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Sample records for nrf2 translocation undermines

  1. Frequency modulated translocational oscillations of Nrf2, a transcription factor functioning like a wireless sensor.

    PubMed

    Xue, Mingzhan; Momiji, Hiroshi; Rabbani, Naila; Bretschneider, Till; Rand, David A; Thornalley, Paul J

    2015-08-01

    The discovery that nuclear factor erythroid 2-related factor 2 (Nrf2) undergoes translocational oscillations from cytoplasm to nucleus in human cells with frequency modulation linked to activation of a stress-stimulated cytoprotective response raises the prospect that the Nrf2 works mechanistically analogous to a wireless sensor. Herein, we consider how this new model of Nrf2 oscillation resolves previous inexplicable experimental findings on Nrf2 regulation and why it is fit-for-purpose. Further investigation is required to assess how generally applicable the oscillatory mechanism is and if characteristics of this regulatory control can be found in vivo. It suggests there are multiple, potentially re-enforcing receptors for Nrf2 activation, indicating that potent Nrf2 activation for improved health and treatment of disease may be achieved through combination of Nrf2 system stimulants. PMID:26551710

  2. 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

  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. Antioxidant-induced modification of INrf2 cysteine 151 and PKC-δ-mediated phosphorylation of Nrf2 serine 40 are both required for stabilization and nuclear translocation of Nrf2 and increased drug resistance

    PubMed Central

    Niture, Suryakant K.; Jain, Abhinav K.; Jaiswal, Anil K.

    2009-01-01

    Summary Antioxidants cause dissociation of nuclear factor erythroid 2-related factor 2 (Nrf2) from inhibitor of Nrf2 (INrf2) and so Nrf2:INrf2 can serve as a sensor of oxidative stress. Nrf2 translocates to the nucleus, binds to antioxidant response element (ARE) and activates defensive gene expression, which protects cells. Controversies exist regarding the role of antioxidant-induced modification of INrf2 cysteine 151 or protein kinase C (PKC)-mediated phosphorylation of Nrf2 serine 40 in the release of Nrf2 from INrf2. In addition, the PKC isoform that phosphorylates Nrf2S40 remains unknown. Here, we demonstrate that antioxidant-induced PKC-δ-mediated phosphorylation of Nrf2S40 leads to release of Nrf2 from INrf2. This was evident from specific chemical inhibitors of PKC isoenzymes in reporter assays, in vitro kinase assays with purified Nrf2 and PKC isoenzymes, in vivo analysis with dominant-negative mutants and siRNA against PKC isoforms, use of PKC-δ+/+ and PKC-δ–/– cells, and use of Nrf2S40 phospho-specific antibody. The studies also showed that antioxidant-induced INrf2C151 modification was insufficient for the dissociation of Nrf2 from INrf2. PKC-δ-mediated Nrf2S40 phosphorylation was also required. Nrf2 and mutant Nrf2S40A both bind to INrf2. However, antioxidant treatment led to release of Nrf2 but not Nrf2S40A from INrf2. In addition, Nrf2 and mutant Nrf2S40A both failed to dissociate from mutant INrf2C151A. Furthermore, antioxidant-induced ubiquitylation of INrf2 in PKC-δ+/+ and PKC-δ–/– cells occurred, but Nrf2 failed to be released in PKC-δ–/– cells. The antioxidant activation of Nrf2 reduced etoposide-mediated DNA fragmentation and promoted cell survival in PKC-δ+/+ but not in PKC-δ–/– cells. These data together demonstrate that both modification of INrf2C151 and PKC-δ-mediated phosphorylation of Nrf2S40 play crucial roles in Nrf2 release from INrf2, antioxidant induction of defensive gene expression, promoting cell

  5. Isoniazid prevents Nrf2 translocation by inhibiting ERK1 phosphorylation and induces oxidative stress and apoptosis

    PubMed Central

    Verma, Ajeet Kumar; Yadav, Arti; Dewangan, Jayant; Singh, Sarvendra Vikram; Mishra, Manisha; Singh, Pradhyumna Kumar; Rath, Srikanta Kumar

    2015-01-01

    Isoniazid is used either alone or in combination with other drugs for the treatment of tuberculosis. It is also used for the prevention of tuberculosis. Chronic treatment of Isoniazid may cause severe liver damage leading to acute liver failure. The mechanism through which Isoniazid causes liver damage is investigated. Isoniazid treatment generates reactive oxygen species and induces apoptosis in Hep3B cells. It induces antioxidative and apoptotic genes leading to increase in mRNA expression and protein levels in Hep3B cells. Whole genome expression analysis of Hep3B cells treated with Isoniazid has resulted in differential expression of various genes playing prime role in regulation of apoptotic, antioxidative, DNA damage, cell signaling, cell proliferation and differentiation pathways. Isoniazid increased cytosolic Nrf2 protein level while decreased nuclear Nrf2 protein level. It also decreased ERK1 phosphorylation and treatment of Hep3B cells with ERK inhibitor followed by Isoniazid resulting in increased apoptosis in these cells. Two dimensional gel electrophoresis results have also shown differential expression of various protein species including heat shock proteins, proteins playing important role in oxidative stress, DNA damage, apoptosis, cell proliferation and differentiation. Results suggest that Isoniazid induces apoptosis through oxidative stress and also prevents Nrf2 translocation into the nucleus by reducing ERK1 phosphorylation thus preventing cytoprotective effect. PMID:26202867

  6. Curcumin Induces Nrf2 Nuclear Translocation and Prevents Glomerular Hypertension, Hyperfiltration, Oxidant Stress, and the Decrease in Antioxidant Enzymes in 5/6 Nephrectomized Rats

    PubMed Central

    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. PMID:22919438

  7. Chebulic acid prevents hepatic fibrosis induced by advanced glycation end-products in LX-2 cell by modulating Nrf2 translocation via ERK pathway.

    PubMed

    Koo, Yun-Chang; Pyo, Min Cheol; Nam, Mi-Hyun; Hong, Chung-Oui; Yang, Sung-Yong; Lee, Kwang-Won

    2016-08-01

    Advanced glycation end-products (AGEs) are formed during normal aging, and at an accelerated rate in metabolic syndrome patients. Nonalcoholic steatohepatitis (NASH) can be caused by the AGEs in plasma, while glyceraldehyde-derived AGEs (glycer-AGEs) are significantly higher in the serum of NASH patients. In this study, we investigated the molecular mechanisms of chebulic acid, isolated from Terminalia chebula Retz., in the inhibition of glycer-AGEs induced production of reactive oxygen species (ROS) and collagen accumulation using the LX-2 cell line. Chebulic acid significantly inhibited the induction of ROS and accumulation of collagen proteins by glycer-AGEs. ERK phosphorylation and total nuclear factor E2-related factor 2 (Nrf2) protein expression were induced by chebulic acid in a dose-dependent manner. Chebulic acid was also found to induce translocation of Nrf2 into the nucleus, which was attenuated by inhibition of ERK phosphorylation through treatment with PD98059. Following translocation of Nrf2, chebulic acid induced the protein expressions of catalytic subunit of γ-glutamylcysteine synthetase and glutathione synthesis. Collagen accumulation was also significantly reduced by chebulic acid treatment. The observed effects of chebulic acid were all inhibited by PD98059 treatment. Taken together, these results suggest that chebulic acid prevents the glycer-AGEs-induced ROS formation of LX-2 cells and collagen accumulation by ERK-phosphorylation-mediated Nrf2 nuclear translocation, which causes upregulation of antioxidant protein production. PMID:27021876

  8. 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. PMID:26968794

  9. Thyroid hormone-induced cytosol-to-nuclear translocation of rat liver Nrf2 is dependent on Kupffer cell functioning.

    PubMed

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

    2012-01-01

    L-3,3',5-triiodothyronine (T(3)) 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 (GdCl(3); 10 mg/kg i.v. 72 h before T(3) [0.1 mg/kg i.p.]) or NADPH oxidase inhibitor apocynin (1.5 mmol/L added to the drinking water for 7 days before T(3)), and determinations were performed 2 h after T(3). T(3) 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 GdCl(3) treatment prior to T(3), 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, T(3)-induced tumor necrosis factor-α (TNF-α) response was eliminated by previous GdCl(3) administration. Similar to GdCl(3), apocynin given before T(3) 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 T(3). This contention is supported by suppression of the respiratory burst activity of Kupffer cells and the associated reactive oxygen species production by GdCl(3) or apocynin given prior to T(3), thus hindering Nrf2 activation. PMID:22649286

  10. Regulation of Nrf2 – An update

    PubMed Central

    Niture, Suryakant K.; Khatri, Raju; Jaiswal, Anil K.

