-01-04

    Activation of NF-E2-related factor 2 (Nrf2) signaling could protect cells from ultra violet (UV) radiation. We aim to provoke Nrf2 activation via downregulating its inhibitor Keap1 by microRNA-141 ("miR-141"). In both human retinal pigment epithelium cells (RPEs) and retinal ganglion cells (RGCs), forced-expression of miR-141 downregulated Keap1, causing Nrf2 stabilization, accumulation and nuclear translocation, which led to transcription of multiple antioxidant-responsive element (ARE) genes (HO1, NOQ1 and GCLC). Further, UV-induced reactive oxygen species (ROS) production and cell death were significantly attenuated in miR-141-expressing RPEs and RGCs. On the other hand, depletion of miR-141 via expressing its inhibitor antagomiR-141 led to Keap1 upregulation and Nrf2 degradation, which aggravated UV-induced death of RPEs and RGCs. Significantly, Nrf2 shRNA knockdown almost abolished miR-141-mediated cytoprotection against UV in RPEs. These results demonstrate that miR-141 targets Keap1 to activate Nrf2 signaling, which protects RPEs and RGCs from UV radiation.

  11. Induction of the pi class of glutathione S-transferase by carnosic acid in rat Clone 9 cells via the p38/Nrf2 pathway.

    PubMed

    Lin, Chia-Yuan; Wu, Chi-Rei; Chang, Shu-Wei; Wang, Yu-Jung; Wu, Jia-Jiuan; Tsai, Chia-Wen

    2015-06-01

    Induction of phase II enzymes is important in cancer chemoprevention. We compared the effect of rosemary diterpenes on the expression of the pi class of glutathione S-transferase (GSTP) in rat liver Clone 9 cells and the signaling pathways involved. Culturing cells with 1, 5, 10, or 20 μM carnosic acid (CA) or carnosol (CS) for 24 h in a dose-dependent manner increased the GSTP expression. CA was more potent than CS. The RNA level and the enzyme activity of GSTP were also enhanced by CA treatment. Treatment with 10 μM CA highly induced the reporter activity of the enhancer element GPEI. Furthermore, CA markedly increased the translocation of nuclear factor erythroid-2 related factor 2 (Nrf2) from the cytosol to the nucleus after 30 to 60 min. CA the stimulated the protein induction of p38, nuclear Nrf2, and GSTP was diminished in the presence of SB203580 (a p38 inhibitor). In addition, SB203580 pretreatment or silencing of Nrf2 by siRNA suppressed the CA-induced GPEI-DNA binding activity and GSTP protein expression. Knockdown of p38 or Nrf2 by siRNA abolished the activation of p38 and Nrf2 as well as the protein induction and enzyme activity of GSTP by CA. These results suggest that CA up-regulates the expression and enzyme activity of GSTP via the p38/Nrf2/GPEI pathway.

  12. Protective effect of tert-butylhydroquinone on the quinolinic-acid-induced toxicity in rat striatal slices: role of the Nrf2-antioxidant response element pathway.

    PubMed

    Tasset, Inmaculada; Pérez-De La Cruz, Verónica; Elinos-Calderón, Diana; Carrillo-Mora, Paul; González-Herrera, Irma Gabriela; Luna-López, Armando; Konigsberg, Mina; Pedraza-Chaverrí, José; Maldonado, Perla D; Ali, Syed F; Túnez, Isaac; Santamaría, Abel

    2010-01-01

    Tert-butylhydroquinone (tBHQ) is a xenobiotic with reported antioxidant properties. tBHQ has been shown to induce nuclear translocation of the transcription factor NF-E2-related factor 2 (Nrf2) to further activate the antioxidant response element (ARE). In turn, the Nrf2/ARE pathway is responsible for the induction of phase 2 antioxidant enzymes that detoxify oxidant promoters from different toxic insults. In this work, the antioxidant and protective actions of tBHQ were explored for the first time on different biomarkers of the neurotoxic model produced by the excitotoxic and pro-oxidant molecule quinolinic acid (QUIN) in rat striatal slices. For comparison purposes, 3-nitropropionic acid was used as reference model. Our results show that tBHQ (25 μM) prevented the QUIN-induced lipid peroxidation and mitochondrial dysfunction. In addition, tBHQ enhanced glutathione-S-transferase activity, partially recovering its depletion induced by QUIN treatment. Our results also demonstrated that tBHQ was able to induce nuclear accumulation of Nrf2 and further antioxidant protection: while QUIN alone decreased the nuclear Nrf2, a treatment with tBHQ preserved the nuclear levels Nrf2 in the presence of QUIN. Therefore, the tBHQ-mediated Nrf2/ARE induction constitutes a signaling-mediated antioxidant strategy and therapeutic tool to be tested in different neurotoxic models. Copyright © 2009 S. Karger AG, Basel.

  13. An Essential Role of NRF2 in Diabetic Wound Healing.

    PubMed

    Long, Min; Rojo de la Vega, Montserrat; Wen, Qing; Bharara, Manish; Jiang, Tao; Zhang, Rui; Zhou, Shiwen; Wong, Pak K; Wondrak, Georg T; Zheng, Hongting; Zhang, Donna D

    2016-03-01

    The high mortality and disability of diabetic nonhealing skin ulcers create an urgent need for the development of more efficacious strategies targeting diabetic wound healing. In the current study, using human clinical specimens, we show that perilesional skin tissues from patients with diabetes are under more severe oxidative stress and display higher activation of the nuclear factor-E2-related factor 2 (NRF2)-mediated antioxidant response than perilesional skin tissues from normoglycemic patients. In a streptozotocin-induced diabetes mouse model, Nrf2(-/-) mice have delayed wound closure rates compared with Nrf2(+/+) mice, which is, at least partially, due to greater oxidative DNA damage, low transforming growth factor-β1 (TGF-β1) and high matrix metalloproteinase 9 (MMP9) expression, and increased apoptosis. More importantly, pharmacological activation of the NRF2 pathway significantly improves diabetic wound healing. In vitro experiments in human immortalized keratinocyte cells confirm that NRF2 contributes to wound healing by alleviating oxidative stress, increasing proliferation and migration, decreasing apoptosis, and increasing the expression of TGF-β1 and lowering MMP9 under high-glucose conditions. This study indicates an essential role for NRF2 in diabetic wound healing and the therapeutic benefits of activating NRF2 in this disease, laying the foundation for future clinical trials using NRF2 activators in treating diabetic skin ulcers. © 2016 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

  14. p62 links autophagy and Nrf2 signaling

    PubMed Central

    Jiang, Tao; Harder, Bryan; Rojo de la Vega, Montserrat; Wong, Pak K.; Chapman, Eli; Zhang, Donna D.

    2015-01-01

    The Nrf2-Keap1-ARE pathway is a redox and xenobiotic sensitive signaling axis that functions to protect cells against oxidative stress, environmental toxicants, and harmful chemicals through the induction of cytoprotective genes. To enforce strict regulation, cells invest a great deal of energy into the maintenance of the Nrf2 pathway to ensure rapid induction upon cellular insult and rapid return to basal levels once the insult is mitigated. Because of the protective role of Nrf2 transcriptional programs, controlled activation of the pathway has been recognized as a means for chemoprevention. On the other hand, constitutive activation of Nrf2, due to somatic mutations of genes that control Nrf2 degradation, promotes carcinogenesis and imparts chemoresistance to cancer cells. Autophagy, a bulk protein degradation process, is another tightly regulated complex cellular process that functions as a cellular quality control system to remove damaged proteins or organelles. Low cellular nutrient levels can also activate autophagy, which acts to restore metabolic homeostasis through the degradation of macromolecules to provide nutrients. Recently, these two cellular pathways were shown to intersect through the direct interaction between p62 (an autophagy adaptor protein) and Keap1 (the Nrf2 substrate adaptor for the Cul3 E3 ubiquitin ligase). Dysregulation of autophagy was shown to result in prolonged Nrf2 activation in a p62-dependent manner. In this review, we will discuss the progress that has been made in dissecting the intersection of these two pathways and the potential tumor-promoting role of prolonged Nrf2 activation. PMID:26117325

  15. NRF2 and p53: Januses in cancer?

    PubMed Central

    Rotblat, Barak; Melino, Gerry; Knight, Richard A.

    2012-01-01

    The transcription factor nuclear factor (erythroid-derived 2)-like 2, also known as NFE2L2 or NRF2, is a master regulator of the anti-oxidative stress response and positively controls the expression of a battery of anti-oxidative stress response proteins and enzymes implicated in detoxification and glutathione generation. Although its detoxifying activity is important in cancer prevention, it has recently been shown that cancer cells also exploit its protective functions to thrive and resist chemotherapy. NRF2 was also shown to the pentose phosphate pathway and glutaminolysis, which promotes purine synthesis for supporting rapid proliferation and glutathione for providing anti-oxidative stress protection. Evidence obtained from cancer patients and cell lines suggest that NRF2 is highly active in a variety of human cancers and is associated with aggressiveness. p53 is a tumor suppressor that also promotes an anti-oxidative stress metabolic program and glutaminolysis. Here we will discuss the similarities between NRF2 and p53 and review evidence that p53 might be exploited by cancer cells to gain protection against oxidative stress, as is the case for NRF2. We discuss findings of co-regulation between these transcription factors and propose possible therapeutic strategies that can be used for treatment of cancers that harbor WT p53 and express high levels of NRF2. PMID:23174755

  16. Oxidative stress responses and NRF2 in human leukaemia.

    PubMed

    Abdul-Aziz, Amina; MacEwan, David J; Bowles, Kristian M; Rushworth, Stuart A

    2015-01-01

    Oxidative stress as a result of elevated levels of reactive oxygen species (ROS) has been observed in almost all cancers, including leukaemia, where they contribute to disease development and progression. However, cancer cells also express increased levels of antioxidant proteins which detoxify ROS. This includes glutathione, the major antioxidant in human cells, which has recently been identified to have dysregulated metabolism in human leukaemia. This suggests that critical balance of intracellular ROS levels is required for cancer cell function, growth, and survival. Nuclear factor (erythroid-derived 2)-like 2 (NRF2) transcription factor plays a dual role in cancer. Primarily, NRF2 is a transcription factor functioning to protect nonmalignant cells from malignant transformation and oxidative stress through transcriptional activation of detoxifying and antioxidant enzymes. However, once malignant transformation has occurred within a cell, NRF2 functions to protect the tumour from oxidative stress and chemotherapy-induced cytotoxicity. Moreover, inhibition of the NRF2 oxidative stress pathway in leukaemia cells renders them more sensitive to cytotoxic chemotherapy. Our improved understanding of NRF2 biology in human leukaemia may permit mechanisms by which we could potentially improve future cancer therapies. This review highlights the mechanisms by which leukaemic cells exploit the NRF2/ROS response to promote their growth and survival.

  17. Repression of the Antioxidant NRF2 Pathway in Premature Aging.

    PubMed

    Kubben, Nard; Zhang, Weiqi; Wang, Lixia; Voss, Ty C; Yang, Jiping; Qu, Jing; Liu, Guang-Hui; Misteli, Tom

    2016-06-02

    Hutchinson-Gilford progeria syndrome (HGPS) is a rare, invariably fatal premature aging disorder. The disease is caused by constitutive production of progerin, a mutant form of the nuclear architectural protein lamin A, leading, through unknown mechanisms, to diverse morphological, epigenetic, and genomic damage and to mesenchymal stem cell (MSC) attrition in vivo. Using a high-throughput siRNA screen, we identify the NRF2 antioxidant pathway as a driver mechanism in HGPS. Progerin sequesters NRF2 and thereby causes its subnuclear mislocalization, resulting in impaired NRF2 transcriptional activity and consequently increased chronic oxidative stress. Suppressed NRF2 activity or increased oxidative stress is sufficient to recapitulate HGPS aging defects, whereas reactivation of NRF2 activity in HGPS patient cells reverses progerin-associated nuclear aging defects and restores in vivo viability of MSCs in an animal model. These findings identify repression of the NRF2-mediated antioxidative response as a key contributor to the premature aging phenotype.

  18. Sulforaphane Inhibits HIV Infection of Macrophages through Nrf2

    PubMed Central

    Furuya, Andrea Kinga Marias; Sharifi, Hamayun J.; Jellinger, Robert M.; Cristofano, Paul; Shi, Binshan; de Noronha, Carlos M. C.

    2016-01-01

    Marburg virus, the Kaposi's sarcoma-associated herpesvirus (KSHV) and Dengue virus all activate, and benefit from, expression of the transcription regulator nuclear erythroid 2-related factor 2 (Nrf2). The impact of Nrf2 activation on human immunodeficiency virus (HIV) infection has not been tested. Sulforaphane (SFN), produced in cruciferous vegetables after mechanical damage, mobilizes Nrf2 to potently reprogram cellular gene expression. Here we show for the first time that SFN blocks HIV infection in primary macrophages but not in primary T cells. Similarly SFN blocks infection in PMA-differentiated promonocytic cell lines, but not in other cell lines tested. siRNA-mediated depletion of Nrf2 boosted HIV infectivity in primary macrophages and reduced the anti-viral effects of SFN treatment. This supports a model in which anti-viral activity is mediated through Nrf2 after it is mobilized by SFN. We further found that, like the type I interferon-induced cellular anti-viral proteins SAMHD1 and MX2, SFN treatment blocks infection after entry, but before formation of 2-LTR circles. Interestingly however, neither SAMHD1 nor MX2 were upregulated. This shows for the first time that Nrf2 action can potently block HIV infection and highlights a novel way to trigger this inhibition. PMID:27093399

  19. Sulforaphane Inhibits HIV Infection of Macrophages through Nrf2.

    PubMed

    Furuya, Andrea Kinga Marias; Sharifi, Hamayun J; Jellinger, Robert M; Cristofano, Paul; Shi, Binshan; de Noronha, Carlos M C

    2016-04-01

    Marburg virus, the Kaposi's sarcoma-associated herpesvirus (KSHV) and Dengue virus all activate, and benefit from, expression of the transcription regulator nuclear erythroid 2-related factor 2 (Nrf2). The impact of Nrf2 activation on human immunodeficiency virus (HIV) infection has not been tested. Sulforaphane (SFN), produced in cruciferous vegetables after mechanical damage, mobilizes Nrf2 to potently reprogram cellular gene expression. Here we show for the first time that SFN blocks HIV infection in primary macrophages but not in primary T cells. Similarly SFN blocks infection in PMA-differentiated promonocytic cell lines, but not in other cell lines tested. siRNA-mediated depletion of Nrf2 boosted HIV infectivity in primary macrophages and reduced the anti-viral effects of SFN treatment. This supports a model in which anti-viral activity is mediated through Nrf2 after it is mobilized by SFN. We further found that, like the type I interferon-induced cellular anti-viral proteins SAMHD1 and MX2, SFN treatment blocks infection after entry, but before formation of 2-LTR circles. Interestingly however, neither SAMHD1 nor MX2 were upregulated. This shows for the first time that Nrf2 action can potently block HIV infection and highlights a novel way to trigger this inhibition.

  20. PHLPP2 down regulation influences nuclear Nrf2 stability via Akt-1/Gsk3β/Fyn kinase axis in acetaminophen induced oxidative renal toxicity: Protection accorded by morin.

    PubMed

    Mathur, Alpana; Rizvi, Fatima; Kakkar, Poonam

    2016-03-01

    NF-E2 p45-related factor 2 (Nrf2) is a cap 'n' collar (CNC) basic region-leucine zipper (bZIP) transcription factor that imparts cellular defence against xenobiotic and oxidative stress evoked responses by inducing an array of cytoprotective genes. Essential factors that regulate Nrf2 activity and stability during analgesic nephropathy are incompletely understood. In this study, we demonstrate that acetaminophen (a classic analgesic) posit nephrotoxicity both in vitro and in vivo via PHLPP2 activation. Enhanced PHLPP2 levels down regulate p-Akt by dephosphorylating it at Ser 473 residue leading to Gsk3β activation. APAP subsided Nrf2 nuclear accumulation by activating Gsk3β which phosphorylates Fyn kinase. p-Fyn kinase translocates into the nucleus and phosphorylates Nrf2 (Tyr 568) leading to its nuclear export, ubiquitination and degradation. Therefore, poor prognosis prevails during analgesic nephrotoxicity because of the defects in Akt-1/Gsk3β/Fyn-Nrf2 signaling pathway. Morin, a bioflavonoid given as co- and pre-treatment with acetaminophen significantly prevented the toxicity induced damage by constitutively stabilizing Nrf2 nuclear retention. Diminished Nrf2 levels by APAP overdose imposed severe proximal tubular damage leading to apoptotic cell death. Morin, as a potent Nrf2 inducer accorded protection against acetaminophen induced renal damages by its molecular intervention with Akt-1/Gsk3β/Fyn kinase pathway via PHLPP2 de-activation.

  1. A purified feverfew extract protects from oxidative damage by inducing DNA repair in skin cells via a PI3-kinase-dependent Nrf2/ARE pathway.

    PubMed

    Rodriguez, Karien J; Wong, Heng-Kuan; Oddos, Thierry; Southall, Michael; Frei, Balz; Kaur, Simarna

    2013-12-01

    Environmental factors such as solar ultraviolet (UV) radiation and other external aggressors provide an oxidative challenge that is detrimental to skin health. The levels of endogenous antioxidants decrease with age, thus resulting in less protection and a greater potential for skin damage. The NF-E2-related factor-2 (Nrf2) - antioxidant response element (ARE) pathway is a primary defense mechanism against oxidative stress, and induces the expression of antioxidant, detoxification and repair genes. Activation of ARE-Nrf2 can help restore oxidative homeostasis of the skin and play a role in inflammatory response and DNA repair mechanisms. To evaluate the role of a purified parthenolide-depleted Feverfew (PD-Feverfew) extract on the ARE-Nrf2 pathway and DNA repair in skin cells. These studies were undertaken in primary human keratinocytes or KB cells using Luciferase Promoter assay, siRNA transfection studies, Western blot analyses, Immunofluorescence microscopy, comet assay and quantitative real-time PCR. PD-Feverfew was found to induce Nrf2 nuclear translocation and to increase ARE activity in a dose dependent manner. Furthermore, knockdown of Nrf2 resulted in suppression of PD-Feverfew-induced ARE activity. PD-Feverfew was also found to induce phosphorylation of Akt, a kinase downstream of PI3K. Inhibition of PI3K via pre-treatment with the selective pharmacological inhibitor, LY294002, abolished PD-Feverfew-induced Nrf2/ARE activation. PD-Feverfew also reduced UV-induced DNA damage in a PI3K and Nrf2-dependent manner. Therefore, by increasing endogenous defense mechanisms and aid in DNA repair of damaged skin cells via activation of a PI3K-dependent Nrf2/ARE pathway, PD-Feverfew may help protect the skin from numerous environmental aggressors. Copyright © 2013 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

  2. Schisandra chinensis regulates drug metabolizing enzymes and drug transporters via activation of Nrf2-mediated signaling pathway

    PubMed Central

    He, Jin-Lian; Zhou, Zhi-Wei; Yin, Juan-Juan; He, Chang-Qiang; Zhou, Shu-Feng; Yu, Yang

    2015-01-01

    Drug metabolizing enzymes (DMEs) and drug transporters are regulated via epigenetic, transcriptional, posttranscriptional, and translational and posttranslational modifications. Phase I and II DMEs and drug transporters play an important role in the disposition and detoxification of a large number of endogenous and exogenous compounds. The nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a critical regulator of a variety of important cytoprotective genes that are involved in disposition and detoxification of xenobiotics. Schisandra chinensis (SC) is a commonly used traditional Chinese herbal medicine that has been primarily used to protect the liver because of its potent antioxidative and anti-inflammatory activities. SC can modulate some DMEs and drug transporters, but the underlying mechanisms are unclear. In this study, we aimed to explore the role of Nrf2 in the regulatory effect of SC extract (SCE) on selected DMEs and drug transporters in human hepatocellular liver carcinoma cell line (HepG2) cells. The results showed that SCE, schisandrin A, and schisandrin B significantly increased the expression of NAD(P)H: Nicotinamide Adenine Dinucleotide Phosphate-oxidase or:quinone oxidoreductase 1, heme oxygenase-1, glutamate–cysteine ligase, and glutathione S-transferase A4 at both transcriptional and posttranscriptional levels. Incubation of HepG2 cells with SCE resulted in a significant increase in the intracellular level of glutathione and total glutathione S-transferase content. SCE significantly elevated the messenger ribonucleic acid and protein levels of P-glycoprotein and multidrug resistance-associated protein 2 and 4, whereas the expression of organic anion transporting peptide 1A2 and 1B1 was significantly downregulated by SCE. Knockdown of Nrf2 by small interfering ribonucleic acid attenuated the regulatory effect of SCE on these DMEs and drug transporters. SCE significantly upregulated Nrf2 and promoted the translocation of Nrf2 from cytoplasm to

  3. EX4 stabilizes and activates Nrf2 via PKCδ, contributing to the prevention of oxidative stress-induced pancreatic beta cell damage.

    PubMed

    Kim, Mi-Hwi; Kim, Eung-Hwi; Jung, Hye Seung; Yang, Dongki; Park, Eun-Young; Jun, Hee-Sook

    2017-01-15

    Oxidative stress in pancreatic beta cells can inhibit insulin secretion and promote apoptotic cell death. Exendin-4 (EX4), a glucagon-like peptide-1 receptor agonist, can suppress beta cell apoptosis, improve beta cell function and protect against oxidative damage. In this study, we investigated the molecular mechanisms for antioxidative effects of EX4 in pancreatic beta cells. INS-1 cells, a rat insulinoma cell line, were pretreated with EX4 and exposed to palmitate or H2O2. Reactive oxygen species (ROS) production, and glutathione and insulin secretion were measured. The mRNA and protein expression levels of antioxidant genes were examined. The level of nuclear factor erythroid 2-related factor 2 (Nrf2), its binding to antioxidant response element (ARE), and its ubiquination in the presence of EX4 were determined. The Nrf2 signaling pathway was determined using rottlerin (protein kinase [PK]Cδ inhibitor), H89 (PKA inhibitor) and LY294002 (phosphatidylinositide 3-kinase [PI3K] inhibitor). EX4 treatment decreased ROS production, recovered cellular glutathione levels and insulin secretion in the presence of oxidative stress in INS-1 cells. The expression levels of glutamate-cysteine ligase catalytic subunit and heme oxygenase-1 were increased by EX4 treatment. EX4 promoted Nrf2 translocation, ARE binding activity and enhanced stabilization of Nrf2 by inhibition of ubiquitination. Knockdown of Nrf2 abolished the effect of EX4 on increased insulin secretion. Inhibition of PKCδ attenuated Nrf2 translocation and antioxidative gene expression by EX4 treatment. We suggest that EX4 activates and stabilizes Nrf2 through PKCδ activation, contributing to the increase of antioxidant gene expression and consequently improving beta cell function in the presence of oxidative stress.

  4. Asiatic acid enhances Nrf2 signaling to protect HepG2 cells from oxidative damage through Akt and ERK activation.

    PubMed

    Qi, Zhimin; Ci, Xinxin; Huang, Jingbo; Liu, Qinmei; Yu, Qinlei; Zhou, Junfeng; Deng, Xuming

    2017-04-01

    Asiatic acid (AA), a natural triterpene isolated from the plant Centella asiatica, have antioxidative potential, but the molecular mechanism of AA against oxidative stress remains unclear. Our study was performed to investigate the antioxidative effect of AA against oxidative stress and the antioxidative mechanism in tert-butyl hydroperoxide (t-BHP) -stimulated the HepG2 cells. The results showed that AA suppressed t-BHP-induced cytotoxicity, apoptosis, and reactive oxygen species (ROS) generation. Additionally, AA activated the nuclear factor erythroid 2-related factor 2 (Nrf2) signal, which was closely related to induction Nrf2 nuclear translocation, reduction the expression of Keap1 and up-regulation the activity of the antioxidant response element (ARE). Meanwhile, activation of Nrf2 signal upregulated the protein expressions of antioxidant genes, including heme oxygenase-1 (HO-1), NAD(P)H: quinone oxidase (NQO-1), and glutamyl cysteine ligase catalytic subunit (GCLC). Excitingly, Knockout of Nrf2 almost abolished AA-mediated antioxidant activity and cytoprotection against t-BHP. Further studies showed the mechanism underlying that AA induced Nrf2 activation in HepG2 cells via Akt and ERK signal activation. We found Akt and ERK inhibitors treatment attenuated AA-mediated Nrf2 nuclear translocation. Furthermore, treatment with either Akt or ERK inhibitor also decreased AA-mediated cytoprotection against t-BHP-induced cellular damage. Collectively, these results presented in this study indicate that AA has the protective effect against t-BHP-induced cellular damage and oxidative stress by modulating Nrf2 signaling through activating the signals of Akt and ERK.

  5. CYP2E1 impairs GLUT4 gene expression and function: NRF2 as a possible mediator.

    PubMed

    Armoni, M; Harel, C; Ramdas, M; Karnieli, E

    2014-06-01

    Impaired GLUT4 function/expression in insulin target tissues is well-documented in diabetes and obesity. Cytochrome P450 isoform 2E1 (CYP2E1) induces oxidative stress, leading to impaired insulin action. CYP2E1 knockout mice are protected against high fat diet-induced insulin resistance and obesity; however the molecular mechanisms are still unclear. We examined whether CYP2E1 impairs GLUT4 gene expression and function in adipose and muscle cells. CYP2E1 overexpression in skeletal muscle-derived L6 cells inhibited insulin-stimulated Glut4 translocation and 2-deoxyglucose uptake, with the latter inhibition being blocked by vitamin E. CYP2E1 overexpression in L6 and primary rat adipose (PRA) cells suppressed GLUT4 gene expression at promoter and mRNA levels, whereas CYP2E1 silencing had opposite effects. In PRA, CYP2E1-induced suppression of GLUT4 expression was blocked by chlormethiazole (CYP2E1-specific inhibitor) and the antioxidants vitamin E and N-acetyl-l-cysteine. CYP2E1 effect was mediated by the transcription factor NF-E2-related factor 2 (NRF2), as evident from its complete reversal by a coexpressed dominant-negative, but not wild-type NRF2. GLUT4 transcription was suppressed by NRF2 overexpression, and enhanced by NRF2 silencing. Promoter and ChIP analysis showed a direct and specific binding of NRF2 to a 58-326 GLUT4 promoter region that was required to maintain CYP2E1 suppression; this binding was enhanced by CYP2E1 overexpression. We suggest a mechanism for CYP2E1 action that involves: a) suppression of GLUT4 gene expression that is mediated by NRF2; b) impairment of insulin-stimulated Glut4 translocation and function. CYP2E1 and NRF2 are introduced as negative regulators of GLUT4 expression and function in insulin-sensitive cells.

  6. Upregulating Nrf2-dependent antioxidant defenses in Pacific oysters Crassostrea gigas: Investigating the Nrf2/Keap1 pathway in bivalves.

    PubMed

    Danielli, Naissa Maria; Trevisan, Rafael; Mello, Danielle Ferraz; Fischer, Kelvis; Deconto, Vanessa Schadeck; da Silva Acosta, Daiane; Bianchini, Adalto; Bainy, Afonso Celso Dias; Dafre, Alcir Luiz

    2017-05-01

    Analysis of the Pacific oyster Crassostrea gigas annotated genome revealed genes with conserved sequences belonging to typical cap 'n' collar Nrf2 domain, a major player in antioxidant protection, and domains belonging to Nrf2 cytoplasmic repressor (Keap1), but little is known about Nrf2/Keap1 induction in bivalves. C. gigas were exposed to waterborne 10 and 30μM curcumin, a known inducer of the mammalian Nrf2. Curcumin disappeared from the seawater after 10h, and accumulated in the gills (10h) and digestive gland (10-96h). A clear induction of glutathione (GSH)-related antioxidant defenses was observed at 96h in the gills of curcumin exposed animals (10 and 30μM), including GSH levels, and the activity of glutathione reductase (GR), glutathione peroxidase (GPx), and glutathione S-transferase (GST). This response was completely absent in the digestive gland, in line with the idea that bivalve gills act as a major site for antioxidant protection under acute exposure. The relative mRNA levels coding glutamate-cysteine ligase, GR, GPx2 and GSTpi were clearly induced by curcumin treatment (30μM, 24h). Curcumin pre-treatment for 96h increased oyster resistance to cumene hydroperoxide, but neither Nrf2 nor Keap1 genes were modulated by curcumin. However, the conserved sequences belonging to typical Nrf2 and Keap1 domains, and the notorious induction of antioxidant defense-related genes known to be controlled by Nrf2 in mammals, indicates a functional Nrf2/Keap1 pathway in bivalves, and curcumin seems to be a new tool to investigate the antioxidant response in bivalves. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. The Chinese herbal formula Free and Easy Wanderer ameliorates oxidative stress through KEAP1-NRF2/HO-1 pathway.

    PubMed

    Hong, Chunlan; Cao, Jingming; Wu, Ching-Fen; Kadioglu, Onat; Schüffler, Anja; Kauhl, Ulrich; Klauck, Sabine M; Opatz, Till; Thines, Eckhard; Paul, Norbert W; Efferth, Thomas

    2017-09-14

    Posttraumatic stress disorder (PTSD) gains a lot of attention due to high prevalence and strong psychological upset, but the etiology remains undefined and effective treatment is quite limited. Growing studies demonstrated the involvement of oxidative stress in various psychiatry diseases, suggesting anti-oxidation therapy might be a strategy for PTSD treatment. Free and Easy Wanderer (FAEW) is a poly-herbal drug clinically used in China for hundreds of years in the treatment of psychiatric disorder. We hypothesized that FAEW exerts clinical effects through the activity against oxidative stress with fluoxetine as antidepressant control drug. Our results revealed that FAEW significantly reduced both endogenous and H2O2-induced exogenous ROS levels in the human glioblastoma T98G and neuroblastoma SH-SY5Y cell lines. Transcriptome-wide microarray analysis indicated NRF2/HO-1 as the common target of FAEW and fluoxetine. Western blotting assay proved that the two drugs promoted NRF2 release from KEAP1 in the cytoplasm and translocation to the nuclei in a KEAP1-dependent manner, the expression of the protein HO-1 increased accordingly, suggesting the participation of KEAP1-NRF2/HO-1 pathway. The chemical constituents of FAEW (i.e. paeoniflorin, baicalin) bound to KEAP1 in silico, which hence might be the effective substances of FAEW. In conclusion, FAEW counteracted H2O2-induced oxidative stress through KEAP1-NRF2/HO-1 pathway.

  8. Protective Effects of Quercetin on Mitochondrial Biogenesis in Experimental Traumatic Brain Injury via the Nrf2 Signaling Pathway

    PubMed Central

    Li, Xiang; Wang, Handong; Gao, Yongyue; Li, Liwen; Tang, Chao; Wen, Guodao; Zhou, Yuan; Zhou, Mengliang; Mao, Lei; Fan, Youwu

    2016-01-01

    The present investigation was carried out to elucidate a possible molecular mechanism related to the protective effect of quercetin administration against oxidative stress on various mitochondrial respiratory complex subunits with special emphasis on the role of nuclear factor erythroid 2-related factor 2 (Nrf2) in mitochondrial biogenesis. Recently, quercetin has been proved to have a protective effect against mitochondria damage after traumatic brain injury (TBI). However, its precise role and underlying mechanisms in traumatic brain injury are not yet fully understood. The aim of the present study was to investigate the effect of quercetin on the potential mechanism of these effects in a weight-drop model of TBI in male mice that were treated with quercetin or vehicle via intraperitoneal injection administrated 30 min after TBI. In this experiment, ICR mice were divided into four groups: A sham group, TBI group, TBI + vehicle group, and TBI + quercetin group. Brain samples were collected 24 h later for analysis. Quercetin treatment resulted in an upregulation of Nrf2 expression and cytochrome c, malondialdehyde (MDA) and superoxide dismutase (SOD) levels were restored by quercetin treatment. Quercetin markedly promoted the translocation of Nrf2 protein from the cytoplasm to the nucleus. These observations suggest that quercetin improves mitochondrial function in TBI models, possibly by activating the Nrf2 pathway. PMID:27780244

  9. Protective Effects of Quercetin on Mitochondrial Biogenesis in Experimental Traumatic Brain Injury via the Nrf2 Signaling Pathway.

    PubMed

    Li, Xiang; Wang, Handong; Gao, Yongyue; Li, Liwen; Tang, Chao; Wen, Guodao; Zhou, Yuan; Zhou, Mengliang; Mao, Lei; Fan, Youwu

    2016-01-01

    The present investigation was carried out to elucidate a possible molecular mechanism related to the protective effect of quercetin administration against oxidative stress on various mitochondrial respiratory complex subunits with special emphasis on the role of nuclear factor erythroid 2-related factor 2 (Nrf2) in mitochondrial biogenesis. Recently, quercetin has been proved to have a protective effect against mitochondria damage after traumatic brain injury (TBI). However, its precise role and underlying mechanisms in traumatic brain injury are not yet fully understood. The aim of the present study was to investigate the effect of quercetin on the potential mechanism of these effects in a weight-drop model of TBI in male mice that were treated with quercetin or vehicle via intraperitoneal injection administrated 30 min after TBI. In this experiment, ICR mice were divided into four groups: A sham group, TBI group, TBI + vehicle group, and TBI + quercetin group. Brain samples were collected 24 h later for analysis. Quercetin treatment resulted in an upregulation of Nrf2 expression and cytochrome c, malondialdehyde (MDA) and superoxide dismutase (SOD) levels were restored by quercetin treatment. Quercetin markedly promoted the translocation of Nrf2 protein from the cytoplasm to the nucleus. These observations suggest that quercetin improves mitochondrial function in TBI models, possibly by activating the Nrf2 pathway.

  10. p97 Negatively Regulates NRF2 by Extracting Ubiquitylated NRF2 from the KEAP1-CUL3 E3 Complex.

    PubMed

    Tao, Shasha; Liu, Pengfei; Luo, Gang; Rojo de la Vega, Montserrat; Chen, Heping; Wu, Tongde; Tillotson, Joseph; Chapman, Eli; Zhang, Donna D

    2017-04-15

    Activation of the stress-responsive transcription factor NRF2 is the major line of defense to combat oxidative or electrophilic insults. Under basal conditions, NRF2 is continuously ubiquitylated by the KEAP1-CUL3-RBX1 E3 ubiquitin ligase complex and is targeted to the proteasome for degradation (the canonical mechanism). However, the path from the CUL3 complex to ultimate proteasomal degradation was previously unknown. p97 is a ubiquitin-targeted ATP-dependent segregase that extracts ubiquitylated client proteins from membranes, protein complexes, or chromatin and has an essential role in autophagy and the ubiquitin proteasome system (UPS). In this study, we show that p97 negatively regulates NRF2 through the canonical pathway by extracting ubiquitylated NRF2 from the KEAP1-CUL3 E3 complex, with the aid of the heterodimeric cofactor UFD1/NPL4 and the UBA-UBX-containing protein UBXN7, for efficient proteasomal degradation. Given the role of NRF2 in chemoresistance and the surging interest in p97 inhibitors to treat cancers, our results indicate that dual p97/NRF2 inhibitors may offer a more potent and long-term avenue of p97-targeted treatment.

  11. Oat avenanthramides induce heme oxygenase-1 expression via Nrf2-mediated signaling in HK-2 cells.

    PubMed

    Fu, Junsheng; Zhu, Yingdong; Yerke, Aaron; Wise, Mitchell L; Johnson, Jodee; Chu, YiFang; Sang, Shengmin

    2015-12-01

    Numerous studies have shown that avenanthramides (AVAs), unique compounds found in oats, are strong antioxidants, though the mechanism of action remains unclear. Here, we investigated whether AVAs affect heme oxygenase-1 (HO-1) expression through the activation of Nrf2 translocation. We investigated the effects AVA 2c, 2f, and 2p on HK-2 cells, and found that AVAs could significantly increase HO-1 expression in both a dose- and time-dependent manner. Furthermore, we found that AVA-induced HO-1 expression is mediated by Nrf2 translocation. The addition of N-acetylcysteine (NAC), but not specific inhibitors of p38 (SB202190), PI3K (LY294002), and MEK1 (PD098059) attenuated AVA-induced HO-1 expression, demonstrating an important role for reactive oxygen species, but not PI3K or MAPK activation, in activating the HO-1 pathway. Moreover, hydrogenation of the double bond of the functional α,β-unsaturated carbonyl group of AVAs eliminated their effects on HO-1 expression, suggesting that this group is crucial for the antioxidant activity of AVAs. Our results suggest a novel mechanism whereby AVAs exert an antioxidant function on human health. Further investigation of these markers in human is warranted to explore the beneficial health effects of whole grain oat intake. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Nitrosopersulfide (SSNO(-)) targets the Keap-1/Nrf2 redox system.

    PubMed

    Cortese-Krott, Miriam M; Pullmann, David; Feelisch, Martin

    2016-11-01

    Nitric oxide (NO), hydrogen sulfide and polysulfides have been proposed to contribute to redox signaling by activating the Keap-1/Nrf2 stress response system. Nitrosopersulfide (SSNO(-)) recently emerged as a bioactive product of the chemical interaction of NO or nitrosothiols with sulfide; upon decomposition it generates polysulfides and free NO, triggering the activation of soluble guanylate cyclase, inducing blood vessel relaxation in vitro and lowering blood pressure in vivo. Whether SSNO(-) itself interacts with the Keap-1/Nrf2 system is unknown. We therefore sought to investigate the ability of SSNO(-) to activate Nrf2-dependent processes in human vascular endothelial cells, and to compare the pharmacological effects of SSNO(-) with those of its precursors NO and sulfide at multiple levels of target engagement. We here demonstrate that SSNO(-) strongly increases nuclear levels, binding activity and transactivation activity of Nrf2, thereby increasing mRNA expression of Hmox-1, the gene encoding for heme oxygenase 1, without adversely affecting cell viability. Under all conditions, SSNO(-) appeared to be more potent than its parent compounds, NO and sulfide. SSNO(-)-induced Nrf2 transactivation activity was abrogated by either NO scavenging with cPTIO or inhibition of thiol sulfuration by high concentrations of cysteine, implying a role for both persulfides/polysulfides and NO in SSNO(-) mediated Nrf2 activation. Taken together, our studies demonstrate that the Keap-1/Nrf2 redox system is a biological target of SSNO(-), enriching the portfolio of bioactivity of this vasoactive molecule to also engage in the regulation of redox signaling processes. The latter suggests a possible role as messenger and/or mediator in cellular sensing and adaptations processes.

  13. The emerging role of Nrf2 in mitochondrial function

    PubMed Central

    Dinkova-Kostova, Albena T.; Abramov, Andrey Y.

    2015-01-01

    The transcription factor NF-E2 p45-related factor 2 (Nrf2; gene name NFE2L2) allows adaptation and survival under conditions of stress by regulating the gene expression of diverse networks of cytoprotective proteins, including antioxidant, anti-inflammatory, and detoxification enzymes as well as proteins that assist in the repair or removal of damaged macromolecules. Nrf2 has a crucial role in the maintenance of cellular redox homeostasis by regulating the biosynthesis, utilization, and regeneration of glutathione, thioredoxin, and NADPH and by controlling the production of reactive oxygen species by mitochondria and NADPH oxidase. Under homeostatic conditions, Nrf2 affects the mitochondrial membrane potential, fatty acid oxidation, availability of substrates (NADH and FADH2/succinate) for respiration, and ATP synthesis. Under conditions of stress or growth factor stimulation, activation of Nrf2 counteracts the increased reactive oxygen species production in mitochondria via transcriptional upregulation of uncoupling protein 3 and influences mitochondrial biogenesis by maintaining the levels of nuclear respiratory factor 1 and peroxisome proliferator-activated receptor γ coactivator 1α, as well as by promoting purine nucleotide biosynthesis. Pharmacological Nrf2 activators, such as the naturally occurring isothiocyanate sulforaphane, inhibit oxidant-mediated opening of the mitochondrial permeability transition pore and mitochondrial swelling. Curiously, a synthetic 1,4-diphenyl-1,2,3-triazole compound, originally designed as an Nrf2 activator, was found to promote mitophagy, thereby contributing to the overall mitochondrial homeostasis. Thus, Nrf2 is a prominent player in supporting the structural and functional integrity of the mitochondria, and this role is particularly crucial under conditions of stress. PMID:25975984

  14. The emerging role of Nrf2 in mitochondrial function.

    PubMed

    Dinkova-Kostova, Albena T; Abramov, Andrey Y

    2015-11-01

    The transcription factor NF-E2 p45-related factor 2 (Nrf2; gene name NFE2L2) allows adaptation and survival under conditions of stress by regulating the gene expression of diverse networks of cytoprotective proteins, including antioxidant, anti-inflammatory, and detoxification enzymes as well as proteins that assist in the repair or removal of damaged macromolecules. Nrf2 has a crucial role in the maintenance of cellular redox homeostasis by regulating the biosynthesis, utilization, and regeneration of glutathione, thioredoxin, and NADPH and by controlling the production of reactive oxygen species by mitochondria and NADPH oxidase. Under homeostatic conditions, Nrf2 affects the mitochondrial membrane potential, fatty acid oxidation, availability of substrates (NADH and FADH2/succinate) for respiration, and ATP synthesis. Under conditions of stress or growth factor stimulation, activation of Nrf2 counteracts the increased reactive oxygen species production in mitochondria via transcriptional upregulation of uncoupling protein 3 and influences mitochondrial biogenesis by maintaining the levels of nuclear respiratory factor 1 and peroxisome proliferator-activated receptor γ coactivator 1α, as well as by promoting purine nucleotide biosynthesis. Pharmacological Nrf2 activators, such as the naturally occurring isothiocyanate sulforaphane, inhibit oxidant-mediated opening of the mitochondrial permeability transition pore and mitochondrial swelling. Curiously, a synthetic 1,4-diphenyl-1,2,3-triazole compound, originally designed as an Nrf2 activator, was found to promote mitophagy, thereby contributing to the overall mitochondrial homeostasis. Thus, Nrf2 is a prominent player in supporting the structural and functional integrity of the mitochondria, and this role is particularly crucial under conditions of stress. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  15. Nrf2 pathway activation contributes to anti-fibrosis effects of ginsenoside Rg1 in a rat model of alcohol- and CCl4-induced hepatic fibrosis

    PubMed Central

    Li, Jian-ping; Gao, Yan; Chu, Shi-feng; Zhang, Zhao; Xia, Cong-yuan; Mou, Zheng; Song, Xiu-yun; He, Wen-bin; Guo, Xiao-feng; Chen, Nai-hong

    2014-01-01

    Aim: To investigate the anti-fibrosis effects of ginsenoside Rg1 on alcohol- and CCl4-induced hepatic fibrosis in rats and to explore the mechanisms of the effects. Methods: Rats were given 6% alcohol in water and injected with CCl4 (2 mL/kg, sc) twice a week for 8 weeks. Rg1 (10, 20 and 40 mg/kg per day, po) was administered in the last 2 weeks. Hepatic fibrosis was determined by measuring serum biochemical parameters, HE staining, Masson's trichromic staining, and hydroxyproline and α-SMA immunohistochemical staining of liver tissues. The activities of antioxidant enzymes, lipid peroxidation, and Nrf2 signaling pathway-related proteins (Nrf2, Ho-1 and Nqo1) in liver tissues were analyzed. Cultured hepatic stellate cells (HSCs) of rats were prepared for in vitro studies. Results: In the alcohol- and CCl4-treated rats, Rg1 administration dose-dependently suppressed the marked increases of serum ALT, AST, LDH and ALP levels, inhibited liver inflammation and HSC activation and reduced liver fibrosis scores. Rg1 significantly increased the activities of antioxidant enzymes (SOD, GSH-Px and CAT) and reduced MDA levels in liver tissues. Furthermore, Rg1 significantly increased the expression and nuclear translocation of Nrf2 that regulated the expression of many antioxidant enzymes. Treatment of the cultured HSCs with Rg1 (1 μmol/L) induced Nrf2 translocation, and suppressed CCl4-induced cell proliferation, reversed CCl4- induced changes in MDA, GPX, PCIII and HA contents in the supernatant fluid and α-SMA expression in the cells. Knockdown of Nrf2 gene diminished these actions of Rg1 in CCl4-treated HSCs in vitro. Conclusion: Rg1 exerts protective effects in a rat model of alcohol- and CCl4-induced hepatic fibrosis via promoting the nuclear translocation of Nrf2 and expression of antioxidant enzymes. PMID:24976156

  16. Halofuginone enhances the chemo-sensitivity of cancer cells by suppressing NRF2 accumulation.

    PubMed

    Tsuchida, Kouhei; Tsujita, Tadayuki; Hayashi, Makiko; Ojima, Asaka; Keleku-Lukwete, Nadine; Katsuoka, Fumiki; Otsuki, Akihito; Kikuchi, Haruhisa; Oshima, Yoshiteru; Suzuki, Mikiko; Yamamoto, Masayuki

    2017-02-01

    The KEAP1-NRF2 system regulates the cellular defence against oxidative and xenobiotic stresses. NRF2 is a transcription factor that activates the expression of cytoprotective genes encoding antioxidative, detoxifying and metabolic enzymes as well as transporters. Under normal conditions, KEAP1 represses NRF2 activity by degrading the NRF2 protein. When cells are exposed to stresses, KEAP1 stops promoting NRF2 degradation, and NRF2 rapidly accumulates and activates the transcription of target genes. Constitutive accumulation of NRF2 via a variety of mechanisms that disrupt KEAP1-mediated NRF2 degradation has been observed in various cancer types. Constitutive NRF2 accumulation confers cancer cells with a proliferative advantage as well as resistance to anti-cancer drugs and radiotherapies. To suppress the chemo- and radio-resistance of cancer cells caused by NRF2 accumulation, we conducted high-throughput chemical library screening for NRF2 inhibitors and identified febrifugine derivatives. We found that application of the less-toxic derivative halofuginone in a low dose range rapidly reduced NRF2 protein levels. Halofuginone induced a cellular amino acid starvation response that repressed global protein synthesis and rapidly depleted NRF2. Halofuginone treatment ameliorated the resistance of NRF2-addicted cancer cells to anti-cancer drugs both in vitro and in vivo. These results provide preclinical proof-of-concept evidence for halofuginone as an NRF2 inhibitor applicable to treatment of chemo- and radio-resistant forms of cancer.

  17. Neutrophils in oral paracoccidioidomycosis and the involvement of Nrf2.

    PubMed

    Araújo, Vera Cavalcanti; Demasi, Ana Paula Dias; Soares, Andresa Borges; Passador-Santos, Fabrício; Napimoga, Marcelo Henrique; Martinez, Elizabeth Ferreira; Freitas, Nadir Severina; Araújo, Ney Soares

    2013-01-01

    Neutrophils have been implicated in granuloma formation in several infectious diseases, in addition to their main phagocytic and pathogen destruction role. It has been demonstrated that Nrf2 regulates antioxidant protection in neutrophils, attenuating inflammation without compromising the hosts bacterial defense. In this study, we analyzed the presence of neutrophils in Paracoccidioides brasiliensis mycosis (PCM), as well as the immunoexpression of Nrf2. Thirty-nine cases of oral PCM were classified according to quantity of fungi and to the presence of loose or well-organized granulomas and microabscesses. An Nrf2 antibody was used for immunohistochemical analysis. The results showed that neutrophils are present in microabscesses and loose granulomas, but were absent in structured granulomas. A greater quantity of fungi was shown in cases with only loose granulomas when compared to loose and well organized granulomas. Nrf2 was observed in the nuclei of neutrophils of loose granulomas and abscesses, with its expression in loose granulomas maintained despite the additional presence of well organized granulomas in the same specimen. This study suggests that neutrophils participate in P. brasiliensis granuloma formation and that Nrf2 has a possible role in neutrophil survival, via modulation of the inflammatory response.

  18. Role of the Nrf2-ARE Pathway in Liver Diseases

    PubMed Central

    Yang, Ji Hye; Ki, Sung Hwan

    2013-01-01

    The liver is a central organ that performs a wide range of functions such as detoxification and metabolic homeostasis. Since it is a metabolically active organ, liver is particularly susceptible to oxidative stress. It is well documented that liver diseases including hepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma are highly associated with antioxidant capacity. NF-E2-related factor-2 (Nrf2) is an essential transcription factor that regulates an array of detoxifying and antioxidant defense genes expression in the liver. It is activated in response to electrophiles and induces its target genes by binding to the antioxidant response element (ARE). Therefore, the roles of the Nrf2-ARE pathway in liver diseases have been extensively investigated. Studies from several animal models suggest that the Nrf2-ARE pathway collectively exhibits diverse biological functions against viral hepatitis, alcoholic and nonalcoholic liver disease, fibrosis, and cancer via target gene expression. In this review, we will discuss the role of the Nrf2-ARE pathway in liver pathophysiology and the potential application of Nrf2 as a therapeutic target to prevent and treat liver diseases. PMID:23766860

  19. Exercise, Nrf2 and Antioxidant Signaling in Cardiac Aging

    PubMed Central

    Narasimhan, Madhusudhanan; Rajasekaran, Namakkal S.

    2016-01-01

    Aging is represented by a progressive decline in cellular functions. The age-related deformities in cardiac behaviors are the loss of cardiac myocytes through apoptosis or programmed cell death. Oxidative stress (OS) and its deleterious consequence contribute to age-related mechanical remodeling, reduced regenerative capacity, and apoptosis in cardiac tissue. The pathogenesis of OS in the elderly can predispose the heart to other cardiac complications such as atherosclerosis, hypertension, ischemic heart disease, cardiac myopathy, and so on. At the molecular level, oxidant-induced activation of Nrf2 (Nuclear erythroid-2-p45-related factor-2), a transcription factor, regulates several genes containing AREs (Antioxidant Response Element) and bring the respective translates to counteract the reactive radicals and establish homeostasis. Myriad of Nrf2 gene knockout studies in various organs such as lung, liver, kidney, brain, etc. have shown that dysregulation of Nrf2 severely affects the oxidant/ROS sensitivity and predispose the system to several pathological changes with aberrant cellular lesions. On the other hand, its gain of function chemical interventions exhibited oxidant stress resistance and cytoprotection. However, thus far, only a few investigations have shown the potential role of Nrf2 and its non-pharmacological induction in cardiac aging. Therefore, here we review the involvement of Nrf2 signaling along with its responses and ramifications on the cascade of OS under acute exercise stress (AES), moderate exercise training (MET), and endurance exercise stress (EES) conditions in the aging heart. PMID:27378947

  20. Intracellular degradation of functionalized carbon nanotube/iron oxide hybrids is modulated by iron via Nrf2 pathway

    PubMed Central

    Elgrabli, Dan; Dachraoui, Walid; Marmier, Hélène de; Ménard-Moyon, Cécilia; Bégin, Dominique; Bégin-Colin, Sylvie; Bianco, Alberto; Alloyeau, Damien; Gazeau, Florence

    2017-01-01

    The in vivo fate and biodegradability of carbon nanotubes is still a matter of debate despite tremendous applications. In this paper we describe a molecular pathway by which macrophages degrade functionalized multi-walled carbon nanotubes (CNTs) designed for biomedical applications and containing, or not, iron oxide nanoparticles in their inner cavity. Electron microscopy and Raman spectroscopy show that intracellularly-induced structural damages appear more rapidly for iron-free CNTs in comparison to iron-loaded ones, suggesting a role of iron in the degradation mechanism. By comparing the molecular responses of macrophages derived from THP1 monocytes to both types of CNTs, we highlight a molecular mechanism regulated by Nrf2/Bach1 signaling pathways to induce CNT degradation via NOX2 complex activation and O2•−, H2O2 and OH• production. CNT exposure activates an oxidative stress-dependent production of iron via Nrf2 nuclear translocation, Ferritin H and Heme oxygenase 1 translation. Conversely, Bach1 was translocated to the nucleus of cells exposed to iron-loaded CNTs to recycle embedded iron. Our results provide new information on the role of oxidative stress, iron metabolism and Nrf2-mediated host defence for regulating CNT fate in macrophages. PMID:28120861

  1. Src subfamily kinases regulate nuclear export and degradation of transcription factor Nrf2 to switch off Nrf2-mediated antioxidant activation of cytoprotective gene expression.

    PubMed

    Niture, Suryakant K; Jain, Abhinav K; Shelton, Phillip M; Jaiswal, Anil K

    2011-08-19

    Nrf2 (NF-E2-related factor 2) is a nuclear transcription factor that in response to chemical and radiation stress regulates coordinated induction of a battery of cytoprotective gene expressions leading to cellular protection. In this study, we investigated the role of Src kinases in the regulation of Nrf2 and downstream signaling. siRNA-mediated inhibition of Fyn, Src, Yes, and Fgr, but not Lyn, in mouse hepatoma Hepa-1 cells, led to nuclear accumulation of Nrf2 and up-regulation of Nrf2 downstream gene expression. Mouse embryonic fibroblasts with combined deficiency of Fyn/Src/Yes/Fgr supported results from siRNA. In addition, steady-state overexpression of Fyn, Src, and Yes phosphorylated Nrf2Tyr568 that triggered nuclear export and degradation of Nrf2 and down-regulation of Nrf2 downstream gene expression. Exposure of cells to antioxidant, oxidant, or UV radiation increased nuclear import of Fyn, Src, and Yes kinases, which phosphorylated Nrf2Tyr568 resulting in nuclear export and degradation of Nrf2. Further analysis revealed that stress-activated GSK3β acted upstream to the Src kinases and phosphorylated the Src kinases, leading to their nuclear localization and Nrf2 phosphorylation. The overexpression of Src kinases in Hepa-1 cells led to decreased Nrf2, increased apoptosis, and decreased cell survival. Mouse embryonic fibroblasts deficient in Src kinases showed nuclear accumulation of Nrf2, induction of Nrf2 and downstream gene expression, reduced apoptosis, and increased cell survival. The studies together demonstrate that Src kinases play a critical role in nuclear export and degradation of Nrf2, thereby providing a negative feedback mechanism to switch off Nrf2 activation and restore normal cellular homeostasis.

  2. Overview of Nrf2 as Therapeutic Target in Epilepsy.

    PubMed

    Carmona-Aparicio, Liliana; Pérez-Cruz, Claudia; Zavala-Tecuapetla, Cecilia; Granados-Rojas, Leticia; Rivera-Espinosa, Liliana; Montesinos-Correa, Hortencia; Hernández-Damián, Jacqueline; Pedraza-Chaverri, José; Sampieri, Aristides; Coballase-Urrutia, Elvia; Cárdenas-Rodríguez, Noemí

    2015-08-07

    Oxidative stress is a biochemical state of imbalance in the production of reactive oxygen and nitrogen species and antioxidant defenses. It is involved in the physiopathology of degenerative and chronic neuronal disorders, such as epilepsy. Experimental evidence in humans and animals support the involvement of oxidative stress before and after seizures. In the past few years, research has increasingly focused on the molecular pathways of this process, such as that involving transcription factor nuclear factor E2-related factor 2 (Nrf2), which plays a central role in the regulation of antioxidant response elements (ARE) and modulates cellular redox status. The aim of this review is to present experimental evidence on the role of Nrf2 in this neurological disorder and to further determine the therapeutic impact of Nrf2 in epilepsy.

  3. Lithium Promotes Longevity through GSK3/NRF2-Dependent Hormesis

    PubMed Central

    Castillo-Quan, Jorge Iván; Li, Li; Kinghorn, Kerri J.; Ivanov, Dobril K.; Tain, Luke S.; Slack, Cathy; Kerr, Fiona; Nespital, Tobias; Thornton, Janet; Hardy, John; Bjedov, Ivana; Partridge, Linda

    2016-01-01

    Summary The quest to extend healthspan via pharmacological means is becoming increasingly urgent, both from a health and economic perspective. Here we show that lithium, a drug approved for human use, promotes longevity and healthspan. We demonstrate that lithium extends lifespan in female and male Drosophila, when administered throughout adulthood or only later in life. The life-extending mechanism involves the inhibition of glycogen synthase kinase-3 (GSK-3) and activation of the transcription factor nuclear factor erythroid 2-related factor (NRF-2). Combining genetic loss of the NRF-2 repressor Kelch-like ECH-associated protein 1 (Keap1) with lithium treatment revealed that high levels of NRF-2 activation conferred stress resistance, while low levels additionally promoted longevity. The discovery of GSK-3 as a therapeutic target for aging will likely lead to more effective treatments that can modulate mammalian aging and further improve health in later life. PMID:27068460

  4. Overview of Nrf2 as Therapeutic Target in Epilepsy

    PubMed Central

    Carmona-Aparicio, Liliana; Pérez-Cruz, Claudia; Zavala-Tecuapetla, Cecilia; Granados-Rojas, Leticia; Rivera-Espinosa, Liliana; Montesinos-Correa, Hortencia; Hernández-Damián, Jacqueline; Pedraza-Chaverri, José; Sampieri, Aristides III; Coballase-Urrutia, Elvia; Cárdenas-Rodríguez, Noemí

    2015-01-01

    Oxidative stress is a biochemical state of imbalance in the production of reactive oxygen and nitrogen species and antioxidant defenses. It is involved in the physiopathology of degenerative and chronic neuronal disorders, such as epilepsy. Experimental evidence in humans and animals support the involvement of oxidative stress before and after seizures. In the past few years, research has increasingly focused on the molecular pathways of this process, such as that involving transcription factor nuclear factor E2-related factor 2 (Nrf2), which plays a central role in the regulation of antioxidant response elements (ARE) and modulates cellular redox status. The aim of this review is to present experimental evidence on the role of Nrf2 in this neurological disorder and to further determine the therapeutic impact of Nrf2 in epilepsy. PMID:26262608

  5. Ethanol Extract of Ganoderma lucidum Augments Cellular Anti-oxidant Defense through Activation of Nrf2/HO-1

    PubMed Central

    Lee, Yoo-hwan; Kim, Jung-hee; Song, Choon-ho; Jang, Kyung-jeon; kim, Cheol-hong; Kang, Ji- Sook; Choi, Yung-hyun

    2016-01-01

    Objectives: The mushroom Ganoderma lucidum has been widely used as a traditional herbal medicine for many years. Although several studies have focused on the anti-oxidative activity of this mushroom, the molecular mechanisms underlying its activity have not yet been clearly established. The present study investigated the cytoprotective effect of ethanol extract of Ganoderma lucidum (EGL) against oxidative stress (hydrogen peroxide, H2O2) and elucidated the underlying mechanisms in a C2C12 myoblast cell line. Methods: Oxidative stress markers were determined by using the comet assay to measure reactive oxygen species (ROS) generation and deoxyribonucleic acid (DNA) damage. Cell viability and Western blotting analyses were employed to evaluate the cellular response to EGL and H2O2 in C2C12 cells. Transfection with nuclear factor erythroid 2-related factor 2 (Nrf2)-specific small interfering ribonucleic acid (siRNA) was conducted to understand the relationship between Nrf2 expression and H2O2-induced growth inhibition. Results: The results showed that EGL effectively inhibited H2O2-induced growth and the generation of ROS. EGL markedly suppressed H2O2-induced comet-like DNA formation and phosphorylation of histone H2AX at serine 139 (p-γH2AX), a widely used marker of DNA damage, suggesting that EGL prevented H2O2-induced DNA damage. Furthermore, the EGL treatment effectively induced the expression of Nrf2, as well as heme oxygenase-1 (HO-1), with parallel phosphorylation and nuclear translocation of Nrf2 in the C2C12 myoblasts. However, zinc protoporphyrin IX, a HO-1 inhibitor, significantly abolished the protective effects of EGL against H2O2-induced accumulation of ROS and reduced cell growth. Notably, transient transfection with Nrf2-specific siRNA attenuated the cytoprotective effects and HO-1 induction by EGL, indicating that EGL induced the expression of HO-1 in an Nrf2-dependent manner. Conclusion: Collectively, these results demonstrate that EGL augments the

  6. Nrf2-driven TERT regulates pentose phosphate pathway in glioblastoma

    PubMed Central

    Ahmad, F; Dixit, D; Sharma, V; Kumar, A; Joshi, S D; Sarkar, C; Sen, E

    2016-01-01

    Given the involvement of telomerase activation and dysregulated metabolism in glioma progression, the connection between these two critical players was investigated. Pharmacological inhibition of human Telomerase reverse transcriptase (hTERT) by Costunolide induced glioma cell apoptosis in a reactive oxygen species (ROS)-dependent manner. Costunolide induced an ROS-dependent increase in p53 abrogated telomerase activity. Costunolide decreased Nrf2 level; and ectopic Nrf2 expression decreased Costunolide-induced ROS generation. While TERT knock-down abrogated Nrf2 levels, overexpression of Nrf2 increased TERT expression. Inhibition of hTERT either by Costunolide, or by siRNA or dominant-negative hTERT (DN-hTERT) abrogated (i) expression of Glucose-6-phosphate dehydrogenase (G6PD) and Transketolase (TKT) – two major nodes in the pentose phosphate (PPP) pathway; and (ii) phosphorylation of glycogen synthase (GS). hTERT knock-down decreased TKT activity and increased glycogen accumulation. Interestingly, siRNA-mediated knock-down of TKT elevated glycogen accumulation. Coherent with the in vitro findings, Costunolide reduced tumor burden in heterotypic xenograft glioma mouse model. Costunolide-treated tumors exhibited diminished TKT activity, heightened glycogen accumulation, and increased senescence. Importantly, glioblastoma multiforme (GBM) patient tumors bearing TERT promoter mutations (C228T and C250T) known to be associated with increased telomerase activity; exhibited elevated Nrf2 and TKT expression and decreased glycogen accumulation. Taken together, our findings highlight the previously unknown (i) role of telomerase in the regulation of PPP and glycogen accumulation and (ii) the involvement of Nrf2-TERT loop in maintaining oxidative defense responses in glioma cells. PMID:27148686

  7. Genetic Evidence of an Evolutionarily Conserved Role for Nrf2 in the Protection against Oxidative Stress

    PubMed Central

    Mukaigasa, Katsuki; Nguyen, Linh T. P.; Li, Li; Nakajima, Hitomi; Yamamoto, Masayuki

    2012-01-01

    Transcription factor Nrf2 is considered a master regulator of antioxidant defense in mammals. However, it is unclear whether this concept is applicable to nonmammalian vertebrates, because no animal model other than Nrf2 knockout mice has been generated to examine the effects of Nrf2 deficiency. Here, we characterized a recessive loss-of-function mutant of Nrf2 (nrf2fh318) in a lower vertebrate, the zebrafish (Danio rerio). In keeping with the findings in the mouse model, nrf2fh318 mutants exhibited reduced induction of the Nrf2 target genes in response to oxidative stress and electrophiles but were viable and fertile, and their embryos developed normally. The nrf2fh318 larvae displayed enhanced sensitivity to oxidative stress and electrophiles, especially peroxides, and pretreatment with an Nrf2-activating compound, sulforaphane, decreased peroxide-induced lethality in the wild type but not nrf2fh318 mutants, indicating that resistance to oxidative stress is highly dependent on Nrf2 functions. These results reveal an evolutionarily conserved role of vertebrate Nrf2 in protection against oxidative stress. Interestingly, there were no significant differences between wild-type and nrf2fh318 larvae with regard to their sensitivity to superoxide and singlet oxygen generators, suggesting that the importance of Nrf2 in oxidative stress protection varies based on the type of reactive oxygen species (ROS). PMID:22949501

  8. Regulation of hemeoxygenase-1 gene expression by Nrf2 and c-Jun in tertiary butylhydroquinone-stimulated rat primary astrocytes

    SciTech Connect

    Park, Jin-Sun; Kim, Hee-Sun

    2014-05-16

    Highlights: • tBHQ increased HO-1 mRNA and protein levels in rat primary astrocytes. • tBHQ enhanced HO-1 gene transcription in an ARE-dependent manner. • tBHQ increased the nuclear translocation and DNA binding of Nrf2 and c-Jun to ARE. • Nrf2 and c-Jun are involved in the differential modulation of HO-1 expression. • Nrf2 and c-Jun regulate HO-1 expression via their coordinated interaction. - Abstract: Hemeoxygenase-1 (HO-1) is a phase II antioxidant enzyme that is primarily involved in detoxification and cytoprotection in a variety of tissues. However, the mechanism underlying HO-1 gene expression remains unclear. In the present study, we investigated the regulation of HO-1 expression in primary cultured astrocytes by using the natural antioxidant compound tertiary butylhydroquinone (tBHQ). We found that tBHQ increased HO-1 mRNA and protein levels. Promoter analysis revealed that tBHQ enhanced HO-1 gene transcription in an antioxidant response element (ARE)-dependent manner. In addition, tBHQ increased the nuclear translocation and DNA binding of Nrf2 and c-Jun to ARE. Small interfering RNA (siRNA) experiments demonstrated that Nrf2 and c-Jun are involved in the differential modulation of HO-1 expression. Thus, Nrf2 knockdown reduced the basal level of HO-1 expression but did not affect the fold induction by tBHQ. On the other hand, knockdown of c-Jun diminished tBHQ-mediated induction of HO-1 without affecting basal expression. The data suggest that Nrf2 generally modulates the basal expression of HO-1, while c-Jun mediates HO-1 induction in response to tBHQ. The results of co-immunoprecipitation assays demonstrated a physical interaction between Nrf2 and c-Jun in tBHQ-treated astrocytes. The results suggest that Nrf2 and c-Jun regulate HO-1 expression via their coordinated interaction in tBHQ-treated rat primary astrocytes.

  9. Dermato-protective properties of ergothioneine through induction of Nrf2/ARE-mediated antioxidant genes in UVA-irradiated Human keratinocytes.

    PubMed

    Hseu, You-Cheng; Lo, Heng-Wei; Korivi, Mallikarjuna; Tsai, Yu-Cheng; Tang, Meng-Ju; Yang, Hsin-Ling

    2015-09-01

    UVA irradiation-induced skin damage and redox imbalance have been shown to be ameliorated by ergothioneine (EGT), a naturally occurring sulfur-containing amino acid. However, the responsible molecular mechanism with nanomolar concentrations of EGT remains unclear. We investigated the dermato protective efficacies of EGT (125-500nM) against UVA irradiation (15J/cm(2)), and elucidated the underlying molecular mechanism in human keratinocyte-derived HaCaT cells. We found that EGT treatment prior to UVA exposure significantly increased the cell viability and prevented lactate dehydrogenase release into the medium. UVA-induced ROS and comet-like DNA formation were remarkably suppressed by EGT with a parallel inhibition of apoptosis, as evidenced by reduced DNA fragmentation (TUNEL), caspase-9/-3 activation, and Bcl-2/Bax dysregulation. Furthermore, EGT alleviated UVA-induced mitochondrial dysfunction. Dose-dependent increases of antioxidant genes, HO-1, NQO-1, and γ-GCLC and glutathione by EGT were associated with upregulated Nrf2 and downregulated Keap-1 expressions. This was confirmed by increased nuclear accumulation of Nrf2 and inhibition of Nrf2 degradation. Notably, augmented luciferase activity of ARE may explain Nrf2/ARE-mediated signaling pathways behind EGT dermato-protective properties. We further demonstrated that Nrf2 translocation was mediated by PI3K/AKT, PKC, or ROS signaling cascades. This phenomenon was confirmed with suppressed nuclear Nrf2 activation, and consequently diminished antioxidant genes in cells treated with respective pharmacological inhibitors (LY294002, GF109203X, and N-acetylcysteine). Besides, increased basal ROS by EGT appears to be crucial for triggering the Nrf2/ARE signaling pathways. Silencing of Nrf2 or OCTN1 (EGT carrier protein) signaling with siRNA showed no such protective effects of EGT against UVA-induced cell death, ROS, and apoptosis, which is evidence of the vitality of Nrf2 translocation and protective efficacy of EGT

  10. Apigenin Attenuates Oxidative Injury in ARPE-19 Cells thorough Activation of Nrf2 Pathway

    PubMed Central

    Li, Min; Chen, Weiwei; Yu, Haitao; Yang, Yan; Hang, Li

    2016-01-01

    The current study was aimed at evaluating the therapeutic implication of apigenin and to elucidate the underlying mechanism. The tert-butyl hydroperoxide (t-BHP) at 200 μM was used to induce oxidative stress-associated injury in ARPE-19 cells. Apigenin at concentrations less than 800 μM did not cause cytotoxic effects on ARPE-19 cells. Cell viability assay showed that apigenin at 200 μM significantly promoted cell survival in t-BHP-treated ARPE-19 cells. Additionally, apigenin at 100 μM significantly protected ARPE-19 cells from t-BHP-induced apoptosis. Molecular examinations demonstrated that apigenin at 400 μM significantly upregulated the mRNA and protein expression of Nrf2 and stimulated its nuclear translocation in ARPE-19 cells treated with or without t-BHP. Apigenin 400 μM also significantly elevated the expression of HO-1, NQO1, and GCLM at both mRNA and protein levels in the presence or absence of t-BHP. Furthermore, apigenin at 400 μM significantly increased the activities of SOD, CAT, GSH-PX, and T-AOC and reduced the levels of ROS and MDA in t-BHP-treated ARPE-19 cells. However, these effects of apigenin were all abolished by being transfected with Nrf2 siRNA. Collectively, our current data indicated that apigenin exerted potent antioxidant properties in ARPE-19 cells challenged with t-BHP, which were dependent on activation of Nrf2 signaling. PMID:27656262

  11. Apigenin Attenuates Oxidative Injury in ARPE-19 Cells thorough Activation of Nrf2 Pathway.

    PubMed

    Xu, Xinrong; Li, Min; Chen, Weiwei; Yu, Haitao; Yang, Yan; Hang, Li

    The current study was aimed at evaluating the therapeutic implication of apigenin and to elucidate the underlying mechanism. The tert-butyl hydroperoxide (t-BHP) at 200 μM was used to induce oxidative stress-associated injury in ARPE-19 cells. Apigenin at concentrations less than 800 μM did not cause cytotoxic effects on ARPE-19 cells. Cell viability assay showed that apigenin at 200 μM significantly promoted cell survival in t-BHP-treated ARPE-19 cells. Additionally, apigenin at 100 μM significantly protected ARPE-19 cells from t-BHP-induced apoptosis. Molecular examinations demonstrated that apigenin at 400 μM significantly upregulated the mRNA and protein expression of Nrf2 and stimulated its nuclear translocation in ARPE-19 cells treated with or without t-BHP. Apigenin 400 μM also significantly elevated the expression of HO-1, NQO1, and GCLM at both mRNA and protein levels in the presence or absence of t-BHP. Furthermore, apigenin at 400 μM significantly increased the activities of SOD, CAT, GSH-PX, and T-AOC and reduced the levels of ROS and MDA in t-BHP-treated ARPE-19 cells. However, these effects of apigenin were all abolished by being transfected with Nrf2 siRNA. Collectively, our current data indicated that apigenin exerted potent antioxidant properties in ARPE-19 cells challenged with t-BHP, which were dependent on activation of Nrf2 signaling.

  12. Lutein Activates the Transcription Factor Nrf2 in Human Retinal Pigment Epithelial Cells.

    PubMed

    Frede, Katja; Ebert, Franziska; Kipp, Anna P; Schwerdtle, Tanja; Baldermann, Susanne

    2017-07-26

    The degeneration of the retinal pigment epithelium caused by oxidative damage is a stage of development in age-related macular degeneration (AMD). The carotenoid lutein is a major macular pigment that may reduce the incidence and progression of AMD, but the underlying mechanism is currently not fully understood. Carotenoids are known to be direct antioxidants. However, carotenoids can also activate cellular pathways resulting in indirect antioxidant effects. Here, we investigate the influence of lutein on the activation of nuclear factor erythroid 2-related factor 2 (Nrf2) target genes in human retinal pigment epithelial cells (ARPE-19 cells) using lutein-loaded Tween40 micelles. The micelles were identified as a suitable delivery system since they were nontoxic in APRE-19 cells up to 0.04% Tween40 and led to a cellular lutein accumulation of 62 μM ± 14 μM after 24 h. Lutein significantly enhanced Nrf2 translocation to the nucleus 1.5 ± 0.4-fold compared to that of unloaded micelles after 4 h. Furthermore, lutein treatment for 24 h significantly increased the transcripts of NAD(P)H:quinone oxidoreductase 1 (NQO1) by 1.7 ± 0.1-fold, glutamate-cysteine ligase regulatory subunit (GCLm) by 1.4 ± 0.1-fold, and heme oxygenase-1 (HO-1) by 1.8 ± 0.3-fold. Moreover, we observed a significant enhancement of NQO1 activity by 1.2 ± 0.1-fold. Collectively, this study indicates that lutein not only serves as a direct antioxidant but also activates Nrf2 in ARPE-19 cells.

  13. Alkaloids from Piper nigrum Exhibit Antiinflammatory Activity via Activating the Nrf2/HO-1 Pathway.

    PubMed

    Ngo, Quynh Mai Thi; Tran, Phuong Thao; Tran, Manh Hung; Kim, Jeong Ah; Rho, Seong Soo; Lim, Chi-Hwan; Kim, Jin-Cheol; Woo, Mi Hee; Choi, Jae Sui; Lee, Jeong-Hyung; Min, Byung Sun

    2017-04-01

    In the present study, ten alkaloids, namely chabamide (1), pellitorine (2), retrofractamide A (3), pyrroperine (4), isopiperolein B (5), piperamide C9:1 (8E) (6), 6,7-dehydrobrachyamide B (7), 4,5-dihydropiperine (8), dehydropipernonaline (9), and piperine (10), were isolated from the fruits of Piper nigrum. Among these, chabamide (1), pellitorine (2), retrofractamide A (3), isopiperolein B (5), and 6,7-dehydrobrachyamide B (7) exhibited significant inhibitory activity on lipopolysaccharide-induced nitric oxide (NO) production in RAW264.7 cells, with IC50 values of 6.8, 14.5, 30.2, 23.7, and 38.5 μM, respectively. Furthermore, compound 1 inhibited lipopolysaccharide-induced NO production in bone marrow-derived macrophages with IC50 value of 9.5 μM. Consistent with NO inhibition, treatment of RAW264.7 cells with chabamide (1), pellitorine (2), and 6,7-dehydrobrachyamide B (7) suppressed expression of inducible NO synthase and cyclooxygenase-2. Chabamide (1), pellitorine (2), and 6,7-dehydrobrachyamide B (7) induced heme-oxygenase-1 expression at the transcriptional level. In addition, compound 1 induced the nuclear translocation of nuclear factor-E2-related factor 2 (Nrf2) and upregulated the expression of Nrf2 target genes, NAD(P)H:quinone oxidoreductase 1 and γ-glutamyl cysteine synthetase catalytic subunit, in a concentration-dependent manner in RAW264.7 cells. These findings suggest that chabamide (1) from P. nigrum exert antiinflammatory effects via the activation of the Nrf2/heme-oxygenase-1 pathway; hence, it might be a promising candidate for the treatment of inflammatory diseases. Copyright © 2017 John Wiley & Sons, Ltd.

  14. Evaluation of the rotenone-induced activation of the Nrf2 pathway in a neuronal model derived from human induced pluripotent stem cells.

    PubMed

    Zagoura, Dimitra; Canovas-Jorda, David; Pistollato, Francesca; Bremer-Hoffmann, Susanne; Bal-Price, Anna

    2016-09-09

    Human induced pluripotent stem cells (hiPSCs) are considered as a powerful tool for drug and chemical screening and development of new in vitro testing strategies in the field of toxicology, including neurotoxicity evaluation. These cells are able to expand and efficiently differentiate into different types of neuronal and glial cells as well as peripheral neurons. These human cells-based neuronal models serve as test systems for mechanistic studies on different pathways involved in neurotoxicity. One of the well-known mechanisms that are activated by chemically-induced oxidative stress is the Nrf2 signaling pathway. Therefore, in the current study, we evaluated whether Nrf2 signaling machinery is expressed in human induced pluripotent stem cells (hiPSCs)-derived mixed neuronal/glial culture and if so whether it becomes activated by rotenone-induced oxidative stress mediated by complex I inhibition of mitochondrial respiration. Rotenone was found to induce the activation of Nrf2 signaling particularly at the highest tested concentration (100 nM), as shown by Nrf2 nuclear translocation and the up-regulation of the Nrf2-downstream antioxidant enzymes, NQO1 and SRXN1. Interestingly, exposure to rotenone also increased the number of astroglial cells in which Nrf2 activation may play an important role in neuroprotection. Moreover, rotenone caused cell death of dopaminergic neurons since a decreased percentage of tyrosine hydroxylase (TH(+)) cells was observed. The obtained results suggest that hiPSC-derived mixed neuronal/glial culture could be a valuable in vitro human model for the establishment of neuronal specific assays in order to link Nrf2 pathway activation (biomarker of oxidative stress) with additional neuronal specific readouts that could be applied to in vitro neurotoxicity evaluation.

  15. Lung endothelial barrier protection by resveratrol involves inhibition of HMGB1 release and HMGB1-induced mitochondrial oxidative damage via an Nrf2-dependent mechanism.

    PubMed

    Dong, Wen-Wen; Liu, Yu-Jian; Lv, Zhou; Mao, Yan-Fei; Wang, Ying-Wei; Zhu, Xiao-Yan; Jiang, Lai

    2015-11-01

    High-mobility group box 1 (HMGB1) contributes to lung vascular hyperpermeability during ventilator-induced lung injury. We aimed to determine whether the natural antioxidant resveratrol protected against HMGB1-induced endothelial hyperpermeability both in vitro and in vivo. We found that HMGB1 decreased vascular endothelial (VE)-cadherin expression and increased endothelial permeability, leading to mitochondrial oxidative damage in primary cultured mouse lung vascular endothelial cells (MLVECs). Both the mitochondrial superoxide dismutase 2 mimetic MnTBAP and resveratrol blocked HMGB1-induced mitochondrial oxidative damage, VE-cadherin downregulation, and endothelial hyperpermeability. In in vivo studies, anesthetized male ICR mice were ventilated for 4h using low tidal volume (6 ml/kg) or high tidal volume (HVT; 30 ml/kg) ventilation. The mice were injected intraperitoneally with resveratrol immediately before the onset of ventilation. We found that resveratrol attenuated HVT-associated lung vascular hyperpermeability and HMGB1 production. HVT caused a significant increase in nuclear factor-erythroid 2-related factor 2 (Nrf2) nuclear translocation and Nrf2 target gene expression in lung tissues, which was further enhanced by resveratrol treatment. HMGB1 had no effect on Nrf2 activation, whereas resveratrol treatment activated the Nrf2 signaling pathway in HMGB1-treated MLVECs. Moreover, Nrf2 knockdown reversed the inhibitory effects of resveratrol on HMGB1-induced mitochondrial oxidative damage and endothelial hyperpermeability. The inhibitory effect of resveratrol on cyclic stretch-induced HMGB1 mRNA expression in primary cultured MLVECs was also abolished by Nrf2 knockdown. In summary, this study demonstrates that resveratrol protects against lung endothelial barrier dysfunction initiated by HVT. Lung endothelial barrier protection by resveratrol involves inhibition of mechanical stretch-induced HMGB1 release and HMGB1-induced mitochondrial oxidative damage

  16. Upregulation of thioredoxin system via Nrf2-antioxidant responsive element pathway in adaptive-retinal neuroprotection in vivo and in vitro.

    PubMed

    Tanito, Masaki; Agbaga, Martin-Paul; Anderson, Robert E

    2007-06-15

    We tested the hypothesis that stress responses mediated by the Nrf2-antioxidant responsive element (ARE) pathway are involved in the initiation of retinal neuroprotection provided by bright-cyclic-light rearing. Albino rats born and raised in dim (5 lux) or bright (400 lux) cyclic light were exposed to damaging light (3000 lux, 6 h). After exposure, the outer nuclear layer thickness and area and the electroretinogram a- and b-wave amplitudes were significantly reduced in the dim-light-reared rats compared to the bright-light-reared rats, demonstrating a light adaptation neuroprotection phenomenon. In bright-cyclic-light-reared rats, the retinal levels of thioredoxin (Trx) (2.4-fold), Trx reductase (TrxR) (2.9-fold), and proteins modified by 4-hydroxynonenal (4-HNE) (1.5-fold) were upregulated by Western blot analyses, and the nuclear translocation of Nrf2 (2.2-fold) and the DNA binding activity of Nrf2, small Maf, and cJun to the ARE were increased as determined by electrophoretic mobility shift assays. In mouse photoreceptor-derived 661W cells, pretreatment with a sublethal dose of 4-HNE protected against H(2)O(2)-induced cell damage. Treatment with 4-HNE upregulated cellular Trx, TrxR, and heme oxygenase-1 (HO-1) levels in addition to DNA binding activity of Nrf2, small Maf, and cJun to the ARE. Downregulation of Nrf2 using RNA interference technology diminished 4-HNE-mediated upregulation of Trx and Trx reductase but did not affect the upregulation of HO-1 by 4-HNE. Cytoprotection by 4-HNE pretreatment against H(2)O(2)-induced cell damage was not observed in 661W cells with a silenced Nrf2 gene. The results suggest that upregulation of the Trx system by 4-HNE via the Nrf2-ARE pathway may be involved in the molecular mechanism of the retinal neuroprotection phenomenon.

  17. Role of nuclear factor-erythroid 2-related factor 2 (Nrf2) in the transcriptional regulation of brain ABC transporters during acute acetaminophen (APAP) intoxication in mice.

    PubMed

    Ghanem, Carolina I; Rudraiah, Swetha; Bataille, Amy M; Vigo, María B; Goedken, Michael J; Manautou, José E

    2015-04-01

    Changes in expression of liver ABC transporters have been described during acute APAP intoxication. However, the effect of APAP on brain ABC transporters is poorly understood. The aim of this study was to evaluate the effect of APAP on brain ABC transporters expression and the role of the oxidative stress sensor Nrf2. Male C57BL/6J mice were administered APAP (400mg/kg) for analysis of brain mRNA and protein expression of Mrp1-6, Bcrp and P-gp. The results show induction of P-gp, Mrp2 and Mrp4 proteins, with no changes in Bcrp, Mrp1 or Mrp5-6. The protein values were accompanied by corresponding changes in mRNA levels. Additionally, brain Nrf2 nuclear translocation and expression of two Nrf2 target genes, quinone oxidoreductase 1 (Nqo1) and Hemoxygenase 1 (Ho-1), was evaluated at 6, 12 and 24h after APAP treatment. Nrf2 nuclear content increased by 58% at 12h after APAP along with significant increments in mRNA and protein expression of Nqo1 and Ho-1. Furthermore, APAP treated Nrf2 knockout mice did not increase mRNA or protein expression of Mrp2 and Mrp4 as observed in wildtypes. In contrast, P-gp induction by APAP was observed in both genotypes. In conclusion, acute APAP intoxication induces protein expression of brain P-gp, Mrp2 and Mrp4. This study also suggests that brain changes in Mrp2 and Mrp4 expression may be due to in situ Nrf2 activation by APAP, while P-gp induction is independent of Nrf2 function. The functional consequences of these changes in brain ABC transporters by APAP deserve further attention. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Quinone-induced activation of Keap1/Nrf2 signaling by aspirin prodrugs masquerading as nitric oxide.

    PubMed

    Dunlap, Tareisha; Piyankarage, Sujeewa C; Wijewickrama, Gihani T; Abdul-Hay, Samer; Vanni, Michael; Litosh, Vladislav; Luo, Jia; Thatcher, Gregory R J

    2012-12-17

    The promising therapeutic potential of the NO-donating hybrid aspirin prodrugs (NO-ASA) includes induction of chemopreventive mechanisms and has been reported in almost 100 publications. One example, NCX-4040 (pNO-ASA), is bioactivated by esterase to a quinone methide (QM) electrophile. In cell cultures, pNO-ASA and QM-donating X-ASA prodrugs that cannot release NO rapidly depleted intracellular GSH and caused DNA damage; however, induction of Nrf2 signaling elicited cellular defense mechanisms including upregulation of NAD(P)H:quinone oxidoreductase-1 (NQO1) and glutamate-cysteine ligase (GCL). In HepG2 cells, the "NO-specific" 4,5-diaminofluorescein reporter, DAF-DA, responded to NO-ASA and X-ASA, with QM-induced oxidative stress masquerading as NO. LC-MS/MS analysis demonstrated efficient alkylation of Cys residues of proteins including glutathione-S-transferase-P1 (GST-P1) and Kelch-like ECH-associated protein 1 (Keap1). Evidence was obtained for alkylation of Keap1 Cys residues associated with Nrf2 translocation to the nucleus, nuclear translocation of Nrf2, activation of antioxidant response element (ARE), and upregulation of cytoprotective target genes. At least in cell culture, pNO-ASA acts as a QM donor, bioactivated by cellular esterase activity to release salicylates, NO(3)(-), and an electrophilic QM. Finally, two novel aspirin prodrugs were synthesized, both potent activators of ARE, designed to release only the QM and salicylates on bioactivation. Current interest in electrophilic drugs acting via Nrf2 signaling suggests that QM-donating hybrid drugs can be designed as informative chemical probes in drug discovery.

  19. Taurine Chloramine Stimulates Efferocytosis Through Upregulation of Nrf2-Mediated Heme Oxygenase-1 Expression in Murine Macrophages: Possible Involvement of Carbon Monoxide.

    PubMed

    Kim, Wonki; Kim, Hoon-Ui; Lee, Ha-Na; Kim, Seung Hyeon; Kim, Chaekyun; Cha, Young-Nam; Joe, Yeonsoo; Chung, Hun Taeg; Jang, Jaebong; Kim, Kyeojin; Suh, Young-Ger; Jin, Hyeon-Ok; Lee, Jin Kyung; Surh, Young-Joon

    2015-07-10

    To examine the pro-resolving effects of taurine chloramine (TauCl). TauCl injected into the peritoneum of mice enhanced the resolution of zymosan A-induced peritonitis. Furthermore, when the macrophages obtained from peritoneal exudates were treated with TauCl, their efferocytic ability was elevated. In the murine macrophage-like RAW264.7 cells exposed to TauCl, the proportion of macrophages engulfing the apoptotic neutrophils was also increased. In these macrophages treated with TauCl, expression of heme oxygenase-1 (HO-1) was elevated along with increased nuclear translocation of the nuclear factor E2-related factor 2 (Nrf2). TauCl binds directly to Kelch-like ECH association protein 1 (Keap1), which appears to retard the Keap1-driven degradation of Nrf2. This results in stabilization and enhanced nuclear translocation of Nrf2 and upregulation of HO-1 expression. TauCl, when treated to peritoneal macrophages isolated from either Nrf2 or HO-1 wild-type mice, stimulated efferocytosis (phagocytic engulfment of apoptotic neutrophils by macrophages), but not in the macrophages from Nrf2 or HO-1 knockout mice. Furthermore, transcriptional expression of some scavenger receptors recognizing the phosphatidylserines exposed on the surface of apoptotic cells was increased in RAW264.7 cells treated with TauCl. Pharmacologic inhibition of HO-1 activity or knockdown of HO-1 gene in RAW264.7 cells abolished the TauCl-induced efferocytosis, whereas both overexpression of HO-1 and treatment with carbon monoxide (CO), the product of HO, potentiated the efferocytic activity of macrophages. This work provides the first evidence that TauCl stimulates efferocytosis by macrophages. The results of this study suggest the therapeutic potential of TauCl in the management of inflammatory disorders. TauCl can facilitate resolution of inflammation by increasing the efferocytic activity of macrophages through Nrf2-mediated HO-1 upregulation and subsequent production of CO.

  20. DL-3-n-Butylphthalide (NBP) Provides Neuroprotection in the Mice Models After Traumatic Brain Injury via Nrf2-ARE Signaling Pathway.

    PubMed

    Liu, Zhengwei; Wang, Handong; Shi, Xiaofeng; Li, Liwen; Zhou, Mengliang; Ding, Hui; Yang, Youqing; Li, Xiang; Ding, Ke

    2017-02-18

    The present study was aimed to evaluate the neuroprotective effects of NBP in the mice models of TBI, as well as the possible role of Nrf2-ARE pathways in the assumptive neuroprotection. In mice,a modified Marmarou's weight-drop model was employed to induce TBI. ICR mice were randomly assigned to four experimental groups: sham, TBI, TBI+vehicle(V) and TBI+NBP. NBP (100 mg/kg) was administered via an intraperitoneal (i.p.) injection at 1 h following TBI. The administration of NBP significantly ameliorated the effects of the brain injury, including neurological deficits, brain water content, and cortical neuronal apoptosis. Furthermore, the level of malondialdehyde and the activity of superoxide dismutase (SOD) paired with glutathione peroxidase (GPx) were restored in the NBP treatment group. NBP promoted the translocation of Nrf2 protein from the cytoplasm to the nucleus markedly, increased the expressions of Nrf2-ARE pathway-related downstream factors, including hemeoxygenase-1(HO-1) and NAD(P)H: quinone oxidoreductase 1 (NQO1), and prevented the decline of antioxidant enzyme activities, including SOD and GPx. NBP enhanced the translocation of Nrf2 to the nucleus from the cytoplasm,verified by a western blot, immunofluorescence. Additionally, it upregulated the expression of the Nrf2 downstream factors such as HO-1 and NQO1 were also confirmed via a western blot and real-time quantitative polymerase chain reaction. In conclusion, NBP administration may increase the activities of antioxidant enzymes and attenuate brain injury in a TBI model, potentially via the mediation of the Nrf2-ARE pathway.

  1. Myofibrillogenesis regulator-1 attenuated hypoxia/reoxygenation-induced apoptosis by inhibiting the PERK/Nrf2 pathway in neonatal rat cardiomyocytes.

    PubMed

    Tao, Tian-Qi; Wang, Xiao-Reng; Liu, Mi; Xu, Fei-Fei; Liu, Xiu-Hua

    2015-03-01

    The purpose of this study was to investigate the role of myofibrillogenesis regulator-1 (MR-1) in cardiomyocyte apoptosis induced by hypoxia/reoxygenation (H/R), through protein kinase R-like ER kinase (PERK)/nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. To address this aim, an H/R model of neonatal rat cardiomyocytes was used. MR-1 was overexpressed using an adenoviral vector system and knocked down using MR-1 specific siRNA. Apoptosis was assessed by using Annexin V/PI double staining, terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling assay, and the Bcl-2/Bax ratio. Western blotting was used to detect the protein levels of MR-1, glucose-regulated protein 78 (GRP78), total and phosphorylated PERK, Nrf2, activating transcription factor 4 (ATF4), C/EBP homologous protein (CHOP), Bcl-2 and Bax. Immunofluorescence staining was used to assess the subcellular location of Nrf2. We found that H/R induced significant apoptosis in neonatal rat cardiomyocytes. MR-1 overexpression attenuated H/R-induced apoptosis, decreased GRP78 (P < 0.01) and CHOP expression (P < 0.05), and increased the Bcl-2/Bax ratio (P < 0.01). MR-1 overexpression suppressed H/R-induced PERK phosphorylation, Nrf2 nuclear translocation, and ATF4 expression (P < 0.01). While MR-1 knockdown aggravated H/R-induced apoptosis, increased expression of GRP78 and CHOP (P < 0.05), and decreased the Bcl-2/Bax ratio (P < 0.01). MR-1 knockdown significantly increased H/R-induced PERK phosphorylation (P < 0.05), Nrf2 nuclear translocation, and ATF4 expression (P < 0.01). These findings suggest that MR-1 alleviates H/R-induced cardiomyocyte apoptosis through inhibition of the PERK/Nrf2 pathway.

  2. Effects of monascin on anti-inflammation mediated by Nrf2 activation in advanced glycation end product-treated THP-1 monocytes and methylglyoxal-treated wistar rats.

    PubMed

    Lee, Bao-Hong; Hsu, Wei-Hsuan; Huang, Tao; Chang, Yu-Ying; Hsu, Ya-Wen; Pan, Tzu-Ming

    2013-02-13

    Hyperglycemia is associated with advanced glycation end products (AGEs). This study was designed to evaluate the inhibitory effects of monascin on receptor for advanced glycation end product (RAGE) signal and THP-1 monocyte inflammation after treatment with S100b, a specific ligand of RAGE. Monascin inhibited cytokine production by S100b-treated THP-1 monocytes via up-regulation of nuclear factor-erythroid 2-related factor-2 (Nrf2) and alleviated p47phox translocation to the membrane. Methylglyoxal (MG, 600 mg/kg bw) was used to induce diabetes in Wistar rats. Inhibitions of RAGE and p47phox by monascin were confirmed by peripheral blood mononuclear cells (PBMCs) of MG-induced rats. Silymarin (SM) was used as a positive control group. It was found that monascin promoted heme oxygenase-1 (HO-1) expression mediated by Nrf2. Suppressions of AGEs, tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-β) in serum of MG-induced rats were attenuated in the monascin administration group treated with retinoic acid (RA). RA treatment resulted in Nrf2 inactivation by increasing RA receptor-α (RARα) activity, suggesting that RA acts as an inhibitor of Nrf2. The results showed that monascin exerted anti-inflammatory and antioxidative effects mediated by Nrf2 to prevent the development of diseases such as type 2 diabetes caused by inflammation.

  3. Anti-inflammatory activity of xanthohumol involves heme oxygenase-1 induction via NRF2-ARE signaling in microglial BV2 cells.

    PubMed

    Lee, Ik-Soo; Lim, Juhee; Gal, Jiyeong; Kang, Jeen Chu; Kim, Hyun Jung; Kang, Bok Yun; Choi, Hyun Jin

    2011-02-01

    Xanthohumol (2',4',4-trihydroxy-6'-methoxy-3'-prenylchalcone) is a major chalcone derivative isolated from hop (Humulus lupulus L.) commonly used in brewing due to its bitter flavors. Xanthohumol has anti-carcinogenic, free radical-scavenging, and anti-inflammatory activities, but its precise mechanisms are not clarified yet. The basic leucine zipper (bZIP) protein NRF2 is a key transcription factor mediating the antioxidant and anti-inflammatory responses in animals. Therefore, we tested whether xanthohumol exerts anti-inflammatory activity in mouse microglial BV2 cells via NRF2 signaling. Xanthohumol significantly inhibited the excessive production of inflammatory mediators NO, IL-1β, and TNF-α, and the activation of NF-κB signaling in LPS-induced stimulated BV2 cells. Xanthohumol up-regulated the transcription of NAD(P)H:quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1), and increased the level of the endogenous antioxidant GSH. In addition, xanthohumol induced nuclear translocation of NRF2 and further activation of ARE promoter-related transcription. The anti-inflammatory response of xanthohumol was attenuated by transfection with NRF2 siRNA and in the presence of the HO-1 inhibitor, ZnPP, but not the NQO1 inhibitor, dicoumarol. Taken together, our study suggests that xanthohumol exerts anti-inflammatory activity through NRF2-ARE signaling and up-regulation of downstream HO-1, and could be an attractive candidate for the regulation of inflammatory responses in the brain.

  4. Role of Nrf2 in Oxidative Stress and Toxicity

    PubMed Central

    Ma, Qiang

    2015-01-01

    Organismal life encounters reactive oxidants from internal metabolism and environmental toxicant exposure. Reactive oxygen and nitrogen species cause oxidative stress and are traditionally viewed as being harmful. On the other hand, controlled production of oxidants in normal cells serves useful purposes to regulate signaling pathways. Reactive oxidants are counterbalanced by complex antioxidant defense systems regulated by a web of pathways to ensure that the response to oxidants is adequate for the body’s needs. A recurrent theme in oxidant signaling and antioxidant defense is reactive cysteine thiol–based redox signaling. The nuclear factor erythroid 2–related factor 2 (Nrf2) is an emerging regulator of cellular resistance to oxidants. Nrf2 controls the basal and induced expression of an array of antioxidant response element–dependent genes to regulate the physiological and pathophysiological outcomes of oxidant exposure. This review discusses the impact of Nrf2 on oxidative stress and toxicity and how Nrf2 senses oxidants and regulates antioxidant defense. PMID:23294312

  5. Role of Nrf2 and Autophagy in Acute Lung Injury

    PubMed Central

    de la Vega, Montserrat Rojo; Dodson, Matthew; Gross, Christine; Manzour, Heidi; Lantz, R. Clark; Chapman, Eli; Wang, Ting; Black, Stephen M.; Garcia, Joe G.N.; Zhang, Donna D.

    2016-01-01

    Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are the clinical manifestations of severe lung damage and respiratory failure. Characterized by severe inflammation and compromised lung function, ALI/ARDS result in very high mortality of affected individuals. Currently, there are no effective treatments for ALI/ARDS, and ironically, therapies intended to aid patients (specifically mechanical ventilation, MV) may aggravate the symptoms. Key events contributing to the development of ALI/ARDS are: increased oxidative and proteotoxic stresses, unresolved inflammation, and compromised alveolar-capillary barrier function. Since the airways and lung tissues are constantly exposed to gaseous oxygen and airborne toxicants, the bronchial and alveolar epithelial cells are under higher oxidative stress than other tissues. Cellular protection against oxidative stress and xenobiotics is mainly conferred by Nrf2, a transcription factor that promotes the expression of genes that regulate oxidative stress, xenobiotic metabolism and excretion, inflammation, apoptosis, autophagy, and cellular bioenergetics. Numerous studies have demonstrated the importance of Nrf2 activation in the protection against ALI/ARDS, as pharmacological activation of Nrf2 prevents the occurrence or mitigates the severity of ALI/ARDS. Another promising new therapeutic strategy in the prevention and treatment of ALI/ARDS is the activation of autophagy, a bulk protein and organelle degradation pathway. In this review, we will discuss the strategy of concerted activation of Nrf2 and autophagy as a preventive and therapeutic intervention to ameliorate ALI/ARDS. PMID:27313980

  6. Brusatol provokes a rapid and transient inhibition of Nrf2 signaling and sensitizes mammalian cells to chemical toxicity-implications for therapeutic targeting of Nrf2.