    2013-01-01

    Nrf2:INrf2 (Keap1) are cellular sensors of oxidative and electrophilic stress. Nrf2 is a nuclear factor that controls the expression and coordinated induction of a battery of genes which encode detoxifying enzymes, drug transporters (MRPs), anti-apoptotic proteins and proteasomes. In the basal state, Nrf2 is constantly degraded in the cytoplasm by its inhibitor, INrf2. INrf2 functions as an adapter for Cul3/Rbx1 E3 ubiquitin ligase mediated degradation of Nrf2. Chemicals including antioxidants, tocopherols including α-tocopherol (vitamin E), phytochemicals and radiations antagonize the Nrf2:INrf2 interaction and leads to the stabilization and activation of Nrf2. The signaling events involve pre-induction, induction and post-induction responses that tightly control Nrf2 activation and repression back to the basal state. Oxidative/electrophilic signals activate unknown tyrosine kinase(s) in a pre-induction response which phosphorylates specific residues on Nrf2 negative-regulators, INrf2, Fyn and Bach1, leading to their nuclear export, ubiquitination and degradation. This prepares nuclei for unhindered import of Nrf2. Oxidative/electrophilic modification of INrf2cysteine151 followed by PKC phosphorylation of Nrf2serine40 in the induction response results in the escape or release of Nrf2 from INrf2. Nrf2 is thus stabilized and translocates to the nucleus resulting in a coordinated activation of gene expression. This is followed by a post-induction response that controls the ‘switching off’ of Nrf2-activated gene expression. GSK3β under the control of AKT and PI3K, phosphorylates Fyn leading to Fyn nuclear localization. Fyn phosphorylates Nrf2Y568 resulting in nuclear export and degradation of Nrf2. The activation and repression of Nrf2 provides protection against oxidative/electrophilic stress and associated diseases, including cancer. However, deregulation of INrf2 and Nrf2 due to mutations may lead to nuclear accumulation of Nrf2 that reduces apoptosis and

  11. 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. PMID:27312421

  12. Berberine activates Nrf2 nuclear translocation and inhibits apoptosis induced by high glucose in renal tubular epithelial cells through a phosphatidylinositol 3-kinase/Akt-dependent mechanism.

    PubMed

    Zhang, Xiuli; Liang, Dan; Lian, Xu; Jiang, Yan; He, Hui; Liang, Wei; Zhao, Yue; Chi, Zhi-Hong

    2016-06-01

    Apoptosis of tubular epithelial cells is a major feature of diabetic kidney disease, and hyperglycemia triggers the generation of free radicals and oxidant stress in tubular cells. Berberine (BBR) is identified as a potential anti-diabetic herbal medicine due to its beneficial effects on insulin sensitivity, glucose metabolism and glycolysis. In this study, the underlying mechanisms involved in the protective effects of BBR on high glucose-induced apoptosis were explored using cultured renal tubular epithelial cells (NRK-52E cells) and human kidney proximal tubular cell line (HK-2 cells). We identified the pivotal role of phosphatidylinositol 3-kinase (PI3K)/Akt in BBR cellular defense mechanisms and revealed the novel effect of BBR on nuclear factor (erythroid-derived 2)-related factor-2 (Nrf2) and heme oxygenase (HO)-1 in NRK-52E and HK-2 cells. BBR attenuated reactive oxygen species production, antioxidant defense (GSH and SOD) and oxidant-sensitive proteins (Nrf2 and HO-1), which also were blocked by LY294002 (an inhibitor of PI3K) in HG-treated NRK-52E and HK-2 cells. Furthermore, BBR improved mitochondrial function by increasing mitochondrial membrane potential. BBR-induced anti-apoptotic function was demonstrated by decreasing apoptotic proteins (cytochrome c, Bax, caspase3 and caspase9). All these findings suggest that BBR exerts the anti-apoptosis effects through activation of PI3K/Akt signal pathways and leads to activation of Nrf2 and induction of Nrf2 target genes, and consequently protecting the renal tubular epithelial cells from HG-induced apoptosis. PMID:26979714

  13. Restoration of Nrf2 Signaling Normalizes the Regenerative Niche.

    PubMed

    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; Ceradini, Daniel J

    2016-03-01

    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

  14. Lipoicmethylenedioxyphenol Reduces Experimental Atherosclerosis through Activation of Nrf2 Signaling

    PubMed Central

    Ying, Zhekang; Chen, Minjie; Xie, Xiaoyun; Wang, Xiaoke; Kherada, Nisharahmed; Desikan, Rajagopal; Mihai, Georgeta; Burns, Patrick; Sun, Qinghua; Rajagopalan, Sanjay

    2016-01-01

    Objective Oxidative stress is implicated in the pathogenesis of atherosclerosis, and Nrf2 is the transcriptional factor central in cellular antioxidant responses. In the present study, we investigate the effect of a dihydrolipoic acid derivative lipoicmethylenedioxyphenol (LMDP) on the progression of atherosclerosis and test whether its effect on atherosclerosis is mediated by Nrf2. Methods and Results Both magnetic resonance imaging (MRI) scanning and en face analysis reveal that 14 weeks of treatment with LMDP markedly reduced atherosclerotic burden in a rabbit balloon vascular injury model. Myograph analyses show decreased aortic contractile response to phenylephrine and increased aortic response to acetylcholine and insulin in LMDP-treated animals, suggesting that LMDP inhibits atherosclerosis through improving vascular function. A role of Nrf2 signaling in mediating the amelioration of vascular function by LMDP was supported by increased Nrf2 translocation into nuclear and increased expression of Nrf2 target genes. Furthermore, chemotaxis analysis with Boydem chamber shows that leukocytes isolated from LMDP-treated rabbits had reduced chemotaxis, and knock-down of Nrf2 significantly reduced the effect of LMDP on the chemotaxis of mouse macrophages. Conclusion Our results support that LMDP has an anti-atherosclerotic effect likely through activation of Nrf2 signaling and subsequent inhibition of macrophage chemotaxis. PMID:26859892

  15. Nrf2-Mediated Cardiac Maladaptive Remodeling and Dysfunction in a Setting of Autophagy Insufficiency.

    PubMed

    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 and switching on Nrf2-mediated cardiac dysfunction. In a murine model of pressure overload-induced cardiac remodeling and dysfunction via transverse aortic arch constriction, 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. Transverse aortic arch constriction -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 the upregulation of angiotensinogen (Agt) only in the autophagy-impaired heart after transverse aortic arch constriction. 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 and 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

  16. 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

  17. 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

    2016-01-01

    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. PMID:27517893

  18. 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.

  19. 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

  20. 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

  1. 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

  2. Glycosylation enables aesculin to activate Nrf2.

    PubMed

    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

  3. 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

  4. 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.

  5. 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

  6. 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.