    PubMed

    Olayanju, Adedamola; Copple, Ian M; Bryan, Holly K; Edge, George T; Sison, Rowena L; Wong, Min Wei; Lai, Zheng-Quan; Lin, Zhi-Xiu; Dunn, Karen; Sanderson, Christopher M; Alghanem, Ahmad F; Cross, Michael J; Ellis, Ewa C; Ingelman-Sundberg, Magnus; Malik, Hassan Z; Kitteringham, Neil R; Goldring, Christopher E; Park, B Kevin

    2015-01-01

    The transcription factor Nrf2 regulates the basal and inducible expression of a battery of cytoprotective genes. Whereas numerous Nrf2-inducing small molecules have been reported, very few chemical inhibitors of Nrf2 have been identified to date. The quassinoid brusatol has recently been shown to inhibit Nrf2 and ameliorate chemoresistance in vitro and in vivo. Here, we show that brusatol provokes a rapid and transient depletion of Nrf2 protein, through a posttranscriptional mechanism, in mouse Hepa-1c1c7 hepatoma cells. Importantly, brusatol also inhibits Nrf2 in freshly isolated primary human hepatocytes. In keeping with its ability to inhibit Nrf2 signaling, brusatol sensitizes Hepa-1c1c7 cells to chemical stress provoked by 2,4-dinitrochlorobenzene, iodoacetamide, and N-acetyl-p-benzoquinone imine, the hepatotoxic metabolite of acetaminophen. The inhibitory effect of brusatol toward Nrf2 is shown to be independent of its repressor Keap1, the proteasomal and autophagic protein degradation systems, and protein kinase signaling pathways that are known to modulate Nrf2 activity, implying the involvement of a novel means of Nrf2 regulation. These findings substantiate brusatol as a useful experimental tool for the inhibition of Nrf2 signaling and highlight the potential for therapeutic inhibition of Nrf2 to alter the risk of adverse events by reducing the capacity of nontarget cells to buffer against chemical and oxidative insults. These data will inform a rational assessment of the risk:benefit ratio of inhibiting Nrf2 in relevant therapeutic contexts, which is essential if compounds such as brusatol are to be developed into efficacious and safe drugs.

  7. RETRACTED: S-allyl cysteine protects against 6-hydroxydopamine-induced neurotoxicity in the rat striatum: involvement of Nrf2 transcription factor activation and modulation of signaling kinase cascades.

    PubMed

    Tobón-Velasco, Julio César; Vázquez-Victorio, Genaro; Macías-Silva, Marina; Cuevas, Elvis; Ali, Syed F; Maldonado, Perla D; González-Trujano, María Eva; Cuadrado, Antonio; Pedraza-Chaverrí, José; Santamaría, Abel

    2012-09-01

    Pharmacological activation at the basal ganglia of the transcription factor Nrf2, guardian of redox homeostasis, holds a strong promise for the slow progression of Parkinson's disease (PD). However, a potent Nrf2 activator in the brain still must be found. In this study, we have investigated the potential use of the antioxidant compound S-allyl cysteine (SAC) in the activation of Nrf2 in 6-hydoxydopamine (6-OHDA)-intoxicated rats. In the rat striatum, SAC by itself promoted the Nrf2 dissociation of Keap-1, its nuclear translocation, the subsequent association with small MafK protein, and further binding of the Nrf2/MafK complex to ARE sequence, as well as the up-regulation of Nrf2-dependent genes encoding the antioxidant enzymes HO-1, NQO-1, GR, and SOD-1. In vivo and in vitro experiments to identify signaling pathways activated by SAC pointed to Akt as the most likely kinase participating in Nrf2 activation by SAC. In PC12 cells, SAC stimulated the activation of Akt and ERK1/2 and inhibited JNK1/2/3 activation. In the rat striatum, the SAC-induced activation of Nrf2 is likely to contribute to inhibit the toxic effects of 6-OHDA evidenced by phase 2 antioxidant enzymes up-regulation, glutathione recovery, and attenuation of reactive oxygen species (ROS), nitric oxide (NO), and lipid peroxides formation. These early protective effects correlated with the long-term preservation of the cellular redox status, the striatal dopamine (DA) and tyrosine hydroxylase (TH) levels, and the improvement of motor skills. Therefore, this study indicates that, in addition to direct scavenging actions, the activation of Nrf2 by SAC might confer neuroprotective responses through the modulation of kinase signaling pathways in rodent models of PD, and suggests that this antioxidant molecule may have a therapeutic value in this human pathology.

  8. Hyperactivation of Nrf2 in early tubular development induces nephrogenic diabetes insipidus

    PubMed Central

    Suzuki, Takafumi; Seki, Shiori; Hiramoto, Keiichiro; Naganuma, Eriko; Kobayashi, Eri H.; Yamaoka, Ayaka; Baird, Liam; Takahashi, Nobuyuki; Sato, Hiroshi; Yamamoto, Masayuki

    2017-01-01

    NF-E2-related factor-2 (Nrf2) regulates cellular responses to oxidative and electrophilic stress. Loss of Keap1 increases Nrf2 protein levels, and Keap1-null mice die of oesophageal hyperkeratosis because of Nrf2 hyperactivation. Here we show that deletion of oesophageal Nrf2 in Keap1-null mice allows survival until adulthood, but the animals develop polyuria with low osmolality and bilateral hydronephrosis. This phenotype is caused by defects in water reabsorption that are the result of reduced aquaporin 2 levels in the kidney. Renal tubular deletion of Keap1 promotes nephrogenic diabetes insipidus features, confirming that Nrf2 activation in developing tubular cells causes a water reabsorption defect. These findings suggest that Nrf2 activity should be tightly controlled during development in order to maintain renal homeostasis. In addition, tissue-specific ablation of Nrf2 in Keap1-null mice might create useful animal models to uncover novel physiological functions of Nrf2. PMID:28233855

  9. Hyperactivation of Nrf2 in early tubular development induces nephrogenic diabetes insipidus.

    PubMed

    Suzuki, Takafumi; Seki, Shiori; Hiramoto, Keiichiro; Naganuma, Eriko; Kobayashi, Eri H; Yamaoka, Ayaka; Baird, Liam; Takahashi, Nobuyuki; Sato, Hiroshi; Yamamoto, Masayuki

    2017-02-24

    NF-E2-related factor-2 (Nrf2) regulates cellular responses to oxidative and electrophilic stress. Loss of Keap1 increases Nrf2 protein levels, and Keap1-null mice die of oesophageal hyperkeratosis because of Nrf2 hyperactivation. Here we show that deletion of oesophageal Nrf2 in Keap1-null mice allows survival until adulthood, but the animals develop polyuria with low osmolality and bilateral hydronephrosis. This phenotype is caused by defects in water reabsorption that are the result of reduced aquaporin 2 levels in the kidney. Renal tubular deletion of Keap1 promotes nephrogenic diabetes insipidus features, confirming that Nrf2 activation in developing tubular cells causes a water reabsorption defect. These findings suggest that Nrf2 activity should be tightly controlled during development in order to maintain renal homeostasis. In addition, tissue-specific ablation of Nrf2 in Keap1-null mice might create useful animal models to uncover novel physiological functions of Nrf2.

  10. Role of Nrf2 activation and NF-κB inhibition in valproic acid induced hepatotoxicity and in diammonium glycyrrhizinate induced protection in mice.

    PubMed

    Jin, Jing; Xiong, Tianqin; Hou, Xiangyu; Sun, Xiaozhe; Liao, Jiayi; Huang, Zhiying; Huang, Min; Zhao, Zhongxiang

    2014-11-01

    Diammonium glycyrrhizinate (DG), an active compound extracted and purified from liquorices root, has been reported to exhibit antioxidant and anti-inflammatory properties. The aim of this study was to investigate the effect and underlying mechanisms of DG on the hepatotoxicity induced by valproic acid (VPA). DG at the dose of 60mg/kg was orally administered with VPA (100mg/kg) to mice once daily for 14 consecutive days. DG treatment attenuated VPA-induced liver dysfunction, structural damage, glutathione depletion and decrease in antioxidant enzymes in BALB/C mice. DG prevented VPA-induced depletion of cytosolic nuclear factor E2-related factor 2 (Nrf2) and suppression of nuclear translocation of Nrf2, which, in turn, up-regulated phase II/antioxidant enzyme activities. The effects of VPA and DG on Nrf2 expression in HepG2 cells were in consistent with that of mice. Furthermore, an increase in the nuclear levels of nuclear factor-kappaB (NF-κB) was observed in the livers of VPA-treated mice that coincided with induction of inflammatory cytokines. In contrast, DG inhibited NF-κB translocation and that subsequently decreased inflammatory cytokines. Taken together, these results demonstrate that DG attenuates VPA-induced liver injury through increasing the expression of Nrf2 mediated phase II/antioxidant enzymes and simultaneously decreasing the expression of inflammatory mediators. Copyright © 2014 Elsevier Ltd. All rights reserved.

  11. Role of Nrf2 in preventing ethanol-induced oxidative stress and lipid accumulation

    SciTech Connect

    Wu, Kai Connie; Liu, Jie; Klaassen, Curtis D.

    2012-08-01

    Oxidative stress and lipid accumulation play important roles in alcohol-induced liver injury. Previous reports showed that, in livers of nuclear factor erythroid 2-related factor 2 (Nrf2)-activated mice, genes involved in antioxidant defense are induced, whereas genes involved in lipid biosynthesis are suppressed. To investigate the role of Nrf2 in ethanol-induced hepatic alterations, Nrf2-null mice, wild-type mice, kelch-like ECH-associated protein 1-knockdown (Keap1-KD) mice with enhanced Nrf2, and Keap1-hepatocyte knockout (Keap1-HKO) mice with maximum Nrf2 activation, were treated with ethanol (5 g/kg, po). Blood and liver samples were collected 6 h thereafter. Ethanol increased alanine aminotransferase and lactate dehydrogenase activities as well as thiobarbituric acid reactive substances in serum of Nrf2-null and wild-type mice, but not in Nrf2-enhanced mice. After ethanol administration, mitochondrial glutathione concentrations decreased markedly in Nrf2-null mice but not in Nrf2-enhanced mice. H{sub 2}DCFDA staining of primary hepatocytes isolated from the four genotypes of mice indicates that oxidative stress was higher in Nrf2-null cells, and lower in Nrf2-enhanced cells than in wild-type cells. Ethanol increased serum triglycerides and hepatic free fatty acids in Nrf2-null mice, and these increases were blunted in Nrf2-enhanced mice. In addition, the basal mRNA and nuclear protein levels of sterol regulatory element-binding protein 1(Srebp-1) were decreased with graded Nrf2 activation. Ethanol further induced Srebp-1 mRNA in Nrf2-null mice but not in Nrf2-enhanced mice. In conclusion, Nrf2 activation prevented alcohol-induced oxidative stress and accumulation of free fatty acids in liver by increasing genes involved in antioxidant defense and decreasing genes involved in lipogenesis. -- Highlights: ► Ethanol depleted mitochondrial GSH in Nrf2-null mice but not in Keap1-KD mice. ► Ethanol increased ROS in hepatocytes isolated from Nrf2-null and wild

  12. Delayed treatment with oleanolic acid attenuates tubulointerstitial fibrosis in chronic cyclosporine nephropathy through Nrf2/HO-1 signaling

    PubMed Central

    2014-01-01

    Background Nuclear factor erythroid-2-related factor-2 (Nrf2) is known to protect against tissue injury by orchestrating antioxidant and detoxification responses to oxidative stress. This study investigated whether upregulation of Nrf2-dependent signaling by oleanolic acid (OA), which is known to activate Nrf2, could attenuate renal inflammation and fibrosis in cyclosporine (CsA)-induced kidney injury. Methods Male ICR mice were divided into four treatment groups: Vehicle (VH, n = 6), VH + OA (n = 6), CsA (n = 8), and CsA + OA (n = 8). For the OA-treated groups, OA (25 mg/kg/day) was administered by intraperitoneal injection for the final week of the 4-week experimental period. Renal function, morphologies and signaling were evaluated at the end of the study. Results Treatment with CsA resulted in decreased kidney function and urine osmolality and increased urine volume and urinary albumin levels. The CsA-induced changes were improved by OA treatment. Specifically, administration of OA decreased tubulointerstitial fibrosis and inflammation scores that were increased in CsA-treated mice. Furthermore, OA treatment decreased urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG) and 8-epi-prostaglandin F2α (8-iso-PGF2α) levels. The beneficial effects of OA were attributed to an increased ratio of nuclear/total Nrf2 and subsequently enhanced expression of heme oxygenase (HO)-1, as well as a stable level of Kelch-like ECH-associated protein 1 (Keap1) expression, indicating that OA enhanced nuclear translocation of Nrf2. Increased apoptotic cell death and a high ratio of B cell leukaemia/lymphoma 2 (Bcl-2)-associated X protein (Bax) to Bcl-2 in CsA-treated mice were also significantly ameliorated by OA treatment. Conclusion Our results suggest that OA activates Nrf2/HO-1 signaling in chronic CsA nephropathy, which may have beneficial effects on inflammation and oxidative stress. PMID:24559268

  13. Effects of Nrf2 silencing on oxidative stress-associated intestinal carcinogenesis in mice.

    PubMed

    Yokoo, Yuh; Kijima, Aki; Ishii, Yuji; Takasu, Shinji; Tsuchiya, Takuma; Umemura, Takashi

    2016-06-01

    To assess the risk of colorectal cancer in humans with inactivation of NRF2, Nrf2-proficient (Nrf2(+/+) ) and -deficient (Nrf2(-/-) ) mice were exposed to potassium bromate (KBrO3 ) at concentrations of 750 or 1500 ppm for 52 weeks. Neoplastic proliferative lesions were observed in the small intestine and exhibited accumulations of β-catenin and cyclin D1. The lesions had characteristics similar to those in experimental models of human hereditary colorectal cancer. An additional 13-week study was performed to examine the role of Nrf2 in the effects of oxidative stress. Significant increase in combined incidences of preneoplastic and neoplastic lesions in Nrf2(-/-) mice administered high-dose KBrO3 . In the short-term study, although 8-hydroxydeoxyguanosine (8-OHdG) levels in the epithelial DNA of Nrf2(-/-) mice at the high dose were significantly lower than those of the corresponding Nrf2(+/+) mice, the difference was very small. mRNA levels of Nrf2-regulated genes were increased in Nrf2(+/+) mice. Overexpression of cyclooxygenase 2 (COX2) and increased numbers of proliferating cell nuclear antigen (PCNA)-positive cells in the jejunal crypts were observed in Nrf2(-/-) mice administered high-dose KBrO3 . Overall, these data suggested that individuals having single-nucleotide polymorphisms in NRF2 may have a risk of colorectal cancer to some extent.

  14. The role of Nrf2 in the attenuation of cardiovascular disease.

    PubMed

    Reuland, Danielle J; McCord, Joe M; Hamilton, Karyn L

    2013-07-01

    Oxidative stress is a component of many human diseases, including cardiovascular diseases (CVD). Exercise and various phytochemicals activate nuclear factor (erythroid-derived 2)-like 2 (Nrf2), the master regulator of antioxidant defenses, and attenuate CVD. This review highlights Nrf2 regulation by exercise and phytochemicals and the role of Nrf2 as a therapeutic target in CVD.

  15. Osteocalcin improves nonalcoholic fatty liver disease in mice through activation of Nrf2 and inhibition of JNK.

    PubMed

    Du, Jing; Zhang, Mingliang; Lu, Junxi; Zhang, Xueli; Xiong, Qin; Xu, Yiting; Bao, Yuqian; Jia, Weiping

    2016-09-01

    Recent studies have demonstrated a protective effect of osteocalcin against nonalcoholic fatty liver disease (NAFLD), although the specific underlying mechanisms remain unclear. Nrf2 and JNK pathways play important roles in the pathogenesis of NAFLD. The present study aimed to investigate whether osteocalcin protects against NAFLD by regulating these pathways. Male C57/BL6J mice were fed a high-fat diet for 12 weeks to induce NAFLD and were treated with recombinant decarboxylate osteocalcin (30 ng/g) or vehicle by daily intraperitoneal injection during this period. Osteocalcin treatment protected mice from diet-induced hepatic triglyceride accumulation and liver injury. Increased levels of malondialdehyde and 8-iso-prostaglandin F2α as well as a higher ratio of oxidized to reduced glutathione in the liver of mice fed a high-fat diet were significantly decreased due to the intervention of osteocalcin. Osteocalcin treatment not only activated Nrf2 nuclear translocation and up-regulated the expression of antioxidant enzyme genes (catalase, SOD, and GPx), but also inhibited the activation of JNK in the liver. GPRC6A, the putative receptor of osteocalcin, was found in the liver. In conclusion, these results suggest that osteocalcin improves NAFLD by activating the Nrf2 pathway to alleviate oxidative stress and inhibiting JNK pathway.

  16. Ginsenoside Rg1 attenuates ultraviolet B-induced glucocortisides resistance in keratinocytes via Nrf2/HDAC2 signalling

    PubMed Central

    Li, Jun; Liu, Dong; Wu, Jinfeng; Zhang, Daniel; Cheng, Binbin; Zhang, Yani; Yin, Zifei; Wang, Yuan; Du, Juan; Ling, Changquan

    2016-01-01

    Oxidative stress, which occurs after ultraviolet (UV) radiation, usually results in Glucocorticoid (GC) resistance and the subsequent development of skin inflammation. One approach to protecting the skin against UV radiation is the use of antioxidants. The ginsenoside Rg1 is a novel natural antioxidant isolated from the medicinal plant Panax ginseng C.A. Mey. We demonstrated that UVB exposure exacerbated inflammation and reduced both the level of the glucocorticoid receptor (GR) and the efficacy of dexamethasone (Dex) in human keratinocytes (HaCaT cells). Pretreatment with Rg1 increased the expression of GR and restored Dex responsiveness to inflammation in UVB-irradiated HaCaT cells. Mechanistically, Rg1 rescued UVB-induced HDAC2 degradation. HDAC2 knockdown partially abolished the Rg1-induced up-regulation of GR and the enhancement of GC sensitivity. In addition, Rg1 reduced the production of reactive oxygen species (ROS), which preceded the up-regulation of HDAC2, and consequent sensitization of cells to Dex. Moreover, Rg1 treatment promoted the translocation and activation of Nrf2. Nrf2 knockdown partially abolished the Rg1-induced decrease of ROS production and increase of HDAC2. Rg1 also potentiated the anti-inflammatory effects of Dex in UVB-irradiated mouse skin. In conclusion, we demonstrated that Rg1 attenuated UVB-induced GC insensitivity. Notably, these effects were partially mediated by the Nrf2/HDAC2 pathway. PMID:27982079

  17. Induction of murine NAD(P)H:quinone oxidoreductase by 2,3,7,8-tetrachlorodibenzo-p-dioxin requires the CNC (cap 'n' collar) basic leucine zipper transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2): cross-interaction between AhR (aryl hydrocarbon receptor) and Nrf2 signal transduction.

    PubMed Central

    Ma, Qiang; Kinneer, Krista; Bi, Yongyi; Chan, Jefferson Y; Kan, Yuet Wai

    2004-01-01

    TCDD (2,3,7,8-tetrachlorodibenzo- p -dixoin) induces phase II drug-metabolizing enzyme NQO1 [NAD(P)H:quinone oxidoreductase; EC 1.6.99.2; DT-diaphorase] in a wide range of mammalian tissues and cells. Here, we analysed the molecular pathway mediating NQO1 induction by TCDD in mouse hepatoma cells. Inhibition of protein synthesis with CHX (cycloheximide) completely blocks induction of NQO1 by TCDD as well as the basal expression and induction by phenolic antioxidant tBHQ (2-t-butylbenzene-1,4-diol), implicating a labile factor in NQO1 mRNA expression. The inhibition is both time- and concentration-dependent, requires inhibition of protein synthesis, and occurs at a transcriptional level. Inhibition of NQO1 transcription by CHX correlates with a rapid reduction of the CNC bZip (cap 'n' collar basic leucine zipper) transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2) through the 26 S proteasome pathway. Moreover, blocking Nrf2 degradation with proteasome inhibitor MG132 increases the amount of Nrf2 and superinduces NQO1 in the presence of TCDD or tBHQ. Finally, genetic experiments using AhR (aryl hydrocarbon receptor)-, Arnt (aryl hydrocarbon receptor nuclear translocator)- or Nrf2-deficient cells reveal that, while induction of NQO1 by TCDD depends on the presence of AhR and Arnt, the basal and inducible expression of NQO1 by either TCDD or tBHQ requires functional Nrf2. The findings demonstrate a novel role of Nrf2 in the induction of NQO1 by TCDD and provide new insights into the mechanism by which Nrf2 regulates the induction of phase II enzymes by both phenolic antioxidants and AhR ligands. PMID:14510636

  18. Chrysin enhances sensitivity of BEL-7402/ADM cells to doxorubicin by suppressing PI3K/Akt/Nrf2 and ERK/Nrf2 pathway.

    PubMed

    Gao, Ai-Mei; Ke, Zun-Ping; Shi, Fang; Sun, Guang-Chun; Chen, Hui

    2013-10-25

    Nuclear factor-E2-related factor 2 (Nrf2) is an important cytoprotective transcription factor which plays a key role in antioxidant and detoxification processes. Recent studies have reported that development of chemoresistance is associated with the constitutive activation of the Nrf2-mediated signaling pathway in many types of cancer cells. Here, we investigated whether Nrf2 was associated with drug resistant in doxorubicin resistant BEL-7402 (BEL-7402/ADM) cells, and if chrysin could reverse drug resistance in BEL-7402/ADM cells. We found that remarkable higher level of Nrf2 and its target proteins in BEL-7402/ADM cells compared to BEL-7402 cells. Similarly, intracellular Nrf2 protein level was significantly decreased and ADM resistance was partially reversed by Nrf2 siRNA in BEL-7402/ADM cells. chrysin is a potent Nrf2 inhibitor which sensitizes BEL-7402/ADM cells to ADM and increases intracellular concentration of ADM. Mechanistically, chrysin significantly reduced Nrf2 expression at both the mRNA and protein levels through down-regulating PI3K-Akt and ERK pathway. Consequently, expression of Nrf2-downstream genes HO-1, AKR1B10, and MRP5 were reduced and the Nrf2-dependent chemoresistance was suppressed. In conclusion, these results clearly indicate that activation of Nrf2 is associated with drug resistance in BEL-7402/ADM cells and chrysin may be an effective adjuvant sensitizer to reduce anticancer drug resistance by down-regulating Nrf2 signaling pathway. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  19. Hydrogen sulfide attenuates calcification of vascular smooth muscle cells via KEAP1/NRF2/NQO1 activation.

    PubMed

    Aghagolzadeh, Parisa; Radpour, Ramin; Bachtler, Matthias; van Goor, Harry; Smith, Edward R; Lister, Adam; Odermatt, Alex; Feelisch, Martin; Pasch, Andreas

    2017-10-01

    Vascular calcification is a common health problem related to oxidative stress, inflammation, and circulating calciprotein particles (CPP). Hydrogen sulfide is an endogenous signaling molecule with antioxidant properties and potential for drug development targeting redox signaling. Yet, its molecular mechanisms of action in vascular smooth muscle cell (VSMC) calcification have not been delineated. We therefore sought to identify key pathways involved in the calcification-inhibitory properties of sulfide employing our recently developed CPP-induced VSMC calcification model. Using next-generation sequencing, we investigated the transcriptomic changes of sodium hydrosulfide-treated versus non-treated calcifying VSMCs. The potential role of candidate genes and/or regulatory pathways in prevention of calcification was investigated by small interfering RNA (siRNA). CPP led to a pronounced accumulation of cell-associated calcium, which was decreased by sulfide in a concentration-dependent manner. Both, CPP-induced hydrogen peroxide production and enhanced pro-inflammatory/oxidative stress-related gene expression signatures were attenuated by sulfide-treatment. Gene ontology enrichment and in silico pathway analysis of our transcriptome data suggested NAD(P)H dehydrogenase [quinone] 1 (NQO1) as potential mediator. Corroborating these findings, silencing of Kelch-like ECH-associated protein 1 (KEAP1), an inhibitor of nuclear factor (erythroid-derived 2)-like 2 (NRF2) nuclear activity, enhanced NQO1 expression, whereas NRF2 silencing reduced the expression of NQO1 and abrogated the calcification-suppressing activity of sulfide. Moreover, immunofluorescence microscopy and Western blot analysis confirmed nuclear translocation of NRF2 by sulfide in VSMC. Sulfide attenuates CPP-induced VSMC calcification in vitro via the KEAP1-NRF2 redox sensing/stress response system by enhancing NQO1 expression. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Sargassum fusiforme polysaccharides activate antioxidant defense by promoting Nrf2-dependent cytoprotection and ameliorate stress insult during aging.

    PubMed

    Chen, Peichao; He, Dan; Zhang, Ya; Yang, Shanshan; Chen, Liujun; Wang, Shengqin; Zou, Huixi; Liao, Zhiyong; Zhang, Xu; Wu, Mingjiang

    2016-11-09

    Aging is a complex issue, which results in a progressive decline process in cellular protection and physiological functions. Illustrating the causes of aging and pharmaceutical interference with the aging process has been a pivotal issue for thousands of years. Sargassum fusiforme (S. fusiforme), a kind of brown alga, is also named the "longevity vegetable" as it is not only a kind of food, but also used as an herb in traditional Chinese Medicine for maintaining health and treatment of thyroid disease, cardiovascular disease and so on. But how S. fusiforme promotes longevity is vastly equivocal. We got clues from S. fusiforme polysaccharides, which exhibited antioxidant activity, but the underlying mechanisms remained unclear. In this study, we evaluated the antioxidant effect and the possible mechanisms that S. fusiforme polysaccharides have against d-galactose-induced aging and chronic aging. We selected the SFPS as the candidate for antioxidant defense evaluation, which is a type of S. fusiforme polysaccharide with strong free radical scavenging activity and non-toxicity. It revealed that the antioxidant defense of the d-galactose-induced mice was markedly recovered when they were intragastrically administrated with the SFPS. However, oxidative damage may not be the only cause of aging. We further evaluated the function of the SFPS in the chronic aging mice. Intriguingly, we even found an obvious aging phenotype in the middle aged male ICR mice, which showed a significant decline in Nrf2-dependent cytoprotection. When 9-month old male ICR mice were treated with the SFPS for 2 months or even 11 months to their mean survival age, experimental measurements showed that the SFPS significantly promoted the antioxidant defense and mitochondrial integrity during aging. Furthermore, we suggest that the SFPS promotes Nrf2-dependent cytoprotection by upregulating the nuclear Nrf2 translocation, which may be mediated by p21 and JNK dependent pathways. These results suggest

  1. Protocatechualdehyde Protects Against Cerebral Ischemia-Reperfusion-Induced Oxidative Injury Via Protein Kinase Cε/Nrf2/HO-1 Pathway.

    PubMed

    Guo, Chao; Wang, Shiquan; Duan, Jialin; Jia, Na; Zhu, Yanrong; Ding, Yi; Guan, Yue; Wei, Guo; Yin, Ying; Xi, Miaomaio; Wen, Aidong

    2017-03-01

    Oxidative stress is closely related to the pathogenesis of ischemic stroke. Protocatechualdehyde (PCA) is a phenolic acid compound that has the putative antioxidant activities. The present study was aimed to investigate the molecular mechanisms involved in the antioxidative effect of PCA against cerebral ischemia/reperfusion (I/R) injury. The experiment stroke model was produced in Sprague-Dawley rats via middle cerebral artery occlusion (MCAO). To model ischemia-like conditions in vitro, differentiated SH-SY5Y cells were exposed to transient oxygen and glucose deprivation (OGD). Treatment with PCA significantly improved neurologic score, reduced infarct volume and necrotic neurons, and also decreased reactive oxygen species (ROS) production, 4-hydroxynonenal (4-HNE), and 8-hydroxy-2'-deoxyguanosine (8-OHdG) contents at 24 h after reperfusion. Meanwhile, PCA significantly increased the transcription nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) expressions in the ischemic cerebral cortex as shown by immunofluorescence staining and Western blot analysis. In vitro experiment showed that PCA protected differentiated SH-SY5Y cells against OGD-induced injury. Likewise, PCA also increased markedly the Nrf2 and HO-1 expressions in a dose-dependent manner. The neuroprotection effect of PCA was abolished by knockdown of Nrf2 and HO-1. Moreover, knockdown of protein kinase Cε (PKCε) also blocked PCA-induced Nfr2 nuclear translocation, HO-1 expression, and neuroprotection. Taken together, these results provide evidences that PCA can protect against cerebral ischemia-reperfusion-induced oxidative injury, and the neuroprotective effect involves the PKCε/Nrf2/HO-1 pathway.

  2. [Schisandrin B protects against nephrotoxicity induced by cisplatin in HK-2 cells via Nrf2-ARE activation].

    PubMed

    Li, Mei; Jin, Jing; Li, Jia; Guan, Cui-Wen; Wang, Wen-Wen; Qiu, Yu-Wen; Huang, Zhi-Ying

    2012-11-01

    This study is to investigate the protection effect of schisandrin B (Sch B) against oxidation stress of HK-2 cells induced by cisplatin and the mechanisms involved. HK-2 cells were cultured and divided into different groups: solvent control group, cisplatin exposure group, positive group, Sch B treatment group. Cell viability and toxicity were evaluated by MTT and LDH assay. GSH level and SOD enzymes activities were also measured. DCFH-DA as fluorescence probe was used to detect ROS level by fluorescence microplate reader. Nrf2 translocation was detected by Western blotting. Real time Q-PCR was used to detect expressions of NQO1, HO-1 and GCLC mRNA level. The results showed that Sch B could significantly inhibit the decline of cell viability induced by cisplatin treatment (P < 0.05) and the protective effect was in a dose dependent manner. Furthermore, Sch B treatment significantly inhibited the increase of ROS level induced by cisplatin and reversed the decrease of GSH level (P < 0.05). When Sch B concentration was up to 5 micromol x L(-1), SOD enzyme activities were also enhanced significantly compared with that of the cisplatin group (P < 0.05). It was shown that Sch B could cause nuclear accumulation of Nrf2 in association with downstream activation of Nrf2 mediated oxidative response genes such as GCLC, NQO1 and HO-1. These results suggested Sch B could protect against the oxidative damage of HK-2 cells induced by cisplatin via the activation of Nrf2/ARE signal pathway.

  3. Melatonin successfully rescues hippocampal bioenergetics and improves cognitive function following drug intoxication by promoting Nrf2-ARE signaling activity.

    PubMed

    Chen, Li-You; Renn, Ting-Yi; Liao, Wen-Chieh; Mai, Fu-Der; Ho, Ying-Jui; Hsiao, George; Lee, Ai-Wei; Chang, Hung-Ming

    2017-09-01

    Prolonged exposure to gamma-hydroxybutyric acid (GHB) would cause drug intoxication in which impaired cognitive function results from enhanced hippocampal oxidative stress may serve as a major symptom in this deficiency. Considering melatonin possesses significant anti-oxidative efficacy, this study aimed to determine whether melatonin would successfully promote the nuclear factor erythroid 2-related factor 2 and antioxidant responsive element (Nrf2-ARE) signaling, depress oxidative stress, and rescue hippocampal bioenergetics and cognitive function following drug intoxication injury. Adolescent rats subjected to 10 days of GHB were received melatonin at doses of either 10 or 100 mg/kg. Time-of-flight secondary ion mass spectrometry, biochemical assay, quantitative histochemistry, [(14) C]-2-deoxyglucose analysis, together with Morris water maze were employed to detect the molecular signaling, oxidative status, bioenergetic level, as well as the cognitive performances, respectively. Results indicated that in GHB-intoxicated rats, enhanced oxidative stress, increased cholesterol level, and decreased anti-oxidative enzymes activities were detected in hippocampal regions. Intense oxidative stress paralleled well with reduced bioenergetics and poor performance in behavioral testing. However, in rats treated with melatonin following GHB intoxication, all above parameters and cognitive function were gradually returned to nearly normal levels. Melatonin also remarkably promoted the translocation of Nrf2 from cytoplasm to nucleus in a dose-dependent manner, thereby increased the Nrf2-ARE signaling-related downstream anti-oxidative enzymes activities. As melatonin effectively rescues hippocampal bioenergetics through depressing the oxidative stress by promoting Nrf2-ARE molecular machinery, this study thus highlights for the first time that clinical use of melatonin may serve as a therapeutic strategy to improve the cognitive function in unsuspecting victims suffered from

  4. Role of NF-E2 related factor 2 (Nrf2) on chemotherapy resistance in acute myeloid leukemia (AML) and the effect of pharmacological inhibition of Nrf2

    PubMed Central

    Karathedath, Sreeja; Rajamani, Bharathi M.; Musheer Aalam, Syed Mohammed; Abraham, Ajay; Varatharajan, Savitha; Krishnamurthy, Partha; Mathews, Vikram; Velayudhan, Shaji Ramachandran

    2017-01-01

    Cytarabine (Ara-C) and Daunorubicin (Dnr) forms the backbone of acute myeloid leukemia (AML) therapy. Drug resistance and toxic side effects pose a major threat to treatment success and hence alternate less toxic therapies are warranted. NF-E2 related factor-2 (Nrf2), a master regulator of antioxidant response is implicated in chemoresistance in solid tumors. However, little is known about the role of Nrf2 in AML chemoresistance and the effect of pharmacological inhibitor brusatol in modulating this resistance. Primary AML samples with high ex-vivo IC50 to Ara-C, ATO, Dnr had significantly high NRF2 RNA expression. Gene-specific knockdown of NRF2 improved sensitivity to these drugs in resistant AML cell lines by decreasing the expression of downstream antioxidant targets of Nrf2 by compromising the cell’s ability to scavenge the ROS. Treatment with brusatol, a pharmacological inhibitor of Nrf2, improved sensitivity to Ara-C, ATO, and Dnr and reduced colony formation capacity. AML cell lines stably overexpressing NRF2 showed increased resistance to ATO, Dnr and Ara-C and increased expression of downstream targets. This study demonstrates that Nrf2 could be an ideal druggable target in AML, more so to the drugs that function through ROS, suggesting the possibility of using Nrf2 inhibitors in combination with chemotherapeutic agents to modulate drug resistance in AML. PMID:28505160

  5. Mitochondrial permeabilization without caspase activation mediates the increase of basal apoptosis in cells lacking Nrf2.

    PubMed

    Ariza, Julia; González-Reyes, José A; Jódar, Laura; Díaz-Ruiz, Alberto; de Cabo, Rafael; Villalba, José Manuel

    2016-06-01

    Nuclear factor E2-related factor-2 (Nrf2) is a cap'n'collar/basic leucine zipper (b-ZIP) transcription factor which acts as sensor of oxidative and electrophilic stress. Low levels of Nrf2 predispose cells to chemical carcinogenesis but a dark side of Nrf2 function also exists because its unrestrained activation may allow the survival of potentially dangerous damaged cells. Since Nrf2 inhibition may be of therapeutic interest in cancer, and a decrease of Nrf2 activity may be related with degenerative changes associated with aging, it is important to investigate how the lack of Nrf2 function activates molecular mechanisms mediating cell death. Murine Embryonic Fibroblasts (MEFs) bearing a Nrf2 deletion (Nrf2KO) displayed diminished cellular growth rate and shortened lifespan compared with wild-type MEFs. Basal rates of DNA fragmentation and histone H2A.X phosphorylation were higher in Nrf2KO MEFs, although steady-state levels of reactive oxygen species were not significantly increased. Enhanced rates of apoptotic DNA fragmentation were confirmed in liver and lung tissues from Nrf2KO mice. Apoptosis in Nrf2KO MEFs was associated with a decrease of Bcl-2 but not Bax levels, and with the release of the mitochondrial pro-apoptotic factors cytochrome c and AIF. Procaspase-9 and Apaf-1 were also increased in Nrf2KO MEFs but caspase-3 was not activated. Inhibition of XIAP increased death in Nrf2KO but not in wild-type MEFs. Mitochondrial ultrastructure was also altered in Nrf2KO MEFs. Our results support that Nrf2 deletion produces mitochondrial dysfunction associated with mitochondrial permeabilization, increasing basal apoptosis through a caspase-independent and AIF-dependent pathway.

  6. Decreased histone deacetylase 2 impairs Nrf2 activation by oxidative stress

    SciTech Connect

    Mercado, Nicolas; Thimmulappa, Rajesh; Thomas, Catherine M.R.; Fenwick, Peter S.; Chana, Kirandeep K.; Donnelly, Louise E.; Biswal, Shyam; Ito, Kazuhiro; Barnes, Peter J.

    2011-03-11

    Research highlights: {yields} Nrf2 anti-oxidant function is impaired when HDAC activity is inhibited. {yields} HDAC inhibition decreases Nrf2 protein stability. {yields} HDAC2 is involved in reduced Nrf2 stability and both correlate in COPD samples. {yields} HDAC inhibition increases Nrf2 acetylation. -- Abstract: Nuclear factor erythroid 2-related factor 2 (Nrf2) plays a crucial role in cellular defence against oxidative stress by inducing the expression of multiple anti-oxidant genes. However, where high levels of oxidative stress are observed, such as chronic obstructive pulmonary disease (COPD), Nrf2 activity is reduced, although the molecular mechanism for this defect is uncertain. Here, we show that down-regulation of histone deacetylase (HDAC) 2 causes Nrf2 instability, resulting in reduced anti-oxidant gene expression and increase sensitivity to oxidative stress. Although Nrf2 protein was clearly stabilized after hydrogen peroxide (H{sub 2}O{sub 2}) stimulation in a bronchial epithelial cell line (BEAS2B), Nrf2 stability was decreased and Nrf2 acetylation increased in the presence of an HDAC inhibitor, trichostatin A (TSA). TSA also reduced Nrf2-regulated heme-oxygenase-1 (HO-1) expression in these cells, and this was confirmed in acute cigarette-smoke exposed mice in vivo. HDAC2 knock-down by RNA interference resulted in reduced H{sub 2}O{sub 2}-induced Nrf2 protein stability and activity in BEAS2B cells, whereas HDAC1 knockdown had no effect. Furthermore, monocyte-derived macrophages obtained from healthy volunteers (non-smokers and smokers) and COPD patients showed a significant correlation between HDAC2 expression and Nrf2 expression (r = 0.92, p < 0.0001). Thus, reduced HDAC2 activity in COPD may account for increased Nrf2 acetylation, reduced Nrf2 stability and impaired anti oxidant defences.

  7. Agmatine Reduces Lipopolysaccharide-Mediated Oxidant Response via Activating PI3K/Akt Pathway and Up-Regulating Nrf2 and HO-1 Expression in Macrophages

    PubMed Central

    Chai, Jianshen; Luo, Li; Hou, Fengyan; Fan, Xia; Yu, Jing; Ma, Wei; Tang, Wangqi; Yang, Xue; Zhu, Junyu; Kang, Wenyuan; Yan, Jun; Liang, Huaping

    2016-01-01

    Macrophages are key responders of inflammation and are closely related with oxidative stress. Activated macrophages can enhance oxygen depletion, which causes an overproduction of reactive oxygen species (ROS) and leads to further excessive inflammatory response and tissue damage. Agmatine, an endogenous metabolite of L-arginine, has recently been shown to have neuroprotective effects based on its antioxidant properties. However, the antioxidant effects of agmatine in peripheral tissues and cells, especially macrophages, remain unclear. In this study we explored the role of agmatine in mediating antioxidant effects in RAW 264.7 cells and studied its antioxidant mechanism. Our data demonstrate that agmatine is an activator of Nrf2 signaling that markedly enhances Nrf2 nuclear translocation, increases nuclear Nrf2 protein level, up-regulates the expression of the Nrf2 downstream effector HO-1, and attenuates ROS generation induced by Lipopolysaccharide (LPS). We further demonstrated that the agmatine-induced activation of Nrf2 is likely through the PI3K/Akt pathway. LY294002, a specific PI3K/Akt inhibitor, abolished agmatine-induced HO-1 up-regulation and ROS suppression significantly. Inhibiting HO-1 pathway significantly attenuated the antioxidant effect of agmatine which the products of HO-1 enzymatic activity contributed to. Furthermore, the common membrane receptors of agmatine were evaluated, revealing that α2-adrenoceptor, I1-imidazoline receptor or I2-imidazoline receptor are not required by the antioxidant properties of agmatine. Taken together, our findings revealed that agmatine has antioxidant activity against LPS-induced ROS accumulation in RAW 264.7 cells involving HO-1 expression induced by Nrf2 via PI3K/Akt pathway activation. PMID:27685463

  8. Regulation of hemeoxygenase-1 gene expression by Nrf2 and c-Jun in tertiary butylhydroquinone-stimulated rat primary astrocytes.

    PubMed

    Park, Jin-Sun; Kim, Hee-Sun

    2014-05-16

    Hemeoxygenase-1 (HO-1) is a phase II antioxidant enzyme that is primarily involved in detoxification and cytoprotection in a variety of tissues. However, the mechanism underlying HO-1 gene expression remains unclear. In the present study, we investigated the regulation of HO-1 expression in primary cultured astrocytes by using the natural antioxidant compound tertiary butylhydroquinone (tBHQ). We found that tBHQ increased HO-1 mRNA and protein levels. Promoter analysis revealed that tBHQ enhanced HO-1 gene transcription in an antioxidant response element (ARE)-dependent manner. In addition, tBHQ increased the nuclear translocation and DNA binding of Nrf2 and c-Jun to ARE. Small interfering RNA (siRNA) experiments demonstrated that Nrf2 and c-Jun are involved in the differential modulation of HO-1 expression. Thus, Nrf2 knockdown reduced the basal level of HO-1 expression but did not affect the fold induction by tBHQ. On the other hand, knockdown of c-Jun diminished tBHQ-mediated induction of HO-1 without affecting basal expression. The data suggest that Nrf2 generally modulates the basal expression of HO-1, while c-Jun mediates HO-1 induction in response to tBHQ. The results of co-immunoprecipitation assays demonstrated a physical interaction between Nrf2 and c-Jun in tBHQ-treated astrocytes. The results suggest that Nrf2 and c-Jun regulate HO-1 expression via their coordinated interaction in tBHQ-treated rat primary astrocytes.

  9. NRF2 mitigates radiation-induced hematopoietic death

    PubMed Central

    Chute, John P.

    2014-01-01

    Fractionated, high-dose total body irradiation (TBI) is used therapeutically to myeloablate and immune suppress patients undergoing hematopoietic stem cell (HSC) transplantation. Acute exposure to ionizing radiation can have fatal effects on the hematopoietic and immune systems. Currently, therapies aimed at ameliorating ionizing radiation–associated toxicities are limited. In the February 2014 issue of the JCI, Kim and colleagues demonstrated that induction of nuclear factor erythroid 2–related factor 2 (NRF2) enhances HSC regeneration and increases survival following ionizing radiation exposure in mice. The results of this study suggest that NRF2 is a novel potential target for the development of therapeutics aimed at mitigating the toxicities of ionizing radiation exposure. PMID:24569364

  10. Nitroxide delivery system for Nrf2 activation and skin protection.

    PubMed

    Ben Yehuda Greenwald, Maya; Frušić-Zlotkin, Marina; Soroka, Yoram; Sasson, Shmuel Ben; Bianco-Peled, Havazelet; Bitton, Ronit; Kohen, Ron

    2015-08-01

    Cyclic nitroxides are a large group of compounds composed of diverse stable radicals also known as synthetic antioxidants. Although nitroxides are valuable for use in several skin conditions, in in vivo conditions they have several drawbacks, such as nonspecific dispersion in normal tissue, preferential renal clearance and rapid reduction of the nitroxide to the corresponding hydroxylamine. However, these drawbacks can be easily addressed by encapsulating the nitroxides within microemulsions. This approach would allow nitroxide activity and therefore their valuable effects (e.g. activation of the Keap1-Nrf2-EpRE pathway) to continue. In this work, nitroxides were encapsulated in a microemulsion composed of biocompatible ingredients. The nanometric size and shape of the vehicle microemulsion and nitroxide microemulsion displayed high similarity, indicating that the stability of the microemulsions was preserved. Our studies demonstrated that nitroxide microemulsions were more potent inducers of the Keap1-Nrf2-EpRE pathway than the free nitroxides, causing the activation of phase II enzymes. Moreover, microemulsions containing nitroxides significantly reduced UVB-induced cytotoxicity in the skin. Understanding the mechanism of this improved activity may expand the usage of many other Nrf2 modulating molecules in encapsulated form, as a skin protection strategy against oxidative stress-related conditions.

  11. Nrf2-dependent protection against acute sodium arsenite toxicity in zebrafish.

    PubMed

    Fuse, Yuji; Nguyen, Vu Thanh; Kobayashi, Makoto

    2016-08-15

    Transcription factor Nrf2 induces a number of detoxifying enzymes and antioxidant proteins to confer protection against the toxic effects of a diverse range of chemicals including inorganic arsenicals. Although a number of studies using cultured cells have demonstrated that Nrf2 has a cell-protective function against acute and high-dose arsenic toxicity, there is no clear in vivo evidence of this effect. In the present study, we genetically investigated the protective role of Nrf2 against acute sodium arsenite toxicity using the zebrafish Nrf2 mutant, nrf2a(fh318). After treatment with 1mM sodium arsenite, the survival of nrf2a(fh318) larvae was significantly shorter than that of wild-type siblings, suggesting that Nrf2 protected the zebrafish larvae against high-dose arsenite exposure. To understand the molecular basis of the Nrf2-dependent protection, we analyzed the gene expression profiles after arsenite exposure, and found that the genes involved in the antioxidative function (prdx1 and gclc), arsenic metabolism (gstp1) and xenobiotic elimination (abcc2) were induced in an Nrf2-dependent manner. Furthermore, pre-treatment with sulforaphane, a well-known Nrf2 activator improved the survival of zebrafish larvae after arsenic exposure. Based on these results, we concluded that Nrf2 plays a fundamental and conserved role in protection against acute sodium arsenite toxicity.

  12. Activation of Nrf2-ARE signaling mitigates cyclophosphamide-induced myelosuppression.

    PubMed

    Que, Linling; He, Liu; Yu, Chenshu; Yin, Wencheng; Ma, Liwen; Cao, Baoshan; Yu, Siwang

    2016-11-16

    Myelosuppression is the most common dose-limiting adverse effect of chemotherapies. In the present study, we investigated the involvement of nuclear erythroid 2-related factor 2 (Nrf2) in cyclophosphamide-induced myelosuppression in mice, and evaluated the potential of activating Nrf2 signaling as a preventive strategy. The whole blood from Nrf2(-/-) mice exhibited decreased antioxidant capacities, while the bone marrow cells, peripheral blood mononuclear cells and granulocytes from Nrf2(-/-) mice were more susceptible to acrolein-induced cytotoxicity than those from wild type mice. Single dosage of cyclophosphamide induced significantly severer acute myelosuppression in Nrf2(-/-) mice than in wild type mice. Furthermore, Nrf2(-/-) mice exhibited greater loss of peripheral blood nucleated cells and recovered slower from myelosuppression nadir upon multiple consecutive dosages of cyclophosphamide than wild type mice did. This was accompanied with decreased antioxidant and detoxifying gene expressions and impaired colony formation ability of Nrf2(-/-) bone marrow cells. More importantly, activation of Nrf2 signaling by CDDO-Me significantly alleviated cyclophosphamide-induced myelosuppression, while this alleviation was diminished in Nrf2(-/-) mice. In conclusion, the present study shows that Nrf2 plays a protective role in cyclophosphamide-induced myelosuppression and activation of Nrf2 is a promising strategy to prevent or treat chemotherapy-induced myelosuppression.