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

    PubMed

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

    2015-12-01

    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

  8. 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

  9. 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

  10. The Nrf2/HO-1 Axis in Cancer Cell Growth and Chemoresistance

    PubMed Central

    Furfaro, A. L.; Traverso, N.; Domenicotti, C.; Piras, S.; Moretta, L.; Marinari, U. M.; Pronzato, M. A.; Nitti, M.

    2016-01-01

    The transcription factor, nuclear factor erythroid 2 p45-related factor 2 (Nrf2), acts as a sensor of oxidative or electrophilic stresses and plays a pivotal role in redox homeostasis. Oxidative or electrophilic agents cause a conformational change in the Nrf2 inhibitory protein Keap1 inducing the nuclear translocation of the transcription factor which, through its binding to the antioxidant/electrophilic response element (ARE/EpRE), regulates the expression of antioxidant and detoxifying genes such as heme oxygenase 1 (HO-1). Nrf2 and HO-1 are frequently upregulated in different types of tumours and correlate with tumour progression, aggressiveness, resistance to therapy, and poor prognosis. This review focuses on the Nrf2/HO-1 stress response mechanism as a promising target for anticancer treatment which is able to overcome resistance to therapies. PMID:26697129

  11. 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

  12. 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.

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

    PubMed Central

    Malhotra, Deepti; Portales-Casamar, Elodie; Singh, Anju; Srivastava, Siddhartha; Arenillas, David; Happel, Christine; Shyr, Casper; Wakabayashi, Nobunao; Kensler, Thomas W.; Wasserman, Wyeth W.; Biswal, Shyam

    2010-01-01

    The Nrf2 (nuclear factor E2 p45-related factor 2) transcription factor responds to diverse oxidative and electrophilic environmental stresses by circumventing repression by Keap1, translocating to the nucleus, and activating cytoprotective genes. Nrf2 responses provide protection against chemical carcinogenesis, chronic inflammation, neurodegeneration, emphysema, asthma and sepsis in murine models. Nrf2 regulates the expression of a plethora of genes that detoxify oxidants and electrophiles and repair or remove damaged macromolecules, such as through proteasomal processing. However, many direct targets of Nrf2 remain undefined. Here, mouse embryonic fibroblasts (MEF) with either constitutive nuclear accumulation (Keap1−/−) or depletion (Nrf2−/−) of Nrf2 were utilized to perform chromatin-immunoprecipitation with parallel sequencing (ChIP-Seq) and global transcription profiling. This unique Nrf2 ChIP-Seq dataset is highly enriched for Nrf2-binding motifs. Integrating ChIP-Seq and microarray analyses, we identified 645 basal and 654 inducible direct targets of Nrf2, with 244 genes at the intersection. Modulated pathways in stress response and cell proliferation distinguish the inducible and basal programs. Results were confirmed in an in vivo stress model of cigarette smoke-exposed mice. This study reveals global circuitry of the Nrf2 stress response emphasizing Nrf2 as a central node in cell survival response. PMID:20460467

  14. Piper betle induces phase I & II genes through Nrf2/ARE signaling pathway in mouse embryonic fibroblasts derived from wild type and Nrf2 knockout cells

    PubMed Central

    2014-01-01

    Background Nuclear factor-erythroid 2 p45 related factor 2 (Nrf2) is a primary transcription factor, protecting cells from oxidative stress by regulating a number of antioxidants and phase II detoxifying enzymes. Dietary components such as sulforaphane in broccoli and quercetin in onions have been shown to be inducers of Nrf2. Piper betle (PB) grows well in tropical climate and the leaves are used in a number of traditional remedies for the treatment of stomach ailments and infections among Asians. The aim of this study was to elucidate the effect of Piper betle (PB) leaves extract in Nrf2 signaling pathway by using 2 types of cells; mouse embryonic fibroblasts (MEFs) derived from wild-type (WT) and Nrf2 knockout (N0) mice. Methods WT and N0 cells were treated with 5 and 10 μg/ml of PB for 10 and 12-h for the determination of nuclear translocation of Nrf2 protein. Luciferase reporter gene activity was performed to evaluate the antioxidant response element (ARE)-induction by PB. Real-time PCR and Western blot were conducted on both WT and N0 cells after PB treatment for the determination of antioxidant enzymes [superoxide dismutase (SOD1) and heme-oxygenase (HO-1)], phase I oxidoreductase enzymes [NAD(P)H: quinone oxidoreductase (NQO1)] and phase II detoxifying enzyme [glutathione S-transferase (GST)]. Results Nuclear translocation of Nrf2 by PB in WT cells was better after 10 h incubation compared to 12 h. Real time PCR and Western blot analysis showed increased expressions of Nrf2, NQO1 and GSTA1 genes with corresponding increases in glutathione, NQO1 and HO-1 proteins in WT cells. Reporter gene ARE was stimulated by PB as shown by ARE/luciferase assay. Interestingly, PB induced SOD1 gene and protein expressions in N0 cells but not in WT cells. Conclusion The results of this study confirmed that PB activated Nrf2-ARE signaling pathway which subsequently induced some phase I oxidoreductase, phase II detoxifying and antioxidant genes expression via ARE reporter

  15. 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.

  16. 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. PMID:26505797

  17. Molecular similarity guided optimization of novel Nrf2 activators with 1,2,4-oxadiazole core.

    PubMed

    Xu, Li-Li; Zhang, Xian; Jiang, Zheng-Yu; You, Qi-Dong

    2016-08-15

    DDO-7204 is a novel Nrf2 activator first identified through screening of in-house database by ARE-luciferase reporter gene assay. To further optimize this kind of Nrf2 activators efficiently, the hit-based substructure search was applied to screen the Specs database virtually. DDO-7204 contains three rings of A, B, C. SAR results showed that: for ring A, the cyclane substituent is beneficial for ARE inductivity. Enhanced flexibility of linker between ring A and ring B is not preferable for the Nrf2 activity. Ring A replaced by heterocyclic aromatic is beneficial for the Nrf2 activity. The resulting compound 7 was more potent than DDO-7204. Compound 7 can induce Nrf2 translocation into nuclear not only in HCT116 cells, but also in three normal cells such as L02, NCM460 and PC12 cells. The Nrf2-regualted genes, γ-GCS, NQO1 and HO-1, were up-regulated at a concentration-dependent manner. In addition, compound 7 showed cytoprotective effects on the three normal cells against the damage of H2O2. PMID:27316545

  18. 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. PMID:26165427

  19. 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

  20. 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

  1. 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. PMID:26765101

  2. 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

  3. Gold nanoparticles induce heme oxygenase-1 expression through Nrf2 activation and Bach1 export in human vascular endothelial cells

    PubMed Central

    Lai, Tsung-Hsuan; Shieh, Jiunn-Min; Tsou, Chih-Jen; Wu, Wen-Bin

    2015-01-01

    It has been reported that increased levels and activity of the heme oxygenase-1 (HO-1) protein ameliorate tissue injuries. In the present study, we investigated the effects and mechanisms of action of gold nanoparticles (AuNPs) on HO-1 protein expression in human vascular endothelial cells (ECs). The AuNPs induced HO-1 protein and mRNA expression in a concentration- and time-dependent manner. The induction was reduced by the thiol-containing antioxidants, including N-acetylcysteine and glutathione, but not by the non-thiol-containing antioxidants and inhibitors that block the enzymes for intracellular reactive oxygen species generation. The AuNPs enhanced Nrf2 protein levels but did not affect Nrf2 mRNA expression. In response to the AuNP treatment, the cytosolic Nrf2 translocated to the nucleus, and, concomitantly, Bach1 exited the nucleus and its tyrosine phosphorylation increased. The chromatin immunoprecipitation assay revealed that the translocated Nrf2 bound to the antioxidant-response element located in the E2 enhancer region of the HO-1 gene promoter and acted as a transcription factor. Although N-acetylcysteine inhibited the AuNP-induced Nrf2 nuclear translocation, the AuNPs did not promote intracellular reactive oxygen species production or endoplasmic reticulum stress in the ECs. Knockdown of Nrf2 expression by RNA interference significantly inhibited AuNP-induced HO-1 expression at the protein and mRNA levels. In summary, AuNPs enhance the levels and nuclear translocation of the Nrf2 protein and Bach1 export/tyrosine phosphorylation, leading to Nrf2 binding to the HO-1 E2 enhancer promoter region to drive HO-1 expression in ECs. This study, together with our parallel findings, demonstrates that AuNPs can act as an HO-1 inducer, which may partially contribute to their anti-inflammatory bioactivity in human vascular ECs. PMID:26445536

  4. 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. PMID:26546103

  5. 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

  6. 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. PMID:27145762

  7. Vitamin A Supplementation Alleviates Extrahepatic Cholestasis Liver Injury through Nrf2 Activation

    PubMed Central

    Wang, Guiyang; Xiu, Peng; Li, Fu; Xin, Cheng; Li, Kewei

    2014-01-01

    Aim. To investigate the role of vitamin A in liver damage induced by bile duct ligation (BDL) in rats. Methods. Thirty male Wistar rats were randomly divided into three groups: SHAM group, BDL group, and BDL + VitA group . The concentrations of retinol and retinyl palmitate in the liver were analyzed using HPLC, and liver function was evaluated by the level of TBIL, ALT, AST, and ALP in serum. Hepatic oxidative status was estimated by measuring T-SOD, CAT, GSH, MDA, and AOPP. Nrf2 expression was assessed using immunohistochemistry and western blotting, and EMSA was performed to determine Nrf2 DNA-binding activity. The expression of the downstream factors such as Ho1 and Nqo1 was also examined using immunohistochemistry and western blotting assays. Results. Vitamin A treatment restored levels of retinoids in liver, improved liver function, alleviated oxidative stress, and facilitated the translocation of Nrf2 to the nucleus in the experimental obstructive jaundice. Vitamin A was also found to increase the expression of Nrf2 downstream proteins such as Ho1 and Nqo1. Conclusion. Vitamin A was here found to ameliorate cholestatic liver injury. This effect may be related to the activation of Nrf2/ARE pathway in bile duct ligation rats. PMID:25126202

  8. 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

  9. 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

  10. 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

  11. 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. PMID:26026609

  12. 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

  13. 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. PMID:26889770

  14. Roles of Nrf2 in cell proliferation and differentiation.

    PubMed

    Murakami, Shohei; Motohashi, Hozumi

    2015-11-01

    The Keap1-Nrf2 system plays pivotal roles in defense mechanisms by regulating cellular redox homeostasis. Nrf2 is an inducible transcription factor that activates a battery of genes encoding antioxidant proteins and phase II enzymes in response to oxidative stress and electrophilic xenobiotics. The activity of Nrf2 is regulated by Keap1, which promotes the ubiquitination and subsequent degradation of Nrf2 under normal conditions and releases the inhibited Nrf2 activity upon exposure to the stresses. Though an impressive contribution of the Keap1-Nrf2 system to the protection from exogenous and endogenous electrophilic insults has been well established, a line of evidence has suggested that the Keap1-Nrf2 system has various novel functions, particularly in cell proliferation and differentiation. Because the proliferation and differentiation of diverse cell types are often influenced and modulated by the cellular redox balance, Nrf2 has been considered to control these cellular processes by regulating the cellular levels of reactive oxygen species (ROS). In addition, analyses of the genome-wide distribution of Nrf2 have identified new sets of Nrf2 target genes whose products are involved in cell proliferation and differentiation but not necessarily in the regulation of oxidative stress. Considering the most characteristic features of Nrf2 as an inducible transcription factor, a newly emerged concept proposes that the Keap1-Nrf2 system translates environmental stresses into regulatory network signals in cell fate determination. In this review, we introduce the contribution of Nrf2 to lineage-specific differentiation, maintenance and differentiation of stem cells, and proliferation of normal and cancer cells, and we discuss how the response to fluctuating environments modulates cell behavior through the Keap1-Nrf2 system. PMID:26119783

  15. 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

  16. Protocatechuic acid induces antioxidant/detoxifying enzyme expression through JNK-mediated Nrf2 activation in murine macrophages.

    PubMed

    Varì, Rosaria; D'Archivio, Massimo; Filesi, Carmelina; Carotenuto, Simona; Scazzocchio, Beatrice; Santangelo, Carmela; Giovannini, Claudio; Masella, Roberta

    2011-05-01

    Protocatechuic acid (PCA) is a main metabolite of anthocyanins, whose daily intake is much higher than that of other polyphenols. PCA has biological effects, e.g., it induces the antioxidant/detoxifying enzyme gene expression. This study was aimed at defining the molecular mechanism responsible for PCA-induced over-expression of glutathione (GSH) peroxidase (GPx) and GSH reductase (GR) in J774 A.1 macrophages. New evidence is provided that PCA increases GPx and GR expression by inducing C-JUN NH(2)-terminal kinase (JNK)-mediated phosphorylation of Nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2). RNA and proteins were extracted from cells treated with PCA (25 μM) for different time points. Quantitative real-time polymerase chain reaction and immunoblotting analyses showed a rapid increase in mRNA (>60%) and protein (>50%) for both the enzymes. This was preceded by the up-regulation of Nrf2, in terms of mRNA and protein, and by its significant activation as assessed by increased Nrf2 phosphorylation and nuclear translocation (+60%). By using specific kinase inhibitors and detecting the activated form, we showed that JNK was the main upstream kinase responsible for Nrf2 activation. Convincing evidence is provided of a causal link between PCA-induced Nrf2 activation and increased enzyme expression. By silencing Nrf2 and using a JNK inhibitor, enzyme enhancement was counteracted. Finally, with the ChIP assay, we demonstrated that PCA-activated Nrf2 specifically bound ARE sequences in enzyme gene promoters. Our study demonstrates for the first time that PCA improves the macrophage endogenous antioxidant potential by a mechanism in which JNK-mediated Nrf2 activation plays an essential role. This knowledge could contribute to novel diet-based approaches aimed at counteracting oxidative injury by reinforcing endogenous defences. PMID:20621462

  17. Chemoprevention of dietary digitoflavone on colitis-associated colon tumorigenesis through inducing Nrf2 signaling pathway and inhibition of inflammation

    PubMed Central

    2014-01-01

    Background Nuclear factor-erythroid 2-related factor 2 (Nrf2) has emerged as a novel target for the prevention of colorectal cancer (CRC). Many chemopreventive compounds associated with Nrf2 activation are effective in preclinical systems and many on-going clinical trials are showing promising findings. In present study we evaluated the cytoprotective effect and chemopreventive properties of dietary digitoflavone. Method A cell based Antioxidant Response Element (ARE)-driven luciferase reporter system was applied to screen potential Nrf2 activators. Activation of Nrf2 by digitoflavone was confirmed through mRNA, protein and GSH level assay in Caco-2 cell line. The cytoprotective effect of digitoflavone was evaluated in H2O2-induced oxidative stress model and further signaling pathways analysis was used to determine the target of digitoflavone induced Nrf2 activation. An AOM-DSS induced colorectal cancer model was used to assess the chemopreventive effect of digitoflavone. Result Micromolarity (10 μM) level of digitoflavone increased Nrf2 expressing, nuclear translocation and expression of downstream phase II antioxidant enzymes. Furthermore, digitoflavone decreased H2O2-induced oxidative stress and cell death via p38 MAPK-Nrf2/ARE pathway. In vivo study, 50 mg/kg digitoflavone significantly reduced AOM-DSS induced tumor incidence, number and size. Conclusion These observations suggest that digitoflavone is a novel Nrf2 pathway activator, and protects against oxidative stress-induced cell injury. The results of the present study add further evidence of the molecular mechanisms that allow digitoflavone to exert protective effects and reaffirm its potential role as a chemopreventive agent in colorectal carcinogenesis. PMID:24602443

  18. 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.