  13. Interplay between VEGF and Nrf2 regulates angiogenesis due to intracranial venous hypertension

    PubMed Central

    Li, Liwen; Pan, Hao; Wang, Handong; Li, Xiang; Bu, Xiaomin; Wang, Qiang; Gao, Yongyue; Wen, Guodao; Zhou, Yali; Cong, Zixiang; Yang, Youqing; Tang, Chao; Liu, Zhengwei

    2016-01-01

    Venous hypertension(VH) plays an important role in the pathogenesis of cerebral arteriovenous malformations (AVMs) and is closely associated with the HIF-1α/VEGF signaling pathway. Nuclear factor erythroid 2-related factor 2(Nrf2) significantly influences angiogenesis; however, the interplay between Nrf2 and VEGF under VH in brain AVMs remains unclear. Therefore, our study aimed to investigate the interplay between Nrf2 and VEGF due to VH in brain AVMs. Immunohistochemistry indicated that Nrf2 and VEGF were highly expressed in human brain AVM tissues. In vivo, we established a VH model in both wild-type (WT) and siRNA-mediated Nrf2 knockdown rats. VH significantly increased the expression of Nrf2 and VEGF. Loss of Nrf2 markedly inhibited the upregulation of VEGF, as determined by Western blot analysis and qRT-PCR. In vitro, primary brain microvascular endothelial cells (BMECs) were isolated from WT and Nrf2−/− mice, and a VEGF-Nrf2 positive feed-back loop was observed in BMECs. By trans well assay and angiogenesis assay, Nrf2 knockout significantly inhibited the migration and vascular tube formation of BMECs. These findings suggest that the interplay between Nrf2 and VEGF can contribute to VH-induced angiogenesis in brain AVMs pathogenesis. PMID:27869147

  14. Melatonin Attenuates Memory Impairment Induced by Klotho Gene Deficiency Via Interactive Signaling Between MT2 Receptor, ERK, and Nrf2-Related Antioxidant Potential

    PubMed Central

    Shin, Eun-Joo; Chung, Yoon Hee; Le, Hoang-Lan Thi; Jeong, Ji Hoon; Dang, Duy-Khanh; Nam, Yunsung; Wie, Myung Bok; Nah, Seung-Yeol; Nabeshima, Yo-Ichi; Nabeshima, Toshitaka; Kim, Hyoung-Chun

    2015-01-01

    Background: We demonstrated that oxidative stress plays a crucial role in cognitive impairment in klotho mutant mice, a genetic model of aging. Since down-regulation of melatonin due to aging is well documented, we used this genetic model to determine whether the antioxidant property of melatonin affects memory impairment. Methods: First, we examined the effects of melatonin on hippocampal oxidative parameters and the glutathione/oxidized glutathione (GSH/GSSG) ratio and memory dysfunction of klotho mutant mice. Second, we investigated whether a specific melatonin receptor is involved in the melatonin-mediated pharmacological response by application with melatonin receptor antagonists. Third, we examined phospho-extracellular-signal-regulated kinase (ERK) expression, nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear translocation, Nrf2 DNA binding activity, and glutamate-cysteine ligase (GCL) mRNA expression. Finally, we examined effects of the ERK inhibitor SL327 in response to antioxidant efficacy and memory enhancement mediated by melatonin. Results: Treatment with melatonin resulted in significant attenuations of oxidative damage, a decrease in the GSH/GSSG ratio, and a significant amelioration of memory impairment in this aging model. These effects of melatonin were significantly counteracted by the selective MT2 receptor antagonist 4-P-PDOT. Importantly, 4-P-PDOT or SL327 also counteracted melatonin-mediated attenuation in response to the decreases in phospho-ERK expression, Nrf2 nuclear translocation, Nrf2 DNA-binding activity, and GCL mRNA expression in the hippocampi of klotho mutant mice. SL327 also counteracted the up-regulation of the GSH/GSSG ratio and the memory enhancement mediated by melatonin in klotho mutant mice. Conclusions: Melatonin attenuates oxidative stress and the associated memory impairment induced by klotho deficiency via signaling interaction between the MT2 receptor and ERK- and Nrf2-related antioxidant potential. PMID

  15. The activation of HO-1/Nrf-2 contributes to the protective effects of diallyl disulfide (DADS) against ethanol-induced oxidative stress.

    PubMed

    Zeng, Tao; Zhang, Cui-Li; Song, Fu-Yong; Zhao, Xiu-Lan; Yu, Li-Hua; Zhu, Zhen-Ping; Xie, Ke-Qin

    2013-10-01

    Diallyl disulfide (DADS) is a garlic-derived organosulfur compound. The current study is designed to evaluate the protective effects of DADS against ethanol-induced oxidative stress, and to explore the underlying mechanisms by examining the HO-1/Nrf-2 pathway. We investigated whether or not DADS could activate the HO-1 in normal human liver cell LO2, and then evaluated the protective effects of DADS against ethanol-induced damage in LO2 cells and in acute ethanol-intoxicated mice. The biochemical parameters were measured using commercial kits. HO-1 mRNA level was determined by RT-PCR. Histopathology and immunofluorescence assay were performed with routine methods. Protein levels were measured by western blot. DADS significantly increased the mRNA and protein levels of HO-1, stimulated the nuclear translocation of Nrf-2 and increased the phosphorylation of MAPK in LO2 cells. The nuclear translocation of Nrf-2 was abrogated by MAPK inhibitors. DADS significantly suppressed ethanol-induced elevation of lactate dehydrogenase (LDH) and aspartate transaminase (AST) activities, decrease of glutathione (GSH) level, increase of malondialdehyde (MDA) levels, and apoptosis of LO2 cells, which were all blocked by ZnPPIX. In mice, DADS effectively suppressed acute ethanol-induced elevation of aminotransferase activities, and improved liver histopathological changes, which might be associated with HO-1 activation. These results demonstrate that DADS could induce the activation of HO-1/Nrf-2 pathway, which may contribute to the protective effects of DADS against ethanol-induced liver injury. DADS may be beneficial for the prevention and treatment of ALD due to significant activation of HO-1/Nrf-2 pathway. © 2013.

  16. Regulation of Ahr signaling by Nrf2 during development: Effects of Nrf2a deficiency on PCB126 embryotoxicity in zebrafish (Danio rerio).

    PubMed

    Rousseau, Michelle E; Sant, Karilyn E; Borden, Linnea R; Franks, Diana G; Hahn, Mark E; Timme-Laragy, Alicia R

    2015-10-01

    The embryotoxicity of co-planar PCBs is regulated by the aryl hydrocarbon receptor (Ahr), and has been reported to involve oxidative stress. Ahr participates in crosstalk with another transcription factor, Nfe2l2, or Nrf2. Nrf2 binds to antioxidant response elements to regulate the adaptive response to oxidative stress. To explore aspects of the crosstalk between Nrf2 and Ahr and its impact on development, we used zebrafish (Danio rerio) with a mutated DNA binding domain in Nrf2a (nrf2a(fh318/fh318)), rendering these embryos more sensitive to oxidative stress. Embryos were exposed to 2 nM or 5 nM PCB126 at 24 h post fertilization (prim-5 stage of pharyngula) and examined for gene expression and morphology at 4 days post fertilization (dpf; protruding - mouth stage). Nrf2a mutant eleutheroembryos were more sensitive to PCB126 toxicity at 4 dpf, and in the absence of treatment also displayed some subtle developmental differences from wildtype embryos, including delayed inflation of the swim bladder and smaller yolk sacs. We used qPCR to measure changes in expression of the nrf gene family, keap1a, keap1b, the ahr gene family, and known target genes. cyp1a induction by PCB126 was enhanced in the Nrf2a mutants (156-fold in wildtypes vs. 228-fold in mutants exposed to 5 nM). Decreased expression of heme oxygenase (decycling) 1 (hmox1) in the Nrf2a mutants was accompanied by increased nrf2b expression. Target genes of Nrf2a and AhR2, NAD(P)H:quinone oxidoreductase 1 (nqo1) and glutathione S-transferase, alpha-like (gsta1), showed a 2-5-fold increase in expression in the Nrf2a mutants as compared to wildtype. This study elucidates the interaction between two important transcription factor pathways in the developmental toxicity of co-planar PCBs.

  17. Curcumin analog 1, 5-bis (2-trifluoromethylphenyl)-1, 4-pentadien-3-one exhibits enhanced ability on Nrf2 activation and protection against acrolein-induced ARPE-19 cell toxicity

    SciTech Connect

    Li, Yuan; Zou, Xuan; Cao, Ke; Xu, Jie; Yue, Tingting; Dai, Fang; Zhou, Bo; Lu, Wuyuan; Feng, Zhihui; Liu, Jiankang

    2013-11-01

    Curcumin, a phytochemical agent in the spice turmeric, has received increasing attention for its anticancer, anti-inflammatory and antioxidant properties. However, application of curcumin has been limited due to its insolubility in water and poor bioavailability both clinically and experimentally. In addition, the protective effects and mechanisms of curcumin in eye diseases have been poorly studied. In the present study, we synthesized a curcumin analog, 1, 5-bis (2-trifluoromethylphenyl)-1, 4-pentadien-3-one (C3), which displayed improved protective effect against acrolein-induced toxicity in a human retinal pigment epithelial cell line (ARPE-19). At 5 μM, curcumin completely protected against acrolein-induced cell oxidative damage and preserved GSH levels and mitochondrial function. Surprisingly, C3 displayed a complete protective effect at 0.5 μM, which was much more efficient than curcumin. Both 0.5 μM C3 and 5 μM curcumin induced Nrf2 nuclear translocation and Nrf2 target genes transcription similarly. Experiments using Nrf2 siRNA showed that the protective effects of curcumin and C3 were eliminated by Nrf2 knockdown. Additionally, both curcumin and C3 activated the PI3/Akt pathway, however, Nrf2 activation was independent of this pathway, and therefore, we hypothesized that both curcumin and C3 activated phase II enzymes via directly disrupting the Nrf2/Keap1 complex and promoting Nrf2's nuclear translocation. Since acrolein challenge of ARPE-19 cells has been used as a model of smoking and age-related macular degeneration (AMD), we concluded that the curcumin analog, C3, may be a more promising drug candidate for its potential application for the prevention and treatment of eye diseases, such as AMD. - Highlights: • We examine toxicity effects of cigarette smoking component acrolein in retina cells. • We report a more efficient curcumin analog (C3) protecting cellular function. • Mitochondrial function and phase II enzyme activation are the major

  18. Nrf2 deficiency improves glucose tolerance in mice fed a high-fat diet.

    PubMed

    Zhang, Yu-Kun Jennifer; Wu, Kai Connie; Liu, Jie; Klaassen, Curtis D

    2012-11-01

    Nrf2, a master regulator of intracellular redox homeostasis, is indicated to participate in fatty acid metabolism in liver. However, its role in diet-induced obesity remains controversial. In the current study, genetically engineered Nrf2-null, wild-type (WT), and Nrf2-activated, Keap1-knockdown (K1-KD) mice were fed either a control or a high-fat Western diet (HFD) for 12 weeks. The results indicate that the absence or enhancement of Nrf2 activity did not prevent diet-induced obesity, had limited effects on lipid metabolism, but affected blood glucose homeostasis. Whereas the Nrf2-null mice were resistant to HFD-induced glucose intolerance, the Nrf2-activated K1-KD mice exhibited prolonged elevation of circulating glucose during a glucose tolerance test even on the control diet. Feeding a HFD did not activate the Nrf2 signaling pathway in mouse livers. Fibroblast growth factor 21 (Fgf21) is a liver-derived anti-diabetic hormone that exerts glucose- and lipid-lowering effects. Fgf21 mRNA and protein were both elevated in livers of Nrf2-null mice, and Fgf21 protein was lower in K1-KD mice than WT mice. The inverse correlation between Nrf2 activity and hepatic expression of Fgf21 might explain the improved glucose tolerance in Nrf2-null mice. Furthermore, a more oxidative cellular environment in Nrf2-null mice could affect insulin signaling in liver. For example, mRNA of insulin-like growth factor binding protein 1, a gene repressed by insulin in hepatocytes, was markedly elevated in livers of Nrf2-null mice. In conclusion, genetic alteration of Nrf2 does not prevent diet-induced obesity in mice, but deficiency of Nrf2 improves glucose homeostasis, possibly through its effects on Fgf21 and/or insulin signaling.

  19. Ectodermal-neural cortex 1 down-regulates Nrf2 at the translational level.

    PubMed

    Wang, Xiao-Jun; Zhang, Donna D

    2009-01-01

    The transcription factor Nrf2 is the master regulator of a cellular defense mechanism against environmental insults. The Nrf2-mediated antioxidant response is accomplished by the transcription of a battery of genes that encode phase II detoxifying enzymes, xenobiotic transporters, and antioxidants. Coordinated expression of these genes is critical in protecting cells from toxic and carcinogenic insults and in maintaining cellular redox homeostasis. Activation of the Nrf2 pathway is primarily controlled by Kelch-like ECH-associated protein 1 (Keap1), which is a molecular switch that turns on or off the Nrf2 signaling pathway according to intracellular redox conditions. Here we report our finding of a novel Nrf2 suppressor ectodermal-neural cortex 1 (ENC1), which is a BTB-Kelch protein and belongs to the same family as Keap1. Transient expression of ENC1 reduced steady-state levels of Nrf2 and its downstream gene expression. Although ENC1 interacted with Keap1 indirectly, the ENC1-mediated down-regulation of Nrf2 was independent of Keap1. The negative effect of ENC1 on Nrf2 was not due to a change in the stability of Nrf2 because neither proteasomal nor lysosomal inhibitors had any effects. Overexpression of ENC1 did not result in a change in the level of Nrf2 mRNA, rather, it caused a decrease in the rate of Nrf2 protein synthesis. These results demonstrate that ENC1 functions as a negative regulator of Nrf2 through suppressing Nrf2 protein translation, which adds another level of complexity in controlling the Nrf2 signaling pathway.

  20. Silencing Nrf2 impairs glioma cell proliferation via AMPK-activated mTOR inhibition

    SciTech Connect

    Jia, Yue; Wang, Handong; Wang, Qiang; Ding, Hui; Wu, Heming; Pan, Hao

    2016-01-15

    Gliomas are the leading cause of death among adults with primary brain malignancies. Treatment for malignant gliomas remains limited, and targeted therapies have been incompletely explored. Nuclear factor erythroid 2-related factor 2 (Nrf2), a key transcription regulator for antioxidant and detoxification enzymes, is abundantly expressed in cancer cells. In this study, the role and mechanism of Nrf2 in cancer cell proliferation was investigated in multiple glioma cell lines. We first evaluated the expression patterns of Nrf2 in four glioma cell lines and found all four cell lines expressed Nrf2, but the highest level was observed in U251 cells. We further evaluated the biological functions of Nrf2 in U251 glioma cell proliferation by specific inhibition of Nrf2 using short hairpin RNA (shRNA). We found that Nrf2 depletion inhibited glioma cell proliferation. Nrf2 depletion also decreased colony formation in U251 cells stably expressing Nrf2 shRNA compared to scrambled control shRNA. Moreover, suppression of Nrf2 expression could lead to ATP depletion (with concomitant rise in AMP/ATP ratio) and consequently to AMPK-activated mTOR inhibition. Finally, activation of adenosine monophosphate–activated protein kinase (AMPK) by treated with phenformin, an AMPK agonist, can mimic the inhibitory effect of Nrf2 knockdown in U251 cells. In conclusion, our findings will shed light to the role and mechanism of Nrf2 in regulating glioma proliferation via ATP-depletion-induced AMPK activation and consequent mTOR inhibition, a novel insight into our understanding the role and mechanism of Nrf2 in glioma pathoetiology. To our knowledge, this is also the first report to provide a rationale for the implication of cross-linking between Nrf2 and mTOR signaling.

  1. Cytoprotection “gone astray”: Nrf2 and its role in cancer

    PubMed Central

    Geismann, Claudia; Arlt, Alexander; Sebens, Susanne; Schäfer, Heiner

    2014-01-01

    Nrf2 has gained great attention with respect to its pivotal role in cell and tissue protection. Primarily defending cells against metabolic, xenobiotic and oxidative stress, Nrf2 is essential for maintaining tissue integrity. Owing to these functions, Nrf2 is regarded as a promising drug target in the chemoprevention of diseases, including cancer. However, much evidence has accumulated that the beneficial role of Nrf2 in cancer prevention essentially depends on the tight control of its activity. In fact, the deregulation of Nrf2 is a critical determinant in oncogenesis and found in many types of cancer. Therefore, amplified Nrf2 activity has profound effects on the phenotype of tumor cells, including radio/chemoresistance, apoptosis protection, invasiveness, antisenescence, autophagy deficiency, and angiogenicity. The deregulation of Nrf2 can result from various epigenetic and genetic alterations directly affecting Nrf2 control or from the complex interplay of Nrf2 with numerous oncogenic signaling pathways. Additionally, alterations of the cellular environment, eg, during inflammation, contribute to Nrf2 deregulation and its persistent activation. Therefore, the status of Nrf2 as anti- or protumorigenic is defined by many different modalities. A better understanding of these modalities is essential for the safe use of Nrf2 as an activation target for chemoprevention on the one hand and as an inhibition target in cancer therapy on the other. The present review mainly addresses the conditions that promote the oncogenic function of Nrf2 and the resulting consequences providing the rationale for using Nrf2 as a target structure in cancer therapy. PMID:25210464

  2. Nrf2 and Nrf2-Related Proteins in Development and Developmental Toxicity: Insights from studies in Zebrafish (Danio rerio)

    PubMed Central

    Hahn, Mark E.; Timme-Laragy, Alicia R.; Karchner, Sibel I.; Stegeman, John J.

    2015-01-01

    Oxidative stress is an important mechanism of chemical toxicity, contributing to developmental toxicity and teratogenesis as well as to cardiovascular and neurodegenerative diseases and diabetic embryopathy. Developing animals are especially sensitive to effects of chemicals that disrupt the balance of processes generating reactive species and oxidative stress, and those anti-oxidant defenses that protect against oxidative stress. The expression and inducibility of anti-oxidant defenses through activation of NFE2-related factor 2 (Nrf2) and related proteins is an essential process affecting the susceptibility to oxidants, but the complex interactions of Nrf2 in determining embryonic response to oxidants and oxidative stress are only beginning to be understood. The zebrafish (Danio rerio) is an established model in developmental biology and now also in developmental toxicology and redox signaling. Here we review the regulation of genes involved in protection against oxidative stress in developing vertebrates, with a focus on Nrf2 and related cap’n’collar (CNC)-basic-leucine zipper (bZIP) transcription factors. Vertebrate animals including zebrafish share Nfe2, Nrf1, Nrf2, and Nrf3 as well as a core set of genes that respond to oxidative stress, contributing to the value of zebrafish as a model system with which to investigate the mechanisms involved in regulation of redox signaling and the response to oxidative stress during embryolarval development. Moreover, studies in zebrafish have revealed nrf and keap1 gene duplications that provide an opportunity to dissect multiple functions of vertebrate NRF genes, including multiple sensing mechanisms involved in chemical-specific effects. PMID:26130508

  3. The involvement of Nrf2 in the protective effects of diallyl disulfide on carbon tetrachloride-induced hepatic oxidative damage and inflammatory response in rats.

    PubMed

    Lee, In-Chul; Kim, Sung-Hwan; Baek, Hyung-Seon; Moon, Changjong; Kang, Seong-Soo; Kim, Sung-Ho; Kim, Yun-Bae; Shin, In-Sik; Kim, Jong-Choon

    2014-01-01

    This study investigated the potential effect of diallyl disulfide (DADS) against carbon tetrachloride (CCl4)-induced oxidative hepatic damage and inflammatory response in rat liver. DADS at doses of 50 and 100 mg/kg/day was administered orally once daily for 5 days, prior to CCl4 administration. Pretreatment with DADS attenuated CCl4-induced elevated serum transaminase activities and histopathological alterations in liver. It prevented the hepatocellular apoptotic changes with induction of Bcl-2-associated X (Bax), cytochrome c, and caspase-3 caused by CCl4. An increase in the nuclear translocation of nuclear factor-kappaB (NF-κB) and phosphorylation of I kappaB alpha (IκBα) was observed in the livers of CCl4-treated rats that coincided with induction of inflammatory mediators or cytokines. In contrast, DADS inhibited NF-κB translocation and IκBα phosphorylation, and that subsequently decreased inflammatory mediators. Furthermore, DADS prevented CCl4-induced depletion of cytosolic nuclear factor E2-related factor 2 (Nrf2) and suppression of nuclear translocation of Nrf2, which, in turn, up-regulated phase II/antioxidant enzyme activities. Taken together, these results demonstrate that DADS increases the expression of phase II/antioxidant enzymes and simultaneously decreases the expression of inflammatory mediators in CCl4-induced liver injury. These findings indicate that DADS induces antioxidant defense mechanism by activating Nrf2 pathway and reduces inflammatory response by inhibiting NF-κB activation. Copyright © 2013 Elsevier Ltd. All rights reserved.

  4. Identification of an adaptor protein that facilitates Nrf2-Keap1 complex formation and modulates antioxidant response.

    PubMed

    Zhang, Yuxue; Hou, Yongfan; Liu, Chunchun; Li, Yinlong; Guo, Weiwei; Wu, Jiu-Lin; Xu, Daqian; You, Xue; Pan, Yi; Chen, Yan

    2016-08-01

    Nrf2 plays a key role in the protection of the body against environmental stress via inducible expression of detoxification and antioxidant enzymes. Keap1 functions as a sensor for oxidative and electrophilic stresses and promotes Nrf2 degradation via its E3 ligase activity. Modulation of the Nrf2-Keap1 pathway has been extensively explored as a strategy to combat against drug toxicity and stress-induced diseases. Here we report a new player that modulates the Nrf2-Keap1 pathway. PAQR3, a membrane protein specifically localized in the Golgi apparatus, negatively regulates the expression of an array of Nrf2 target genes and alters cellular level of reactive oxygen species. PAQR3 tethers Nrf2 and Keap1, but not small MAF proteins to the Golgi apparatus. PAQR3 interacts with both Nrf2 and Keap1 and facilitates the interaction of Nrf2 with Keap1. PAQR3 promotes ubiquitination and degradation of Nrf2. Disruption of PAQR3 interaction with Nrf2 and Keap1 by a synthetic peptide reduces Nrf2 ubiquitination and elevates expression of Nrf2 target genes. At the animal level, deletion of PAQR3 increases Nrf2 protein level and the expression of Nrf2 target genes. In conclusion, our study pinpoints that PAQR3 functions as an adaptor protein to promote Nrf2-Keap1 complex formation, thereby modulating the Nrf2-Keap2 pathway and playing an important role in controlling antioxidant response of the cell.

  5. Safranal prevents rotenone-induced oxidative stress and apoptosis in an in vitro model of Parkinson's disease through regulating Keap1/Nrf2 signaling pathway.

    PubMed

    Pan, P-K; Qiao, L-Y; Wen, X-N

    2016-12-30

    Safranal, a major constituent of saffron, possesses antioxidant and anti-apoptotic properties showing considerable neuroprotective effects. However, whether safranal shows therapeutic effect on Parkinson's disease (PD) remains unknown. In this study, we aimed to investigate the potential effect of safranal on PD using an in vitro model of PD induced by rotenone. We found that safranal significantly inhibited rotenone-induced cell death in a dose-dependent manner. Moreover, safranal also markedly suppressed the reactive oxygen species (ROS) generation and cell apoptosis induced by rotenone. Further investigation showed that safranal inhibited the expression of kelch-like ECH-associated protein 1 (Keap1) and promoted the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) in rotenone-induced dopaminergic neurons. Meanwhile, the downstream antioxidant enzyme genes of Nrf2 including glutathione S transferase (GST), glutamate-cysteine ligase catalytic subunit (GCLc), NADPH-quinone oxidoreductase 1 (NQO1) and heme oxygenase1 (HO-1) were also induced by safranal in rotenone-induced dopaminergic neurons. However, the knockdown of Nrf2 significantly abrogated the protective effect of safranal on rotenone-induced neurotoxicity. Taken together, our study suggests that safranal protects against rotenone-induced neurotoxicity associated with Nrf2 signaling pathway implying that safranal may serve as a potent and promising therapeutic drug for the treatment of PD.

  6. Activation of Nrf2 target enzymes conferring protection against oxidative stress in PC12 cells by ginger principal constituent 6-shogaol.

    PubMed

    Peng, Shoujiao; Yao, Juan; Liu, Yaping; Duan, Dongzhu; Zhang, Xiaolong; Fang, Jianguo

    2015-08-01

    Natural compounds containing phenoxyl groups and/or Michael acceptor units appear to possess antioxidant and cytoprotective properties. The ginger principal constituent 6-shogaol (6-S) represents one of such compounds. In this study, we reported that 6-S efficiently scavenges various free radicals in vitro, and displays remarkable cytoprotection against oxidative stress-induced cell damage in the neuron-like rat pheochromocytoma cell line, PC12 cells. Pretreatment of PC12 cells with 6-S significantly upregulates a series of phase II antioxidant molecules, such as glutathione, heme oxygenase 1, NAD(P)H: quinone oxidoreductase 1, thioredoxin reductase 1, and thioredoxin 1. A mechanistic study revealed that 6-S enhanced the translocation of Nrf2 from the cytosol to the nucleus and knockdown of Nrf2 abolished such protection, indicating that this cytoprotection is mediated by the activation of the transcription factor Nrf2. Another ginger constituent 6-gingerol (6-G), having a similar structure of 6-S but lacking the alpha,beta-unsaturated ketone structure (Michael acceptor moiety), failed to shelter PC12 cells from oxidative stress. Our results demonstrate that 6-S is a novel small molecule activator of Nrf2 in PC12 cells, and suggest that 6-S might be a potential candidate for the prevention of oxidative stress-mediated neurodegenerative disorders.

  7. Induction of Nrf2-mediated genes by Antrodia salmonea inhibits ROS generation and inflammatory effects in lipopolysaccharide-stimulated RAW264.7 macrophages.

    PubMed

    Yang, Hsin-Ling; Lin, Shu-Wei; Lee, Chuan-Chen; Lin, Kai-Yuan; Liao, Chun-Huei; Yang, Ting-Yu; Wang, Hui-Min; Huang, Hui-Chi; Wu, Chi-Rei; Hseu, You-Cheng

    2015-01-01

    Antrodia salmonea (AS), a well-known medicinal mushroom in Taiwan, has been reported to exhibit anti-oxidant, anti-angiogenic, anti-atherogenic, and anti-inflammatory effects. In the present study, we investigated the activation of Nrf2-mediated antioxidant genes in RAW264.7 macrophages by the fermented culture broth of AS, studied the resulting protection against lipopolysaccharide (LPS)-stimulated inflammation, and revealed the molecular mechanisms underlying these protective effects. We found that non-cytotoxic concentrations of AS (25-100 μg mL⁻¹) protected macrophages from LPS-induced cell death and ROS generation in a dose-dependent manner. The antioxidant potential of AS was directly correlated with the increased expression of the antioxidant genes HO-1, NQO-1, and γ-GCLC, as well as the level of intracellular GSH followed by an increase in the nuclear translocation and transcriptional activation of the Nrf2-ARE pathway. Furthermore, Nrf2 knockdown diminished the protective effects of AS, as evidenced by the increased production of pro-inflammatory cytokines and chemokines, including PGE₂, NO, TNF-α, and IL-1β, in LPS-stimulated macrophages. Notably, AS treatment significantly inhibited LPS-induced ICAM-1 expression in macrophages. Our data suggest that the anti-inflammatory potential of Antrodia salmonea is mediated by the activation of Nrf2-dependent antioxidant defense mechanisms. Results support the traditional usage of this beneficial mushroom for the treatment of free radical-related diseases and inflammation.

  8. Glycyrrhizin reduces HMGB1 secretion in lipopolysaccharide-activated RAW 264.7 cells and endotoxemic mice by p38/Nrf2-dependent induction of HO-1.

    PubMed

    Kim, Young Min; Kim, Hye Jung; Chang, Ki Churl

    2015-05-01

    High mobility group box 1 (HMGB1) is now recognized as a late mediator of sepsis. Although glycyrrhizin was known as inhibitor of HMGB1, it is not yet clear underlying mechanism(s). We found that glycyrrhizin activates translocation of Nrf2 from cytosol to nucleus and induces heme oxygenase (HO)-1 expression in RAW 264.7 cells. In addition, deletion of Nrf2 by siRNA significantly reduced mRNA expression of NQO1 and HO-1 suggesting that glycyrrhizin targets Nrf2 gene. The expression of iNOS protein and release of HMGB1 in LPS activated cells were significantly reduced by glycyrrhizin and cells transfected with mouse HO-1 expression vector. The p38MAPK inhibitor (SB203580) but not JNK inhibitor (SP600125) or ERK inhibitor (PD98059) significantly inhibited HO-1 induction by glycyrrhizin, which was confirmed by showing that siP38 transfected cells significantly reduced HO-1 induction. Pretreatment with SB203580 significantly reversed the expression of iNOS and release of NO and HMGB1 in LPS-activated cells. Most importantly, administration of glycyrrhizin (200mg/kg, i.p) significantly reduced hepatic injury and serum HMGB1 in a ZnPPIX-sensitive manner. Thus, it is concluded that glycyrrhizin reduces HMGB1 secretion in lipopolysaccharide-activated RAW 264.7 cells and endotoxemic mice by p38/Nrf2-dependent induction of HO-1. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Coniferaldehyde inhibits LPS-induced apoptosis through the PKC α/β II/Nrf-2/HO-1 dependent pathway in RAW264.7 macrophage cells.

    PubMed

    Kim, Ki Mo; Heo, Deok Rim; Kim, Young-A; Lee, Jun; Kim, No Soo; Bang, Ok-Sun

    2016-12-01

    Coniferaldehyde (CA) exerts anti-inflammatory properties by inducing heme oxygenase-1 (HO-1). To define the regulation mechanism by which CA induces a cytoprotective function and HO-1 expression, the up-stream regulations involved in the activation of nuclear transcription factor-erythroid 2-related factor (Nrf)-2/HO-1 pathway were investigated. CA dramatically increased the Nrf-2 nuclear translocation and HO-1 expression. Lipopolysaccharide (LPS)-induced expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2, and cell death were down-regulated by CA, which were reversed by inhibition of HO-1 activity. Furthermore, CA specifically enhanced the phosphorylation of protein kinase C (PKC) α/β II. Selective inhibition of PKC α/β II using Go6976 or siRNA abolished the CA-induced Nrf-2/HO-1 signaling, and consequently suppressed the cytoprotective activity of CA on the LPS-induced cell death. Together, our results elucidate the regulatory mechanism of PKC α/β II as the upstream molecule of Nrf-2 required for HO-1 expression during CA-induced anti-inflammatory cytoprotective function in LPS stimulated macrophages. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. IL-11 contribution to tumorigenesis in an NRF2 addiction cancer model.

    PubMed

    Kitamura, H; Onodera, Y; Murakami, S; Suzuki, T; Motohashi, H

    2017-07-17

    The interaction between cancer cells and their microenvironment is an important determinant of the pathological nature of cancers, particularly their tumorigenic abilities. The KEAP1-NRF2 system, originally identified as a critical defense mechanism against oxidative stress, is often dysregulated in various human cancers forming solid tumors, resulting in the aberrant activation of NRF2. Increased accumulation of NRF2 in cancers is strongly associated with the poor prognoses of cancer patients, including those with lung and breast cancers. Multiple lines of evidence suggest that aberrantly activated NRF2 in cancer cells drives their malignant progression and that the cancer cells consequently develop 'NRF2 addiction.' Although the downstream effectors of NRF2 that are responsible for cancer malignancy have been extensively studied, mechanisms of how NRF2 activation contributes to the aggressive tumorigenesis remains to be elucidated. In this study, we found a significant correlation between NRF2 and IL-11 status in breast cancer patients. Based on a recent report demonstrating that IL-11 is induced downstream of NRF2, we examined the significance of IL-11 in NRF2-driven tumorigenesis with a newly established NRF2 addiction cancer model. Expression of Il11 was elevated during the tumorigenesis of the NRF2 addiction cancer model, but intriguingly, it was hardly detected when the cancer model cells were cultured in vitro. These results imply that a signal originating from the microenvironment cooperates with NRF2 to activate Il11. To the best of our knowledge, this is the first report showing the influence of the microenvironment on the NRF2 pathway in cancer cells and the contribution of NRF2 to the secretory phenotypes of cancers. Disruption of Il11 in the NRF2 addiction cancer model remarkably inhibited the tumorigenesis, suggesting an essential role of IL-11 in NRF2-driven tumorigenesis. Thus, this study suggests that IL-11 is a potential therapeutic target for

  11. Nrf2 deficiency improves glucose tolerance in mice fed a high-fat diet

    SciTech Connect

    Zhang, Yu-Kun Jennifer; Wu, Kai Connie; Liu, Jie; Klaassen, Curtis D.

    2012-11-01

    Nrf2, a master regulator of intracellular redox homeostasis, is indicated to participate in fatty acid metabolism in liver. However, its role in diet-induced obesity remains controversial. In the current study, genetically engineered Nrf2-null, wild-type (WT), and Nrf2-activated, Keap1-knockdown (K1-KD) mice were fed either a control or a high-fat Western diet (HFD) for 12 weeks. The results indicate that the absence or enhancement of Nrf2 activity did not prevent diet-induced obesity, had limited effects on lipid metabolism, but affected blood glucose homeostasis. Whereas the Nrf2-null mice were resistant to HFD-induced glucose intolerance, the Nrf2-activated K1-KD mice exhibited prolonged elevation of circulating glucose during a glucose tolerance test even on the control diet. Feeding a HFD did not activate the Nrf2 signaling pathway in mouse livers. Fibroblast growth factor 21 (Fgf21) is a liver-derived anti-diabetic hormone that exerts glucose- and lipid-lowering effects. Fgf21 mRNA and protein were both elevated in livers of Nrf2-null mice, and Fgf21 protein was lower in K1-KD mice than WT mice. The inverse correlation between Nrf2 activity and hepatic expression of Fgf21 might explain the improved glucose tolerance in Nrf2-null mice. Furthermore, a more oxidative cellular environment in Nrf2-null mice could affect insulin signaling in liver. For example, mRNA of insulin-like growth factor binding protein 1, a gene repressed by insulin in hepatocytes, was markedly elevated in livers of Nrf2-null mice. In conclusion, genetic alteration of Nrf2 does not prevent diet-induced obesity in mice, but deficiency of Nrf2 improves glucose homeostasis, possibly through its effects on Fgf21 and/or insulin signaling. -- Highlights: ► Nrf2 deficiency improves glucose tolerance in mice fed a high-fat diet. ► The anti-diabetic hormone, Fgf21, is highly expressed in livers of Nrf2-null mice. ► The absence of Nrf2 increases the insulin-regulated Igfbp-1 mRNA in liver.

  12. PECAM-1-dependent heme oxygenase-1 regulation via an Nrf2-mediated pathway in endothelial cells.

    PubMed

    Saragih, Hendry; Zilian, Eva; Jaimes, Yarúa; Paine, Ananta; Figueiredo, Constanca; Eiz-Vesper, Britta; Blasczyk, Rainer; Larmann, Jan; Theilmeier, Gregor; Burg-Roderfeld, Monika; Andrei-Selmer, Luminita-Cornelia; Becker, Jan Ulrich; Santoso, Sentot; Immenschuh, Stephan

    2014-06-01

    The antioxidant enzyme heme oxygenase (HO)-1, which catalyses the first and rate-limiting step of heme degradation, has major anti-inflammatory and immunomodulatory effects via its cell-type-specific functions in the endothelium. In the current study, we investigated whether the key endothelial adhesion and signalling receptor PECAM-1 (CD31) might be involved in the regulation of HO-1 gene expression in human endothelial cells (ECs). To this end PECAM-1 expression was down-regulated in human umbilical vein ECs (HUVECs) by an adenoviral vector-based knockdown approach. PECAM-1 knockdown markedly induced HO-1, but not the constitutive HO isoform HO-2. Nuclear translocation of the transcription factor NF-E2-related factor-2 (Nrf2), which is a master regulator of the inducible antioxidant cell response, and intracellular levels of reactive oxygen species (ROS) were increased in PECAM-1-deficient HUVECs, respectively. PECAM-1-dependent HO-1 regulation was also examined in PECAM-1 over-expressing Chinese hamster ovary and murine L-cells. Endogenous HO-1 gene expression and reporter gene activity of transiently transfected luciferase HO-1 promoter constructs with Nrf2 target sequences were decreased in PECAM-1 over-expressing cells. Moreover, a regulatory role of ROS for HO-1 regulation in these cells is demonstrated by studies with the antioxidant N-acetylcysteine and exogenous hydrogenperoxide. Finally, direct interaction of PECAM-1 with a native complex of its binding partner NB1 (CD177) and serine proteinase 3 (PR3) from human neutrophils, markedly induced HO-1 expression in HUVECs. Taken together, we demonstrate a functional link between HO-1 gene expression and PECAM-1 in human ECs, which might play a critical role in the regulation of inflammation.

  13. The Keap1-Nrf2-ARE Pathway As a Potential Preventive and Therapeutic Target: An Update.

    PubMed

    Lu, Meng-Chen; Ji, Jian-Ai; Jiang, Zheng-Yu; You, Qi-Dong

    2016-09-01

    The Keap1-Nrf2-ARE ((Kelch-like ECH-Associating protein 1) nuclear factor erythroid 2 related factor 2-antioxidant response element) pathway is one of the most important defense mechanisms against oxidative and/or electrophilic stresses, and it is closely associated with inflammatory diseases, including cancer, neurodegenerative diseases, cardiovascular diseases, and aging. In recent years, progress has been made in strategies aimed at modulating the Keap1-Nrf2-ARE pathway. The Nrf2 activator DMF (Dimethylfumarates) has been approved by the FDA as a new first-line oral drug to treat patients with relapsing forms of multiple sclerosis, while a phase 3 study of another promising candidate, CDDO-Me, was terminated for safety reasons. Directly inhibiting Keap1-Nrf2 protein-protein interactions as a novel Nrf2-modulating strategy has many advantages over using electrophilic Nrf2 activators. The development of Keap1-Nrf2 protein-protein interaction inhibitors has become a topic of intense research, and potent inhibitors of this target have been identified. In addition, inhibiting Nrf2 activity has attracted an increasing amount of attention because it may provide an alternative cancer therapy. This review summarizes the molecular mechanisms and biological functions of the Keap1-Nrf2-ARE system. The main focus of this review is on recent progress in studies of agents that target the Keap1-Nrf2-ARE pathway and the therapeutic applications of such agents.

  14. Artemisitene activates the Nrf2-dependent antioxidant response and protects against bleomycin-induced lung injury.

    PubMed

    Chen, Weimin; Li, Shanshan; Li, Jinwei; Zhou, Wen; Wu, Shouhai; Xu, Shengmei; Cui, Ke; Zhang, Donna D; Liu, Bo

    2016-07-01

    The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) is a crucial regulator of the cellular antioxidant response and xenobiotic metabolism. Activation of the Nrf2 signaling pathway has been demonstrated to confer protection against environmental insults and prevent disease or inhibit the progression of diseases related to oxidative stress. In an attempt to identify novel improved Nrf2 inducers for systemic protection against tissue damage by environmental insults, we identified artemisitene as a novel Nrf2 activator using antioxidant responsive element luciferase assay in MDA-MB-231 cells. Further studies suggest that artemisitene activates Nrf2 by decreasing Nrf2 ubiquitination and increasing its stability. In Nrf2 wild-type mice, systemic administration of artemisitene strongly inhibits bleomycin-induced lung damage. Artemisitene represents a novel class of Nrf2 inducer, and artemisitene-based therapeutic approach targeting Nrf2 may also provide antioxidant protection for humans against tissue damage by toxic chemicals.-Chen, W., Li, S., Li, J., Zhou, W., Wu, S., Xu, S., Cui, K., Zhang, D. D., Liu, B. Artemisitene activates the Nrf2-dependent antioxidant response and protects against bleomycin-induced lung injury.

  15. Small Molecule Modulators of Keap1-Nrf2-ARE Pathway as Potential Preventive and Therapeutic Agents$

    PubMed Central

    Magesh, Sadagopan; Chen, Yu; Hu, Longqin

    2012-01-01

    Keap1-Nrf2-ARE pathway represents one of the most important cellular defense mechanisms against oxidative stress and xenobiotic damage. Activation of Nrf2 signaling induces the transcriptional regulation of ARE-dependent expression of various detoxifying and antioxidant defense enzymes and proteins. Keap1-Nrf2-ARE signaling has become an attractive target for the prevention and treatment of oxidative stress-related diseases and conditions including cancer, neurodegenerative, cardiovascular, metabolic and inflammatory diseases. Over the last few decades, numerous Nrf2 inducers have been developed and some of them are currently undergoing clinical trials. Recently, over-activation of Nrf2 has been implicated in cancer progression as well as in drug resistance to cancer chemotherapy. Thus, Nrf2 inhibitors could potentially be used to improve the effectiveness of cancer therapy. Herein, we review the signaling mechanism of Keap1-Nrf2-ARE pathway, its disease relevance, and currently known classes of small molecule modulators. We also discuss several aspects of Keap1-Nrf2 interaction, Nrf2-based peptide inhibitor design, and the screening assays currently used for the discovery of direct inhibitors of Keap1-Nrf2 interaction. PMID:22549716

  16. NRF2 immunolocalization in human breast cancer patients as a prognostic factor.

    PubMed

    Onodera, Yoshiaki; Motohashi, Hozumi; Takagi, Kiyoshi; Miki, Yasuhiro; Shibahara, Yukiko; Watanabe, Mika; Ishida, Takanori; Hirakawa, Hisashi; Sasano, Hironobu; Yamamoto, Masayuki; Suzuki, Takashi

    2014-04-01

    Nuclear factor erythroid 2-related factor 2 (NRF2 (NFE2L2)) is an important transcriptional activator involved in the cellular defense mechanisms against electrophilic and oxidative stress. Recent studies have demonstrated that the expression of NRF2 protein is upregulated in several human malignancies and is associated with worse prognosis in these patients. However, the pathological and clinical significance of NRF2 has remained largely unknown in breast cancer patients. Therefore, in this study, we immunolocalized NRF2 in 106 breast carcinoma cases. NRF2 immunoreactivity was mainly detected in the nucleus of the breast carcinoma cells and it was positive in 44% of the cases. NRF2 status was significantly associated with histological grade, Ki-67 labeling index, p62 immunoreactivity, and quinone oxidoreductase 1 (NQO1) immunoreactivity, and the results of multivariate analyses revealed that NRF2 status was an independent adverse prognostic factor for both recurrence and disease-free survival of the patients. Subsequent in vitro studies demonstrated that the expression of NRF2 significantly increased the proliferation activity of MCF7 and SK-BR-3 breast carcinoma cells. These results indicate that nuclear NRF2 protein plays important roles in the proliferation and/or progression of breast carcinoma, and nuclear NRF2 immunoreactivity is therefore considered a potent prognostic factor in breast cancer patients.

  17. Conservation of the Nrf2-Mediated Gene Regulation of Proteasome Subunits and Glucose Metabolism in Zebrafish

    PubMed Central

    Fuse, Yuji; Tamaoki, Junya; Akiyama, Shin-ichi; Muratani, Masafumi

    2016-01-01

    The Keap1-Nrf2 system is an evolutionarily conserved defense mechanism against oxidative and xenobiotic stress. Besides the exogenous stress response, Nrf2 has been found to regulate numerous cellular functions, including protein turnover and glucose metabolism; however, the evolutionary origins of these functions remain unknown. In the present study, we searched for novel target genes associated with the zebrafish Nrf2 to answer this question. A microarray analysis of zebrafish embryos that overexpressed Nrf2 revealed that 115 candidate genes were targets of Nrf2, including genes encoding proteasome subunits and enzymes involved in glucose metabolism. A real-time quantitative PCR suggested that the expression of 3 proteasome subunits (psma3, psma5, and psmb7) and 2 enzymes involved in glucose metabolism (pgd and fbp1a) were regulated by zebrafish Nrf2. We thus next examined the upregulation of these genes by an Nrf2 activator, diethyl maleate, using Nrf2 mutant zebrafish larvae. The results of real-time quantitative PCR and whole-mount in situ hybridization showed that all of these 5 genes were upregulated by diethyl maleate treatment in an Nrf2-dependent manner, especially in the liver. These findings implied that the Nrf2-mediated regulation of the proteasome subunits and glucose metabolism is evolutionarily conserved among vertebrates. PMID:28116036

  18. Vitamin E prevents NRF2 suppression by allergens in asthmatic alveolar macrophages in vivo.

    PubMed

    Dworski, Ryszard; Han, Wei; Blackwell, Timothy S; Hoskins, Aimee; Freeman, Michael L

    2011-07-15

    Asthma is a chronic inflammatory airway disease associated with increased generation of reactive oxidant species and disturbed antioxidant defenses. NRF2 is the master transcription factor that regulates the expression of Phase II antioxidant and detoxifying enzymes. Disruption of NRF2 augments oxidative stress and inflammation in a mouse model of asthma, suggesting a protective role for NRF2 in the lungs in vivo. Yet, little is known about the regulation and function of NRF2 in human asthmatics. Using segmental allergen challenge, a well-established experimental model of IgE-mediated asthma exacerbation in human atopic asthmatics, we investigated the effects of a specific allergen and the modulatory role of vitamin E on NRF2 and a NRF2-target gene, superoxide dismutase, in alveolar macrophages recovered from the airways at 24h after allergen instillation in vivo. Allergen-provoked airway inflammation in sensitive asthmatics caused a profound inhibition of macrophage NRF2 activity and superoxide dismutase, rendering them incapable of responding to the NRF2 inducers. Prolonged treatment with high doses of the antioxidant vitamin E lessened this allergen-induced drop in alveolar macrophage NRF2. These results are the first to demonstrate that NRF2 expression in human asthmatics is compromised upon allergen challenge but can be rescued by vitamin E in vivo.