  19. 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

    2016-01-01

    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. PMID:27561946

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

    PubMed Central

    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

    2016-01-01

    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. PMID:27561946

  1. 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

    2016-02-01

    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. PMID:26102008

  2. Functional Role of NRF2 in Cervical Carcinogenesis

    PubMed Central

    Jiao, Shu-Juan; Zheng, Jian-He; xiao, Jing-Bao; Hasim, Ayshamgul

    2015-01-01

    Nuclear factor erythroid-2-related factor 2 (NFE2L2) is a transcription factor associated with resistance to chemotherapy and increased tumor growth. NRF2 is repressed by the inhibitor Keap1. The Keap1-NRF2 pathway is dysfunctional in multiple tumor types. Among Uighur women, the incidence of cervical squamous cell carcinoma (CSCC) and cervical intraepithelial neoplasia (CIN) was associated with elevated nuclear expression of NRF2 and decreased cytoplasmic expression of Keap1. Up-regulation of nuclear NRF2 was significantly associated with reduced cytoplasmic Keap1 expression. NRF2 positivity and Keap1 negativity were frequently associated with more advanced tumors (i.e., higher histological grade, lymph node involvement, and higher tumor stages) (p<0.05 for all). Methylated CpG islands in the Keap1 gene promoter in cervical cancer tissue were identified using MassARRAY. Moreover, promoter hypermethylation of this gene was significantly associated with decreased protein expression and increased nuclear NRF2 expression in cervical cancer tissues. Overexpression and knockdown of NRF2 in CSCC cell lines showed that NRF2 promotes proliferation, inhibits apoptosis, and enhances migration and invasion. These studies support the concept that epigenetic changes regulate expression of Keap1 in cervical cancer tissues. The association of NRF2 expression with aggressive tumor behavior suggests that NRF2 may be a marker of poor prognosis in patients with cervical cancer. PMID:26247201

  3. Myocardial ischemic reperfusion induces de novo Nrf2 protein translation

    PubMed Central

    Xu, Beibei; Zhang, Jack; Strom, Joshua; Lee, Sang; Chen, Qin M.

    2016-01-01

    Nrf2 is a bZIP transcription factor regulating the expression of antioxidant and detoxification genes. We have found that Nrf2 knockout mice have an increased infarction size in response to regional ischemic reperfusion and have a reduced degree of cardiac protection by means of ischemic preconditioning. With cycles of brief ischemia and reperfusion (5′I/5′R) that induce cardiac protection in wild type mice, an elevated Nrf2 protein was observed without prior increases of Nrf2 mRNA. When an mRNA species is being translated into a protein, it is occupied by multiple ribosomes. The level of ribosome-associated Nrf2 mRNA increased following cycles of 5′I/5′R, supporting de novo Nrf2 protein translation. A dicistronic reporter assay indicated a role of the 5′ untranslated region (5′ UTR) of Nrf2 mRNA in oxidative stress induced Nrf2 protein translation in isolated cardiomyocytes. Western blot analyses after isolation of proteins binding to biotinylated Nrf2 5′ UTR from the myocardium or cultured cardiomyocytes demonstrated that cycles of 5′I/5′R or oxidants caused an increased association of La protein with Nrf2 5′ UTR. Ribonucleoprotein complex immunoprecipitation assays confirmed such association indeed occurring in vivo. Knocking down La using siRNA was able to prevent Nrf2 protein elevation by oxidants in cultured cardiomyocytes and by cycles of 5′I/5′R in the myocardium. Our data point out a novel mechanism of cardiac protection by de novo Nrf2 protein translation involving interaction of La protein with 5′ UTR of Nrf2 mRNA in cardiomyocytes. PMID:24915518

  4. Nrf2 Enhances Cholangiocyte Expansion in Pten-Deficient Livers

    PubMed Central

    Taguchi, Keiko; Hirano, Ikuo; Itoh, Tohru; Tanaka, Minoru; Miyajima, Atsushi; Suzuki, Akira

    2014-01-01

    Keap1-Nrf2 system plays a central role in the stress response. While Keap1 ubiquitinates Nrf2 for degradation under unstressed conditions, this Keap1 activity is abrogated in response to oxidative or electrophilic stresses, leading to Nrf2 stabilization and coordinated activation of cytoprotective genes. We recently found that nuclear accumulation of Nrf2 is significantly increased by simultaneous deletion of Pten and Keap1, resulting in the stronger activation of Nrf2 target genes. To clarify the impact of the cross talk between the Keap1-Nrf2 and Pten–phosphatidylinositide 3-kinase–Akt pathways on the liver pathophysiology, in this study we have conducted closer analysis of liver-specific Pten::Keap1 double-mutant mice (Pten::Keap1-Alb mice). The Pten::Keap1-Alb mice were lethal by 1 month after birth and displayed severe hepatomegaly with abnormal expansion of ductal structures comprising cholangiocytes in a Nrf2-dependent manner. Long-term observation of Pten::Keap1-Alb::Nrf2+/− mice revealed that the Nrf2-heterozygous mice survived beyond 1 month but developed polycystic liver fibrosis by 6 months. Gsk3 directing the Keap1-independent degradation of Nrf2 was heavily phosphorylated and consequently inactivated by the double deletion of Pten and Keap1 genes. Thus, liver-specific disruption of Keap1 and Pten augments Nrf2 activity through inactivation of Keap1-dependent and -independent degradation of Nrf2 and establishes the Nrf2-dependent molecular network promoting the hepatomegaly and cholangiocyte expansion. PMID:24379438

  5. 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

  6. Translational control of Nrf2 within the open reading frame

    PubMed Central

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

    2013-01-01

    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. PMID:23806685

  7. 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

  8. 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.

  9. 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.

  10. Oxidative stress and dysfunctional NRF2 underlie pachyonychia congenita phenotypes.

    PubMed

    Kerns, Michelle L; Hakim, Jill M C; Lu, Rosemary G; Guo, Yajuan; Berroth, Andreas; Kaspar, Roger L; Coulombe, Pierre A

    2016-06-01

    Palmoplantar keratoderma (PPK) are debilitating lesions that arise in individuals with pachyonychia congenita (PC) and feature upregulation of danger-associated molecular patterns and skin barrier regulators. The defining features of PC-associated PPK are reproduced in mice null for keratin 16 (Krt16), which is commonly mutated in PC patients. Here, we have shown that PPK onset is preceded by oxidative stress in footpad skin of Krt16-/- mice and correlates with an inability of keratinocytes to sustain nuclear factor erythroid-derived 2 related factor 2-dependent (NRF2-dependent) synthesis of the cellular antioxidant glutathione (GSH). Additionally, examination of plantar skin biopsies from individuals with PC confirmed the presence of high levels of hypophosphorylated NRF2 in lesional tissue. In Krt16-/- mice, genetic ablation of Nrf2 worsened spontaneous skin lesions and accelerated PPK development in footpad skin. Hypoactivity of NRF2 in Krt16-/- footpad skin correlated with decreased levels or activity of upstream NRF2 activators, including PKCδ, receptor for activated C kinase 1 (RACK1), and p21. Topical application of the NRF2 activator sulforaphane to the footpad of Krt16-/- mice prevented the development of PPK and normalized redox balance via regeneration of GSH from existing cellular pools. Together, these findings point to oxidative stress and dysfunctional NRF2 as contributors to PPK pathogenesis, identify K16 as a regulator of NRF2 activation, and suggest that pharmacological activation of NRF2 should be further explored for PC treatment. PMID:27183391

  11. 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

  12. Nitro-linoleic acid inhibits vascular smooth muscle cell proliferation via the Keap1/Nrf2 signaling pathway

    PubMed Central

    Villacorta, Luis; Zhang, Jifeng; Garcia-Barrio, Minerva T.; Chen, Xi-lin; Freeman, Bruce A.; Chen, Yuqing E.; Cui, Taixing

    2007-01-01

    Nitroalkenes, the nitration products of unsaturated fatty acids formed via NO-dependent oxidative reactions, have been demonstrated to exert strong biological actions in endothelial cells and monocytes/macrophages; however, little is known about their effects on vascular smooth muscle cells (VSMCs). The present study examined the role of nitro-linoleic acid (LNO2) in the regulation of VSMC proliferation. We observed that LNO2 inhibited VSMC proliferation in a dose-dependent manner. In addition, LNO2 induced growth arrest of VSMCs in the G1/S phase of the cell cycle with an upregulation of the cyclin-dependent kinase inhibitor p27kip1. Furthermore, LNO2 triggered nuclear factor-erythroid 2-related factor 2 (Nrf2) nuclear translocation and activation of the antioxidant-responsive element-driven transcriptional activity via impairing Kelch-like ECH-associating protein 1 (Keap1)-mediated negative control of Nrf2 activity in VSMCs. LNO2 upregulated the expression of Nrf2 protein levels, but not mRNA levels, in VSMCs. A forced activation of Nrf2 led to an upregulation of p27kip1 and growth inhibition of VSMCs. In contrast, knock down of Nrf2 using an Nrf2 siRNA approach reversed the LNO2-induced upregulation of p27kip1 and inhibition of cellular proliferation in VSMCs. These studies provide the first evidence that nitroalkene LNO2 inhibits VSMC proliferation through activation of the Keap1/Nrf2 signaling pathway, suggesting an important role of nitroalkenes in vascular biology. PMID:17468336

  13. Inhibition of cytochrome P450 2E1 and activation of transcription factor Nrf2 are renoprotective in myoglobinuric acute kidney injury.

    PubMed

    Wang, Zhe; Shah, Sudhir V; Liu, Hua; Baliga, Radhakrishna

    2014-08-01

    Rhabdomyolysis accounts for ∼10% of acute kidney injuries. In glycerol-induced myoglobinuric acute kidney injury, we found an increase in the nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear protein, a key redox-sensitive transcription factor, and Nrf2-regulated genes and proteins including upregulation of heme oxygenase-1. In in vitro studies, pretreatment of LLC-PK1 cells with an activator of Nrf2 before myoglobin exposure significantly decreased oxidant generation and cytotoxicity, whereas Nrf2 inhibition and gene silencing exacerbated the injury. Chlormethiazole, a specific CYP2E1 transcription inhibitor, prevented an increase in catalytic iron in the kidneys, decreased oxidative stress, blocked nuclear translocation of the Nrf2 protein, decreased heme oxygenase-1 upregulation, and provided functional and histological protection against acute kidney injury. CYP2E1 inhibitors and gene silencing in renal tubular epithelial cells significantly decreased reactive oxygen species generation and provided marked protection against myoglobin-induced cytotoxicity. Thus, during CYP2E1-induced oxidative stress, the transcription factor Nrf2 has a pivotal role in the early adaptive response. Inhibition of CYP2E1 coupled with the prior induction of Nrf2 may be a valuable tool to reduce CYP2E1-mediated rhabdomyolysis-induced acute kidney injury. PMID:24717297

  14. Withaferin A induces heme oxygenase (HO-1) expression in endothelial cells via activation of the Keap1/Nrf2 pathway.

    PubMed

    Heyninck, Karen; Sabbe, Linde; Chirumamilla, Chandra Sekhar; Szarc Vel Szic, Katarzyna; Vander Veken, Pieter; Lemmens, Kristien J A; Lahtela-Kakkonen, Maija; Naulaerts, Stefan; Op de Beeck, Ken; Laukens, Kris; Van Camp, Guy; Weseler, Antje R; Bast, Aalt; Haenen, Guido R M M; Haegeman, Guy; Vanden Berghe, Wim

    2016-06-01

    Withaferin A (WA), a natural phytochemical derived from the plant Withania somnifera, is a well-studied bioactive compound exerting a broad spectrum of health promoting effects. To gain better insight in the potential therapeutic capacity of WA, we evaluated the transcriptional effects of WA on primary human umbilical vein endothelial cells (HUVECs) and an endothelial cell line (EA.hy926). RNA microarray analysis of WA treated HUVEC cells demonstrated increased expression of the antioxidant gene heme oxygenase (HO-1). Transcriptional regulation of this gene is strongly dependent on the transcription factor NF-E2-related factor 2 (Nrf2), which senses chemical changes in the cell and coordinates transcriptional responses to maintain chemical homeostasis via expression of antioxidant genes and cytoprotective Phase II detoxifying enzymes. Under normal conditions, Nrf2 is kept in the cytoplasm by Kelch-like ECH-associated protein 1 (Keap1), an adaptor protein controlling the half-life of Nrf2 via constant proteasomal degradation. In this study we demonstrate that WA time- and concentration-dependently induces HO-1 expression in endothelial cells via upregulation and increased nuclear translocation of Nrf2. According to the crucial negative regulatory role of Keap1 in Nrf2 expression levels, a direct interaction of WA with Keap1 could be demonstrated. In vitro and in silico evaluations suggest that specific cysteine residues in Keap1 might be involved in the interaction with WA. PMID:27045103

  15. Performance of the N/TERT epidermal model for skin sensitizer identification via Nrf2-Keap1-ARE pathway activation.

    PubMed

    Alloul-Ramdhani, Mariam; Tensen, Cornelis P; El Ghalbzouri, Abdoelwaheb

    2014-08-01

    Animal testing of chemical ingredients for cosmetic purposes is prohibited. Therefore there is an urgent need for in vitro models to identify chemical allergens. In human skin, keratinocytes (KCs) are abundantly present and are key players in initiation of allergic contact dermatitis. One of the pathways that has been shown to be induced by sensitizers is the Keap1-Nrf2-ARE pathway. In this study we compared the response of four keratinocyte-based models including (a) primary human KCs, (b) N/TERT monolayer cultures, (c) the Leiden Epidermal models (LEMs) and (d) the N/TERT epidermal models (NEMs). All keratinocyte-based models were subjected to chemical exposure of the sensitizer 2,4-dinitrochlorobenzene (DNCB) and irritant Sodium dodecyl sulfate (SDS) at nontoxic concentrations. Activation of the Keap1-Nrf2-ARE pathway was evaluated by measuring Nrf2 protein levels as well as nuclear translocation and activation of transcriptional targets of Nrf2. Results show that the Keap1-Nrf2-ARE pathway is activated by the sensitizer DNCB in monolayer keratinocytes and as well as the LEMs and NEMs and not by the irritant SDS. Collectively our data demonstrate that the N/TERT models respond similarly as primary KCs and could therefore serve as an alternative model for skin sensitizer identification, thereby overcoming the need for primary skin tissue. PMID:24794257

  16. 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 ...