  19. The complexity of the Nrf2 pathway: Beyond the antioxidant response

    PubMed Central

    Huang, Ying; Li, Wenji; Su, Zheng-yuan; Kong, Ah-Ng Tony

    2016-01-01

    The NF-E2-related factor 2 (Nrf2)-mediated signaling pathway provides living organisms an efficient and pivotal line of defensive to counteract environmental insults and endogenous stressors. Nrf2 coordinates the basal and inducible expression of antioxidant and phase II detoxification enzymes to adapt to different stress conditions. The stability and cellular distribution of Nrf2 is tightly controlled by its inhibitory binding protein Kelch-like ECH-associated protein 1 (Keap1). Nrf2 signaling is also regulated by post-translational, transcriptional, translational and epigenetic mechanisms, as well as by other protein partners, including p62, p21 and IQ motif-containing GTPase activating protein 1 (IQGAP1). Many studies have demonstrated that Nrf2 is a promising target for preventing carcinogenesis and other chronic diseases, including cardiovascular diseases, neurodegenerative diseases, and pulmonary injury. However, constitutive activation of Nrf2 in advanced cancer cells may confer drug resistance. Here, we review the molecular mechanisms of Nrf2 signaling, the diverse classes of Nrf2 activators, including bioactive nutrients and other chemicals and the cellular functions and disease relevance of Nrf2 and discuss the dual role of Nrf2 in different contexts. PMID:26419687

  20. Are Astrocytes the Predominant Cell Type for Activation of Nrf2 in Aging and Neurodegeneration?

    PubMed Central

    2017-01-01

    Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that regulates hundreds of antioxidant genes, and is activated in response to oxidative stress. Given that many neurodegenerative diseases including Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, Huntington’s disease and multiple sclerosis are characterised by oxidative stress, Nrf2 is commonly activated in these diseases. Evidence demonstrates that Nrf2 activity is repressed in neurons in vitro, and only cultured astrocytes respond strongly to Nrf2 inducers, leading to the interpretation that Nrf2 signalling is largely restricted to astrocytes. However, Nrf2 activity can be observed in neurons in post-mortem brain tissue and animal models of disease. Thus this interpretation may be false, and a detailed analysis of the cell type expression of Nrf2 in neurodegenerative diseases is required. This review describes the evidence for Nrf2 activation in each cell type in prominent neurodegenerative diseases and normal aging in human brain and animal models of neurodegeneration, the response to pharmacological and genetic modulation of Nrf2, and clinical trials involving Nrf2-modifying drugs. PMID:28820437

  1. WNT-3A Regulates an Axin1/NRF2 Complex That Regulates Antioxidant Metabolism in Hepatocytes

    PubMed Central

    Rada, Patricia; Rojo, Ana I.; Offergeld, Anika; Feng, Gui Jie; Velasco-Martín, Juan P.; González-Sancho, José Manuel; Valverde, Ángela M.; Dale, Trevor; Regadera, Javier

    2015-01-01

    Abstract Aims: Nuclear factor (erythroid-derived 2)-like 2 (NRF2) is a master regulator of oxidant and xenobiotic metabolism, but it is unknown how it is regulated to provide basal expression of this defense system. Here, we studied the putative connection between NRF2 and the canonical WNT pathway, which modulates hepatocyte metabolism. Results: WNT-3A increased the levels of NRF2 and its transcriptional signature in mouse hepatocytes and HEK293T cells. The use of short interfering RNAs in hepatocytes and mouse embryonic fibroblasts which are deficient in the redox sensor Kelch-like ECH-associated protein 1 (KEAP1) indicated that WNT-3A activates NRF2 in a β-Catenin- and KEAP1-independent manner. WNT-3A stabilized NRF2 by preventing its GSK-3-dependent phosphorylation and subsequent SCF/β-TrCP-dependent ubiquitination and proteasomal degradation. Axin1 and NRF2 were physically associated in a protein complex that was regulated by WNT-3A, involving the central region of Axin1 and the Neh4/Neh5 domains of NRF2. Axin1 knockdown increased NRF2 protein levels, while Axin1 stabilization with Tankyrase inhibitors blocked WNT/NRF2 signaling. The relevance of this novel pathway was assessed in mice with a conditional deletion of Axin1 in the liver, which showed upregulation of the NRF2 signature in hepatocytes and disruption of liver zonation of antioxidant metabolism. Innovation: NRF2 takes part in a protein complex with Axin1 that is regulated by the canonical WNT pathway. This new WNT-NRF2 axis controls the antioxidant metabolism of hepatocytes. Conclusion: These results uncover the participation of NRF2 in a WNT-regulated signalosome that participates in basal maintenance of hepatic antioxidant metabolism. Antioxid. Redox Signal. 22, 555–571. PMID:25336178

  2. Nrf2 Expression and Apoptosis in Quercetin-treated Malignant Mesothelioma Cells.

    PubMed

    Lee, Yoon-Jin; Lee, David M; Lee, Sang-Han

    2015-05-01

    NF-E2-related factor 2 (Nrf2), a basic leucine zipper transcription factor, has recently received a great deal of attention as an important molecule that enhances antioxidative defenses and induces resistance to chemotherapy or radiotherapy. In this study, we investigated the apoptosis-inducing and Nrf2-upregulating effects of quercetin on malignant mesothelioma (MM) MSTO-211H and H2452 cells. Quercetin treatment inhibited cell growth and led to upregulation of Nrf2 at both the mRNA and protein levels without altering the ubiquitination and extending the half-life of the Nrf2 protein. Following treatment with quercetin, analyses of the nuclear level of Nrf2, Nrf2 antioxidant response element-binding assay, Nrf2 promoter-luc assay, and RT-PCR toward the Nrf2-regulated gene, heme oxygenase-1, demonstrated that the induced Nrf2 is transcriptionally active. Knockdown of Nrf2 expression with siRNA enhanced cytotoxicity due to the induction of apoptosis, as evidenced by an increase in the level of proapoptotic Bax, a decrease in the level of antiapoptotic Bcl-2 with enhanced cleavage of caspase-3 and PARP proteins, the appearance of a sub-G0/G1 peak in the flow cytometric assay, and increased percentage of apoptotic propensities in the annexin V binding assay. Effective reversal of apoptosis was observed following pretreatment with the pan-caspase inhibitor Z-VAD. Moreover, Nrf2 knockdown exhibited increased sensitivity to the anticancer drug, cisplatin, presumably by potentiating the oxidative stress induced by cisplatin. Collectively, our data demonstrate the importance of Nrf2 in cytoprotection, survival, and drug resistance with implications for the potential significance of targeting Nrf2 as a promising strategy for overcoming resistance to chemotherapeutics in MM.

  3. Trafficking of the Transcription Factor Nrf2 to Promyelocytic Leukemia-Nuclear Bodies

    PubMed Central

    Malloy, Melanie Theodore; McIntosh, Deneshia J.; Walters, Treniqka S.; Flores, Andrea; Goodwin, J. Shawn; Arinze, Ifeanyi J.

    2013-01-01

    Ubiquitylation of Nrf2 by the Keap1-Cullin3/RING box1 (Cul3-Rbx1) E3 ubiquitin ligase complex targets Nrf2 for proteasomal degradation in the cytoplasm and is an extensively studied mechanism for regulating the cellular level of Nrf2. Although mechanistic details are lacking, reports abound that Nrf2 can also be degraded in the nucleus. Here, we demonstrate that Nrf2 is a target for sumoylation by both SUMO-1 and SUMO-2. HepG2 cells treated with As2O3, which enhances attachment of SUMO-2/3 to target proteins, increased SUMO-2/3-modification (polysumoylation) of Nrf2. We show that Nrf2 traffics, in part, to promyelocytic leukemia-nuclear bodies (PML-NBs). Cell fractions harboring key components of PML-NBs did not contain biologically active Keap1 but contained modified Nrf2 as well as RING finger protein 4 (RNF4), a poly-SUMO-specific E3 ubiquitin ligase. Overexpression of wild-type RNF4, but not the catalytically inactive mutant, decreased the steady-state levels of Nrf2, measured in the PML-NB-enriched cell fraction. The proteasome inhibitor MG-132 interfered with this decrease, resulting in elevated levels of polysumoylated Nrf2 that was also ubiquitylated. Wild-type RNF4 accelerated the half-life (t½) of Nrf2, measured in PML-NB-enriched cell fractions. These results suggest that RNF4 mediates polyubiquitylation of polysumoylated Nrf2, leading to its subsequent degradation in PML-NBs. Overall, this work identifies Nrf2 as a target for sumoylation and provides a novel mechanism for its degradation in the nucleus, independent of Keap1. PMID:23543742

  4. Nrf2 is critical in defense against high glucose-induced oxidative damage in cardiomyocytes.

    PubMed

    He, Xiaoqing; Kan, Hong; Cai, Lu; Ma, Qiang

    2009-01-01

    Exposure to high levels of glucose induces the production of reactive oxygen species (ROS) in cardiomyocytes that may contribute to the development of cardiomyopathy in diabetes. Nuclear factor erythroid 2-related factor 2 (Nrf2) controls the antioxidant response element (ARE)-dependent gene regulation in response to oxidative stress. The role of Nrf2 in defense against high glucose-induced oxidative damage in cardiomyocytes was investigated. Glucose at high concentrations induced ROS production in both primary neonatal and adult cardiomyocytes from the Nrf2 wild type (WT) mouse heart, whereas, in Nrf2 knockout (KO) cells, ROS was significantly higher under basal conditions and high glucose markedly further increased ROS production in concentration and time-dependent manners. Concomitantly, high glucose induced significantly higher levels of apoptosis at lower concentrations and in shorter time in Nrf2 KO cells than in WT cells. Primary adult cardiomyocytes from control and diabetic mice also showed dependence on Nrf2 function for isoproterenol-stimulated contraction. Additionally, cardiomyocytes from Nrf2 KO mice exhibited increased sensitivity to 3-nitropropionic acid, an inhibitor of mitochondrial respiratory complex II, for both ROS production and apoptosis compared with Nrf2 WT cells, further emphasizing the role of Nrf2 in ROS defense in the cells. Mechanistically, Nrf2 was shown to mediate the basal expression and induction of ARE-controlled cytoprotective genes, Nqo1 and Ho1, at both mRNA and protein levels in cardiomyocytes, as both the basal and inducible expressions of the genes were lost in Nrf2 KO cells or largely reduced by Nrf2 SiRNA. The findings, for the first time, established Nrf2 as a critical regulator of defense against ROS in normal and diabetic hearts.

  5. Mechanism of progestin resistance in endometrial precancer/cancer through Nrf2-survivin pathway

    PubMed Central

    Fan, Rujia; Wang, Yiying; Wang, Yue; Wei, Li; Zheng, Wenxin

    2017-01-01

    Progestin is commonly used for young patients suffering from endometrial hyperplasia or cancer. However, there is approximately 30% failure rate with unclear mechanism. We investigated if Nrf2-survivin pathway contributes the progestin resistance (PR) in this setting. Current study detected Nrf2 and survivin protein expression in post progestin treated endometrial tissue samples by using immunohistochemistry. Transfection of Nrf2 and survivin into endometrial cancer cells in vitro was done to determine the roles of Nrf2 and survivin in progestin resistance. Silence of survivin was then performed to explore if Nrf2-driven progestin resistance is mediated by survivin. Medorxyprogesterone acetate (MPA) and metformin were applied to examine the cellular proliferations under the controlled conditions. Overexpression of survivin and Nrf2 were found in progestin-resistant endometrial samples as well as in those areas with only partial responses after MPA treatment. In contrast, all responded endometrial tissue with complete decidualization showed negative expression of these two biomarkers. Exogenous overexpression of Nrf2 and survivin resulted in progestin resistance. In addition, reduction of survivin in endometrial cancer cells overcame the Nrf2 overexpression induced progestin resistance. Furthermore, Nrf2 and survivin expressions were effectively suppressed after withdrawal of MPA. Interestingly, metformin increased the progestin sensitivity by down regulation of Nrf2 and survivin. The findings suggest that dysregulation of Nrf2-survivin may represent part of the molecular mechanisms of progestin resistance in endometrial cancer. Detecting survivin and Nrf2 may predict progestin resistance, while targeting Nrf2 and survivin may represent a promising prevention and treatment strategy for endometrial cancer. PMID:28386373

  6. Nrf2 Expression and Apoptosis in Quercetin-treated Malignant Mesothelioma Cells

    PubMed Central

    Lee, Yoon-Jin; Lee, David M.; Lee, Sang-Han

    2015-01-01

    NF-E2-related factor 2 (Nrf2), a basic leucine zipper transcription factor, has recently received a great deal of attention as an important molecule that enhances antioxidative defenses and induces resistance to chemotherapy or radiotherapy. In this study, we investigated the apoptosis-inducing and Nrf2-upregulating effects of quercetin on malignant mesothelioma (MM) MSTO-211H and H2452 cells. Quercetin treatment inhibited cell growth and led to upregulation of Nrf2 at both the mRNA and protein levels without altering the ubiquitination and extending the half-life of the Nrf2 protein. Following treatment with quercetin, analyses of the nuclear level of Nrf2, Nrf2 antioxidant response element-binding assay, Nrf2 promoter-luc assay, and RT-PCR toward the Nrf2-regulated gene, heme oxygenase-1, demonstrated that the induced Nrf2 is transcriptionally active. Knockdown of Nrf2 expression with siRNA enhanced cytotoxicity due to the induction of apoptosis, as evidenced by an increase in the level of proapoptotic Bax, a decrease in the level of antiapoptotic Bcl-2 with enhanced cleavage of caspase-3 and PARP proteins, the appearance of a sub-G0/G1 peak in the flow cytometric assay, and increased percentage of apoptotic propensities in the annexin V binding assay. Effective reversal of apoptosis was observed following pretreatment with the pan-caspase inhibitor Z-VAD. Moreover, Nrf2 knockdown exhibited increased sensitivity to the anticancer drug, cisplatin, presumably by potentiating the oxidative stress induced by cisplatin. Collectively, our data demonstrate the importance of Nrf2 in cytoprotection, survival, and drug resistance with implications for the potential significance of targeting Nrf2 as a promising strategy for overcoming resistance to chemotherapeutics in MM. PMID:25896339

  7. Oncogene-induced Nrf2 transcription promotes ROS detoxification and tumorigenesis

    PubMed Central

    DeNicola, Gina M.; Karreth, Florian A.; Humpton, Timothy J.; Gopinathan, Aarthi; Wei, Cong; Frese, Kristopher; Mangal, Dipti; Yu, Kenneth H.; Yeo, Charles J.; Calhoun, Eric S.; Scrimieri, Francesca; Winter, Jordan M.; Hruban, Ralph H.; Iacobuzio-Donahue, Christine; Kern, Scott E.; Blair, Ian A.; Tuveson, David A.

    2012-01-01

    Reactive oxygen species (ROS) are mutagenic and may thereby promote cancer1. Normally, ROS levels are tightly controlled by an inducible antioxidant program that responds to cellular stressors and is predominantly regulated by the transcription factor Nrf2 and its repressor protein Keap12-5. In contrast to the acute physiological regulation of Nrf2, in neoplasia there is evidence for increased basal activation of Nrf2. Indeed, somatic mutations that disrupt the Nrf2-Keap1 interaction to stabilize Nrf2 and increase the constitutive transcription of Nrf2 target genes were recently identified, suggesting that enhanced ROS detoxification and additional Nrf2 functions may in fact be pro-tumorigenic6. Here, we investigated ROS metabolism in primary murine cells following the expression of endogenous oncogenic alleles of K-Ras, B-Raf and Myc, and find that ROS are actively suppressed by these oncogenes. K-RasG12D, B-RafV619E and MycERT2 each increased the transcription of Nrf2 to stably elevate the basal Nrf2 antioxidant program and thereby lower intracellular ROS and confer a more reduced intracellular environment. Oncogene-directed increased expression of Nrf2 is a novel mechanism for the activation of the Nrf2 antioxidant program, and is evident in primary cells and tissues of mice expressing K-RasG12D and B-RafV619E, and in human pancreatic cancer. Furthermore, genetic targeting of the Nrf2 pathway impairs K-RasG12D-induced proliferation and tumorigenesis in vivo. Thus, the Nrf2 antioxidant and cellular detoxification program represents a previously unappreciated mediator of oncogenesis. PMID:21734707

  8. Vascular Protective Role of Samul-Tang in HUVECs: Involvement of Nrf2/HO-1 and NO

    PubMed Central

    Choi, Eun Sik; Lee, Yun Jung; Seo, Chang Seob; Yoon, Jung Joo; Han, Byung Hyuk; Park, Min Cheol; Kang, Dae Gill; Lee, Ho Sub

    2016-01-01

    Samul-Tang (Si-Wu-Tang, SMT), composed of four medicinal herbs, is a well-known herbal formula treating hematological disorder or gynecologic disease. However, vascular protective effects of SMT and its molecular mechanisms on the vascular endothelium, known as the central spot of vascular inflammatory process, are not reported. The aim of this study was to investigate vascular protective effects of SMT water extract in human umbilical vein endothelial cells (HUVECs). Water extract of SMT was prepared and identified by HPLC-PDA analysis. Expression of cell adhesion molecules (CAMs) and heme oxygenase-1 (HO-1) and translocation of nuclear factor-kappa B (NF-κB) and nuclear factor-erythroid 2-related factor 2 (Nrf2) were determined by western blot. Nuclear localization of NF-κB and Nrf2 was visualized by immunofluorescence and DNA binding activity of NF-κB was measured. ROS production, HL-60 monocyte adhesion, and intracellular nitric oxide (NO) were also measured using a fluorescent indicator. SMT suppressed NF-κB translocation and activation as well as expression of CAMs, monocyte adhesion, and ROS production induced by TNF-α in HUVECs. SMT treated HUVECs showed upregulation of HO-1 and NO which are responsible for vascular protective action. Our study suggests that SMT, a traditionally used herbal formula, protects the vascular endothelium from inflammation and might be used as a promising vascular protective drug. PMID:27366195

  9. Upregulation of heme oxygenase-1 expression by dehydrodiconiferyl alcohol (DHCA) through the AMPK–Nrf2 dependent pathway

    SciTech Connect

    Lee, Junghun; Kim, Sunyoung

    2014-11-15

    Oxidative stress is induced by the accumulation of free radicals, resulting in an imbalanced cellular redox state, which has been implicated in a variety of human diseases. Dehydrodiconiferyl alcohol (DHCA), a lignan compound isolated from Cucurbita moschata, has previously been reported to contain anti-adipogenic and anti-lipogenic effects on 3T3-L1 cells and primary MEFs (Abraham and Kappas, 2008). In this study, it was tested whether DHCA could affect the expression of HO-1, using Raw264.7 mouse macrophage cell line. DHCA increased the protein and RNA levels of HO-1 and upregulated its promoter activity. Data from transient transfection assays indicated that ARE located in the E1 region of the HO-1 promoter are important in this DHCA-mediated induction of HO-1 expression. DHCA was also shown to enhance the nuclear translocation and binding of Nrf2 to the respective DNA sequences. The upregulation of HO-1 expression by DHCA was also observed in primary macrophages derived from wild type animals, but not in those from Nrf2 KO mice. Effects of DHCA on HO-1 and Nrf2 were reduced when cells were treated with an AMPK inhibitor, Compound C, but not by PI3K/Akt or MAPK inhibitors. Data from an experiment using a specific siRNA or chemical inhibitor for HO-1 suggested that the DHCA-mediated induction of the HO-1 protein could suppress the LPS-stimulated production of NO. Taken together, our data suggest that DHCA induces the expression of HO-1 by controlling its promoter activity through the AMPK–Nrf2 pathway, eventually leading to the reduction of NO production, and may thus have potential as an effective antioxidant. - Highlights: • Dehydrodiconiferyl alcohol (DHCA) induced the expression of heme oxygenase (HO)-1. • The AMPK–Nrf2 pathway is critically involved in the DHCA-mediated induction of HO-1. • DHCA increased the expression of HO-1, Gclc and Gclm in primary macrophages. • DHCA-mediated induction of HO-1 contributed to the suppression of NO production.

  10. NRF2 Activation as Target to Implement Therapeutic Treatments

    NASA Astrophysics Data System (ADS)

    Bocci, Velio; Valacchi, Giuseppe

    2015-02-01

    A chronic increase of oxidative stress is typical of serious pathologies such as myocardial infarction, stroke, chronic limb ischemia, chronic obstructive pulmonary disease (COPD), type II-diabetes, age-related macular degeneration leads to an epic increase of morbidity and mortality in all countries of the world. The initial inflammation followed by an excessive release of reactive oxygen species (ROS) implies a diffused cellular injury that needs to be corrected by an inducible expression of the innate detoxifying and antioxidant system. The transcription factor Nrf2, when properly activated, is able to restore a redox homeostasis and possibly improve human health.

  11. Nrf2 activation as target to implement therapeutic treatments

    PubMed Central

    Bocci, Velio; Valacchi, Giuseppe

    2015-01-01

    A chronic increase of oxidative stress is typical of serious pathologies such as myocardial infarction, stroke, chronic limb ischemia, chronic obstructive pulmonary disease (COPD), type II-diabetes, age-related macular degeneration leads to an epic increase of morbidity and mortality in all countries of the world. The initial inflammation followed by an excessive release of reactive oxygen species (ROS) implies a diffused cellular injury that needs to be corrected by an inducible expression of the innate detoxifying and antioxidant system. The transcription factor Nrf2, when properly activated, is able to restore a redox homeostasis and possibly improve human health. PMID:25699252

  12. The Anti-Inflammatory Activity of Eucommia ulmoides Oliv. Bark. Involves NF-κB Suppression and Nrf2-Dependent HO-1 Induction in BV-2 Microglial Cells

    PubMed Central

    Kwon, Seung-Hwan; Ma, Shi-Xun; Hwang, Ji-Young; Ko, Yong-Hyun; Seo, Ji-Yeon; Lee, Bo-Ram; Lee, Seok-Yong; Jang, Choon-Gon

    2016-01-01

    In the present study, we investigated the anti-inflammatory properties of Eucommia ulmoides Oliv. Bark. (EUE) in lipopolysaccharide (LPS)-stimulated microglial BV-2 cells and found that EUE inhibited LPS-mediated up-regulation of pro-inflammatory response factors. In addition, EUE inhibited the elevated production of pro-inflammatory cytokines, mediators, and reactive oxygen species (ROS) in LPS-stimulated BV-2 microglial cells. Subsequent mechanistic studies revealed that EUE suppressed LPS-induced phosphorylation of mitogen-activated protein kinases (MAPKs), phosphoinositide-3-kinase (PI3K)/Akt, glycogen synthase kinase-3β (GSK-3β), and their downstream transcription factor, nuclear factor-kappa B (NF-κB). EUE also blocked the nuclear translocation of NF-κB and inhibited its binding to DNA. We next demonstrated that EUE induced the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and upregulated heme oxygenase-1 (HO-1) expression. We determined that the significant up-regulation of HO-1 expression by EUE was a consequence of Nrf2 nuclear translocation; furthermore, EUE increased the DNA binding of Nrf2. In contrast, zinc protoporphyrin (ZnPP), a specific HO-1 inhibitor, blocked the ability of EUE to inhibit NO and PGE2 production, indicating the vital role of HO-1. Overall, our results indicate that EUE inhibits pro-inflammatory responses by modulating MAPKs, PI3K/Akt, and GSK-3β, consequently suppressing NF-κB activation and inducing Nrf2-dependent HO-1 activation. PMID:27068259

  13. Redox regulation by Keap1 and Nrf2 controls intestinal stem cell proliferation in Drosophila

    PubMed Central

    Hochmuth, Christine E.; Biteau, Benoit; Bohmann, Dirk; Jasper, Heinrich

    2010-01-01

    In Drosophila, intestinal stem cells (ISCs) respond to oxidative challenges and inflammation by increasing proliferation rates. This phenotype is part of a regenerative response, but can lead to hyperproliferation and epithelial degeneration in the aging animal. Here we show that Nrf2, a master regulator of the cellular redox state, specifically controls the proliferative activity of ISCs, promoting intestinal homeostasis. We find that Nrf2 is constitutively active in ISCs, and that repression of Nrf2 by its negative regulator Keap1 is required for ISC proliferation. We further show that Nrf2 and Keap1 exert this function in ISCs by regulating the intracellular redox balance. Accordingly, loss of Nrf2 in ISCs causes accumulation of reactive oxygen species and accelerates age-related degeneration of the intestinal epithelium. Our findings establish Keap1 and Nrf2 as a critical redox management system that regulates stem cell function in high-turnover tissues. PMID:21295275

  14. Diabetic Wound Healing and Activation of Nrf2 by Herbal Medicine

    PubMed Central

    Senger, Donald R.; Cao, Shugeng

    2016-01-01

    Nrf2 defense is a very important cellular mechanism to control oxidative stress, which is implicated in wound healing. Nrf2 can induce many cytoprotective genes, including HO-1, NQO1 and G6PD. Among many natural products that have been reported as Nrf2 activators, sulforaphane and curcumin have been studied more widely than any others, and both are in clinical trials for non-cancerous disorders. Recently, we reported 4-ethyl catechol and 4-vinyl catechol as Nrf2 co-factors that can induce Nrf2 as potently as sulforaphane and curcumin. These new Nrf2 co-factors were identified in hot aqueous extract of an herbal medicine Barleria lupulina, and fermented Noni (Morinda citrifolia) juice, which are used traditionally for diabetic wound healing. PMID:27868087

  15. Naphthazarin protects against glutamate-induced neuronal death via activation of the Nrf2/ARE pathway

    SciTech Connect

    Son, Tae Gen; Kawamoto, Elisa M.; Yu, Qian-Sheng; Greig, Nigel H.; Mattson, Mark P.; Camandola, Simonetta

    2013-04-19

    Highlights: •Naphthazarin activates the Nrf2/ARE pathway. •Naphthazarin induces Nrf2-driven genes in neurons and astrocytes. •Naphthazarin protects neurons against excitotoxicity. -- Abstract: Nuclear factor E2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway is an important cellular stress response pathway involved in neuroprotection. We previously screened several natural phytochemicals and identified plumbagin as a novel activator of the Nrf2/ARE pathway that can protect neurons against ischemic injury. Here we extended our studies to natural and synthetic derivatives of plumbagin. We found that 5,8-dimethoxy-1,4-naphthoquinone (naphthazarin) is a potent activator of the Nrf2/ARE pathway, up-regulates the expression of Nrf2-driven genes in primary neuronal and glial cultures, and protects neurons against glutamate-induced excitotoxicity.

  16. Induction of Mrp3 and Mrp4 transporters during acetaminophen hepatotoxicity is dependent on Nrf2

    SciTech Connect

    Aleksunes, Lauren M. Slitt, Angela L. Maher, Jonathan M. Augustine, Lisa M. Goedken, Michael J. Chan, Jefferson Y. Cherrington, Nathan J. Klaassen, Curtis D. Manautou, Jose E.

    2008-01-01

    The transcription factor NFE2-related factor 2 (Nrf2) mediates detoxification and antioxidant gene transcription following electrophile exposure and oxidative stress. Mice deficient in Nrf2 (Nrf2-null) are highly susceptible to acetaminophen (APAP) hepatotoxicity and exhibit lower basal and inducible expression of cytoprotective genes, including NADPH quinone oxidoreductase 1 (Nqo1) and glutamate cysteine ligase (catalytic subunit, or Gclc). Administration of toxic APAP doses to C57BL/6J mice generates electrophilic stress and subsequently increases levels of hepatic Nqo1, Gclc and the efflux multidrug resistance-associated protein transporters 1-4 (Mrp1-4). It was hypothesized that induction of hepatic Mrp1-4 expression following APAP is Nrf2 dependent. Plasma and livers from wild-type (WT) and Nrf2-null mice were collected 4, 24 and 48 h after APAP. As expected, hepatotoxicity was greater in Nrf2-null compared to WT mice. Gene and protein expression of Mrp1-4 and the Nrf2 targets, Nqo1 and Gclc, was measured. Induction of Nqo1 and Gclc mRNA and protein after APAP was dependent on Nrf2 expression. Similarly, APAP treatment increased hepatic Mrp3 and Mrp4 mRNA and protein in WT, but not Nrf2-null mice. Mrp1 was induced in both genotypes after APAP, suggesting that elevated expression of this transporter was independent of Nrf2. Mrp2 was not induced in either genotype at the mRNA or protein levels. These results show that Nrf2 mediates induction of Mrp3 and Mrp4 after APAP but does not affect Mrp1 or Mrp2. Thus coordinated regulation of detoxification enzymes and transporters by Nrf2 during APAP hepatotoxicity is a mechanism by which hepatocytes may limit intracellular accumulation of potentially toxic chemicals.

  17. Nrf2 pathway regulates multidrug-resistance-associated protein 1 in small cell lung cancer.

    PubMed

    Ji, Lili; Li, Hui; Gao, Pan; Shang, Guoguo; Zhang, Donna D; Zhang, Nong; Jiang, Tao

    2013-01-01

    Although multidrug-resistance-associated protein-1 (MRP1) is a major contributor to multi-drug resistance (MDR), the regulatory mechanism of Mrp1 still remains unclear. Nrf2 is a transcription factor that regulates cellular defense response through antioxidant response elements (AREs) in normal tissues. Recently, Nrf2 has emerged as an important contributor to chemo-resistance in tumor tissues. In the present study, the role of Nrf2-ARE pathway on regulation of Mrp1 was investigated. Compared with H69 lung cancer cells, H69AR cells with MDR showed significantly higher Nrf2-ARE pathway activity and expression of Mrp1 as well. When Nrf2 was knocked down in H69AR cells, MRP1's expression decreased accordingly. Moreover, those H69AR cells with reduced Nrf2 level restored sensitivity to chemo-drugs. To explore how Nrf2-ARE pathway regulates Mrp1, the promoter of Mrp1 gene was searched, and two putative AREs--ARE1 and ARE2--were found. Using reporter gene and ChIP assay, both ARE1 and ARE2 showed response to and interaction with Nrf2. In 40 cases of cancer tissues, the expression of Nrf2 and MRP1 was measured by immunohistochemistry (IHC). As the quantitive data of IHC indicated, both Nrf2 and MRP1 showed significantly higher expression in tumor tissue than adjacent non-tumor tissue. And more important, the correlation analysis of the two genes proved that their expression was correlative. Taken together, theses data suggested that Nrf2-ARE pathway is required for the regulatory expression of Mrp1 and implicated Nrf2 as a new therapeutic target for MDR.

  18. Nrf2 protects against diquat-induced liver and lung injury.

    PubMed

    Wu, Kai Connie; Zhang, Youcai; Klaassen, Curtis D

    2012-10-01

    Diquat is an herbicide that generates superoxide anions through redox cycling. Nuclear factor erythroid-derived 2- like 2 (Nrf2) is a transcription factor that up-regulates cytoprotective genes in response to oxidative stress. To investigate the protective effect of Nrf2 against diquat-induced toxicity, wild-type, Nrf2-null and Kelch-like ECH-associated protein 1-knockdown (Keap1-KD) mice with enhanced Nrf2 activity were treated with diquat dibromide (125 mg/kg, i.p.). Blood and tissues were collected at 1, 2, 4 and 6 hours after treatment. Administration of diquat resulted in lipid peroxidation and lethality in wild-type mice, which were more in Nrf2-null mice and less in Keap1-KD mice. Diquat produced liver injury in Nrf2-null mice, as evidenced by increased serum ALT activity and extensive hepatic necrosis, but not in wild-type and Keap1-KD mice. Diquat produced more severe lung injury in Nrf2-null than in wild-type mice, as evidenced by increased lung weight and alveolar collapse. In contrast, Keap1-KD mice had attenuated lung edema and no histopathological alterations. To further investigate the mechanism of the protective effects of Nrf2, lung and liver glutathione (GSH) concentrations were quantified. Diquat decreased GSH in lung and liver in wild-type mice, and the decrease was more in Nrf2-null mice, and less in Keap1-KD mice. After diquat treatment, the mRNA of the GSH synthesis enzyme Gclc was increased in Keap1-KD, but not in Nrf2-null mice. Collectively, Nrf2 plays an important role in preventing diquat-induced liver and lung injury, and this protective effect results from Nrf2-regulated elevation of cellular GSH and expression of GSH synthetic genes.

  19. RS9, a novel Nrf2 activator, attenuates light-induced death of cells of photoreceptor cells and Müller glia cells.

    PubMed

    Inoue, Yuki; Shimazawa, Masamitsu; Noda, Yasuhiro; Nagano, Ryota; Otsuka, Tomohiro; Kuse, Yoshiki; Nakano, Yukimichi; Tsuruma, Kazuhiro; Nakagami, Yasuhiro; Hara, Hideaki

    2017-06-01

    The retina is highly sensitive to oxidative stress because of its high consumption of oxygen associated with the phototransductional processes. Recent findings have suggested that oxidative stress is involved in the pathology of age-related macular degeneration, a progressive degeneration of the central retina. A well-known environmental risk factor is light exposure, as excessive and continuous light exposure can damage photoreceptors. Nuclear factor-erythroid 2-related factor 2 (Nrf2) is a transcriptional factor that controls antioxidative responses and phase 2 enzymes. Thus, we hypothesized that RS9, a specific activator of Nrf2, decreases light-induced retinal cell death in vivo and in vitro. Nrf2 was detected in the nucleus of the 661W cells exposed to RS9 and also after light exposure, and the Nrf2-antioxidant response element binding was increased in 661W cells after exposure to RS9. Consequentially, the expression of the phase 2 enzyme's mRNAs of Ho-1, Nqo-1, and Gclm genes was increased in 661W cells after exposure to RS9. Furthermore, RS9 decreased the light-induced death of 661W cells (2500 lux, 24 h), and also reduced the functional damages and the histological degeneration of the nuclei in the outer nuclear layer or the retina in the in vivo studies (8000 lux, 3 h). Heme oxygenase-1 was increased after light exposure, and Nrf2 was translocated into the nucleus after light exposure in vivo. Silencing of Ho-1 reduced the protective effects of RS9 against light-induced death of 661W cells. These findings indicate that RS9 has therapeutic potential for retinal diseases that are aggravated by light exposure. © 2017 International Society for Neurochemistry.

  20. Andrographolide protects liver cells from H2O2 induced cell death by upregulation of Nrf-2/HO-1 mediated via adenosine A2a receptor signalling.

    PubMed

    Mittal, Smriti P K; Khole, Swati; Jagadish, Nidhi; Ghosh, Debjani; Gadgil, Vijay; Sinkar, Vilas; Ghaskadbi, Saroj S

    2016-11-01

    Andrographolide, principle constituent of Andrographis paniculata Nees is used in traditional medicine in Southeast Asia and is known to exhibit various biological activities. Its antioxidant activity is due to its ability to activate one of the antioxidant enzymes, heme oxygenase-1 (HO-1) which is regulated transcriptionally through Nrf-2. However, molecular mechanism underlying activation of Nrf-2/HO-1 has not yet been clearly understood. Protective effect of andrographolide against H2O2 induced cell death, reactive oxygen species and lipid peroxidation was observed in HepG2 cells. Ability of andrographolide to modulate G-protein coupled receptor (GPCR) mediated signalling was determined using in silico docking and gene expression was analyzed by qRT-PCR, confocal microscopy and western blot analysis. We clearly show that andrographolide via adenosine A2A receptor signalling leads to activation of p38 MAP kinase, resulting in upregulation of Nrf-2, its translocation to nucleus and activation of HO-1. Additionally, it activates adenylate cyclase resulting in cAMP formation which in turn activates protein kinase A leading to inhibition of GSK-3β by phosphorylation. Inactivated GSK-3β leads to retention of Nrf-2 in the nucleus leading to sustained expression of HO-1 by binding to its antioxidant response element (ARE). Thus, andrographolide probably by binding to adenosine A2a receptor activates Nrf-2 transcription and also inhibits its exclusion from the nucleus by inactivating GSK-3β, together resulting in activation of HO-1. We speculate that andrographolide can be used as a therapeutic drug to combat oxidative stress implicated in pathogenesis of various diseases such as diabetes, osteoporosis, neurodegenerative diseases etc. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Hawthorn (Crataegus oxyacantha L.) bark extract regulates antioxidant response element (ARE)-mediated enzyme expression via Nrf2 pathway activation in normal hepatocyte cell line.

    PubMed

    Krajka-Kuźniak, Violetta; Paluszczak, Jarosław; Oszmiański, Jan; Baer-Dubowska, Wanda

    2014-04-01

    Hawthorn (Crataegus oxyacantha L.), a plant used in traditional medicine, is a rich source of procyanidins which have been reported to exhibit antioxidant and anti-carcinogenic activity. In this study, we assessed the effect of hawthorn bark extract (HBE) on Nrf2 pathway activation in THLE-2 and HepG2 cells. Treatment with 1.1 µg/mL, 5.5 µg/mL and 11 µg/mL of HBE resulted in the translocation of Nrf2 from the cytosol to the nucleus in both cell lines; however, the accumulation of phosphorylated Nrf2 was observed only in THLE-2. Accordingly, treatment of cells with HBE was associated with an increase in the mRNA and protein level of such Nrf2-dependent genes as glutathione S-transferases (GSTA, GSTP, GSTM, GSTT), NAD(P)H:quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1) (0.2-1.1-fold change, p < 0.05), however, only in normal THLE-2 hepatocytes. The induction of NQO1 correlated with an increased level of p53 (0.21-0.42-fold change, p < 0.05). These effects may be related to induction of phosphorylation of upstream ERK and JNK kinases. Collectively, the results suggest that the Nrf2/ARE pathway may play an important role in the regulation of procyanidin-mediated antioxidant/detoxifying effects in hepatocytes, and this may explain the hepatoprotective and chemopreventive properties of these phytochemicals. Copyright © 2013 John Wiley & Sons, Ltd.

  2. Dimerumic acid attenuates receptor for advanced glycation endproducts signal to inhibit inflammation and diabetes mediated by Nrf2 activation and promotes methylglyoxal metabolism into d-lactic acid.

    PubMed

    Lee, Bao-Hong; Hsu, Wei-Hsuan; Hsu, Ya-Wen; Pan, Tzu-Ming

    2013-07-01

    This study was designed to evaluate the effects of dimerumic acid (DMA) on receptor for advanced glycation endproducts (RAGE) signal activation and THP-1 monocyte inflammation treated with S100b, a specific ligand of RAGE. We found that DMA inhibited inflammatory cytokine production via upregulation of nuclear factor-erythroid 2-related factor 2 (Nrf2) and alleviated oxidative stress through attenuation of p47phox translocation to the membrane of S100b-treated THP-1 monocytes. We found that DMA activated Nrf2 mediated by the p38 kinase pathway in THP-1 monocytes. However, anti-inflammatory activity of DMA was attenuated by Nrf2 siRNA treatment. In an animal model, methylglyoxal (MG; 200mg/kg bw) was chosen to induce diabetes in Balb/C mice (6 weeks) in this work. The in vivo verification of anti-inflammation in peripheral blood mononuclear cells by DMA treatment was confirmed by tumor necrosis factor-α and interleukin-1β measurements. Oral glucose tolerance test, insulin tolerance test, hyperinsulinemia, and hyperglycemia were improved in MG-treated mice by DMA treatment and these effects were greater than those of silymarin and N-acetylcysteine. Furthermore, DMA increased hepatic glyoxalase mRNA and glutathione mediated by Nrf2 activation to metabolize MG into d-lactic acid, thereby reducing serum and hepatic AGE levels and suppressing inflammatory factor generation in MG-treated mice. However, DMA did not exert the antiglycation activity in MG-bovine serum albumin incubation. Taken together, the results indicate that DMA is a novel antioxidant and Nrf2 activator that lowers AGE levels and may prove to be an effective treatment for diabetes.

  3. Activation of AKT pathway by Nrf2/PDGFA feedback loop contributes to HCC progression.

    PubMed

    Liu, Danyang; Zhang, Yonglong; Wei, Yingze; Liu, Guoyuan; Liu, Yufeng; Gao, Qiongmei; Zou, Liping; Zeng, Wenjiao; Zhang, Nong

    2016-10-04

    Nuclear factor erythroid-2-related factor 2 (Nrf2), a master transcription factor in the antioxidant response, has been found to be ubiquitously expressed in various cancer cells and in the regulation tumor proliferation, invasion, and chemoresistance activities. The regulatory roles of Nrf2 in controlling Hepatocellular carcinoma (HCC) progression remain unclear. In this study, we demonstrated that Nrf2 was significantly elevated in HCC cells and tissues and was correlated with poor prognosis of HCCs. Consistently, Nrf2 significantly promoted HCC cell growth both in vitro and in vivo. Further investigation suggested a novel association of Nrf2 with Platelet-Derived Growth Factor-A (PDGFA). Nrf2 promoted PDGFA transcription by recruiting specificity protein 1 (Sp1) to its promoter, resulting in increased activation of the AKT/p21 pathway and cell cycle progression of HCC cells. As a feedback loop, PDGFA enhanced Nrf2 expression and activation in an AKT dependent manner. In line with these findings, expression of Nrf2 and PDGFA were positively correlated in HCC tissues. Taken together, this study uncovers a novel mechanism of the Nrf2/PDGFA regulatory loop that is crucial for AKT-dependent HCC progression, and thereby provides potential targets for HCC therapy.

  4. Activation of AKT pathway by Nrf2/PDGFA feedback loop contributes to HCC progression

    PubMed Central

    Wei, Yingze; Liu, Guoyuan; Liu, Yufeng; Gao, Qiongmei; Zou, Liping; Zeng, Wenjiao; Zhang, Nong

    2016-01-01

    Nuclear factor erythroid-2-related factor 2 (Nrf2), a master transcription factor in the antioxidant response, has been found to be ubiquitously expressed in various cancer cells and in the regulation tumor proliferation, invasion, and chemoresistance activities. The regulatory roles of Nrf2 in controlling Hepatocellular carcinoma (HCC) progression remain unclear. In this study, we demonstrated that Nrf2 was significantly elevated in HCC cells and tissues and was correlated with poor prognosis of HCCs. Consistently, Nrf2 significantly promoted HCC cell growth both in vitro and in vivo. Further investigation suggested a novel association of Nrf2 with Platelet-Derived Growth Factor-A (PDGFA). Nrf2 promoted PDGFA transcription by recruiting specificity protein 1 (Sp1) to its promoter, resulting in increased activation of the AKT/p21 pathway and cell cycle progression of HCC cells. As a feedback loop, PDGFA enhanced Nrf2 expression and activation in an AKT dependent manner. In line with these findings, expression of Nrf2 and PDGFA were positively correlated in HCC tissues. Taken together, this study uncovers a novel mechanism of the Nrf2/PDGFA regulatory loop that is crucial for AKT-dependent HCC progression, and thereby provides potential targets for HCC therapy. PMID:27588483

  5. NRF2 and cancer: the good, the bad and the importance of context.

    PubMed

    Sporn, Michael B; Liby, Karen T

    2012-07-19

    Many studies of chemopreventive drugs have suggested that their beneficial effects on suppression of carcinogenesis and many other chronic diseases are mediated through activation of the transcription factor NFE2-related factor 2 (NRF2). More recently, genetic analyses of human tumours have indicated that NRF2 may conversely be oncogenic and cause resistance to chemotherapy. It is therefore controversial whether the activation, or alternatively the inhibition, of NRF2 is a useful strategy for the prevention or treatment of cancer. This Opinion article aims to rationalize these conflicting perspectives by critiquing the context dependence of NRF2 functions and the experimental methods behind these conflicting data.

  6. The transcription factor NF-E2-related Factor 2 (Nrf2): a protooncogene?

    PubMed Central

    Shelton, Phillip; Jaiswal, Anil K.

    2013-01-01

    The transcription factor Nrf2 is responsible for regulating a battery of antioxidant and cellular protective genes, primarily in response to oxidative stress. A member of the cap 'n' collar family of transcription factors, Nrf2 activation is tightly controlled by a series of signaling events. These events can be separated into the basal state, a preinduction response, gene induction, and finally a postinduction response, culminating in the restoration of redox homeostasis. However, despite the immensely intricate level of control the cellular environment imposes on Nrf2 activity, there are many opportunities for perturbations to arise in the signaling events that favor carcinogenesis and, therefore, implicate Nrf2 as both a tumor suppressor and a protooncogene. Herein, we highlight the ways in which Nrf2 is regulated, and discuss some of the Nrf2-inducible antioxidant (NQO1, NQO2, HO-1, GCLC), antiapoptotic (Bcl-2), metabolic (G6PD, TKT, PPARγ), and drug efflux transporter (ABCG2, MRP3, MRP4) genes. In addition, we focus on how Nrf2 functions as a tumor suppressor under normal conditions and how its ability to detoxify the cellular environment makes it an attractive target for other oncogenes either via stabilization or degradation of the transcription factor. Finally, we discuss some of the ways in which Nrf2 is being considered as a therapeutic target for cancer treatment.—Shelton, P., Jaiswal, A. K. The transcription factor NF-E2-related factor 2 (Nrf2): a protooncogene? PMID:23109674

  7. An overview of the mechanisms and novel roles of Nrf2 in cardiovascular diseases.

    PubMed

    Jiang, Shuai; Yang, Yang; Li, Tian; Ma, Zhiqiang; Hu, Wei; Deng, Chao; Fan, Chongxi; Lv, Jianjun; Sun, Yang; Yi, Wei

    2016-12-01

    Nuclear factor erythroid 2-related factor 2 (Nrf2) is a basic leucine zipper (bZIP) transcription factor of the cap'n'collar (CNC) family that is present in various organs. The cardiovascular system (CVS) is susceptible to a spectrum of diseases that are strongly associated with increased risks of mortality and morbidity, and studies have demonstrated that Nrf2 has a pivotal role in protection against cardiovascular diseases (CVDs). Areas covered: Nrf2 is a basic protective molecule that guards against CVD by attenuating oxidative stress, mitochondrial dysfunction, and inflammation. Initially, we briefly introduce the biological characteristics of Nrf2 and the newly discovered Neh7 domain. Next, we discuss the concrete roles of Nrf2 in the CVS and enumerate some related upstream molecules and downstream targets. Lastly, we expand our focus to the behaviors of Nrf2 in CVDs and discuss potential research directions. Expert opinion: Although certain studies have cast doubt on the positive actions of Nrf2 in the CVS, Nrf2 is a pivotal endogenous molecule for defense against CVD. The review compiled here may serve as a broad and comprehensive reference for the roles of Nrf2 in the CVS, with the aim of facilitating the design of new drugs and clinical therapies for CVDs.

  8. Targeting the transcription factor Nrf2 to ameliorate oxidative stress and inflammation in chronic kidney disease.

    PubMed

    Ruiz, Stacey; Pergola, Pablo E; Zager, Richard A; Vaziri, Nosratola D

    2013-06-01

    Oxidative stress and inflammation are mediators in the development and progression of chronic kidney disease (CKD) and its complications, and they are inseparably linked as each begets and amplifies the other. CKD-associated oxidative stress is due to increased production of reactive oxygen species (ROS) and diminished antioxidant capacity. The latter is largely caused by impaired activation of Nrf2, the transcription factor that regulates genes encoding antioxidant and detoxifying molecules. Protective effects of Nrf2 are evidenced by amelioration of oxidative stress, inflammation, and kidney disease in response to natural Nrf2 activators in animal models, while Nrf2 deletion amplifies these pathogenic pathways and leads to autoimmune nephritis. Given the role of impaired Nrf2 activity in CKD-induced oxidative stress and inflammation, interventions aimed at restoring Nrf2 may be effective in retarding CKD progression. Clinical trials of the potent Nrf2 activator bardoxolone methyl showed significant improvement in renal function in CKD patients with type 2 diabetes. However, due to unforeseen complications the BEACON trial, which was designed to investigate the effect of this drug on time to end-stage renal disease or cardiovascular death in patients with advanced CKD, was prematurely terminated. This article provides an overview of the role of impaired Nrf2 activity in the pathogenesis of CKD-associated oxidative stress and inflammation and the potential utility of targeting Nrf2 in the treatment of CKD.

  9. Th2 skewing by activation of Nrf2 in CD4+ T cells1

    PubMed Central

    Rockwell, Cheryl E.; Zhang, Mingcai; Fields, Patrick E.; Klaassen, Curtis D.

    2012-01-01

    Nuclear factor erythroid 2 related factor 2 (Nrf2) is a transcription factor that mediates the upregulation of a battery of cytoprotective genes in response to cell stress. Recent studies have shown that Nrf2 also modulates immune responses and exhibits anti-inflammatory activity. In this report, we demonstrate that a common food preservative, tBHQ, can activate Nrf2 in T cells, as evidenced by Nrf2 binding to the antioxidant response element (ARE) and the subsequent upregulation of Nrf2 target genes. The activation of Nrf2 suppresses IFNγ production, while inducing the production of the Th2 cytokines, IL-4, IL-5, and IL-13. Nrf2 activation also suppresses T-bet DNA binding and promotes GATA-3 DNA binding. Collectively, the present studies suggest that Nrf2 activation skews CD4+ T cells toward Th2 differentiation and thus represents a novel regulatory mechanism in CD4+ T cells. Further studies will be needed to determine whether the commercial use of Nrf2 activators as food preservatives promotes food allergies in humans. PMID:22250088

  10. NRF2 deficiency replicates transcriptomic changes in Alzheimer's patients and worsens APP and TAU pathology.

    PubMed

    Rojo, Ana I; Pajares, Marta; Rada, Patricia; Nuñez, Angel; Nevado-Holgado, Alejo J; Killik, Richard; Van Leuven, Fred; Ribe, Elena; Lovestone, Simon; Yamamoto, Masayuki; Cuadrado, Antonio

    2017-10-01

    Failure to translate successful neuroprotective preclinical data to a clinical setting in Alzheimer's disease (AD) indicates that amyloidopathy and tauopathy alone provide an incomplete view of disease. We have tested here the relevance of additional homeostatic deviations that result from loss of activity of transcription factor NRF2, a crucial regulator of multiple stress responses whose activity declines with ageing. A transcriptomic analysis demonstrated that NRF2-KO mouse brains reproduce 7 and 10 of the most dysregulated pathways of human ageing and AD brains, respectively. Then, we generated a mouse that combines amyloidopathy and tauopathy with either wild type (AT-NRF2-WT) or NRF2-deficiency (AT-NRF2-KO). AT-NRF2-KO brains presented increased markers of oxidative stress and neuroinflammation as well as higher levels of insoluble phosphorylated-TAU and Aβ*56 compared to AT-NRF2-WT mice. Young adult AT-NRF2-KO mice exhibited deficits in spatial learning and memory and reduced long term potentiation in the perforant pathway. This study demonstrates the relevance of normal homeostatic responses that decline with ageing, such as NRF2 activity, in the protection against proteotoxic, inflammatory and oxidative stress and provide a new strategy to fight AD. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  11. Astrocyte-specific overexpression of Nrf2 protects striatal neurons from mitochondrial complex II inhibition.

    PubMed

    Calkins, Marcus J; Vargas, Marcelo R; Johnson, Delinda A; Johnson, Jeffrey A

    2010-06-01

    Nuclear factor E2-related factor 2 (Nrf2) is a transcription factor that is known to regulate a variety of cytoprotective genes through the antioxidant response element (ARE). This endogenous response is one of the major pathways by which cells are protected from xenobiotic or innate oxidative insults. Furthermore, in neural systems, astrocyte-specific activation of Nrf2 is known to protect neurons. In previous work, our laboratory found that Nrf2 protects from intrastriatal injections of the mitochondrial complex II inhibitor malonate. Here, we extend these results to show that multiple methods of astrocyte-specific Nrf2 overexpression provide protection from neurotoxicity in vivo. GFAP-Nrf2 transgenic mice are significantly more resistant to malonate lesioning. This outcome is associated with an increased basal resistance, but more so, an enhanced Nrf2 response to lesioning that attenuated the ensuing neurotoxicity. Furthermore, striatal transplantation of neuroprogenitor cells overexpressing Nrf2 that differentiate into astrocytes after grafting also significantly reduced malonate toxicity. Overall, these data establish that enhanced astrocytic Nrf2 response and Nrf2 preconditioning are both sufficient to protect from acute lesions from mitochondrial complex II inhibition.

  12. Oxidative stress targets in pulmonary emphysema: focus on the Nrf2 pathway.

    PubMed

    Boutten, A; Goven, D; Boczkowski, J; Bonay, M

    2010-03-01

    Oxidative stress has been implicated in the pathogenesis of pulmonary emphysema. Nuclear factor erythroid-2-related factor 2 (Nrf2) a major antioxidant transcription factor could play a protective role in pulmonary emphysema. Nrf2 is ubiquitously expressed throughout the lung, but is predominantly found in epithelium and alveolar macrophages. Evidence suggests that Nrf2 and several Nrf2 downstream genes have an essential protective role in the lung against oxidative stress from environmental pollutants and toxicants such as cigarette smoke, a major causative factor for the development and progression of pulmonary emphysema. Application of Nrf2-deficient mice identified an extensive range of protective roles for Nrf2 against the pathogenesis of pulmonary emphysema. Therefore, Nrf2 promises to be an attractive therapeutic target for intervention and prevention strategies. In this review, we discuss recent findings on the association of oxidative stress with pulmonary emphysema. We also address the mechanisms of Nrf2 lung protection against oxidative stress based on emerging evidence from experimental oxidative disease models and human studie. The current literature suggests that among oxidative stress targets, Nrf2 is a valuable therapeutic target in pulmonary emphysema.

  13. Astrocyte-Specific Overexpression of Nrf2 Protects Striatal Neurons from Mitochondrial Complex II Inhibition

    PubMed Central

    Calkins, Marcus J.; Vargas, Marcelo R.; Johnson, Delinda A.; Johnson, Jeffrey A.

    2010-01-01

    Nuclear factor E2-related factor 2 (Nrf2) is a transcription factor that is known to regulate a variety of cytoprotective genes through the antioxidant response element (ARE). This endogenous response is one of the major pathways by which cells are protected from xenobiotic or innate oxidative insults. Furthermore, in neural systems, astrocyte-specific activation of Nrf2 is known to protect neurons. In previous work, our laboratory found that Nrf2 protects from intrastriatal injections of the mitochondrial complex II inhibitor malonate. Here, we extend these results to show that multiple methods of astrocyte-specific Nrf2 overexpression provide protection from neurotoxicity in vivo. GFAP-Nrf2 transgenic mice are significantly more resistant to malonate lesioning. This outcome is associated with an increased basal resistance, but more so, an enhanced Nrf2 response to lesioning that attenuated the ensuing neurotoxicity. Furthermore, striatal transplantation of neuroprogenitor cells overexpressing Nrf2 that differentiate into astrocytes after grafting also significantly reduced malonate toxicity. Overall, these data establish that enhanced astrocytic Nrf2 response and Nrf2 preconditioning are both sufficient to protect from acute lesions from mitochondrial complex II inhibition. PMID:20211941

  14. NRF2 and cancer: the good, the bad and the importance of context

    PubMed Central

    Sporn, Michael B.; Liby, Karen T.

    2013-01-01

    Many studies of chemopreventive drugs have suggested that their beneficial effects on suppression of carcinogenesis and many other chronic diseases are mediated through activation of the transcription factor NFE2- related factor 2 (NRF2). More recently, genetic analyses of human tumours have indicated that NRF2 may conversely be oncogenic and cause resistance to chemotherapy. It is therefore controversial whether the activation, or alternatively the inhibition, of NRF2 is a useful strategy for the prevention or treatment of cancer. This Opinion article aims to rationalize these conflicting perspectives by critiquing the context dependence of NRF2 functions and the experimental methods behind these conflicting data. PMID:22810811

  15. Direct Keap1-Nrf2 disruption as a potential therapeutic target for Alzheimer’s disease

    PubMed Central

    Sofola-Adesakin, Oyinkan; Gomez Perez-Nievas, Beatriz; Bertrand, Hélène C.; Snoeren, Inge; Cochemé, Helena M.; Adcott, Jennifer; Khericha, Mobina; Castillo-Quan, Jorge Iván; Wells, Geoffrey; Thornton, Janet

    2017-01-01

    Nrf2, a transcriptional activator of cell protection genes, is an attractive therapeutic target for the prevention of neurodegenerative diseases, including Alzheimer’s disease (AD). Current Nrf2 activators, however, may exert toxicity and pathway over-activation can induce detrimental effects. An understanding of the mechanisms mediating Nrf2 inhibition in neurodegenerative conditions may therefore direct the design of drugs targeted for the prevention of these diseases with minimal side-effects. Our study provides the first in vivo evidence that specific inhibition of Keap1, a negative regulator of Nrf2, can prevent neuronal toxicity in response to the AD-initiating Aβ42 peptide, in correlation with Nrf2 activation. Comparatively, lithium, an inhibitor of the Nrf2 suppressor GSK-3, prevented Aβ42 toxicity by mechanisms independent of Nrf2. A new direct inhibitor of the Keap1-Nrf2 binding domain also prevented synaptotoxicity mediated by naturally-derived Aβ oligomers in mouse cortical neurons. Overall, our findings highlight Keap1 specifically as an efficient target for the re-activation of Nrf2 in AD, and support the further investigation of direct Keap1 inhibitors for the prevention of neurodegeneration in vivo. PMID:28253260

  16. Gain of Nrf2 function in non-small-cell lung cancer cells confers radioresistance.

    PubMed

    Singh, Anju; Bodas, Manish; Wakabayashi, Nobunao; Bunz, Fred; Biswal, Shyam

    2010-12-01

    Nuclear factor erythroid-2 related factor 2 (Nrf2), a redox-sensitive transcription factor, regulates the expression of antioxidant enzymes and several anti-apoptotic proteins, which confer cytoprotection against oxidative stress and apoptosis. Constitutive activation of Nrf2 in lung cancer cells promotes tumorigenicity and contributes to chemoresistance by upregulation of glutathione, thioredoxin, and the drug efflux pathways involved in detoxification of electrophiles and broad spectrum of drugs. In this study, we show that RNAi-mediated lowering of Nrf2 levels in non-small-cell lung cancer (NSCLC) cell lines (A549 and H460) led to a dramatic increase in endogenous reactive oxygen species (ROS) levels. Similarly, γ-irradiation-induced formation of protein carbonyls were significantly higher in Nrf2-depleted lung cancer cells, suggesting increased lethality of ionizing radiation in the absence of Nrf2. Radiation-induced protein oxidation in Nrf2shRNA cells correlated with reduced survival as measured by clonogenic assay. Radiation-induced cell death was abrogated by pretreatment with antioxidants such as N-acetyl-L-cysteine, glutathione, and vitamin-E, highlighting the importance of antioxidants in conferring protection against radiation injury. Using genetically-modified gain and loss of function models of Nrf2, in mouse embryonic fibroblasts, we establish that constitutive activation of Nrf2 protects against ionizing radiation toxicity and confers radioresistance. Thus, targeting Nrf2 activity in radioresistant tumors could be a promising strategy to circumvent radioresistance.

  17. Gain of Nrf2 Function in Non-Small-Cell Lung Cancer Cells Confers Radioresistance

    PubMed Central

    Singh, Anju; Bodas, Manish; Wakabayashi, Nobunao; Bunz, Fred

    2010-01-01

    Abstract Nuclear factor erythroid-2 related factor 2 (Nrf2), a redox-sensitive transcription factor, regulates the expression of antioxidant enzymes and several anti-apoptotic proteins, which confer cytoprotection against oxidative stress and apoptosis. Constitutive activation of Nrf2 in lung cancer cells promotes tumorigenicity and contributes to chemoresistance by upregulation of glutathione, thioredoxin, and the drug efflux pathways involved in detoxification of electrophiles and broad spectrum of drugs. In this study, we show that RNAi-mediated lowering of Nrf2 levels in non-small-cell lung cancer (NSCLC) cell lines (A549 and H460) led to a dramatic increase in endogenous reactive oxygen species (ROS) levels. Similarly, γ-irradiation-induced formation of protein carbonyls were significantly higher in Nrf2-depleted lung cancer cells, suggesting increased lethality of ionizing radiation in the absence of Nrf2. Radiation-induced protein oxidation in Nrf2shRNA cells correlated with reduced survival as measured by clonogenic assay. Radiation-induced cell death was abrogated by pretreatment with antioxidants such as N-acetyl-L-cysteine, glutathione, and vitamin-E, highlighting the importance of antioxidants in conferring protection against radiation injury. Using genetically-modified gain and loss of function models of Nrf2, in mouse embryonic fibroblasts, we establish that constitutive activation of Nrf2 protects against ionizing radiation toxicity and confers radioresistance. Thus, targeting Nrf2 activity in radioresistant tumors could be a promising strategy to circumvent radioresistance. Antioxid. Redox Signal. 13, 1627–1637. PMID:20446773

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

    PubMed Central

    Zou, Yuhong; Hu, Min; Lee, Joonyong; Nambiar, Shashank Manohar; Garcia, Veronica; Bao, Qi; Chan, Jefferson Y.

    2014-01-01

    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. PMID:25524062

  19. Nrf2 inhibits epithelial-mesenchymal transition by suppressing snail expression during pulmonary fibrosis

    PubMed Central

    Zhou, Wencheng; Mo, Xiaoting; Cui, Wenhui; Zhang, Zhihui; Li, Delin; Li, Liucheng; Xu, Liang; Yao, Hongwei; Gao, Jian

    2016-01-01

    Epithelial-mesenchymal transition (EMT) is a phenotype conversion that plays a critical role in the development of pulmonary fibrosis (PF). It is known that snail could regulate the progression of EMT. Nuclear factor erythroid 2 related factor 2 (Nrf2), a key regulator of antioxidant defense system, protects cells against oxidative stress. However, it is not known whether Nrf2 regulates snail thereby modulating the development of PF. Here, bleomycin (BLM) was intratracheally injected into both Nrf2-knockout (Nrf2−/−) and wild-type mice to compare the development of PF. Rat type II alveolar epithelial cells (RLE-6TN) were treated with a specific Nrf2 activator sulforaphane, or transfected with Nrf2 and snail siRNAs to determine their effects on transforming growth factor β1 (TGF-β1)-induced EMT. We found that BLM-induced EMT and lung fibrosis were more severe in Nrf2−/− mice compared to wild-type mice. In vitro, sulforaphane treatment attenuated TGF-β1-induced EMT, accompanied by the down-regulation of snail. Inversely, silencing Nrf2 by siRNA enhanced TGF-β1-induced EMT along with increased expression of snail. Interestingly, when snail was silenced by siRNA, sulforaphane treatment was unable to reduce the progression of EMT in RLE-6TN cells. These findings suggest that Nrf2 attenuates EMT and fibrosis process by regulating the expression of snail in PF. PMID:27982105

  20. Deletion of Nrf2 reduces skeletal mechanical properties and decreases load-driven bone formation.

    PubMed

    Sun, Yong-Xin; Li, Lei; Corry, Kylie A; Zhang, Pei; Yang, Yang; Himes, Evan; Mihuti, Cristina Layla; Nelson, Cecilia; Dai, Guoli; Li, Jiliang

    2015-05-01

    Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor expressed in many cell types, including osteoblasts, osteocytes, and osteoclasts. Nrf2 has been considered a master regulator of cytoprotective genes against oxidative and chemical insults. The lack of Nrf2 can induce pathologies in multiple organs. The aim of this study was to investigate the role of Nrf2 in load-driven bone metabolism using Nrf2 knockout (KO) mice. Compared to age-matched littermate wild-type controls, Nrf2 KO mice have significantly lowered femoral bone mineral density (-7%, p<0.05), bone formation rate (-40%, p<0.05), as well as ultimate force (-11%, p<0.01). The ulna loading experiment showed that Nrf2 KO mice were less responsive than littermate controls, as indicated by reduction in relative mineralizing surface (rMS/BS, -69%, p<0.01) and relative bone formation rate (rBFR/BS, -84%, p<0.01). Furthermore, deletion of Nrf2 suppressed the load-driven gene expression of antioxidant enzymes and Wnt5a in cultured primary osteoblasts. Taken together, the results suggest that the loss-of-function mutation of Nrf2 in bone impairs bone metabolism and diminishes load-driven bone formation. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. 15d-PGJ{sub 2} stimulates HO-1 expression through p38 MAP kinase and Nrf-2 pathway in rat vascular smooth muscle cells

    SciTech Connect

    Lim, Hyun-Joung; Lee, Kuy-Sook; Lee, Seahyoung; Park, Jin-Hee; Choi, Hye-Eun; Go, Sang Hee; Kwak, Hyun-Jeong; Park, Hyun-Young

    2007-08-15

    15d-PGJ{sub 2}, a potent endogenous ligand for peroxisome proliferators activated receptor-{gamma}, is a cyclopentenone-type prostaglandin produced by many different types of cells. Pertinent to its effect on vascular smooth muscle cell (VSMC), antiproliferative effects have been most frequently reported. In the present study, we investigated the effect of 15d-PGJ{sub 2} on HO-1 expression that has been reported to inhibit VSMC proliferation. According to our data, 15d-PGJ{sub 2} significantly induced ROS/NO production and HO-1 expression in rVSMCs. We also observed 15d-PGJ{sub 2}-induced translocation of Nrf-2. In addition, ROS scavenger pretreatment suppressed 15d-PGJ{sub 2}-induced HO-1 expression while PPAR{gamma} antagonist did not, suggesting nuclear translocation of Nrf-2 and subsequent HO-1 expression was ROS dependent rather than PPAR{gamma} dependent. Furthermore, an inhibitor of p38 MAPK abolished 15d-PGJ{sub 2}-induced HO-1 expression. These data suggest that 15d-PGJ{sub 2}-induced up-regulation of HO-1 is independent of PPAR{gamma} but dependent of ROS and p38 MAPK pathway. The present study reports for the first time that 15d-PGJ{sub 2} induces HO-1 expression possibly using Nrf-2 pathway as a response to ROS in VSMCs.

  2. Involvement of Nrf2-mediated heme oxygenase-1 expression in anti-inflammatory action of chitosan oligosaccharides through MAPK activation in murine macrophages.

    PubMed

    Hyung, Jun-Ho; Ahn, Chang-Bum; Il Kim, Boo; Kim, Kyunghoi; Je, Jae-Young

    2016-12-15

    Chitosan and its derivatives have been reported to have anti-inflammatory effects in vitro and in vivo. It is also suggested that chitosan and its derivatives could be up-regulating heme oxygenase-1 (HO-1) in different models. However, the up-regulation of HO-1 by chitosan oligosaccharides (COS) remains unexplored in regard to anti-inflammatory action in lipopolysaccharide (LPS)-stimulated murine macrophages (RAW264.7 cells). Treatment with COS induced HO-1 expression in LPS-stimulated RAW264.7 cells, whereas the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) was decreased. Pretreatment with ZnPP, a HO-1 inhibitor, reduced the COS-mediated anti-inflammatory action. HO-1 induction is mediated by activating the nuclear translocation of NF-E2-related factor 2 (Nrf2) using COS. Moreover, COS increased the phosphorylation of extracellular signal regulated kinase (ERK1/2), c-Jun N-terminal kinase/stress-activated protein kinase (JNK), and p38 MAPK. However, specific inhibitors of ERK, JNK, and p38 reduced COS-mediated nuclear translocation of Nrf2. Therefore, HO-1 induction also decreased in RAW264.7 cells. Collectively, COS exert an anti-inflammatory effect through Nrf2/MAPK-mediated HO-1 induction.

  3. Nitric oxide sets off an antioxidant response in adrenal cells: involvement of sGC and Nrf2 in HO-1 induction.

    PubMed

    Astort, F; Mercau, M; Giordanino, E; Degese, M S; Caldareri, L; Coso, O; Cymeryng, C B

    2014-02-15

    Induction of microsomal heme oxygenase 1 (HO-1) activity is considered a cytoprotective mechanism in different cell types. In adrenal cells, HO-1 induction by ACTH exerts a modulatory effect on steroid production as well. As nitric oxide (NO) has been also regarded as an autocrine/paracrine modulator of adrenal steroidogenesis we sought to study the effects of NO on the induction of HO-1 and the mechanism involved. We hereby analyzed the time and dose-dependent effect of a NO-donor (DETA/NO) on HO-1 induction in a murine adrenocortical cell line. We showed that this effect is mainly exerted at a transcriptional level as it is inhibited by actinomycin D and HO-1 mRNA degradation rates were not affected by DETA/NO treatment. HO-1 induction by NO does not appear to involve the generation of oxidative stress as it was not affected by antioxidant treatment. We also demonstrated that NO-treatment results in the nuclear translocation of the nuclear factor-erythroid 2-related factor (Nrf2), an effect that is attenuated by transfecting the cells with a dominant negative isoform of Nrf2. We finally show that the effects of the NO-donor are reproduced by a permeable analog of cGMP and that a soluble guanylate cyclase specific inhibitor blocked both the induction of HO-1 by NO and the nuclear translocation of Nrf2.

  4. Nuclear Respiratory Factor 2β (NRF-2β) recruits NRF-2α to the nucleus by binding to importin-α:β via an unusual monopartite-type nuclear localization signal.

    PubMed

    Hayashi, Rippei; Takeuchi, Nono; Ueda, Takuya

    2013-09-23

    Nuclear respiratory factor 2 (NRF-2) is a mammalian transcription factor composed of two distinct and unrelated proteins: NRF-2α, which binds to DNA through its Ets domain, and NRF-2β, which contains the transcription activation domain. The activity of NRF-2 in neurons is regulated by nuclear localization; however, the mechanism by which NRF-2 is imported into the nucleus remains unknown. By using in vitro nuclear import assays and immuno-cytofluorescence, we dissect the nuclear import pathways of NRF-2. We show that both NRF-2α and NRF-2β contain intrinsic nuclear localization signals (NLSs): the Ets domain within NRF-2α and the NLS within NRF-2β (amino acids 311/321: EEPPAKRQCIE) that is recognized by importin-α:β. When NRF-2α and NRF-2β form a complex, the nuclear import of NRF-2αβ becomes strictly dependent on the NLS within NRF-2β. Therefore, the nuclear import mechanism of NRF-2 is unique among Ets factors. The NRF-2β NLS contains only two lysine/arginine residues, unlike other known importin-α:β-dependent NLSs. Using ELISA-based binding assays, we show that it is bound by importin-α in almost the same manner and with similar affinity to that of the classical monopartite NLSs, such as c-myc and SV40 T-antigen NLSs. However, the part of the tryptophan array of importin-α that is essential for the recognition of classical monopartite NLSs by generating apolar pockets for the P3 and the P5 lysine/arginine side chains is not required for the recognition of the NRF-2β NLS. We conclude that the NRF-2β NLS is an unusual but is, nevertheless, a bona fide monopartite-type NLS.

  5. Trolox contributes to Nrf2-mediated protection of human and murine primary alveolar type II cells from injury by cigarette smoke.

    PubMed

    Messier, E M; Bahmed, K; Tuder, R M; Chu, H W; Bowler, R P; Kosmider, B

    2013-04-04

    Cigarette smoke (CS) is a main risk factor for chronic obstructive pulmonary disease (COPD). Oxidative stress induced by CS causes DNA and lung damage. Oxidant/antioxidant imbalance occurs in the distal air spaces of smokers and in patients with COPD. We studied the effect of oxidative stress generated by CS both in vivo and in vitro on murine primary alveolar type II (ATII) cells isolated from nuclear erythroid 2-related factor-2 (Nrf2)(-/-) mice. We determined human primary ATII cell injury by CS in vitro and analyzed ATII cells isolated from smoker and non-smoker lung donors ex vivo. We also studied whether trolox (water-soluble derivative of vitamin E) could protect murine and human ATII cells against CS-induced DNA damage and/or decrease injury. We analyzed oxidative stress by 4-hydroxynonenal expression, reactive oxygen species (ROS) generation by Amplex Red Hydrogen Peroxide Assay, Nrf2, heme oxygenase 1, p53 and P53-binding protein 1 (53BP1) expression by immonoblotting, Nrf2 nuclear translocation, Nrf2 and p53 DNA-binding activities, apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay and cytokine production by ELISA. We found that ATII cells isolated from Nrf2(-/-) mice are more susceptible to CS-induced oxidative DNA damage mediated by p53/53BP1 both in vivo and in vitro compared with wild-type mice. Therefore, Nrf2 activation is a key factor to protect ATII cells against injury by CS. Moreover, trolox abolished human ATII cell injury and decreased DNA damage induced by CS in vitro. Furthermore, we found higher inflammation and p53 mRNA expression by RT-PCR in ATII cells isolated from smoker lung donors in comparison with non-smokers ex vivo. Our results indicate that the Nrf2 and p53 cross talk in ATII cells affect the susceptibility of these cells to injury by CS. Trolox can protect against oxidative stress, genotoxicity and inflammation induced by CS through ROS scavenging mechanism, and serve as a potential

  6. Seaweed extracts and unsaturated fatty acid constituents from the green alga Ulva lactuca as activators of the cytoprotective Nrf2-ARE pathway.

    PubMed

    Wang, Rui; Paul, Valerie J; Luesch, Hendrik

    2013-04-01

    1, which showed ARE-activating effects similar to those observed in vitro. This could be owing to this fraction's ability to stabilize Nrf2 through inhibition of Keap1-mediated Nrf2 ubiquitination and the subsequent accumulation and nuclear translocation of Nrf2. The induction of many ARE-driven antioxidant genes in vivo and most prominently in the heart agreed with the commonly recognized cardioprotective properties of MUFAs. A significant increase in Nqo1 transcript levels was also found in other mouse tissues such as the brain, lung, and stomach. Collectively, this study provides new insight into why consumption of dietary seaweed may have health benefits, and the identified compounds add to the list of chemopreventive dietary unsaturated fatty acids.

  7. 4-Ketopinoresinol, a novel naturally occurring ARE activator, induces the Nrf2/HO-1 axis and protects against oxidative stress-induced cell injury via activation of PI3K/AKT signaling.

    PubMed

    Chen, Huang-Hui; Chen, Yu-Tsen; Huang, Yen-Wen; Tsai, Hui-Ju; Kuo, Ching-Chuan

    2012-03-15

    The Nrf2/ARE pathway plays an important role in inducing phase II detoxifying enzymes and antioxidant proteins and has been considered a potential target for cancer chemoprevention because it eliminates harmful reactive oxygen species or reactive intermediates generated from carcinogens. The objectives of this study were to identify novel Nrf2/ARE activators and to investigate the mechanistic signaling pathway involved in the activation of Nrf2-mediated cytoprotective effects against oxidative-induced cell injury. A stable ARE-driven luciferase reporter cell line was established to screen a potentially cytoprotective compound. 4-Ketopinoresinol (4-KPR), the (α-γ) double-cyclized type of lignan obtained from adlay (Coix lachryma-jobi L. var. ma-yuen Stapf), activates ARE-driven luciferase activity more effectively than the classical ARE activator tert-butylhydroquinone. 4-KPR treatment resulted in a transient increase in AKT phosphorylation and subsequent phosphorylation and nuclear translocation of Nrf2, along with increased expression of ARE-dependent cytoprotective genes, such as heme oxygenase-1 (HO-1), aldo-keto reductases, and glutathione synthetic enzyme. 4-KPR suppresses oxidative stress-induced DNA damage and cell death via upregulation of HO-1. Inhibition of PI3K/AKT signaling by chemical inhibitors or RNA interference not only suppressed 4-KPR-induced Nrf2/HO-1 activation, but also eliminated the cytoprotective effect against oxidative damage. These observations in an ARE-regulated gene system suggest that 4-KPR is a novel Nrf2/ARE-mediated transcription activator, activates the Nrf2/HO-1 axis, and protects against oxidative stress-induced cell injury via activation of PI3K/AKT signaling.

  8. Nrf2 deficiency impairs the barrier function of mouse esophageal epithelium

    PubMed Central

    Chen, Hao; Hu, Yuhui; Fang, Yu; Djukic, Zorka; Yamamoto, Masayuki; Shaheen, Nicholas J.; Orlando, Roy C.; Chen, Xiaoxin

    2013-01-01

    Objective As a major cellular defense mechanism, the Nrf2/Keap1 pathway regulates expression of genes involved in detoxification and stress response. Our previous study revealed activation of the Nrf2/Keap1 pathway at the maturation phase during mouse esophageal development, suggesting a potential function in epithelial defense. Here we hypothesize that Nrf2 is involved in the barrier function of esophageal epithelium, and plays a protective role against gastroesophageal reflux disease (GERD). Design Human esophageal biopsy samples, mouse surgical models and Nrf2-/- mice were used to assess the role of the Nrf2/Keap1 pathway in esophageal mucosal barrier function. Trans-epithelial electrical resistance (TEER) was measured with mini-Ussing chambers. Hematoxylin and eosin (HE) staining and transmission electron microscopy were used to examine cell morphology, while gene microarray, immunohistochemistry, Western blotting and ChIP analysis were used to assess the expression of pathway genes. Results Nrf2 was expressed in normal esophageal epithelium and activated in GERD of both humans and mice. Nrf2 deficiency and gastroesophageal reflux in mice, either alone or in combination, reduced TEER and increased intercellular space diameter in esophageal epithelium. Nrf2 target genes and gene sets associated with oxidoreductase activity, mitochondrial biogenesis and energy production were down-regulated in the esophageal epithelium of Nrf2-/- mice. Consistent with the antioxidative function of Nrf2, a DNA oxidative damage marker (8OHdG) dramatically increased in esophageal epithelial cells of Nrf2-/- mice compared with those of wild-type mice. Interestingly, ATP biogenesis, Cox IV (a mitochondrial protein) and Claudin-4 (Cldn4) expression were down-regulated in the esophageal epithelium of Nrf2-/- mice, suggesting that energy-dependent tight junction integrity was subject to Nrf2 regulation. ChIP analysis confirmed the binding of Nrf2 to Cldn4 promoter. Conclusion Nrf2

  9. Dihydro-CDDO-trifluoroethyl amide suppresses inflammatory responses in macrophages via activation of Nrf2

    SciTech Connect

    Li, Bin; Abdalrahman, Akram; Lai, Yimu; Janicki, Joseph S.; Ward, Keith W.; Meyer, Colin J.; Wang, Xing Li; Tang, Dongqi; Cui, Taixing

    2014-02-21

    Highlights: • Dh404 suppresses the expression of a selected set of pro-inflammatory cytokines in inflamed macrophages via activating Nrf2. • Dh404 activates Nrf2 while keeping Keap1 function intact in macrophages. • Dh404 minimally regulates NF-κB pathway in macrophages. - Abstract: Nuclear factor erythroid 2-related factor (Nrf2) is the major regulator of cellular defenses against various pathological stresses in a variety of organ systems, thus Nrf2 has evolved to be an attractive drug target for the treatment and/or prevention of human disease. Several synthetic oleanolic triterpenoids including dihydro-CDDO-trifluoroethyl amide (dh404) appear to be potent activators of Nrf2 and exhibit chemopreventive promises in multiple disease models. While the pharmacological efficacy of Nrf2 activators may be dependent on the nature of Nrf2 activation in specific cell types of target organs, the precise role of Nrf2 in mediating biological effects of Nrf2 activating compounds in various cell types remains to be further explored. Herein we report a unique and Nrf2-dependent anti-inflammatory profile of dh404 in inflamed macrophages. In lipopolysaccharide (LPS)-inflamed RAW264.7 macrophages, dh404 dramatically suppressed the expression of pro-inflammatory cytokines including inducible nitric oxide synthase (iNOS), monocyte chemotactic protein-1 (MCP-1), and macrophage inflammatory protein-1 beta (MIP-1β), while minimally regulating the expression of interleulin-6 (IL-6), IL-1β, and tumor necrosis factor alpha (TNFα). Dh404 potently activated Nrf2 signaling; however, it did not affect LPS-induced NF-κB activity. Dh404 did not interrupt the interaction of Nrf2 with its endogenous inhibitor Kelch-like ECH associating protein 1 (Keap1) in macrophages. Moreover, knockout of Nrf2 blocked the dh404-induced anti-inflammatory responses in LPS-inflamed macrophages. These results demonstrated that dh404 suppresses pro-inflammatory responses in macrophages via an activation

  10. Insulin Inhibits Nrf2 Gene Expression via Heterogeneous Nuclear Ribonucleoprotein F/K in Diabetic Mice.

    PubMed

    Ghosh, Anindya; Abdo, Shaaban; Zhao, Shuiling; Wu, Chin-Han; Shi, Yixuan; Lo, Chao-Sheng; Chenier, Isabelle; Alquier, Thierry; Filep, Janos G; Ingelfinger, Julie R; Zhang, Shao-Ling; Chan, John S D

    2017-01-23

    Oxidative stress induces endogenous antioxidants via nuclear factor erythroid 2-related factor 2 (Nrf2), potentially preventing tissue injury. We investigated whether insulin affects renal Nrf2 expression in type 1 diabetes (T1D) and studied its underlying mechanism. Insulin normalized hyperglycemia, hypertension, oxidative stress and renal injury, inhibited renal Nrf2 and angiotensinogen (Agt) gene expression and up-regulated heterogeneous nuclear ribonucleoprotein F (hnRNP F) and hnRNP K expression in Akita mice with T1D. In immortalized rat renal proximal tubular cells, insulin suppressed Nrf2 and Agt but stimulated hnRNP F and hnRNP K gene transcription in high glucose via p44/42 mitogen-activated protein kinase signalling. Transfection with small interfering RNAs of p44/42 MAPK, hnRNP F or hnRNP K blocked insulin inhibition of Nrf2 gene transcription. Insulin curbed Nrf2 promoter activity via a specific DNA-responsive element that binds hnRNP F/K, and hnRNP F/K overexpression curtailed Nrf2 promoter activity. In hyperinsulinemic-euglycemic mice, renal Nrf2 and Agt expression was down-regulated, whereas hnRNP F/K expression was up-regulated. Thus, the beneficial actions of insulin in diabetic nephropathy appear to be mediated, in part, by suppressing renal Nrf2 and Agt gene transcription and preventing Nrf2 stimulation of Agt expression via hnRNP F/K. These findings identify hnRNP F/K and Nrf2 as potential therapeutic targets in diabetes.

  11. Nrf2 protects human bladder urothelial cells from arsenite and monomethylarsonous acid toxicity

    SciTech Connect

    Wang Xiaojun; Sun Zheng; Chen Weimin; Eblin, Kylee E.; Gandolfi, Jay A.; Zhang, Donna D.

    2007-12-01

    Arsenic is widely spread in our living environment and imposes a big challenge on human health worldwide. Arsenic damages biological systems through multiple mechanisms including the generation of reactive oxygen species. The transcription factor Nrf2 regulates the cellular antioxidant response that protects cells from various insults. In this study, the protective role of Nrf2 in arsenic toxicity was investigated in a human bladder urothelial cell line, UROtsa. Using a UROtsa cell line stably infected with Nrf2-siRNA, we clearly demonstrate that compromised Nrf2 expression sensitized the cells to As(III)- and MMA(III)-induced toxicity. On the other hand, the activation of the Nrf2 pathway by tert-butylhydroquinone (tBHQ) and sulforaphane (SF), the known Nrf2-inducers, rendered UROtsa cells more resistant to As(III) and MMA(III). Furthermore, the wild-type mouse embryo fibroblast (WT-MEF) cells were protected from As(III)- and MMA(III)-induced toxicity following Nrf2 activation by tBHQ or SF, whereas neither tBHQ nor SF conferred protection in the Nrf2{sup -/-}MEF cells, demonstrating that tBHQ- or SF-mediated protection against As(III)- and MMA(III)-induced toxicity depends on Nrf2 activation. These results, obtained by both loss of function and gain of function analyses, clearly demonstrate the protective role of Nrf2 in arsenic-induced toxicity. The current work lays the groundwork for using Nrf2 activators for therapeutic and dietary interventions against adverse effects of arsenic.

  12. Nrf2 protects human bladder urothelial cells from arsenite and monomethylarsonous acid toxicity

    PubMed Central

    Wang, Xiao-Jun; Sun, Zheng; Chen, Weimin; Eblin, Kylee E.; Gandolfi, A. Jay; Zhang, Donna D.

    2007-01-01

    Arsenic is widely spread in our living environment and imposes a big challenge on human health worldwide. Arsenic damages biological systems through multiple mechanisms including the generation of reactive oxygen species. The transcription factor Nrf2 regulates the cellular antioxidant response that protects cells from various insults. In this study, the protective role of Nrf2 in arsenic toxicity was investigated in a human bladder urothelial cell line, UROtsa. Using an UROtsa cell line stably infected with Nrf2-siRNA, we clearly demonstrate that compromised Nrf2 expression sensitized the cells to As(III)- and MMA(III)-induced toxicity. On the other hand, the activation of the Nrf2 pathway by tert-butylhydroquinone (tBHQ) and sulforaphane (SF), the known Nrf2-inducers, rendered UROtsa cells more resistant to As(III)- and MMA(III). Furthermore, the wild type mouse embryo fibroblast (WT-MEF) cells were protected from As(III)- and MMA(III)-induced toxicity following Nrf2 activation by tBHQ or SF whereas neither tBHQ nor SF conferred protection in the Nrf2−/−-MEF cells, demonstrating that tBHQ- or SF-mediated protection against As(III)- and MMA(III)-induced toxicity depends on Nrf2 activation. These results, obtained by both loss of function and gain of function analyses, clearly demonstrate the protective role of Nrf2 in arsenic-induced toxicity. The current work lays the groundwork for using Nrf2 activators for therapeutic and dietary interventions against adverse effects of arsenic. PMID:17765279

  13. tert-Butylhydroquinone mobilizes intracellular-bound zinc to stabilize Nrf2 through inhibiting phosphatase activity.

    PubMed

    Chen, Yunfang; Wang, Sheng; Fu, Xin; Zhou, Wenqu; Hong, Wei; Zou, Dongting; Li, Xichong; Liu, Jinbao; Ran, Pixin; Li, Bing

    2015-08-01

    The nuclear factor erythroid 2-related factor 2 (Nrf2) is required to combat increases in oxidative stress. The chemical compound tert-butylhydroquinone (tBHQ) can downregulate Kelch-like ECH-associated protein 1 (Keap1), a repressor of Nrf2, thus maintaining the stability of Nrf2. tBHQ can also increase intracellular "free" zinc in human bronchial epithelial (16HBE) cells. We aim to investigate whether the intracellular free zinc change plays a role in Nrf2 activation. tBHQ exposure dose-dependently increases intracellular free zinc concentrations within 30 min in 16HBE cells by mobilizing intracellular zinc pools. Active Nrf2 and the antioxidant enzyme heme oxygenase-1 (HO-1) increase at 3 h after tBHQ treatment. Chelating intracellular free zinc with tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN) during tBHQ exposure partially abrogates the tBHQ-induced activation of Nrf2 and HO-1 expression, while Keap1 is further decreased. These results indicate that tBHQ-induced stability of Nrf2 is associated with the intracellular free zinc level. Because the activated Nrf2 is phosphorylated, the serine/threonine protein phosphatase activity, which is known to be inhibited by zinc, is assayed. The results showed that tBHQ treatment can suppress cellular protein phosphatase-2A (PP2A) and protein phosphatase-2C (PP2C) activity, which can be abrogated by adding TPEN. This finding is verified in a cell-free protein extract experiment by supplying zinc or by chelating zinc with TPEN. These results provide a novel mechanistic insight into Nrf2 activation in antioxidant enzyme induction involving zinc signaling. The increase of intracellular free zinc may be one mechanism for Nrf2 activation. The inhibition of PP2A and PP2C activity may be involved in Nrf2 phosphorylation modulation. Copyright © 2015 the American Physiological Society.

  14. Molecular basis of electrophilic and oxidative defense: promises and perils of Nrf2.

    PubMed

    Ma, Qiang; He, Xiaoqing

    2012-10-01

    Induction of drug-metabolizing enzymes through the antioxidant response element (ARE)-dependent transcription was initially implicated in chemoprevention against cancer by antioxidants. Recent progress in understanding the biology and mechanism of induction revealed a critical role of induction in cellular defense against electrophilic and oxidative stress. Induction is mediated through a novel signaling pathway via two regulatory proteins, the nuclear factor erythroid 2-related factor 2 (Nrf2) and the Kelch-like erythroid cell-derived protein with CNC homology-associated protein 1 (Keap1). Nrf2 binds to Keap1 at a two site-binding interface and is ubiquitinated by the Keap1/cullin 3/ring box protein-1-ubiquitin ligase, resulting in a rapid turnover of Nrf2 protein. Electrophiles and oxidants modify critical cysteine thiols of Keap1 and Nrf2 to inhibit Nrf2 ubiquitination, leading to Nrf2 activation and induction. Induction increases stress resistance critical for cell survival, because knockout of Nrf2 in mice increased susceptibility to a variety of toxicity and disease processes. Collateral to diverse functions of Nrf2, genome-wide search has led to the identification of a plethora of ARE-dependent genes regulated by Nrf2 in an inducer-, tissue-, and disease-dependent manner to control drug metabolism, antioxidant defense, stress response, proteasomal degradation, and cell proliferation. The protective nature of Nrf2 could also be hijacked in a number of pathological conditions by means of somatic mutation, epigenetic alteration, and accumulation of disruptor proteins, promoting drug resistance in cancer and pathologic liver features in autophagy deficiency. The repertoire of ARE inducers has expanded enormously; the therapeutic potential of the inducers has been examined beyond cancer prevention. Developing potent and specific ARE inducers and Nrf2 inhibitors holds certain new promise for the prevention and therapy against cancer, chronic disease, and toxicity.

  15. Eriodictyol-7-O-glucoside activates Nrf2 and protects against cerebral ischemic injury

    SciTech Connect

    Jing, Xu; Ren, Dongmei; Wei, Xinbing; Shi, Huanying; Zhang, Xiumei; Perez, Ruth G.; Lou, Haiyan; Lou, Hongxiang

    2013-12-15

    Stroke is a complex disease that may involve oxidative stress-related pathways in its pathogenesis. The nuclear factor erythroid-2-related factor 2/antioxidant response element (Nrf2/ARE) pathway plays an important role in inducing phase II detoxifying enzymes and antioxidant proteins and thus has been considered a potential target for neuroprotection in stroke. The aim of the present study was to determine whether eriodictyol-7-O-glucoside (E7G), a novel Nrf2 activator, can protect against cerebral ischemic injury and to understand the role of the Nrf2/ARE pathway in neuroprotection. In primary cultured astrocytes, E7G increased the nuclear localization of Nrf2 and induced the expression of the Nrf2/ARE-dependent genes. Exposure of astrocytes to E7G provided protection against oxygen and glucose deprivation (OGD)-induced oxidative insult. The protective effect of E7G was abolished by RNA interference-mediated knockdown of Nrf2 expression. In vivo administration of E7G in a rat model of focal cerebral ischemia significantly reduced the amount of brain damage and ameliorated neurological deficits. These data demonstrate that activation of Nrf2/ARE signaling by E7G is directly associated with its neuroprotection against oxidative stress-induced ischemic injury and suggest that targeting the Nrf2/ARE pathway may be a promising approach for therapeutic intervention in stroke. - Highlights: • E7G activates Nrf2 in astrocytes. • E7G stimulates expression of Nrf2-mediated cytoprotective proteins in astrocytes. • E7G protects astrocytes against OGD-induced cell death and apoptosis. • The neuroprotective effect of E7G involves the Nrf2/ARE pathway. • E7G protects rats against cerebral ischemic injury.

  16. Daphnetin-mediated Nrf2 antioxidant signaling pathways ameliorate tert-butyl hydroperoxide (t-BHP)-induced mitochondrial dysfunction and cell death.

    PubMed

    Lv, Hongming; Liu, Qinmei; Zhou, Junfeng; Tan, Guangyun; Deng, Xuming; Ci, Xinxin

    2017-05-01

    Daphnetin (Daph), a natural coumarin derivative isolated from plants of the Genus Daphne, possesses abundant biological activities, such as anti-inflammatory, antioxidant and anticancer properties. In the present study, we focused on investigating the protective effect of Daph against tert-butyl hydroperoxide (t-BHP)-induced oxidative damage, mitochondrial dysfunction and the involvement of underlying molecular mechanisms. Our findings indicated that Daph effectively inhibited t-BHP-stimulated cytotoxicity, cell apoptosis, and mitochondrial dysfunction, which are associated with suppressed reactive oxygen species (ROS) generation, decreased malondialdehyde (MDA) formation, increased superoxide dismutase (SOD) levels and glutathione (GSH)/GSSG (oxidized GSH) ratio. Further investigation indicated that Daph significantly suppressed cytochrome c release and NLRP3 inflammasome activation and modulated apoptosis-related protein Bcl-2, Bax, and caspase-3 expression. Moreover, Daph dramatically induced the expression of the glutamate-cysteine ligase modifier (GCLM) subunit and the glutamate-cysteine ligase catalytic (GCLC) subunit, heme oxygenase-1 (HO-1), and NAD (P) H: quinone oxidoreductase (NQO1), which is largely dependent on upregulating the nuclear factor-erythroid 2-related factor 2 (Nrf2) nuclear translocation, reducing the Keap1 protein expression, and strengthening the antioxidant response element (ARE) promoter activity. Additionally, Daph remarkably activated a c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) phosphorylation, but ERK and JNK inhibitor pretreatment exhibited an evident decrease of the level of Daph-enhanced Nrf2 nuclear translocation. Furthermore, Daph exposure suppressed t-BHP-induced cytotoxicity and ROS overproduction, which are mostly blocked in Nrf2 knockout RAW 264.7 cells and peritoneal macrophages. Accordingly, Daph exhibited protective roles against t-BHP-triggered oxidative damage and mitochondrial

  17. Regulation of Ahr signaling by Nrf2 during development: Effects of Nrf2a deficiency on PCB126 embryotoxicity in zebrafish (Danio rerio)

    PubMed Central

    Rousseau, Michelle E.; Sant, Karilyn E.; Borden, Linnea R.; Franks, Diana G.; Hahn, Mark E.; Timme-Laragy, Alicia R.

    2015-01-01

    The embryotoxicity of co-planar PCBs is regulated by the aryl hydrocarbon receptor (Ahr), and has been reported to involve oxidative stress. Ahr participates in crosstalk with another transcription factor, Nfe2l2, or Nrf2. Nrf2 binds to antioxidant response elements to regulate the adaptive response to oxidative stress. To explore aspects of the crosstalk between Nrf2 and Ahr and its impact on development, we used zebrafish (Danio rerio) with a mutated DNA binding domain in Nrf2a (nrf2afh318/fh318), rendering these embryos more sensitive to oxidative stress. Embryos were exposed to 2 nM or 5 nM PCB126 at 24 hours post fertilization (prim-5 stage of pharyngula) and examined for gene expression and morphology at 4 days post fertilization (dpf; protruding –mouth stage). Nrf2a mutant eleutheroembryos were more sensitive to PCB126 toxicity at 4 dpf, and in the absence of treatment also displayed some subtle developmental differences from wildtype embryos, including delayed inflation of the swim bladder and smaller yolk sacs. We used qPCR to measure changes in expression of the nrf gene family, keap1a, keap1b, the ahr gene family, and known target genes. cyp1a induction by PCB126 was enhanced in the Nrf2a mutants (156-fold in wildtypes vs. 228-fold in mutants exposed to 5 nM). Decreased expression of heme oxygenase (decycling) 1 (hmox1) in the Nrf2a mutants was accompanied by increased nrf2b expression. Target genes of Nrf2a and AhR2, NAD(P)H:quinone oxidoreductase 1 (nqo1) and glutathione S-transferase, alpha-like (gsta1), showed a 2-5-fold increase in expression in the Nrf2a mutants as compared to wildtype. This study elucidates the interaction between two important transcription factor pathways in the developmental toxicity of co-planar PCBs. PMID:26325326

  18. 20C, a bibenzyl compound isolated from Gastrodia elata, protects PC12 cells against rotenone-induced apoptosis via activation of the Nrf2/ARE/HO-1 signaling pathway.

    PubMed

    Huang, Ju-Yang; Yuan, Yu-He; Yan, Jia-Qing; Wang, Ya-Nan; Chu, Shi-Feng; Zhu, Cheng-Gen; Guo, Qing-Lan; Shi, Jian-Gong; Chen, Nai-Hong

    2016-06-01

    Our preliminary study shows that a bibenzyl compound isolated from Gastrodia elata, 2-[4-hydroxy-3-(4-hydroxybenzyl)benzyl]-4-(4-hydroxybenzyl)phenol (designated 20C), protects PC12 cells against H2O2-induced injury. In this study we investigated whether 20C exerted neuroprotective action in a cell model of Parkinson's disease. A cell model of Parkinson's disease was established in PC12 cells by exposure to rotenone (4 μmol/L) for 48 h. Cell viability and apoptosis were assessed, and intracellular ROS level and the mitochondrial membrane potential (MMP) were detected. The expression of apoptosis-related proteins Bax, Bcl-2, cytochrome c, cleaved caspase-3, and oxidative stress-related proteins Nrf2, HO-1 and NQO1 were examined using Western blotting. The mRNA levels of HO-1 and NQO1 were determined with RT-PCR. The nuclear translocation of Nrf2 was observed with immunofluorescence staining. Treatment with rotenone significantly increased the number of apoptotic cells, accompanied by marked increases in the Bax/Bcl-2 ratio, cytochrome c release and caspase-3 activation. Rotenone also increased ROS accumulation, reduced MMP, and increased the nuclear translocation of Nrf2 as well as the mRNA and protein levels of the Nrf2 downstream target genes HO-1 and NQO1 in PC12 cells. Co-treatment with 20C (0.01-1 μmol/L) dose-dependently attenuated rotenone-induced apoptosis and oxidative stress in PC12 cells. Nrf2 knockdown by siRNA partially reversed the protective effects of 20C in rotenone-treated PC12 cells. The bibenzyl compound 20C protects PC12 cells from rotenone-induced apoptosis, at least in part, via activation of the Nrf2/ARE/HO-1 signaling pathway.

  19. 20C, a bibenzyl compound isolated from Gastrodia elata, protects PC12 cells against rotenone-induced apoptosis via activation of the Nrf2/ARE/HO-1 signaling pathway

    PubMed Central

    Huang, Ju-yang; Yuan, Yu-he; Yan, Jia-qing; Wang, Ya-nan; Chu, Shi-feng; Zhu, Cheng-gen; Guo, Qing-lan; Shi, Jian-gong; Chen, Nai-hong

    2016-01-01

    Aim: Our preliminary study shows that a bibenzyl compound isolated from Gastrodia elata, 2-[4-hydroxy-3-(4-hydroxybenzyl)benzyl]-4-(4-hydroxybenzyl)phenol (designated 20C), protects PC12 cells against H2O2-induced injury. In this study we investigated whether 20C exerted neuroprotective action in a cell model of Parkinson's disease. Methods: A cell model of Parkinson's disease was established in PC12 cells by exposure to rotenone (4 μmol/L) for 48 h. Cell viability and apoptosis were assessed, and intracellular ROS level and the mitochondrial membrane potential (MMP) were detected. The expression of apoptosis-related proteins Bax, Bcl-2, cytochrome c, cleaved caspase-3, and oxidative stress-related proteins Nrf2, HO-1 and NQO1 were examined using Western blotting. The mRNA levels of HO-1 and NQO1 were determined with RT-PCR. The nuclear translocation of Nrf2 was observed with immunofluorescence staining. Results: Treatment with rotenone significantly increased the number of apoptotic cells, accompanied by marked increases in the Bax/Bcl-2 ratio, cytochrome c release and caspase-3 activation. Rotenone also increased ROS accumulation, reduced MMP, and increased the nuclear translocation of Nrf2 as well as the mRNA and protein levels of the Nrf2 downstream target genes HO-1 and NQO1 in PC12 cells. Co-treatment with 20C (0.01–1 μmol/L) dose-dependently attenuated rotenone-induced apoptosis and oxidative stress in PC12 cells. Nrf2 knockdown by siRNA partially reversed the protective effects of 20C in rotenone-treated PC12 cells. Conclusion: The bibenzyl compound 20C protects PC12 cells from rotenone-induced apoptosis, at least in part, via activation of the Nrf2/ARE/HO-1 signaling pathway. PMID:27180985

  20. Geraniin exerts cytoprotective effect against cellular oxidative stress by upregulation of Nrf2-mediated antioxidant enzyme expression via PI3K/AKT and ERK1/2 pathway.