  17. Nrf2 links epidermal barrier function with antioxidant defense.

    PubMed

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

    2012-05-01

    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

  18. The emerging role of Nrf2 in dermatotoxicology

    PubMed Central

    Tan, Nguan S; Wahli, Walter

    2014-01-01

    The nuclear factor erythroid 2-related factor 2 (Nrf2) is best known for its role in resistance to oxidant stress. In this issue of EMBO Molecular Medicine, Nrf2-prolonged genetic activation is shown with devastating effects on skin homeostasis. The study provides novel molecular insights into poison-induced chloracne and metabolizing acquired dioxin-induced skin hamartomas or MADISH. PMID:24521743

  19. 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. PMID:26718502

  20. 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. PMID:22781654

  1. 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. PMID:27208483

  2. Carnosic acid protects against acetaminophen-induced hepatotoxicity by potentiating Nrf2-mediated antioxidant capacity in mice

    PubMed Central

    Guo, Qi; Shen, Zhiyang; Yu, Hongxia; Lu, Gaofeng; Yu, Yong; Liu, Xia

    2016-01-01

    Acetaminophen (APAP) overdose is one of the most common causes of acute liver failure. The study aimed to investigate the protective effect of carnosic acid (CA) on APAP-induced acute hepatotoxicity and its underlying mechanism in mice. To induce hepatotoxicity, APAP solution (400 mg/kg) was administered into mice by intraperitoneal injection. Histological analysis revealed that CA treatment significantly ameliorated APAP-induced hepatic necrosis. The levels of both alanine aminotransferase (ALT) and aspartate transaminase (AST) in serum were reduced by CA treatment. Moreover, CA treatment significantly inhibited APAP-induced hepatocytes necrosis and lactate dehydrogenase (LDH) releasing. Western blot analysis showed that CA abrogated APAP-induced cleaved caspase-3, Bax and phosphorylated JNK protein expression. Further results showed that CA treatment markedly inhibited APAP-induced pro-inflammatory cytokines TNF-α, IL-1β, IL-6 and MCP-1 mRNA expression and the levels of phosphorylated IκBα and p65 protein in the liver. In addition, CA treatment reduced APAP- induced hepatic malondialdehyde (MDA) contents and reactive oxygen species (ROS) accumulation. Conversely, hepatic glutathione (GSH) level was increased by administration of CA in APAP-treated mice. Mechanistically, CA facilitated Nrf2 translocation into nuclear through blocking the interaction between Nrf2 and Keap1, which, in turn, upregulated anti-oxidant genes mRNA expression. Taken together, our results indicate that CA facilitates Nrf2 nuclear translocation, causing induction of Nrf2-dependent genes, which contributes to protection from acetaminophen hepatotoxicity. PMID:26807019

  3. 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

  4. Isoorientin induces Nrf2 pathway-driven antioxidant response through phosphatidylinositol 3-kinase signaling.

    PubMed

    Lim, Ju Hee; Park, Hae-Suk; Choi, Jung-Kap; Lee, Ik-Soo; Choi, Hyun Jin

    2007-12-01

    Because oxidative stress is involved in the pathogenesis of various chronic diseases and the aging process, antioxidants that can increase the intrinsic antioxidant potency are proposed as desirable therapeutic agents to counteract oxidative stress-related diseases. NF-E2-related factor-2 (Nrf2) is a transcription factor that regulates important antioxidant and phase II detoxification genes, and therefore, the molecule that regulates nuclear translocation of Nrf2 and the induction of antioxidative proteins is thought to be a promising candidate as a cytoprotective agent for oxidative stress. In the present study, we show that isoorientin (luteolin 6-C-beta-D-glucoside) obtained from the leaves of Sasa borealis upregulates and activates Nrf2, and has protective ability against oxidative damage caused by reactive oxygen intermediates in HepG2 cells. Isoorientin induces increase in the level of antioxidant enzyme proteins, especially NQO1, and the cytoprotective and antioxidative effects of isoorientin are PI3K/Akt pathway-dependent. Together with direct radical scavenging activity, the novel effect of isoorientin on the regulation of antioxidative gene expression provides attractive strategy to prevent diseases associated with oxidative stress and attenuate the progress of the diseases. PMID:18254247

  5. Nrf2 protects mitochondrial decay by oxidative stress.

    PubMed

    Strom, Joshua; Xu, Beibei; Tian, Xiuqing; Chen, Qin M

    2016-01-01

    Sublethal levels of oxidative stress are commonly associated with various pathophysiological conditions. Cardiomyocytes have the highest content of mitochondria among all cell types, allowing the study of mitochondria in cells surviving oxidative stress and address whether nuclear factor-erythroid-derived 2-related factor 2 (Nrf2) can reverse these changes. Mitochondria normally exist in elaborated networks, which were replaced by predominately individual punctuate mitochondria 24 h after exposure to a nonlethal dose of H2O2. Electron microscopy revealed that cells surviving H2O2 show swelling of mitochondria with disorganized cristae and areas of condensation. Measurements of functional mitochondria showed a H2O2 dose-dependent decrease over a course of 5 d. At the protein and mRNA levels, cells surviving H2O2 treatment show a reduction of mitochondrial components, cytochrome c, and cytochrome b. Nrf2 overexpression prevented H2O2 from inducing mitochondria morphologic changes and reduction of cytochrome b/c. Although Nrf2 is known as a transcription factor regulating antioxidant and detoxification genes, Nrf2 overexpression did not significantly reduce the level of protein oxidation. Instead, Nrf2 was found to associate with the outer mitochondrial membrane. Mitochondria prepared from the myocardium of Nrf2 knockout mice are more sensitive to permeability transition. Our data suggest that Nrf2 protects mitochondria from oxidant injury likely through direct interaction with mitochondria. PMID:26340923

  6. 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. PMID:27075625

  7. 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. PMID:26117322

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

    PubMed

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

    2016-01-01

    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

  9. 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

  10. 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

  11. Nrf2-Mediated Regulation of Skeletal Muscle Glycogen Metabolism.

    PubMed

    Uruno, Akira; Yagishita, Yoko; Katsuoka, Fumiki; Kitajima, Yasuo; Nunomiya, Aki; Nagatomi, Ryoichi; Pi, Jingbo; Biswal, Shyam S; Yamamoto, Masayuki

    2016-06-01

    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

  12. Oxidative stress and dysfunctional NRF2 underlie pachyonychia congenita phenotypes

    PubMed Central

    Kerns, Michelle L.; Hakim, Jill M.C.; Lu, Rosemary G.; Guo, Yajuan; Berroth, Andreas; Kaspar, Roger L.

    2016-01-01

    Palmoplantar keratoderma (PPK) are debilitating lesions that arise in individuals with pachyonychia congenita (PC) and feature upregulation of danger-associated molecular patterns and skin barrier regulators. The defining features of PC-associated PPK are reproduced in mice null for keratin 16 (Krt16), which is commonly mutated in PC patients. Here, we have shown that PPK onset is preceded by oxidative stress in footpad skin of Krt16–/– mice and correlates with an inability of keratinocytes to sustain nuclear factor erythroid–derived 2 related factor 2–dependent (NRF2-dependent) synthesis of the cellular antioxidant glutathione (GSH). Additionally, examination of plantar skin biopsies from individuals with PC confirmed the presence of high levels of hypophosphorylated NRF2 in lesional tissue. In Krt16–/– mice, genetic ablation of Nrf2 worsened spontaneous skin lesions and accelerated PPK development in footpad skin. Hypoactivity of NRF2 in Krt16–/– footpad skin correlated with decreased levels or activity of upstream NRF2 activators, including PKCδ, receptor for activated C kinase 1 (RACK1), and p21. Topical application of the NRF2 activator sulforaphane to the footpad of Krt16–/– mice prevented the development of PPK and normalized redox balance via regeneration of GSH from existing cellular pools. Together, these findings point to oxidative stress and dysfunctional NRF2 as contributors to PPK pathogenesis, identify K16 as a regulator of NRF2 activation, and suggest that pharmacological activation of NRF2 should be further explored for PC treatment. PMID:27183391