    PubMed

    Wang, Peng; Peng, Xiao; Wei, Zuo-Fu; Wei, Fu-Yao; Wang, Wei; Ma, Wei-Dong; Yao, Li-Ping; Fu, Yu-Jie; Zu, Yuan-Gang

    2015-09-01

    Geraniin, an active compound with remarkable antioxidant activity, was isolated from Geranium sibiricum. The present study aimed to investigate whether geraniin has the ability to activate Nrf2, induce antioxidant enzyme expression and protect cells from oxidative damage. The cells were pretreated with geraniin for 24h and exposed to hydrogen peroxide (H₂O₂) for 4h. Intracellular reactive oxygen species (ROS) levels, mitochondrial membrane potential and apoptosis were measured. We also investigated intracellular glutathione (GSH) levels and changes in nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated signaling cascade in cells treated with geraniin. We investigated the protective effects of geraniin against H₂O₂-induced apoptosis in HepG2 cells. Geraniin significantly reduced H₂O₂-induced oxidative damage in a dose dependent manner. Further, geraniin induced the expression of heme oxygenase-1 (HO-1), NAD(P)H quinone oxidoreductase-1 (NQO1) and level of glutathione (GSH) in a concentration- and time-dependent manner, and increased Nrf2 nuclear translocation. The Nrf2-related cytoprotective effects of geraniin were PI3K/AKT and extracellular signal-regulated protein kinase1/2 (ERK1/2) pathway-dependent. However, inhibitors of PI3K/AKT and ERK1/2 (LY294002 or U0126) not only suppressed geraniin-induced nuclear translocation of Nrf2 but also abolished the expression of HO-1, NQO1 and GSH. These results demonstrated that geraniin induced Nrf2-mediated expression of antioxidant enzymes HO-1 and NQO1, presumably via PI3K/AKT and ERK1/2 signaling pathways, thereby protecting cells from H₂O₂-induced oxidative cell death. Geraniin, at least in part, offers an antioxidant defense capacity to protect cells from the oxidative stress-related diseases. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Ferulic Acid Protects Against Lead Acetate-Induced Inhibition of Neurite Outgrowth by Upregulating HO-1 in PC12 Cells: Involvement of ERK1/2-Nrf2 Pathway.

    PubMed

    Yu, Chun-Lei; Zhao, Xue-Mei; Niu, Ying-Cai

    2016-11-01

    Prenatal lead exposure is associated with poor intellectual development in children. However, there are few breakthroughs in therapeutic intervention of developmental lead neurotoxicity. The aim of this study is to evaluate the hypothesis that ferulic acid-mediated promotion of neurite outgrowth following lead exposure might mainly result from its antioxidant capability by extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation of nuclear factor erythroid 2-related factor 2 (Nrf2). Exposure of PC12 cells to lead acetate inhibits neurite outgrowth and causes oxidative stress as measured by ROS, LPO, GSH/GSSG, and NAD(+)/NADH. FA treatment significantly, although not completely, protected the cells against lead acetate-induced neurite outgrowth inhibition. The effects of FA could be blocked by PD98059, zinc protoporphyrin (Zn-PP), and Nrf2 shRNA. In addition, FA induced heme oxygenase 1 (HO-1) gene expression, enhanced antioxidant response element (ARE) promoter activity, promoted ERK1/2 phosphorylation, and Nrf2 translocation in PC12 cells exposed to lead acetate. ERK1/2 locate upstream of Nrf2 and regulate Nrf2-dependent HO-1 expression in antioxidative effects of FA. Our results suggest that FA is a promising candidate for treatment of developmental lead neurotoxicity. These promising findings warrant future investigation evaluating the FA-mediated potentiation of neurite outgrowth following lead exposure in vivo.

  2. The cytoprotective effects of ethanol extract of Ecklonia cava against oxidative stress are associated with upregulation of Nrf2-mediated HO-1 and NQO-1 expression through activation of the MAPK pathway.

    PubMed

    Choi, Yung Hyun

    2016-01-01

    The aim of the present study was to examine the cytoprotective effect of Ecklonia cava against oxidative stress in C2C12 myoblasts. The ethanol extract of E. cava (EEEC) prevented hydrogen peroxide (H₂O₂)-induced inhibition of the growth of C2C12 myoblasts and exhibited scavenging activity against intracellular reactive oxygen species (ROS) induced by H₂O₂. EEEC treatment attenuated H2O2-induced comet tail formation and phospho-histone γH2A.X expression. Furthermore, EEEC treatment enhanced the level of the phosphorylated form of nuclear factor erythroid 2- related factor 2 (Nrf2) and its nuclear translocation, which was associated with the induction of heme oxygenase-1 (HO-1) and NADPH-quinone oxidoreductase 1 (NQO-1). Zinc protoporphyrin IX, a HO-1 competitive inhibitor, significantly abolished the protective effects of EEEC against H₂O₂-induced ROS generation and growth inhibition in C2C12 myoblasts. Transient transfection with Nrf2-specific small interfering RNA restored the elevated HO-1 and NQO-1 expression and the phosphorylation of Nrf2 to near normal levels. The EEEC treatment also induced the activation of mitogen-activated protein kinases (MAPKs), and specific inhibitors of MAPKs abolished upregulated HO-1 and NQO-1, as well as the phosphorylation of Nrf2. Taken together, these data suggest that EEEC attenuates oxidative stress by activating Nrf2-mediated HO-1 and inducing NQO-1 via the activation of MAPK signaling pathways.

  3. Nrf2 protects the lung against inflammation induced by titanium dioxide nanoparticles: A positive regulator role of Nrf2 on cytokine release.

    PubMed

    Delgado-Buenrostro, Norma L; Medina-Reyes, Estefany I; Lastres-Becker, Isabel; Freyre-Fonseca, Verónica; Ji, Zhaoxia; Hernández-Pando, Rogelio; Marquina, Brenda; Pedraza-Chaverri, José; Espada, Sandra; Cuadrado, Antonio; Chirino, Yolanda I

    2015-07-01

    Titanium dioxide nanoparticles (TiO2 NPs) have been classified as possibly carcinogenic to humans and they are an important nanomaterial widely used in pharmaceutical and paint industries. Inhalation is one of the most important routes of exposure in occupational settings. Several experimental models have shown that oxidative stress and inflammation are key mediators of cell damage. In this regard, Nrf2 modulates cytoprotection against oxidative stress and inflammation, however, its role in inflammation induced by TiO2 NPs exposure has been less investigated. The aim of this work was to investigate the role of Nrf2 in the cytokines produced after 4 weeks of TiO2 NPs exposure (5 mg/kg/2 days/week) using wild-type and Nrf2 knockout C57bl6 mice. Results showed that Nrf2 protects against inflammation and oxidative damage induced by TiO2 NPs exposure, however, Nrf2 is a positive mediator in the expression of IFN-γ, TNF-α, and TGF-β in bronchial epithelium and alveolar space after 4 weeks of exposure. These results suggest that Nrf2 has a central role in up-regulation of cytokines released during inflammation induced by TiO2 NPs and those cytokines are needed to cope with histological alterations in lung tissue.

  4. AAV-Nrf2 Promotes Protection and Recovery in Animal Models of Oxidative Stress.

    PubMed

    Liang, Katharine J; Woodard, Kenton T; Weaver, Mark A; Gaylor, John Paul; Weiss, Ellen R; Samulski, R Jude

    2017-03-01

    NRF2 is a transcription factor that drives antioxidant gene expression in multiple organ systems. We hypothesized that Nrf2 overexpression could be therapeutically applied toward diseases in which redox homeostasis is disrupted. In this study, adeno-associated virus (AAV)-Nrf2 was tested in a mouse model of acute acetaminophen-induced liver toxicity and successfully conferred protection from hepatotoxicity, validating the vector design and early onset of NRF2-mediated protection. Furthermore, therapeutic potential of AAV-Nrf2 in chronic disease also was tested in a light-induced mouse model of age-related macular degeneration. Adult BALB/c mice were intravitreally injected with AAV-Nrf2 and subject to light damage following injection. Retinal thickness and function were monitored following light damage using optical coherence tomography and electroretinography, respectively. By 3 months post-damage, injected eyes had greater retinal thickness compared to uninjected controls. At 1 month post-damage, AAV-Nrf2 injection facilitated full functional recovery from light damage. Our results suggest a therapeutic potential for Nrf2 overexpression in acute and long-term capacities in multiple organ systems, opening up doors for combination gene therapy where replacement gene therapy requires additional therapeutic support to prevent further degeneration.

  5. Neuronal development is promoted by weakened intrinsic antioxidant defences due to epigenetic repression of Nrf2

    PubMed Central

    Bell, Karen F.S.; Al-Mubarak, Bashayer; Martel, Marc-André; McKay, Sean; Wheelan, Nicola; Hasel, Philip; Márkus, Nóra M.; Baxter, Paul; Deighton, Ruth F.; Serio, Andrea; Bilican, Bilada; Chowdhry, Sudhir; Meakin, Paul J.; Ashford, Michael L.J.; Wyllie, David J.A.; Scannevin, Robert H.; Chandran, Siddharthan; Hayes, John D.; Hardingham, Giles E.

    2015-01-01

    Forebrain neurons have weak intrinsic antioxidant defences compared with astrocytes, but the molecular basis and purpose of this is poorly understood. We show that early in mouse cortical neuronal development in vitro and in vivo, expression of the master-regulator of antioxidant genes, transcription factor NF-E2-related-factor-2 (Nrf2), is repressed by epigenetic inactivation of its promoter. Consequently, in contrast to astrocytes or young neurons, maturing neurons possess negligible Nrf2-dependent antioxidant defences, and exhibit no transcriptional responses to Nrf2 activators, or to ablation of Nrf2's inhibitor Keap1. Neuronal Nrf2 inactivation seems to be required for proper development: in maturing neurons, ectopic Nrf2 expression inhibits neurite outgrowth and aborization, and electrophysiological maturation, including synaptogenesis. These defects arise because Nrf2 activity buffers neuronal redox status, inhibiting maturation processes dependent on redox-sensitive JNK and Wnt pathways. Thus, developmental epigenetic Nrf2 repression weakens neuronal antioxidant defences but is necessary to create an environment that supports neuronal development. PMID:25967870

  6. Phytochemical activation of Nrf2 protects human coronary artery endothelial cells against an oxidative challenge.

    PubMed

    Donovan, Elise L; McCord, Joe M; Reuland, Danielle J; Miller, Benjamin F; Hamilton, Karyn L

    2012-01-01

    Activation of NF-E2-related factor 2 (Nrf2) is a potential therapeutic intervention against endothelial cell oxidative stress and associated vascular disease. We hypothesized that treatment with the phytochemicals in the patented dietary supplement Protandim would induce Nrf2 nuclear localization and phase II antioxidant enzyme protein in human coronary artery endothelial cells (HCAECs), protecting against an oxidant challenge in an Nrf2- dependent manner. Protandim treatment induced Nrf2 nuclear localization, and HO-1 (778% of control ± 82.25 P < 0.01), SOD1 (125.9% of control ± 6.05 P < 0.01), NQO1 (126% of control ± 6.5 P < 0.01), and GR (119.5% of control ± 7.00 P < 0.05) protein expression in HCAEC. Treatment of HCAEC with H(2)O(2) induced apoptosis in 34% of cells while pretreatment with Protandim resulted in only 6% apoptotic cells (P < 0.01). Nrf2 silencing significantly decreased the Protandim-induced increase in HO-1 protein (P < 0.01). Nrf2 silencing also significantly decreased the protection afforded by Protandim against H(2)O(2)- induced apoptosis (P < 0.01 compared to no RNA, and P < 0.05 compared to control RNA). These results show that Protandim induces Nrf2 nuclear localization and antioxidant enzyme expression, and protection of HCAEC from an oxidative challenge is Nrf2 dependent.

  7. Phytochemical Activation of Nrf2 Protects Human Coronary Artery Endothelial Cells against an Oxidative Challenge

    PubMed Central

    Donovan, Elise L.; McCord, Joe M.; Reuland, Danielle J.; Miller, Benjamin F.; Hamilton, Karyn L.

    2012-01-01

    Activation of NF-E2-related factor 2 (Nrf2) is a potential therapeutic intervention against endothelial cell oxidative stress and associated vascular disease. We hypothesized that treatment with the phytochemicals in the patented dietary supplement Protandim would induce Nrf2 nuclear localization and phase II antioxidant enzyme protein in human coronary artery endothelial cells (HCAECs), protecting against an oxidant challenge in an Nrf2- dependent manner. Protandim treatment induced Nrf2 nuclear localization, and HO-1 (778% of control ± 82.25 P < 0.01), SOD1 (125.9% of control ± 6.05 P < 0.01), NQO1 (126% of control ± 6.5 P < 0.01), and GR (119.5% of control ± 7.00 P < 0.05) protein expression in HCAEC. Treatment of HCAEC with H2O2 induced apoptosis in 34% of cells while pretreatment with Protandim resulted in only 6% apoptotic cells (P < 0.01). Nrf2 silencing significantly decreased the Protandim-induced increase in HO-1 protein (P < 0.01). Nrf2 silencing also significantly decreased the protection afforded by Protandim against H2O2- induced apoptosis (P < 0.01 compared to no RNA, and P < 0.05 compared to control RNA). These results show that Protandim induces Nrf2 nuclear localization and antioxidant enzyme expression, and protection of HCAEC from an oxidative challenge is Nrf2 dependent. PMID:22685617

  8. Genetic polymorphism in the NRF2 gene as a prognosis marker for cancer chemotherapy.

    PubMed

    Ishikawa, Toshihisa

    2014-01-01

    NF-E2-related factor 2 (NRF2) is a transcription factor that controls the expression of a variety of antioxidant and detoxification genes. Accumulating evidence strongly suggests that NRF2 mediates cancer cell proliferation and drug resistance, as well. Single nucleotide polymorphism (SNP) -617C > A in the anti-oxidant response element-like loci of the human NRF2 gene play a pivotal role in the positive feedback loop of transcriptional activation of the NRF2 gene. Since the SNP (-617A) reportedly decreases the binding affinity to the transcription factors of NRF2/small multiple alignment format (MafK), the homozygous -617A/A allele may attenuate the positive feedback loop of transcriptional activation of the NRF2 gene and reduce the NRF2 protein level. As the consequence, cancer cells are considered to become more sensitive to therapy and less aggressive than cancer cells harboring the -617C (WT) allele. Indeed, Japanese lung cancer patients carrying SNP homozygous alleles (c. -617A/A) exhibited remarkable survival over 1,700 days after surgical operation (log-rank p = 0.021). The genetic polymorphism in the human NRF2 gene is considered as one of prognosis markers for cancer therapy.

  9. Nrf2: A Novel Biomarker of Disease Severity and Target for Therapeutic Intervention in Multiple Sclerosis

    DTIC Science & Technology

    2014-10-01

    suppression of disease symptoms in EAE (Experimental Autoimmune Encephalomyelitis). In the current report we: 1) characterize the Nrf2-targeted antioxidant ...including their potential to suppress disease symptoms in EAE. In current report we first characterized the Nrf2 targeted antioxidant properties of...sclerosis, rheumatoid arthritis4 and skin inflammation.5 Further it has been reported that Celastrol can inhibit inflammatory reactions between

  10. Nonhematopoietic Nrf2 dominantly impedes adult progression of sickle cell anemia in mice

    PubMed Central

    Ghosh, Samit; Ihunnah, Chibueze A.; Hazra, Rimi; Walker, Aisha L.; Hansen, Jason M.; Archer, David R.; Owusu-Ansah, Amma T.; Ofori-Acquah, Solomon F.

    2016-01-01

    The prevention of organ damage and early death in young adults is a major clinical concern in sickle cell disease (SCD). However, mechanisms that control adult progression of SCD during the transition from adolescence are poorly defined with no cognate prophylaxis. Here, we demonstrate in a longitudinal cohort of homozygous SCD (SS) mice a link between intravascular hemolysis, vascular inflammation, lung injury, and early death. Prophylactic Nrf2 activation in young SS mice stabilized intravascular hemolysis, reversed vascular inflammation, and attenuated lung edema in adulthood. Enhanced Nrf2 activation in endothelial cells in vitro concurred with the dramatic effect on vascular inflammation in the mice. BM chimeric SS mice lacking Nrf2 expression in nonhematopoietic tissues were created to dissect the role of nonerythroid Nrf2 in SCD progression. The SS chimeras developed severe intravascular hemolysis despite having erythroid Nrf2. In addition, they developed premature vascular inflammation and pulmonary edema and died younger than donor littermates with intact nonhematopoietic Nrf2. Our results reveal a dominant protective role for nonhematopoietic Nrf2 against tissue damage in both erythroid and nonerythroid tissues in SCD. Furthermore, we show that prophylactic augmentation of Nrf2-coordinated cytoprotection effectively impedes onset of the severe adult phenotype of SCD in mice. PMID:27158670

  11. NRF2 Regulates HER2 and HER3 Signaling Pathway to Modulate Sensitivity to Targeted Immunotherapies

    PubMed Central

    Khalil, Hilal S.; Langdon, Simon P.; Kankia, Ibrahim H.; Bown, James; Deeni, Yusuf Y.

    2016-01-01

    NF-E2 related factor-2 (NRF2) is an essential transcription factor for multiple genes encoding antioxidants and detoxification enzymes. NRF2 is implicated in promoting cancer therapeutic resistance by its detoxification function and crosstalk with proproliferative pathways. However, the exact mechanism of this intricate connectivity between NRF2 and growth factor induced proliferative pathway remains elusive. Here, we have demonstrated that pharmacological activation of NRF2 by tert-butylhydroquinone (tBHQ) upregulates the HER family receptors, HER2 and HER3 expression, elevates pAKT levels, and enhances the proliferation of ovarian cancer cells. Preactivation of NRF2 also attenuates the combined growth inhibitory effects of HER2 targeting monoclonal antibodies, Pertuzumab and Trastuzumab. Further, tBHQ caused transcriptional induction of HER2 and HER3, while SiRNA-mediated knockdown of NRF2 prevented this and further caused transcriptional repression and enhanced cytotoxicity of the HER2 inhibitors. Hence, NRF2 regulates both HER2 and HER3 receptors to influence cellular responses to HER2 targeting monoclonal antibodies. This deciphered crosstalk mechanism reinforces the role of NRF2 in drug resistance and as a relevant anticancer target. PMID:26770651

  12. Bixin protects mice against ventilation-induced lung injury in an NRF2-dependent manner

    PubMed Central

    Tao, Shasha; Rojo de la Vega, Montserrat; Quijada, Hector; Wondrak, Georg T.; Wang, Ting; Garcia, Joe G. N.; Zhang, Donna D.

    2016-01-01

    Mechanical ventilation (MV) is a therapeutic intervention widely used in the clinic to assist patients that have difficulty breathing due to lung edema, trauma, or general anesthesia. However, MV causes ventilator-induced lung injury (VILI), a condition characterized by increased permeability of the alveolar-capillary barrier that results in edema, hemorrhage, and neutrophil infiltration, leading to exacerbated lung inflammation and oxidative stress. This study explored the feasibility of using bixin, a canonical NRF2 inducer identified during the current study, to ameliorate lung damage in a murine VILI model. In vitro, bixin was found to activate the NRF2 signaling pathway through blockage of ubiquitylation and degradation of NRF2 in a KEAP1-C151 dependent manner; intraperitoneal (IP) injection of bixin led to pulmonary upregulation of the NRF2 response in vivo. Remarkably, IP administration of bixin restored normal lung morphology and attenuated inflammatory response and oxidative DNA damage following MV. This observed beneficial effect of bixin derived from induction of the NRF2 cytoprotective response since it was only observed in Nrf2+/+ but not in Nrf2−/− mice. This is the first study providing proof-of-concept that NRF2 activators can be developed into pharmacological agents for clinical use to prevent patients from lung injury during MV treatment. PMID:26729554

  13. The Keap1-Nrf2 system prevents onset of diabetes mellitus.

    PubMed

    Uruno, Akira; Furusawa, Yuki; Yagishita, Yoko; Fukutomi, Toshiaki; Muramatsu, Hiroyuki; Negishi, Takaaki; Sugawara, Akira; Kensler, Thomas W; Yamamoto, Masayuki

    2013-08-01

    Transcription factor Nrf2 (NF-E2-related factor 2) regulates a broad cytoprotective response to environmental stresses. Keap1 (Kelch-like ECH-associated protein 1) is an adaptor protein for cullin3-based ubiquitin E3 ligase and negatively regulates Nrf2. Whereas the Keap1-Nrf2 system plays important roles in oxidative stress response and metabolism, the roles Nrf2 plays in the prevention of diabetes mellitus remain elusive. Here we show that genetic activation of Nrf2 signaling by Keap1 gene hypomorphic knockdown (Keap1flox/-) markedly suppresses the onset of diabetes. When Keap1flox/- mice were crossed with diabetic db/db mice, blood glucose levels became lower through improvement of both insulin secretion and insulin resistance. Keap1flox/- also prevented high-calorie-diet-induced diabetes. Oral administration of the Nrf2 inducer CDDO-Im {oleanolic acid 1-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl] imidazole} also attenuated diabetes in db/db mice. Nrf2 induction altered antioxidant-, energy consumption-, and gluconeogenesis-related gene expression in metabolic tissues. Thus, the Keap1-Nrf2 system is a critical target for preventing the onset of diabetes mellitus.

  14. NRF2 Regulates HER2 and HER3 Signaling Pathway to Modulate Sensitivity to Targeted Immunotherapies.

    PubMed

    Khalil, Hilal S; Langdon, Simon P; Kankia, Ibrahim H; Bown, James; Deeni, Yusuf Y

    2016-01-01

    NF-E2 related factor-2 (NRF2) is an essential transcription factor for multiple genes encoding antioxidants and detoxification enzymes. NRF2 is implicated in promoting cancer therapeutic resistance by its detoxification function and crosstalk with proproliferative pathways. However, the exact mechanism of this intricate connectivity between NRF2 and growth factor induced proliferative pathway remains elusive. Here, we have demonstrated that pharmacological activation of NRF2 by tert-butylhydroquinone (tBHQ) upregulates the HER family receptors, HER2 and HER3 expression, elevates pAKT levels, and enhances the proliferation of ovarian cancer cells. Preactivation of NRF2 also attenuates the combined growth inhibitory effects of HER2 targeting monoclonal antibodies, Pertuzumab and Trastuzumab. Further, tBHQ caused transcriptional induction of HER2 and HER3, while SiRNA-mediated knockdown of NRF2 prevented this and further caused transcriptional repression and enhanced cytotoxicity of the HER2 inhibitors. Hence, NRF2 regulates both HER2 and HER3 receptors to influence cellular responses to HER2 targeting monoclonal antibodies. This deciphered crosstalk mechanism reinforces the role of NRF2 in drug resistance and as a relevant anticancer target.

  15. Amelioration of inflammation and tissue damage in sickle cell model mice by Nrf2 activation.

    PubMed

    Keleku-Lukwete, Nadine; Suzuki, Mikiko; Otsuki, Akihito; Tsuchida, Kouhei; Katayama, Saori; Hayashi, Makiko; Naganuma, Eriko; Moriguchi, Takashi; Tanabe, Osamu; Engel, James Douglas; Imaizumi, Masue; Yamamoto, Masayuki

    2015-09-29

    Sickle cell disease (SCD) is an inherited disorder caused by a point mutation in the β-globin gene, leading to the production of abnormally shaped red blood cells. Sickle cells are prone to hemolysis and thereby release free heme into plasma, causing oxidative stress and inflammation that in turn result in damage to multiple organs. The transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2) is a master regulator of the antioxidant cell-defense system. Here we show that constitutive Nrf2 activation by ablation of its negative regulator Keap1 (kelch-like ECH-associated protein 1) significantly improves symptoms in SCD model mice. SCD mice exhibit severe liver damage and lung inflammation associated with high expression levels of proinflammatory cytokines and adhesion molecules compared with normal mice. Importantly, these symptoms subsided after Nrf2 activation. Although hemolysis and stress erythropoiesis did not change substantially in the Nrf2-activated SCD mice, Nrf2 promoted the elimination of plasma heme released by sickle cells' hemolysis and thereby reduced oxidative stress and inflammation, demonstrating that Nrf2 activation reduces organ damage and segregates inflammation from prevention of hemolysis in SCD mice. Furthermore, administration of the Nrf2 inducer CDDO-Im (2-cyano-3, 12 dioxooleana-1, 9 diene-28-imidazolide) also relieved inflammation and organ failure in SCD mice. These results support the contention that Nrf2 induction may be an important means to protect organs from the pathophysiology of sickle cell-induced damage.

  16. The influence of Nrf2 on cardiac responses to environmental stressors

    EPA Science Inventory

    Nrf2 protects the lung from adverse responses to oxidants, including 100% oxygen (hyperoxia) and airborne pollutants like particulate matter (PM) exposure, but the role of Nrf2 on heart rate (HR) and heart rate variability (HRV) responses is not known. We hypothesized that genet...

  17. Nrf2: a potential therapeutic target for naturally occurring anticancer drugs?

    PubMed

    Catanzaro, Elena; Calcabrini, Cinzia; Turrini, Eleonora; Sestili, Piero; Fimognari, Carmela

    2017-08-01

    Nuclear factor (erythroid-derived-2)-like 2 is one of the most efficient cytoprotective rheostats against exogenous or endogenous oxidative insults. At present, the modulation of the Nrf2 pathway represents an interesting and highly explored strategy in the oncological area. Area covered: In this review, we present and discuss the different modulation of the Nrf2 pathway by some natural compounds with a well demonstrated anticancer activity, and critically analyze the challenges associated with the development of an Nrf2-based anticancer strategy. Expert opinion: Many natural compounds with a well-defined anticancer activity are able to modulate this pathway. Both Nrf2 inducers and inhibitors can be useful as anticancer strategy. However, since Nrf2 modulates many networks potentially involved in the detoxification process of anticancer drugs, its activation in cancer cells could lead to chemoresistance. The switch between a beneficial or detrimental role of Nrf2 in cancer cells essentially depends on the tight control of its activity, the specific conditions of tumor microenvironment, and cell type. In line with the paucity of clear data related to the mechanisms underpinning the role of Nrf2 in cancer development and chemoresistance, discovery and development of Nrf2-based strategies is one of the most critical and challenging assignments for fighting cancers.

  18. Nrf2 protects against As(III)-induced damage in mouse liver and bladder

    SciTech Connect

    Jiang Tao; Huang Zheping; Chan, Jefferson Y.; Zhang, Donna D.

    2009-10-01

    Arsenic compounds are classified as toxicants and human carcinogens. Environmental exposure to arsenic imposes a big health issue worldwide. Arsenic elicits its toxic efforts through many mechanisms, including generation of reactive oxygen species (ROS). Nrf2 is the primary transcription factor that controls expression of a main cellular antioxidant response, which is required for neutralizing ROS and thus defending cells from exogenous insults. Previously, we demonstrated a protective role of Nrf2 against arsenic-induced toxicity using a cell culture model. In this report, we present evidence that Nrf2 protects against liver and bladder injury in response to six weeks of arsenic exposure in a mouse model. Nrf2{sup -/-} mice displayed more severe pathological changes in the liver and bladder, compared to Nrf2{sup +/+} mice. Furthermore, Nrf2{sup -/-} mice were more sensitive to arsenic-induced DNA hypomethylation, oxidative DNA damage, and apoptotic cell death. These results indicate a protective role of Nrf2 against arsenic toxicity in vivo. Hence, this work demonstrates the feasibility of using dietary compounds that target activation of the Nrf2 signaling pathway to alleviate arsenic-induced damage.

  19. Overexpression of Nrf2 Protects Cerebral Cortical Neurons from Ethanol-Induced Apoptotic DeathS⃞

    PubMed Central

    Narasimhan, Madhusudhanan; Mahimainathan, Lenin; Rathinam, Mary Latha; Riar, Amanjot Kaur

    2011-01-01

    Ethanol (ETOH) can cause apoptotic death of neurons by depleting GSH with an associated increase in oxidative stress. The current study illustrates a means to overcome this ETOH-induced neurotoxicity by enhancing GSH through boosting Nrf2, a transcription factor that controls GSH homeostasis. ETOH treatment caused a significant increase in Nrf2 protein, transcript expression, Nrf2-DNA binding activity, and expression of its transcriptional target, NQO1, in primary cortical neuron (PCNs). However, this increase in Nrf2 did not maintain GSH levels in response to ETOH, and apoptotic death still occurred. To elucidate this phenomenon, we silenced Nrf2 in neurons and found that ETOH-induced GSH depletion and the increase in superoxide levels were exacerbated. Furthermore, Nrf2 knockdown resulted in significantly increased (P < 0.05) caspase 3 activity and apoptosis. Adenovirus-mediated overexpression of Nrf2 prevented ETOH-induced depletion of GSH from the medium and high GSH subpopulations and prevented ETOH-related apoptotic death. These studies illustrate the importance of Nrf2-dependent maintenance of GSH homeostasis in cerebral cortical neurons in the defense against oxidative stress and apoptotic death elicited by ETOH exposure. PMID:21873460

  20. The influence of Nrf2 on cardiac responses to environmental stressors

    EPA Science Inventory

    Nrf2 protects the lung from adverse responses to oxidants, including 100% oxygen (hyperoxia) and airborne pollutants like particulate matter (PM) exposure, but the role of Nrf2 on heart rate (HR) and heart rate variability (HRV) responses is not known. We hypothesized that genet...

  1. Amelioration of inflammation and tissue damage in sickle cell model mice by Nrf2 activation

    PubMed Central

    Keleku-Lukwete, Nadine; Suzuki, Mikiko; Otsuki, Akihito; Tsuchida, Kouhei; Katayama, Saori; Hayashi, Makiko; Naganuma, Eriko; Moriguchi, Takashi; Tanabe, Osamu; Engel, James Douglas; Imaizumi, Masue; Yamamoto, Masayuki

    2015-01-01

    Sickle cell disease (SCD) is an inherited disorder caused by a point mutation in the β-globin gene, leading to the production of abnormally shaped red blood cells. Sickle cells are prone to hemolysis and thereby release free heme into plasma, causing oxidative stress and inflammation that in turn result in damage to multiple organs. The transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2) is a master regulator of the antioxidant cell-defense system. Here we show that constitutive Nrf2 activation by ablation of its negative regulator Keap1 (kelch-like ECH-associated protein 1) significantly improves symptoms in SCD model mice. SCD mice exhibit severe liver damage and lung inflammation associated with high expression levels of proinflammatory cytokines and adhesion molecules compared with normal mice. Importantly, these symptoms subsided after Nrf2 activation. Although hemolysis and stress erythropoiesis did not change substantially in the Nrf2-activated SCD mice, Nrf2 promoted the elimination of plasma heme released by sickle cells’ hemolysis and thereby reduced oxidative stress and inflammation, demonstrating that Nrf2 activation reduces organ damage and segregates inflammation from prevention of hemolysis in SCD mice. Furthermore, administration of the Nrf2 inducer CDDO-Im (2-cyano-3, 12 dioxooleana-1, 9 diene-28-imidazolide) also relieved inflammation and organ failure in SCD mice. These results support the contention that Nrf2 induction may be an important means to protect organs from the pathophysiology of sickle cell-induced damage. PMID:26371321

  2. Genetic silencing of Nrf2 enhances X-ROS in dysferlin-deficient muscle

    PubMed Central

    Kombairaju, Ponvijay; Kerr, Jaclyn P.; Roche, Joseph A.; Pratt, Stephen J. P.; Lovering, Richard M.; Sussan, Thomas E.; Kim, Jung-Hyun; Shi, Guoli; Biswal, Shyam; Ward, Christopher W.

    2014-01-01

    Oxidative stress is a critical disease modifier in the muscular dystrophies. Recently, we discovered a pathway by which mechanical stretch activates NADPH Oxidase 2 (Nox2) dependent ROS generation (X-ROS). Our work in dystrophic skeletal muscle revealed that X-ROS is excessive in dystrophin-deficient (mdx) skeletal muscle and contributes to muscle injury susceptibility, a hallmark of the dystrophic process. We also observed widespread alterations in the expression of genes associated with the X-ROS pathway and redox homeostasis in muscles from both Duchenne muscular dystrophy patients and mdx mice. As nuclear factor erythroid 2-related factor 2 (Nrf2) plays an essential role in the transcriptional regulation of genes involved in redox homeostasis, we hypothesized that Nrf2 deficiency may contribute to enhanced X-ROS signaling by reducing redox buffering. To directly test the effect of diminished Nrf2 activity, Nrf2 was genetically silenced in the A/J model of dysferlinopathy—a model with a mild histopathologic and functional phenotype. Nrf2-deficient A/J mice exhibited significant muscle-specific functional deficits, histopathologic abnormalities, and dramatically enhanced X-ROS compared to control A/J and WT mice, both with functional Nrf2. Having identified that reduced Nrf2 activity is a negative disease modifier, we propose that strategies targeting Nrf2 activation may address the generalized reduction in redox homeostasis to halt or slow dystrophic progression. PMID:24600403

  3. Lonchocarpine Increases Nrf2/ARE-Mediated Antioxidant Enzyme Expression by Modulating AMPK and MAPK Signaling in Brain Astrocytes

    PubMed Central

    Jeong, Yeon-Hui; Park, Jin-Sun; Kim, Dong-Hyun; Kim, Hee-Sun

    2016-01-01

    Lonchocarpine is a phenylpropanoid compound isolated from Abrus precatorius that has anti-bacterial, anti-inflammatory, antiproliferative, and antiepileptic activities. In the present study, we investigated the antioxidant effects of lonchocarpine in brain glial cells and analyzed its molecular mechanisms. We found that lonchocarpine suppressed reactive oxygen species (ROS) production and cell death in hydrogen peroxide-treated primary astrocytes. In addition, lonchocarpine increased the expression of antioxidant enzymes, such as heme oxygenase-1 (HO-1), NAD(P)H:quinone oxidoreductase 1 (NQO1), and manganese superoxide dismutase (MnSOD), which are all under the control of Nrf2/antioxidant response element (ARE) signaling. Further, mechanistic studies showed that lonchocarpine increases the nuclear translocation and DNA binding of Nrf2 to ARE as well as ARE-mediated transcriptional activities. Moreover, lonchocarpine increased the phosphorylation of AMP-activated protein kinase (AMPK) and three types of mitogen-activated protein kinases (MAPKs). By treating astrocytes with each signaling pathway-specific inhibitor, AMPK, c-jun N-terminal protein kinase (JNK), and p38 MAPK were identified to be involved in lonchocarpine-induced HO-1 expression and ARE-mediated transcriptional activities. Therefore, lonchocarpine may be a potential therapeutic agent for neurodegenerative diseases that are associated with oxidative stress. PMID:27737527

  4. Curcumin attenuates dimethylnitrosamine-induced liver injury in rats through Nrf2-mediated induction of heme oxygenase-1.

    PubMed

    Farombi, E Olatunde; Shrotriya, Sangeeta; Na, Hye-Kyung; Kim, Sung-Hoon; Surh, Young-Joon

    2008-04-01

    Curcumin (diferuloymethane), a yellow colouring agent present in the rhizome of Curcuma longa Linn (Zingiberaceae), has been reported to possess anti-inflammatory, antioxidant, antimutagenic and anticarcinogenic activities. Curcumin exerts its chemoprotective and chemopreventive effects via multiple mechanisms. It has been reported to induce expression of the antioxidant enzymes in various cell lines. Heme oxygenase-1 (HO-1) is an important antioxidant enzyme that plays a pivotal role in cytoprotection against noxious stimuli of both endogenous and exogenous origin. In the present study, we found that oral administration of curcumin at 200mg/kg dose for four consecutive days not only protected against dimethylnitrosamine (DMN)-induced hepatic injury, but also resulted in more than three-fold induction of HO-1 protein expression as well as activity in rat liver. Inhibition of HO-1 activity by zinc protoporphyrin-IX abrogated the hepatoprotective effect of curcumin against DMN toxicity. NF-E2-related factor 2 (Nrf2) plays a role in the cellular protection against oxidative stress through antioxidant response element (ARE)-directed induction of several phase-2 detoxifying and antioxidant enzymes including HO-1. Curcumin administration resulted in enhanced nuclear translocation and ARE-binding of Nrf2. Taken together, these findings suggest that curcumin protects against DMN-induced hepatotoxicity, at least in part, through ARE-driven induction of HO-1 expression.

  5. Endothelial HO-1 induction by model TG-rich lipoproteins is regulated through a NOX4-Nrf2 pathway.

    PubMed

    Latham Birt, Sally H; Purcell, Robert; Botham, Kathleen M; Wheeler-Jones, Caroline P D

    2016-07-01

    Circulating levels of chylomicron remnants (CMRs) increase postprandially and their composition directly reflects dietary lipid intake. These TG-rich lipoproteins likely contribute to the development of endothelial dysfunction, albeit via unknown mechanisms. Here, we investigated how the FA composition of CMRs influences their actions on human aortic endothelial cells (HAECs) by comparing the effects of model CMRs-artificial TG-rich CMR-like particles (A-CRLPs)-containing TGs extracted from fish, DHA-rich algal, corn, or palm oils. HAECs responded with distinct transcriptional programs according to A-CRLP TG content and oxidation status, with genes involved in antioxidant defense and cytoprotection most prominently affected by n-3 PUFA-containing A-CRLPs. These particles were significantly more efficacious inducers of heme oxygenase-1 (HO-1) than n-6 PUFA corn or saturated FA-rich palm CRLPs. Mechanistically, HO-1 induction by all CRLPs requires NADPH oxidase 4, with PUFA-containing particles additionally dependent upon mitochondrial reactive oxygen species. Activation of both p38 MAPK and PPARβ/δ culminates in increased nuclear factor erythroid 2-related factor 2 (Nrf2) expression/nuclear translocation and HO-1 induction. These studies define new molecular pathways coupling endothelial cell activation by model CMRs with adaptive regulation of Nrf2-dependent HO-1 expression and may represent key mechanisms through which dietary FAs differentially impact progression of endothelial dysfunction. Copyright © 2016 by the American Society for Biochemistry and Molecular Biology, Inc.

  6. Nrf2-mediated mucoprotective and anti-inflammatory actions of Artemisia extracts led to attenuate stress related mucosal damages

    PubMed Central

    Park, Jong-Min; Han, Young-Min; Lee, Jin-Seok; Ko, Kwang Hyun; Hong, Sung-Pyo; Kim, Eun-Hee; Hahm, Ki-Baik

    2015-01-01

    The aim of this study was to compare biological actions between isopropanol and ethanol extracts of Artemisia including antioxidant, anti-inflammatory, and cytoprotective actions. Antioxidant activities were evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH) method and confocal microscopy on lipopolysaccharide-induced RGM1 cells, cytoprotection effects evaluated by detecting heme oxygenase-1 (HO-1), Nf-E2 related factor2 (Nrf2) and heat shock protein 70 (HSP70), and anti-inflammatory effects investigated by measuring inflammatory mediators. Water immersion restraint stress was imposed to provoke stress related mucosal damages (SRMD) in rats. Isopropanol extracts of Artemisia showed the higher DPPH radical scavenging activity and lesser LPS-induced reactive oxygen species productions and increased HO-1 expression through increased nuclear translocation of Nrf2 transcription factor compared to ethanol extracts. The increased expression of HSP70 and decreased expression of endothelin-1 were only increased with isopropanol extracts. A concentration-dependent inhibition of LPS-induced COX-2 and iNOS even at a rather lower concentration than ethanol extract was achieved with isopropanol extracts. Cytokine protein array revealed Artemisia extracts significantly attenuated the levels of CXCL-1, CXCL-16, and MCP-1. These orchestrated actions led to significant rescue from SRMD. Conclusively, Artemisia extracts imposed significant antioxidant and anti-inflammatory activity against SRMD and isopropanol extracts were superior to ethanol extracts in these beneficiary actions of Artemisia. PMID:25759519

  7. Ankaflavin: a natural novel PPARγ agonist upregulates Nrf2 to attenuate methylglyoxal-induced diabetes in vivo.

    PubMed

    Lee, Bao-Hong; Hsu, Wei-Hsuan; Chang, Yu-Ying; Kuo, Hsuan-Fu; Hsu, Ya-Wen; Pan, Tzu-Ming

    2012-12-01

    Ankaflavin (AK) is an active compound having anti-inflammatory, anti-cancer, antiatherosclerotic, and hypolipidemic effects. We have previously reported that AK acts as an antioxidant and antidiabetic drug; however, the mechanism by which AK prevents diabetes remains unknown. Hyperglycemia is associated with protein glycation, which produces advanced glycation end-products (AGEs). Methylglyoxal (MG)-a metabolite of carbohydrates-is believed to cause insulin resistance by inducing inflammation and pancreas damage. In this work, diabetes was induced in Wistar rats (4 weeks of age) by treating them with MG (600 mg/kg bw) for 4 weeks. We observed that AK (10mg/kg bw) exerted peroxisome proliferator-activated receptor-γ (PPARγ) agonist activity, thereby enhancing insulin sensitivity (as indicated by hepatic GLUT2 translocation, PTP1B suppression, and glucose uptake) by downregulating blood glucose and upregulating pancreatic and duodenal homeobox-1 and Maf-A expression and increasing insulin production in MG-induced rats. However, these effects were abolished by the administration of GW9662 (PPARγ antagonist), but the expression of hepatic heme oxygenase-1 (HO-1) and glutamate-cysteine ligase (GCL) was not suppressed in MG-induced rats. Therefore, the nuclear factor erythroid-related factor-2 (Nrf2) activation was investigated. AK did not affect hepatic Nrf2 mRNA or protein expression but significantly increased Nrf2 phosphorylation (serine 40), which was accompanied by increased transcriptional activation of hepatic HO-1 and GCL. These data indicated that AK protected rats from oxidative stress resulting from MG-induced insulin resistance. In contrast, these effects were not detected when the rats were treated with the antidiabetic drug rosiglitazone (10mg/kg bw). Moreover, we found that AK did not inhibit the generation of AGEs in vitro; however, the glutathione (GSH) levels in liver and pancreas of MG-induced rats were elevated in rats administered AK. Therefore

  8. Nrf2 inhibition sensitizes cholangiocarcinoma cells to cytotoxic and antiproliferative activities of chemotherapeutic agents.

    PubMed

    Samatiwat, Papavee; Prawan, Auemduan; Senggunprai, Laddawan; Kukongviriyapan, Upa; Kukongviriyapan, Veerapol

    2016-08-01

    Nuclear factor erythroid 2-related factor 2 (Nrf2), a key transcription factor regulating antioxidant, cytoprotective, and metabolic enzymes, plays important roles in drug resistance and proliferation in cancer cells. The present study was aimed to examine the expression of Nrf2 in connection with chemotherapeutic drug sensitivity on cholangiocarcinoma (CCA) cells. The basal levels of Nrf2 protein in cytosol and nuclear fractions of CCA cells were determined using Western blot analysis. Nrf2 mRNA expression of KKU-M156 and KKU-100 cells, representatives of low and high-Nrf2-expressing CCA cells, were silenced using siRNA. After knockdown of Nrf2, the sensitivity of those cells to the cytotoxicity of cisplatin (Cis) was enhanced in association with the increased release of AIF and downregulation of Bcl-xl in both cells. Also, knockdown of Nrf2 suppressed the replicative capability of those cells in colony-forming assay and enhanced their sensitivity to antiproliferative activity of Cis and 5-fluorouracil. The chemosensitizing effect was associated with the suppressed expression of Nrf2-regulated and Cis-induced antioxidant and metabolic genes including NQO1, HO-1, GCLC, TXN, MRP2, TKT, and G6PD. In cell cycle analysis, Nrf2 knockdown cells were arrested at G0/G1 phase and combination with Cis increased the accumulation of cells at S phase. The suppression of KKU-M156 cell proliferation was associated with the downregulation of cyclin D1 and increased level of p21. Inhibition of Nrf2 could be a novel strategy in enhancing antitumor activity of chemotherapeutic agent in control of resistant cancer.

  9. Nrf2 promotes reparative angiogenesis through regulation of NADPH oxidase-2 in oxygen-induced retinopathy.

    PubMed

    Wei, Yanhong; Gong, Junsong; Xu, Zhenhua; Duh, Elia J

    2016-10-01

    Revascularization of ischemic tissue is a highly desirable outcome in multiple diseases, including cardiovascular diseases and ischemic retinopathies. Oxidative stress and inflammation are both known to play a role in suppressing reparative angiogenesis in ischemic disease models including oxygen-induced retinopathy (OIR), but the regulatory molecules governing these pathophysiologic processes in retinal ischemia are largely unknown. Nrf2 is a major stress-response transcription factor that has been implicated in regulating ischemic angiogenesis in the retina and other tissue beds. Using Nrf2-deficient mice, we investigated the effects of Nrf2 in regulating revascularization and modulating the retinal tissue milieu during ischemia. Strikingly, Nrf2's beneficial effect on reparative angiogenesis only became manifested in the later phase of ischemia in OIR, from postnatal day 14 (P14) to P17. This was temporally associated with a reduction in both oxidative stress and inflammatory mediators in wild-type compared to Nrf2(-/-) mice. Nrf2(-/-) retinas exhibited an increase in VEGF but also induction of anti-angiogenic Dll4/Notch signaling. NADPH oxidase (NOX), and especially NOX2, is a major pathogenic molecule and a particularly important contributor to oxidative stress in multiple retinal disease processes. Nrf2(-/-) mice exhibited a significant exacerbation of NOX2 induction in OIR that manifested in the later phases of ischemia. Pharmacologic inhibition of NADPH oxidase abrogated the adverse effect of Nrf2 deficiency on reparative angiogenesis. Taken together, this suggests that Nrf2 is an important regulator of the retinal milieu during tissue ischemia, and that the Nrf2/NOX2 balance may play a critical role in determining the fate of ischemic revascularization.

  10. Withaferin A induces Nrf2-dependent protection against liver injury: Role of Keap1-independent mechanisms.

    PubMed

    Palliyaguru, Dushani L; Chartoumpekis, Dionysios V; Wakabayashi, Nobunao; Skoko, John J; Yagishita, Yoko; Singh, Shivendra V; Kensler, Thomas W

    2016-12-01

    Small molecules of plant origin offer presumptively safe opportunities to prevent carcinogenesis, mutagenesis and other forms of toxicity in humans. However, the mechanisms of action of such plant-based agents remain largely unknown. In recent years the stress responsive transcription factor Nrf2 has been validated as a target for disease chemoprevention. Withania somnifera (WS) is a herb used in Ayurveda (an ancient form of medicine in South Asia). In the recent past, withanolides isolated from WS, such as Withaferin A (WA) have been demonstrated to be preventive and therapeutic against multiple diseases in experimental models. The goals of this study are to evaluate withanolides such as WA as well as Withania somnifera root extract as inducers of Nrf2 signaling, to probe the underlying signaling mechanism of WA and to determine whether prevention of acetaminophen (APAP)-induced hepatic toxicity in mice by WA occurs in an Nrf2-dependent manner. We observed that WA profoundly protects wild-type mice but not Nrf2-disrupted mice against APAP hepatotoxicity. WA is a potent inducer of Nrf2-dependent cytoprotective enzyme expression both in vivo and in vitro. Unexpectedly, WA induces Nrf2 signaling at least in part, in a Keap1-independent, Pten/Pi3k/Akt-dependent manner in comparison to prototypical Nrf2 inducers, sulforaphane and CDDO-Im. The identification of WA as an Nrf2 inducer that can signal through a non-canonical, Keap1-independent pathway provides an opportunity to evaluate the role of other regulatory partners of Nrf2 in the dietary and pharmacological induction of Nrf2-mediated cytoprotection.

  11. Oxidative stress, mammospheres and Nrf2-new implication for breast cancer therapy?

    PubMed

    Wu, Tongde; Harder, Bryan G; Wong, Pak K; Lang, Julie E; Zhang, Donna D

    2015-11-01

    Mammosphere culture of breast cancer cell lines is an important approach used for enrichment of cancer stem cells (CSCs), which exhibit high tumorigenicity and chemoresistance features. Evidence shows that CSCs maintain lower ROS levels due to elevated expression of ROS-scavenging molecules and antioxidative enzymes, which favors the survival of the CSCs and their chemoresistance. The transcription factor NF-E2-related factor 2 (Nrf2) has emerged as the master regulator of cellular redox homeostasis, by up-regulating antioxidant response element (ARE)-bearing genes products. Although Nrf2 has long-term been regarded as a beneficial defense mechanism, accumulating studies have revealed the "dark side" of Nrf2. High constitutive levels of Nrf2 was observed in many types of tumors and cancer cell lines promoting their resistance to chemotherapeutics. In this study, we report a high expression of Nrf2 and its target genes in mammospheres compared to corresponding adherent cells. In MCF-7 and MDA-MB-231 mammmosphere cells, the Nrf2-mediated cellular protective response is significantly elevated which is associated with increased resistance to taxol and anchorage-independent growth. Brusatol, an inhibitor of the Nrf2 pathway, suppressed the protein level of Nrf2 and its target genes, enhanced intracellular ROS and sensitized mammospheres to taxol, and reduced the anchorage-independent growth. These results suggest that mammospheres rely on abnormal up-regulation of Nrf2 to maintain low intracellular ROS levels. Nrf2 inhibitors, such as brusatol, have the potential to be developed into novel adjuvant chemotherapeutic drug combinations in order to combat refractory tumor initiating CSCs.

  12. The rise of antioxidant signaling-The evolution and hormetic actions of Nrf2

    SciTech Connect

    Maher, Jonathan; Yamamoto, Masayuki

    2010-04-01

    Organisms have evolved sophisticated and redundant mechanisms to manage oxidative and electrophilic challenges that arise from internal metabolism or xenobiotic challenge for survival. NF-E2-related factor 2 (Nrf2) is a transcription factor that has evolved over millennia from primitive origins, with homologues traceable back to invertebrate Caenorhabditis and Drosophila species. The ancestry of Nrf2 clearly has deep-seated roots in hematopoiesis, yet has diversified into a transcription factor that can mediate a multitude of antioxidant signaling and detoxification genes. In higher organisms, a more sophisticated means of tightly regulating Nrf2 activity was introduced via the cysteine-rich kelch-like ECH-associated protein 1 (Keap1), thus suggesting a need to modulate Nrf2 activity. This is evidenced in Keap1{sup -/-} mice, which succumb to juvenile mortality due to hyperkeratosis of the gastrointestinal tract. Although Nrf2 activation protects against acute toxicity and prevents or attenuates several disease states, constitutive activation in some tumors leads to poor clinical outcomes, suggesting Nrf2 has evolved in response to a multitude of selective pressures. The purpose of this review is to examine the origins of Nrf2, while highlighting the versatility and protective abilities elicited upon activation. Various model systems in which Nrf2 is normally beneficial but in which exaggerated pharmacology exacerbates a physiological or pathological condition will be addressed. Although Darwinian principles have selected Nrf2 activity for maximal beneficial effect based on environmental and oxidative challenge, both sub- or super-physiological effects have been noted to be detrimental. The functions of Nrf2 thus suggest a hormetic factor that has evolved empirically over time.

  13. Metallothionein Is Downstream of Nrf2 and Partially Mediates Sulforaphane Prevention of Diabetic Cardiomyopathy.

    PubMed

    Gu, Junlian; Cheng, Yanli; Wu, Hao; Kong, Lili; Wang, Shudong; Xu, Zheng; Zhang, Zhiguo; Tan, Yi; Keller, Bradley B; Zhou, Honglan; Wang, Yuehui; Xu, Zhonggao; Cai, Lu

    2017-02-01

    We have reported that sulforaphane (SFN) prevented diabetic cardiomyopathy in both type 1 and type 2 diabetes (T2DM) animal models via the upregulation of nuclear transcription factor erythroid 2-related factor 2 (Nrf2) and metallothionein (MT). In this study, we tested whether SFN protects the heart from T2DM directly through Nrf2, MT, or both. Using Nrf2-knockout (KO), MT-KO, and wild-type (WT) mice, T2DM was induced by feeding a high-fat diet for 3 months followed by a small dose of streptozotocin. Age-matched controls were given a normal diet. Both T2DM and control mice were then treated with or without SFN for 4 months by continually feeding a high-fat or normal diet. SFN prevented diabetes-induced cardiac dysfunction as well as diabetes-associated cardiac oxidative damage, inflammation, fibrosis, and hypertrophy, with increases in Nrf2 and MT expressions in the WT mice. Both Nrf2-KO and MT-KO diabetic mice exhibited greater cardiac damage than WT diabetic mice. SFN did not provide cardiac protection in Nrf2-KO mice, but partially or completely protected the heart from diabetes in MT-KO mice. SFN did not induce MT expression in Nrf2-KO mice, but stimulated Nrf2 function in MT-KO mice. These results suggest that Nrf2 plays the indispensable role for SFN cardiac protection from T2DM with significant induction of MT and other antioxidants. MT expression induced by SFN is Nrf2 dependent, but is not indispensable for SFN-induced cardiac protection from T2DM.

  14. Brusatol enhances the efficacy of chemotherapy by inhibiting the Nrf2-mediated defense mechanism.

    PubMed

    Ren, Dongmei; Villeneuve, Nicole F; Jiang, Tao; Wu, Tongde; Lau, Alexandria; Toppin, Henry A; Zhang, Donna D

    2011-01-25

    The major obstacle in cancer treatment is the resistance of cancer cells to therapies. Nrf2 is a transcription factor that regulates a cellular defense response and is ubiquitously expressed at low basal levels in normal tissues due to Keap1-dependent ubiquitination and proteasomal degradation. Recently, Nrf2 has emerged as an important contributor to chemoresistance. High constitutive expression of Nrf2 was found in many types of cancers, creating an environment conducive for cancer cell survival. Here, we report the identification of brusatol as a unique inhibitor of the Nrf2 pathway that sensitizes a broad spectrum of cancer cells and A549 xenografts to cisplatin and other chemotherapeutic drugs. Mechanistically, brusatol selectively reduces the protein level of Nrf2 through enhanced ubiquitination and degradation of Nrf2. Consequently, expression of Nrf2-downstream genes is reduced and the Nrf2-dependent protective response is suppressed. In A549 xenografts, brusatol and cisplatin cotreatment induced apoptosis, reduced cell proliferation, and inhibited tumor growth more substantially when compared with cisplatin treatment alone. Additionally, A549-K xenografts, in which Nrf2 is expressed at very low levels due to ectopic expression of Keap1, do not respond to brusatol treatment, demonstrating that brusatol-mediated sensitization to cisplatin is Nrf2 dependent. Moreover, a decrease in drug detoxification and impairment in drug removal may be the primary mechanisms by which brusatol enhances the efficacy of chemotherapeutic drugs. Taken together, these results clearly demonstrate the effectiveness of using brusatol to combat chemoresistance and suggest that brusatol can be developed into an adjuvant chemotherapeutic drug.

  15. Astrocytes Prevent Ethanol Induced Apoptosis of Nrf2 Depleted Neurons by Maintaining GSH Homeostasis

    PubMed Central

    Narasimhan, Madhusudhanan; Rathinam, Marylatha; Patel, Dhyanesh; Henderson, George; Mahimainathan, Lenin

    2013-01-01

    Glutathione (GSH), a major cellular antioxidant protects cells against oxidative stress injury. Nuclear factor erythroid 2-related factor 2 (NFE2L2/Nrf2) is a redox sensitive master regulator of battery of antioxidant enzymes including those involved in GSH antioxidant machinery. Earlier we reported that ethanol (ETOH) elicits apoptotic death of primary cortical neurons (PCNs) which in partly due to depletion of intracellular GSH levels. Further a recent report from our laboratory illustrated that ETOH exacerbated the dysregulation of GSH and caspase mediated cell death of cortical neurons that are compromised in Nrf2 machinery (Narasimhan et al., 2011). In various experimental models of neurodegeneration, neuronal antioxidant defenses mainly GSH has been shown to be supported by astrocytes. We therefore sought to determine whether astrocytes can render protection to neurons against ETOH toxicity, particularly when the function of Nrf2 is compromised in neurons. The experimental model consisted of co-culturing primary cortical astrocytes (PCA) with Nrf2 downregulated PCNs that were exposed with 4 mg/mL ETOH for 24 h. Monochlorobimane (MCB) staining followed by FACS analysis showed that astrocytes blocked ETOH induced GSH decrement in Nrf2-silenced neurons as opposed to exaggerated GSH depletion in Nrf2 downregulated PCNs alone. Similarly, the heightened activation of caspase 3/7 observed in Nrf2-compromised neurons was attenuated when co-cultured with astrocytes as measured by luminescence based caspase Glo assay. Furthermore, annexin-V-FITC staining followed by FACS analysis revealed that Nrf2 depleted neurons showed resistance to ETOH induced neuronal apoptosis when co-cultured with astrocytes. Thus, the current study identifies ETOH induced dysregulation of GSH and associated apoptotic events observed in Nrf2-depleted neurons can be blocked by astrocytes. Further our results suggest that this neuroprotective effect of astrocyte despite dysfunctional Nrf2 system

  16. Astrocytes Prevent Ethanol Induced Apoptosis of Nrf2 Depleted Neurons by Maintaining GSH Homeostasis.

    PubMed

    Narasimhan, Madhusudhanan; Rathinam, Marylatha; Patel, Dhyanesh; Henderson, George; Mahimainathan, Lenin

    2012-07-01

    Glutathione (GSH), a major cellular antioxidant protects cells against oxidative stress injury. Nuclear factor erythroid 2-related factor 2 (NFE2L2/Nrf2) is a redox sensitive master regulator of battery of antioxidant enzymes including those involved in GSH antioxidant machinery. Earlier we reported that ethanol (ETOH) elicits apoptotic death of primary cortical neurons (PCNs) which in partly due to depletion of intracellular GSH levels. Further a recent report from our laboratory illustrated that ETOH exacerbated the dysregulation of GSH and caspase mediated cell death of cortical neurons that are compromised in Nrf2 machinery (Narasimhan et al., 2011). In various experimental models of neurodegeneration, neuronal antioxidant defenses mainly GSH has been shown to be supported by astrocytes. We therefore sought to determine whether astrocytes can render protection to neurons against ETOH toxicity, particularly when the function of Nrf2 is compromised in neurons. The experimental model consisted of co-culturing primary cortical astrocytes (PCA) with Nrf2 downregulated PCNs that were exposed with 4 mg/mL ETOH for 24 h. Monochlorobimane (MCB) staining followed by FACS analysis showed that astrocytes blocked ETOH induced GSH decrement in Nrf2-silenced neurons as opposed to exaggerated GSH depletion in Nrf2 downregulated PCNs alone. Similarly, the heightened activation of caspase 3/7 observed in Nrf2-compromised neurons was attenuated when co-cultured with astrocytes as measured by luminescence based caspase Glo assay. Furthermore, annexin-V-FITC staining followed by FACS analysis revealed that Nrf2 depleted neurons showed resistance to ETOH induced neuronal apoptosis when co-cultured with astrocytes. Thus, the current study identifies ETOH induced dysregulation of GSH and associated apoptotic events observed in Nrf2-depleted neurons can be blocked by astrocytes. Further our results suggest that this neuroprotective effect of astrocyte despite dysfunctional Nrf2 system

  17. Novel Nrf2 activators from microbial transformation products inhibit blood–retinal barrier permeability in rabbits

    PubMed Central

    Nakagami, Yasuhiro; Masuda, Kayoko; Hatano, Emiko; Inoue, Tatsuya; Matsuyama, Takuya; Iizuka, Mayumi; Ono, Yasunori; Ohnuki, Takashi; Murakami, Yoko; Iwasaki, Masaru; Yoshida, Kazuhiro; Kasuya, Yuji; Komoriya, Satoshi

    2015-01-01

    Background and Purpose Nuclear factor erythroid 2-related factor 2 (Nrf2) is a redox-sensitive transcription factor that binds to antioxidant response elements located in the promoter region of genes encoding many antioxidant enzymes and phase II detoxifying enzymes. Activation of the Nrf2 pathway seems protective for many organs, and although a well-known Nrf2 activator, bardoxolone methyl, was evaluated clinically for treating chronic kidney disease, it was found to induce adverse events. Many bardoxolone methyl derivatives, mostly derived by chemical modifications, have already been studied. However, we adopted a biotransformation technique to obtain a novel Nrf2 activator. Experimental Approach The potent novel Nrf2 activator, RS9, was obtained from microbial transformation products. Its Nrf2 activity was evaluated by determining NADPH:quinone oxidoreductase-1 induction activity in Hepa1c1c7 cells. We also investigated the effects of RS9 on oxygen-induced retinopathy in rats and glycated albumin-induced blood–retinal barrier permeability in rabbits because many ocular diseases are associated with oxidative stress and inflammation. Key Results Bardoxolone methyl doubled the specific activity of Nrf2 in Hepa1c1c7 cells at a much higher concentration than RS9. Moreover, the induction of Nrf2-targeted genes was observed at a one-tenth lower concentration of RS9. Interestingly, the cytotoxicity of RS9 was substantially reduced compared with bardoxolone methyl. Oral and intravitreal administration of RS9 ameliorated the pathological scores and leakage in the models of retinopathy in rats and ocular inflammation in rabbits respectively. Conclusion and Implications Nrf2 activators are applicable for treating ocular diseases and novel Nrf2 activators have potential as a unique method for prevention and treatment of retinovascular disease. PMID:25363737

  18. Lico A Enhances Nrf2-Mediated Defense Mechanisms against t-BHP-Induced Oxidative Stress and Cell Death via Akt and ERK Activation in RAW 264.7 Cells.

    PubMed

    Lv, Hongming; Ren, Hua; Wang, Lidong; Chen, Wei; Ci, Xinxin

    2015-01-01

    Licochalcone A (Lico A) exhibits various biological properties, including anti-inflammatory and antioxidant activities. In this study, we investigated the antioxidative potential and mechanisms of Lico A against tert-butyl hydroperoxide- (t-BHP-) induced oxidative damage in RAW 264.7 cells. Our results indicated that Lico A significantly inhibited t-BHP-induced cytotoxicity, apoptosis, and reactive oxygen species (ROS) generation and reduced glutathione (GSH) depletion but increased the glutamate-cysteine ligase modifier (GCLM) subunit and the glutamate-cysteine ligase catalytic (GCLC) subunit genes expression. Additionally, Lico A dramatically upregulated the antioxidant enzyme heme oxygenase 1 (HO-1) and nuclear factor erythroid 2-related factor 2 (Nrf2), which were associated with inducing Nrf2 nuclear translocation, decreasing Keap1 protein expression and increasing antioxidant response element (ARE) promoter activity. Lico A also obviously induced the activation of serine/threonine kinase (Akt) and extracellular signal-regulated kinase (ERK), but PI3K/Akt and ERK inhibitors treatment displayed clearly decreased levels of LicoA-induced Nrf2 nuclear translocation and HO-1 expression, respectively. Furthermore, Lico A treatment markedly attenuated t-BHP-induced oxidative damage, which was reduced by treatment with PI3K/Akt, ERK, and HO-1 inhibitors. Therefore, Lico A might have a protective role against t-BHP-induced cytotoxicity by modulating HO-1 and by scavenging ROS via the activation of the PI3K/Akt and ERK/Nrf2 signaling pathways.

  19. Trianthema portulacastrum Linn. displays anti-inflammatory responses during chemically induced rat mammary tumorigenesis through simultaneous and differential regulation of NF-κB and Nrf2 signaling pathways.

    PubMed

    Mandal, Animesh; Bishayee, Anupam

    2015-01-22

    Trianthema portulacastrum, a medicinal and dietary plant, has gained substantial importance due to its various pharmacological properties, including anti-inflammatory and anticarcinogenic activities. We have recently reported that a characterized T. portulacastrum extract (TPE) affords a considerable chemoprevention of 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary tumorigenesis though the underlying mechanisms are not completely understood. The objective of this study was to investigate anti-inflammatory mechanisms of TPE during DMBA mammary carcinogenesis in rats by monitoring cyclooxygenase-2 (COX-2), heat shock protein 90 (HSP90), nuclear factor-kappaB (NF-κB) and nuclear factor erythroid 2-related factor 2 (Nrf2). Mammary tumors were harvested from our previous study in which TPE (50-200 mg/kg) was found to inhibit mammary tumorigenesis in a dose-response manner. The expressions of intratumor COX-2, HSP90, NF-κB, inhibitory kappaB-alpha (IκBα) and Nrf2 were determined by immunohistochemistry. TPE downregulated the expression of COX-2 and HSP90, blocked the degradation of IκBα, hampered the translocation of NF-κB from cytosol to nucleus and upregulated the expression and nuclear translocation of Nrf2 during DMBA mammary carcinogenesis. These results in conjunction with our previous findings suggest that TPE prevents DMBA-induced breast neoplasia by anti-inflammatory mechanisms mediated through simultaneous and differential modulation of two interconnected molecular circuits, namely NF-κB and Nrf2 signaling pathways.

  20. Lico A Enhances Nrf2-Mediated Defense Mechanisms against t-BHP-Induced Oxidative Stress and Cell Death via Akt and ERK Activation in RAW 264.7 Cells

    PubMed Central

    Lv, Hongming; Ren, Hua; Wang, Lidong; Chen, Wei; Ci, Xinxin

    2015-01-01

    Licochalcone A (Lico A) exhibits various biological properties, including anti-inflammatory and antioxidant activities. In this study, we investigated the antioxidative potential and mechanisms of Lico A against tert-butyl hydroperoxide- (t-BHP-) induced oxidative damage in RAW 264.7 cells. Our results indicated that Lico A significantly inhibited t-BHP-induced cytotoxicity, apoptosis, and reactive oxygen species (ROS) generation and reduced glutathione (GSH) depletion but increased the glutamate-cysteine ligase modifier (GCLM) subunit and the glutamate-cysteine ligase catalytic (GCLC) subunit genes expression. Additionally, Lico A dramatically upregulated the antioxidant enzyme heme oxygenase 1 (HO-1) and nuclear factor erythroid 2-related factor 2 (Nrf2), which were associated with inducing Nrf2 nuclear translocation, decreasing Keap1 protein expression and increasing antioxidant response element (ARE) promoter activity. Lico A also obviously induced the activation of serine/threonine kinase (Akt) and extracellular signal-regulated kinase (ERK), but PI3K/Akt and ERK inhibitors treatment displayed clearly decreased levels of LicoA-induced Nrf2 nuclear translocation and HO-1 expression, respectively. Furthermore, Lico A treatment markedly attenuated t-BHP-induced oxidative damage, which was reduced by treatment with PI3K/Akt, ERK, and HO-1 inhibitors. Therefore, Lico A might have a protective role against t-BHP-induced cytotoxicity by modulating HO-1 and by scavenging ROS via the activation of the PI3K/Akt and ERK/Nrf2 signaling pathways. PMID:26576227

  1. Anthocyanins from purple sweet potato attenuate dimethylnitrosamine-induced liver injury in rats by inducing Nrf2-mediated antioxidant enzymes and reducing COX-2 and iNOS expression.

    PubMed

    Hwang, Yong Pil; Choi, Jae Ho; Yun, Hyo Jeong; Han, Eun Hee; Kim, Hyung Gyun; Kim, Jin Young; Park, Bong Hwan; Khanal, Tilak; Choi, Jun Min; Chung, Young Chul; Jeong, Hye Gwang

    2011-01-01

    Anthocyanins of the purple sweet potato exhibit antioxidant and hepatoprotective activities via a multitude of biochemical mechanisms. However, the signaling pathways involved in the actions of anthocyanin-induced antioxidant enzymes against chronic liver injury are not fully understood. We examined whether an anthocyanin fraction (AF) from purple sweet potato may prevent dimethylnitrosamine (DMN)-induced liver injury by inducing antioxidants via nuclear erythroid 2-related factor 2 (Nrf2) pathways and by reducing inflammation. Treatment with AF attenuated the DMN-induced increased serum alanine aminotransferase and aspartate aminotransferase activities. It also prevented the formation of hepatic malondialdehyde and the depletion of glutathione and maintained normal glutathione-S-transferase (GST) activity in the livers of DMN-intoxicated rats. Furthermore, AF increased the expression of Nrf2, NADPH:quinine oxidoreductase-1, heme oxygenase-1, and GSTα, which were reduced by DMN, and decreased the expression of cyclooxygenase-2 and inducible nitric oxide synthase. An increase in the nuclear translocation of nuclear factor kappa B (NF-κB) was observed in the DMN-induced liver injury group, but AF inhibited this translocation. Taken together, these results demonstrate that AF increases the expression of antioxidant enzymes and Nrf2 and at the same time decreases the expression of inflammatory mediators in DMN-induced liver injury. These data imply that AF induces antioxidant defense via the Nrf2 pathway and reduces inflammation via NF-κB inhibition.

  2. Metallothionein-III protects against 6-hydroxydopamine-induced oxidative stress by increasing expression of heme oxygenase-1 in a PI3K and ERK/Nrf2-dependent manner

    SciTech Connect

    Hwang, Yong Pil; Kim, Hyung Gyun; Han, Eun Hee; Jeong, Hye Gwang

    2008-09-15

    The zinc-binding protein metallothionein-III (MT-III) is associated with resistance to neuronal injury. However, the underlying mechanism for its effects is unclear. In this study, we demonstrate that MT-III prevents the accumulation of reactive oxygen species (ROS) in dopaminergic SH-SY5Y cells challenged with the Parkinson's disease-related neurotoxin 6-hydroxydopamine (6-OHDA) by a mechanism that involves phosphatidylinositol 3-kinase (PI3K) and ERK kinase/NF-E2-related factor 2 (Nrf2) dependent induction of the stress response protein heme oxygenase-1 (HO-1). Pretreatment of SH-SY5Y cells with MT-III significantly reduced 6-OHDA-induced generation of ROS, caspase-3 activation, and subsequent cell death. Also, MT-III up-regulates HO-1 expression and this expression confers neuroprotection against oxidative injury induced by 6-OHDA. Moreover, MT-III induces Nrf2 nuclear translocation, which is upstream of MT-III-induced HO-1 expression, and PI3K and ERK1/2 activation, a pathway that is involved in induced Nrf2 nuclear translocation, HO-1 expression and neuroprotection. Taken together, these results suggest that the PI3K and ERK/Nrf2 signaling pathway controls the intracellular levels of ROS by regulating the expression of the antioxidant enzyme HO-1.

  3. G-Quadruplex in the NRF2 mRNA 5' Untranslated Region Regulates De Novo NRF2 Protein Translation under Oxidative Stress.

    PubMed

    Lee, Sang C; Zhang, Jack; Strom, Josh; Yang, Danzhou; Dinh, Thai Nho; Kappeler, Kyle; Chen, Qin M

    2017-01-01

    Inhibition of protein synthesis serves as a general measure of cellular consequences of chemical stress. A few proteins are translated selectively and influence cell fate. How these proteins can bypass the general control of translation remains unknown. We found that low to mild doses of oxidants induce de novo translation of the NRF2 protein. Here we demonstrate the presence of a G-quadruplex structure in the 5' untranslated region (UTR) of NRF2 mRNA, as measured by circular dichroism, nuclear magnetic resonance, and dimethylsulfate footprinting analyses. Such a structure is important for 5'-UTR activity, since its removal by sequence mutation eliminated H2O2-induced activation of the NRF2 5' UTR. Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics revealed elongation factor 1 alpha (EF1a) as a protein binding to the G-quadruplex sequence. Cells responded to H2O2 treatment by increasing the EF1a protein association with NRF2 mRNA, as measured by RNA-protein interaction assays. The EF1a interaction with small and large subunits of ribosomes did not appear to change due to H2O2 treatment, nor did posttranslational modifications, as measured by two-dimensional (2-D) Western blot analysis. Since NRF2 encodes a transcription factor essential for protection against tissue injury, our data have revealed a novel mechanism of cellular defense involving de novo NRF2 protein translation governed by the EF1a interaction with the G-quadruplex in the NRF2 5' UTR during oxidative stress. Copyright © 2016 American Society for Microbiology.

  4. 7-Methoxy-(9H-β-Carbolin-1-il)-(E)-1-Propenoic Acid, a β-Carboline Alkaloid From Eurycoma longifolia, Exhibits Anti-Inflammatory Effects by Activating the Nrf2/Heme Oxygenase-1 Pathway.

    PubMed

    Nguyen, Hai Dang; Choo, Young-Yeon; Nguyen, Tien Dat; Nguyen, Hoai Nam; Chau, Van Minh; Lee, Jeong-Hyung

    2016-03-01

    Eurycoma longifolia is an herbal medicinal plant popularly used in Southeast Asian countries. In the present study, we show that 7-methoxy-(9H-β-carbolin-1-il)-(E)-1-propenoic acid (7-MCPA), a β-carboline alkaloid isolated from E. longifolia, exerted anti-inflammatory effects by activating the nuclear factor-E2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway. 7-MCPA inhibited lipopolysaccharide (LPS)-induced production of nitric oxide (NO), prostaglandin E2 (PGE2 ), and interleukin-6 (IL-6) in RAW264.7 cells and rescued C57BL/6 mice from LPS-induced lethality in vivo. LPS-induced expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and IL-6 was also significantly suppressed by treatment of 7-MCPA in RAW264.7 cells. 7-MCPA induced nuclear translocation of Nrf2 and increased transcription of its target genes, such as HO-1. Treating RAW264.7 cells with 7-MCPA increased the intracellular level of reactive oxygen species (ROS) and the phosphorylation level of p38 mitogen-activated protein kinase (MAPK); however, co-treatment with the antioxidant N-acetyl-cysteine (NAC) blocked 7-MCPA-induced p38 MAPK phosphorylation. Moreover, NAC or SB203580 (p38 MAPK inhibitor) blocked 7-MCPA-induced nuclear translocation of Nrf2, suggesting that 7-MCPA activated Nrf2 via a ROS-dependent p38 pathway. 7-MCPA induced HO-1 protein and mRNA expression and knockdown of Nrf2 with siRNA or SB203580 blocked 7-MCPA-mediated induction of HO-1 expression. Inhibiting Nrf2 or HO-1 abrogated the anti-inflammatory effects of 7-MCPA in LPS-stimulated RAW264.7 cells. We also demonstrated that 7-MCPA suppressed LPS-induced nuclear factor κB (NF-κB) activation. These results provide the first evidence that 7-MCPA exerts its anti-inflammatory effect by modulating the Nrf2 and NF-κB pathways and may be a potential Nrf2 activator to prevent or treat inflammatory diseases.

  5. Salidroside inhibits oxygen glucose deprivation (OGD)/re-oxygenation-induced H9c2 cell necrosis through activating of Akt-Nrf2 signaling.

    PubMed

    Zheng, Koulong; Sheng, Zhenqiang; Li, Yefei; Lu, Huihe

    2014-08-15

    Oxygen glucose deprivation (OGD)/re-oxygenation has been applied to cultured cardiomyocytes to create a cellular model of ischemic heart damage. In the current study, we explored the potential role of salidroside against OGD/re-oxygenation-induced damage in H9c2 cardiomyocytes, and studied the underlying mechanisms. We found that OGD/re-oxygenation primarily induced necrosis in H9c2 cells, which was inhibited by salidroside. Salidroside suppressed OGD/re-oxygenation-induced reactive oxygen species (ROS) production, p53 mitochondrial translocation and cyclophilin D (Cyp-D) association as well as mitochondrial membrane potential (MMP) decrease in H9c2 cells. Meanwhile, salidroside activated Akt and promoted transcription of NF-E2-related factor 2 (Nrf2)-regulated genes (heme oxygenase-1 (HO-1) and quinone oxidoreductase 1 (NQO-1)). Significantly, Nrf2 shRNA knockdown or Akt inhibitors (LY 294002 and wortmannin) not only prevented salidroside-induced HO-1/NQO-1 transcription, but also alleviated salidroside-mediated cytoprotective effect against OGD/re-oxygenation in H9c2 cells. These observations suggest that salidroside activates Nrf2-regulated anti-oxidant signaling, and protects against OGD/re-oxygenation-induced H9c2 cell necrosis via activation of Akt signaling. Copyright © 2014 Elsevier Inc. All rights reserved.

  6. Caffeic acid phenethyl ester up-regulates antioxidant levels in hepatic stellate cell line T6 via an Nrf2-mediated mitogen activated protein kinases pathway

    PubMed Central

    Yang, Ning; Shi, Juan-Juan; Wu, Feng-Ping; Li, Mei; Zhang, Xin; Li, Ya-Ping; Zhai, Song; Jia, Xiao-Li; Dang, Shuang-Suo

    2017-01-01

    AIM To investigate the antioxidant effect of caffeic acid phenethyl ester (CAPE) in hepatic stellate cell-T6 (HSC-T6) cells cultured in vitro and the potential mechanisms. METHODS HSC-T6 cells were cultured in vitro and treated with various concentrations of CAPE for 24, 48 and 72 h, respectively. Cell proliferation was investigated using the MTT assay, and cell ultrastructural alterations were observed by transmission electron microscopy. Flow cytometry was employed to investigate the effects of CAPE on apoptosis and the levels of reactive oxygen species in HSC-T6 cells cultured in vitro. An enzyme immunoassay instrument was used to evaluate antioxidant enzyme expression. The effect on α-smooth muscle actin was shown using immunofluorescence. Gene and protein levels of Nrf2, related factors, and mitogen activated protein kinases (MAPKs), in HSC-T6 cells were detected using RT-PCR and Western blot, respectively. RESULTS CAPE inhibited the proliferation and activation of HSC-T6 cells cultured in vitro. CAPE increased the antioxidant levels and the translocation of Nrf2 from the cytoplasm to the nucleus in HSC-T6 cells. Moreover, the phosphorylation of MAPKs in cells decreased in response to CAPE. Interestingly, CAPE-induced oxidative stress in the cells was significantly attenuated by pretreatment with MAPKs inhibitors. CONCLUSION CAPE inhibits cell proliferation and up-regulates the antioxidant levels in HSC-T6 cells partly through the Nrf2-MAPKs signaling pathway. PMID:28275300

  7. The enhancement of phase 2 enzyme activities by sodium butyrate in normal intestinal epithelial cells is associated with Nrf2 and p53.

    PubMed

    Yaku, Keisuke; Enami, Yuka; Kurajyo, Chika; Matsui-Yuasa, Isao; Konishi, Yotaro; Kojima-Yuasa, Akiko

    2012-11-01

    Dietary fiber fermentation by the colonic bacterial flora produces short-chain fatty acids, acetate, propionate and butyrate. Among them, butyrate is considered to be the major energy substrate for colonocytes and, at least in rats, seems to protect against colonic carcinogenesis. In this study, we examined the effect and the mechanisms of short-chain fatty acids on the activity of phase 2 enzymes. Sodium butyrate increased phase 2 enzyme activities in normal rat small intestine epithelial cells, Glutathione S-transferase and NAD(P)H:quinone oxidoreductase (NQO) in a dose-dependent manner(;) however, other short-chain fatty acids did not increase them. The mechanism of the induction of phase 2 enzymes with sodium butyrate sodium butyrate, but not other short-chain fatty acids was related to the increase of NF-E2-related factor 2 (Nrf2) nuclear translocation and the decrease in the levels of nuclear fraction p53. Sodium butyrate also caused enhancement of Nrf2 mRNA levels and suppression of p53 mRNA levels. Sodium butyrate enhances the activities of phase 2 enzymes via an increase in the Nrf2 protein levels in the nucleus and a decrease in the mRNA and protein levels of p53.

  8. The activation of the Nrf2/ARE pathway in HepG2 hepatoma cells by phytochemicals and subsequent modulation of phase II and antioxidant enzyme expression.

    PubMed

    Krajka-Kuźniak, Violetta; Paluszczak, Jarosław; Szaefer, Hanna; Baer-Dubowska, Wanda

    2015-06-01

    Previous studies have shown that naturally occurring phytochemicals, indole-3-carbinol, phenethyl isothiocyanate, protocatechuic acid, and tannic acid increased the activity and protein level of hepatic phase II enzymes in animal models. In order to further explore the mechanism of this activity, we investigated the effect of these compounds on the activation of nuclear factor erythroid-2-related factor 2 (Nrf2)-regulated transcription in human hepatocellular carcinoma HepG2 cells. Treatment with all the tested compounds resulted in the translocation from the cytosol and nuclear accumulation of active phosphorylated Nrf2. Furthermore, phenethyl isothiocyanate and indole-3-carbinol increased the transcript and protein levels of GSTA, GSTP, GSTM, GSTT, and NQO1. On the other hand, protocatechuic and tannic acids enhanced only the expression of GSTA, GSTM, and GSTT. The expression of genes encoding antioxidant enzymes CAT, SOD, GR, and GPx was increased after the treatment with all the tested phytochemicals. These results indicate that isothiocyanates/indoles and protocatechuic and tannic acids induce phase II and antioxidant gene expression in HepG2 cells through the Nrf2-Keap1-ARE signaling pathway. Moreover, the results of this study confirmed that the degradation products of glucosinolates are more effective inducers of phase II and antioxidant enzymes than protocatechuic and tannic acids.

  9. Antioxidant effects of hydrogen sulfide on left ventricular remodeling in smoking rats are mediated via PI3K/Akt-dependent activation of Nrf2.

    PubMed

    Zhou, Xiang; Zhao, Liangping; Mao, Jinning; Huang, Jian; Chen, Jianchang

    2015-03-01

    There is growing evidence that oxidative stress plays critical roles in the pathogenesis of cardiac remodeling. In the present study, we established a rat model of passive smoking and investigated the antioxidant effects of hydrogen sulfide (H2S) on smoking-induced left ventricular remodeling. Cardiac structure and function were evaluated using 2-dimensional echocardiography. Myocardial fibrosis was detected by Masson's trichrome staining and immunohistochemistry. Oxidative stress was assessed by measuring malondialdehyde levels, superoxide dismutase and glutathione peroxidase activities, and reactive oxygen species generation in the myocardium. Neonatal rat cardiomyocytes transfected with specific siRNA and exposed to cigarette smoke condensate and H2S donor sodium hydrosulfide were used to confirm the involvement of Nrf2 and PI3K/Akt signaling in the antioxidant effects of H2S. Our results indicated that H2S could protect against left ventricular remodeling in smoking rats via attenuation of oxidative stress. Moreover, H2S was also found to increase the phosphorylation of Akt and GSK3β and decrease the nuclear expression of Fyn, which consequently leads to nuclear translocation of Nrf2 and elevated expression of HO-1 and NQO1. In conclusion, H2S may exert antioxidant effects on left ventricular remodeling in smoking rats via PI3K/Akt-dependent activation of Nrf2 signaling.

  10. Cytoprotective Effect of Makgeolli Lees on Paraquat Induced Oxidative Stress in A549 Cells via Activation of NRF2 and Antioxidant Genes.

    PubMed

    Jeon, Miso; Rahman, Naimur; Kim, Yong-Sik

    2016-02-01

    Makgeolli lees (ML) has several physiological effects such as antioxidant, antidiabetic, and anticancer properties, but its biological functions have not been determined definitively. Here, we tested whether ML has a cytoprotective effect on paraquat (PQ)-induced oxidative stress in the human lung carcinoma cell line A549. At 0.1 mg/ml ML, viability of PQ-exposed A549 cells was restored by 12.4%, 18.5%, and 48.6% after 24, 48, and 72 h, respectively. ML also reduced production of the intracellular reactive oxygen species (ROS) that were generated by PQ treatment. Further experiments revealed that ML treatment enhanced the expression and nuclear translocation of nuclear factor erythroid 2-related factor 2 (NRF2) as well as ARE-GFP reporter activity. ML treatment also effectively increased the expression of NRF2's target genes NAD(P)H dehydrogenase quinone 1 (NQO1) and heme oxygenase 1 (HO-1). Moreover, we found that expression of cytoprotective genes, including glutathione peroxidases (GPXs), superoxide dismutase (SOD1), catalase (CAT), peroxiredoxin 3 (PRDX3), and peroxiredoxin 4 (PRDX4), was greatly enhanced by treatment with ML during PQ exposure. Taken together, the data suggest that treatment of PQ-exposed A549 cells with ML ameliorates cytotoxicity through induction of NRF2 expression and its target genes HO-1, NQO1, and other antioxidant genes. Thus, ML may serve as a functional food applicable to ROS-mediated human diseases.

  11. Ferrous Iron Induces Nrf2 Expression in Mouse Brain Astrocytes to Prevent Neurotoxicity.

    PubMed

    Cui, Zhenwen; Zhong, Zhihong; Yang, Yong; Wang, Baofeng; Sun, Yuhao; Sun, Qingfang; Yang, Guo-Yuan; Bian, Liuguan

    2016-08-01

    Free radical damage caused by ferrous iron is involved in the pathogenesis of secondary brain injury after intracerebral hemorrhage (ICH). NF-E2-related factor 2 (Nrf2), a major phase II gene regulator that binds to antioxidant response element, represents an important cellular cytoprotective mechanism against oxidative damage. We hypothesized that Nrf2 might protect astrocytes from damage by Fe(2+) . Therefore, we examined cytotoxicity in primary astrocytes induced by iron overload and evaluated the effects of Fe(2+) on Nrf2 expression. The results demonstrated that 24-h Fe(2+) exposure exerted time- and concentration-dependent cytotoxicity in astrocytes. Furthermore, Fe(2+) exposure in astrocytes resulted in time- and concentration-dependent increases in Nrf2 expression, which preceded Fe(2+) toxicity. Nrf2-specific siRNA further knocked down Nrf2 levels, resulting in greater Fe(2+) -induced astrocyte cytotoxicity. These data indicate that induction of Nrf2 expression could serve as an adaptive self-defense mechanism, although it is insufficient to completely protect primary astrocytes from Fe(2+) -induced neurotoxicity.

  12. Discovery of a novel Nrf2 inhibitor that induces apoptosis of human acute myeloid leukemia cells.

    PubMed

    Zhang, JinFeng; Su, Le; Ye, Qing; Zhang, ShangLi; Kung, HsiangFu; Jiang, Fan; Jiang, GuoSheng; Miao, JunYing; Zhao, BaoXiang

    2017-01-31

    Nuclear factor-erythroid 2-related factor 2 (Nrf2) is persistently activated in many human tumors including acute myeloid leukemia (AML). Therefore, inhibition of Nrf2 activity may be a promising target in leukemia therapy. Here, we used an antioxidant response element-luciferase reporter system to identify a novel pyrazolyl hydroxamic acid derivative, 1-(4-(tert-Butyl)benzyl)-3-(4-chlorophenyl)-N-hydroxy-1H pyrazole-5-carboxamide (4f), that inhibited Nrf2 activity. 4f had a profound growth-inhibitory effect on three AML cell lines, THP-1, HL-60 and U937, and a similar anti-growth effect in a chick embryo model. Moreover, flow cytometry of AML cells revealed increased apoptosis with 4f (10 μM) treatment for 48 h. The protein levels of cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase were enhanced in all three AML cell types. Furthermore, Nrf2 protein level was downregulated by 4f. Upregulation of Nrf2 by tert-butylhydroquinone (tBHQ) or Nrf2 overexpression could ameliorate 4f-induced growth inhibition and apoptosis. Treatment with 4f reduced both B-cell lymphoma-2 (Bcl-2) expression and Bcl-2/Bcl-2-associated X protein (Bax) ratio, which indicated that 4f induced apoptosis, at least in part, via mitochondrial-dependent signaling. Therefore, as an Nrf2 inhibitor, the pyrazolyl hydroxamic acid derivative 4f may be a promising agent in AML therapy.

  13. Nuclear transcription factor Nrf2 suppresses prostate cancer cells growth and migration through upregulating ferroportin.

    PubMed

    Xue, Dong; Zhou, Cuixing; Shi, Yunbo; Lu, Hao; Xu, Renfang; He, Xiaozhou

    2016-11-29

    VTo investigate the effect of nuclear transcription factor Nrf2 on the transcription of Ferroportin (FPN) in prostate cancer cells, and the regulation mechanisms of FPN on cell viability, migration and apoptosis of prostate cancer cells.Empty vectors, pEGFPC1-Nrf2, pEGFPC1-FPN, Si-FPN and Si-Nrf2 were transfected into prostate cancer cell line PC3. The expression of mRNA and protein were measured by real time-PCR (RT-PCR) and western blot. Cell viability, migration, cycle and apoptosis were tested by CCK-8 assay, wound healing and flow cytometry, respectively. The interaction between FPN and Nrf2 was confirmed by chromatin immunoprecipitation (CHIP) assay.The viability, migration and mitosis of PC3 cells could be repressed by over-expressed FPN, with decreased intracellular ferritin. The CHIP assay demonstrated that Nrf2 is one transcription factor of FPN and promotes its transcription. With the increase of Nrf2 in PC3 cells, the viability, migration ability and concentration of ferritin were suppressed, while the apoptosis rate was increased. The above effects were counteracted by down-regulating FPN.FPN could inhibit the prostate cancer cell viability, migration and mitosis, which is also related to a decrease of intracellular ferritin content. In conclusion, Nrf2 suppresses prostate cancer cells viability, migration, and mitosis through upregulating FPN.

  14. Gene regulatory effects of disease-associated variation in the NRF2 network.

    PubMed

    Lacher, Sarah E; Slattery, Matthew

    2016-12-01

    Reactive oxygen species (ROS), which are both a natural byproduct of oxidative metabolism and an undesirable byproduct of many environmental stressors, can damage all classes of cellular macromolecules and promote diseases from cancer to neurodegeneration. The actions of ROS are mitigated by the transcription factor NRF2, which regulates expression of antioxidant genes via its interaction with cis-regulatory antioxidant response elements (AREs). However, despite the seemingly straightforward relationship between the opposing forces of ROS and NRF2, regulatory precision in the NRF2 network is essential. Genetic variants that alter NRF2 stability or alter ARE sequences have been linked to a range of diseases. NRF2 hyperactivating mutations are associated with tumorigenesis. On the subtler end of the spectrum, single nucleotide variants (SNVs) that alter individual ARE sequences have been linked to neurodegenerative disorders including progressive supranuclear palsy and Parkinson's disease, as well as other diseases. Although the human health implications of NRF2 dysregulation have been recognized for some time, a systems level view of this regulatory network is beginning to highlight key NRF2-targeted AREs consistently associated with disease.

  15. Persistent activation of Nrf2 through p62 in hepatocellular carcinoma cells

    PubMed Central

    Inami, Yoshihiro; Waguri, Satoshi; Sakamoto, Ayako; Kouno, Tsuguka; Nakada, Kazuto; Hino, Okio; Watanabe, Sumio; Ando, Jin; Iwadate, Manabu; Yamamoto, Masayuki; Lee, Myung-Shik; Tanaka, Keiji

    2011-01-01

    Suppression of autophagy is always accompanied by marked accumulation of p62, a selective autophagy substrate. Because p62 interacts with the Nrf2-binding site on Keap1, which is a Cullin 3–based ubiquitin ligase adapter protein, autophagy deficiency causes competitive inhibition of the Nrf2–Keap1 interaction, resulting in stabilization of Nrf2 followed by transcriptional activation of Nrf2 target genes. Herein, we show that liver-specific autophagy-deficient mice harbor adenomas linked to both the formation of p62- and Keap1-positive cellular aggregates and induction of Nrf2 targets. Importantly, similar aggregates were identified in more than 25% of human hepatocellular carcinomas (HCC), and induction of Nrf2 target genes was recognized in most of these tumors. Gene targeting of p62 in an HCC cell line markedly abrogates the anchorage-independent growth, whereas forced expression of p62, but not a Keap1 interaction-defective mutant, resulted in recovery of the growth defect. These results indicate the involvement of persistent activation of Nrf2 through the accumulation of p62 in hepatoma development. PMID:21482715

  16. Myeloid-Derived Suppressor Cell Survival and Function Are Regulated by the Transcription Factor Nrf2.

    PubMed

    Beury, Daniel W; Carter, Kayla A; Nelson, Cassandra; Sinha, Pratima; Hanson, Erica; Nyandjo, Maeva; Fitzgerald, Phillip J; Majeed, Amry; Wali, Neha; Ostrand-Rosenberg, Suzanne

    2016-04-15

    Tumor-induced myeloid-derived suppressor cells (MDSC) contribute to immune suppression in tumor-bearing individuals and are a major obstacle to effective immunotherapy. Reactive oxygen species (ROS) are one of the mechanisms used by MDSC to suppress T cell activation. Although ROS are toxic to most cells, MDSC survive despite their elevated content and release of ROS. NF erythroid 2-related factor 2 (Nrf2) is a transcription factor that regulates a battery of genes that attenuate oxidative stress. Therefore, we hypothesized that MDSC resistance to ROS may be regulated by Nrf2. To test this hypothesis, we used Nrf2(+/+)and Nrf2(-/-)BALB/c and C57BL/6 mice bearing 4T1 mammary carcinoma and MC38 colon carcinoma, respectively. Nrf2 enhanced MDSC suppressive activity by increasing MDSC production of H2O2, and it increased the quantity of tumor-infiltrating MDSC by reducing their oxidative stress and rate of apoptosis. Nrf2 did not affect circulating levels of MDSC in tumor-bearing mice because the decreased apoptotic rate of tumor-infiltrating MDSC was balanced by a decreased rate of differentiation from bone marrow progenitor cells. These results demonstrate that Nrf2 regulates the generation, survival, and suppressive potency of MDSC, and that a feedback homeostatic mechanism maintains a steady-state level of circulating MDSC in tumor-bearing individuals.

  17. Chemical and biological mechanisms of phytochemical activation of Nrf2 and importance in disease prevention

    PubMed Central

    Eggler, Aimee L.; Savinov, Sergey N.

    2016-01-01

    Plants are an incredibly rich source of compounds that activate the Nrf2 transcription factor, leading to upregulation of a battery of cytoprotective genes. This perspective surveys established and proposed molecular mechanisms of Nrf2 activation by phytochemicals with a special emphasis on a common chemical property of Nrf2 activators: the ability as “soft” electrophiles to modify cellular thiols, either directly or as oxidized biotransformants. In addition, the role of reactive oxygen/nitrogen species as secondary messengers in Nrf2 activation is discussed. While the uniquely reactive C151 of Keap1, an Nrf2 repressor protein, is highlighted as a key target of cytoprotective phytochemicals, also reviewed are other stress-responsive proteins, including kinases, which play non-redundant roles in the activation of Nrf2 by plant-derived agents. Finally, the perspective presents two key factors accounting for the enhanced therapeutic windows of effective phytochemical activators of the Keap1–Nrf2 axis: enhanced selectivity toward sensor cysteines and reversibility of addition to thiolate molecules. PMID:26855455

  18. Activation of Nrf2 in keratinocytes causes chloracne (MADISH)-like skin disease in mice

    PubMed Central

    Schäfer, Matthias; Willrodt, Ann-Helen; Kurinna, Svitlana; Link, Andrea S; Farwanah, Hany; Geusau, Alexandra; Gruber, Florian; Sorg, Olivier; Huebner, Aaron J; Roop, Dennis R; Sandhoff, Konrad; Saurat, Jean-Hilaire; Tschachler, Erwin; Schneider, Marlon R; Langbein, Lutz; Bloch, Wilhelm; Beer, Hans-Dietmar; Werner, Sabine

    2014-01-01

    The transcription factor Nrf2 is a key regulator of the cellular stress response, and pharmacological Nrf2 activation is a promising strategy for skin protection and cancer prevention. We show here that prolonged Nrf2 activation in keratinocytes causes sebaceous gland enlargement and seborrhea in mice due to upregulation of the growth factor epigen, which we identified as a novel Nrf2 target. This was accompanied by thickening and hyperkeratosis of hair follicle infundibula. These abnormalities caused dilatation of infundibula, hair loss, and cyst development upon aging. Upregulation of epigen, secretory leukocyte peptidase inhibitor (Slpi), and small proline-rich protein 2d (Sprr2d) in hair follicles was identified as the likely cause of infundibular acanthosis, hyperkeratosis, and cyst formation. These alterations were highly reminiscent to the phenotype of chloracne/“metabolizing acquired dioxin-induced skin hamartomas” (MADISH) patients. Indeed, SLPI, SPRR2, and epigen were strongly expressed in cysts of MADISH patients and upregulated by dioxin in human keratinocytes in an NRF2-dependent manner. These results identify novel Nrf2 activities in the pilosebaceous unit and point to a role of NRF2 in MADISH pathogenesis. PMID:24503019

  19. Knockout of the transcription factor Nrf2 disrupts spermatogenesis in an age-dependent manner

    PubMed Central

    Nakamura, Brooke N.; Lawson, Gregory; Chan, Jefferson Y.; Banuelos, Jésus; Cortés, Mabel M.; Hoang, Yvonne D.; Ortiz, Laura; Rau, Bogdan A.; Luderer, Ulrike

    2010-01-01

    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</