  13. Kaposi's Sarcoma-Associated Herpesvirus Induces Nrf2 during De Novo Infection of Endothelial Cells to Create a Microenvironment Conducive to Infection

    PubMed Central

    Gjyshi, Olsi; Bottero, Virginie; Veettil, Mohanan Valliya; Dutta, Sujoy; Singh, Vivek Vikram; Chikoti, Leela; Chandran, Bala

    2014-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma (KS) and primary effusion B-cell lymphoma. KSHV induces reactive oxygen species (ROS) early during infection of human dermal microvascular endothelial (HMVEC-d) cells that are critical for virus entry. One of the downstream targets of ROS is nuclear factor E2-related factor 2 (Nrf2), a transcription factor with important anti-oxidative functions. Here, we show that KS skin lesions have high Nrf2 activity compared to healthy skin tissue. Within 30 minutes of de novo KSHV infection of HMVEC-d cells, we observed Nrf2 activation through ROS-mediated dissociation from its inhibitor Keap1, Ser-40 phosphorylation, and subsequent nuclear translocation. KSHV binding and consequent signaling through Src, PI3-K and PKC-ζ were also important for Nrf2 stability, phosphorylation and transcriptional activity. Although Nrf2 was dispensable for ROS homeostasis, it was essential for the induction of COX-2, VEGF-A, VEGF-D, Bcl-2, NQO1, GCS, HO1, TKT, TALDO and G6PD gene expression in KSHV-infected HMVEC-d cells. The COX-2 product PGE2 induced Nrf2 activity through paracrine and autocrine signaling, creating a feed-forward loop between COX-2 and Nrf2. vFLIP, a product of KSHV latent gene ORF71, induced Nrf2 and its target genes NQO1 and HO1. Activated Nrf2 colocalized with the KSHV genome as well as with the latency protein LANA-1. Nrf2 knockdown enhanced ORF73 expression while reducing ORF50 and other lytic gene expression without affecting KSHV entry or genome nuclear delivery. Collectively, these studies for the first time demonstrate that during de novo infection, KSHV induces Nrf2 through intricate mechanisms involving multiple signal molecules, which is important for its ability to manipulate host and viral genes, creating a microenvironment conducive to KSHV infection. Thus, Nrf2 is a potential attractive target to intervene in KSHV infection and the associated maladies. PMID:25340789

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

    PubMed

    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

  15. Activation of the NRF2-ARE signalling pathway by the Lentinula edodes polysaccharose LNT alleviates ROS-mediated cisplatin nephrotoxicity.

    PubMed

    Chen, Qian; Peng, Huixia; Dong, Lei; Chen, Lijuan; Ma, Xiaobin; Peng, Yuping; Dai, Shejiao; Liu, Qiang

    2016-07-01

    The nephrotoxicity of cisplatin (cis-DDP) limits its general clinical applications. Lentinan (LNT), a dextran extracted from the mushroom Lentinula edodes, has been shown to have multiple pharmacological activities. The primary objective of the current study was to determine whether and how LNT alleviates cis-DDP- induced cytotoxicity in HK-2 cells and nephrotoxicity in mice. LNT did not interfere with cisplatin's anti-tumour efficacy in vitro and functioned cooperatively with cis-DDP to inhibit activity in HeLa and A549 tumour cells. LNT alleviated the cis-DDP-induced decrease in HK-2 cell viability, caspase-3 activation and cleavage of the DNA repair enzyme PARP, decreased HK-2 cell apoptosis and inhibited reactive oxygen species (ROS) accumulation in HK-2 cells. The inhibitor of ROS (N-acetyl-L-cysteine, NAC) could decreased the apoptosis of HK-2 cell. In addition, LNT significantly prevented cis-DDP-induced kidney injury in vivo. LNT itself could not eliminate ROS levels in vitro. Further studies demonstrated that LNT induced NF-E2 p45-related factor 2 (Nrf2) protein and mRNA expression in a time- and dose-dependent manner. LNT promoted Nrf2 translocation to the nucleus and binding to the antioxidant-response element (ARE) sequence and induced the transcription and translation of heme oxygenase 1 (HO-1), aldo-keto reductases 1C1 and 1C2 (AKR1C), and NADP(H):quinone oxidoreductase 1 (NQO1). Finally, we used hNrf2 siRNA and an Nrf2 agonist (tBHQ) to inhibit or enhance Nrf2 expression. The results demonstrated that the LNT-mediated alleviation of cis-DDP-induced nephrotoxicity was achieved by preventing the accumulation of ROS in a manner that depended on the activation of the Nrf2-ARE signalling pathway. PMID:27093515

  16. 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. PMID:22946519

  17. 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. PMID:25974399

  18. 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

  19. 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

    2016-06-01

    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. PMID:27259148

  20. 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

  1. NRF2 Regulates PINK1 Expression under Oxidative Stress Conditions

    PubMed Central

    Murata, Hitoshi; Takamatsu, Hitoshi; Liu, Sulai; Kataoka, Ken; Huh, Nam-ho; Sakaguchi, Masakiyo

    2015-01-01

    Mutations of the PTEN-induced putative kinase 1 (PINK1) gene are a cause of autosomal recessive forms of Parkinson’s disease. Recent studies have revealed that PINK1 is an essential factor for controlling mitochondrial quality, and that it protects cells from oxidative stresses. Although there has been considerable progress in the elucidation of various aspects of PINK1 protein regulation such as activation, stability and degradation, the transcriptional regulation of PINK1 mRNA under stress conditions remains unclear. In this study, we found that nuclear factor (erythroid-derived 2)-like 2 (NRF2), an antioxidant transcription factor, regulates PINK1 expression under oxidative stress conditions. Damaged mitochondria arising from stress conditions induced NRF2-dependent transcription of the PINK1 gene through production of reactive oxygen species (ROS). Either an ROS scavenger or forced expression of KEAP1, a potent inhibitory partner to NRF2, restricted PINK1 expression induced by activated NRF2. Transcriptionally up-regulated PINK1 diminished oxidative stress-associated cell death. The results indicate that PINK1 expression is positively regulated by NRF2 and that the NRF2-PINK1 signaling axis is deeply involved in cell survival. PMID:26555609

  2. 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

  3. Oncogenic KRAS confers chemoresistance by upregulating NRF2

    PubMed Central

    Tao, Shasha; Wang, Shue; Moghaddam, Seyed Javad; Ooi, Aikseng; Chapman, Eli; Wong, Pak K.; Zhang, Donna D.

    2014-01-01

    Oncogenic KRAS mutations found in 20–30% of all non-small cell lung cancers (NSCLC) are associated with chemoresistance and poor prognosis. Here we demonstrate that activation of the cell protective stress response gene NRF2 by KRAS is responsible for its ability to promote drug resistance. RNAi-mediated silencing of NRF2 was sufficient to reverse resistance to cisplatin elicited by ectopic expression of oncogenic KRAS in NSCLC cells. Mechanistically, KRAS increased NRF2 gene transcription through a TPA response element (TRE) located in the NRF2 promoter. In a mouse model of mutant KrasG12D-induced lung cancer, we found that suppressing the NRF2 pathway with the chemical inhibitor brusatol enhanced the antitumor efficacy of cisplatin. Co-treatment reduced tumor burden and improved survival. Our findings illuminate the mechanistic details of KRAS-mediated drug resistance and provide a preclinical rationale to improve the management of lung tumors harboring KRAS mutations with NRF2 pathway inhibitors. PMID:25339352

  4. Role of Nrf2 in chronic liver disease

    PubMed Central

    Tang, Wei; Jiang, Yong-Fang; Ponnusamy, Murugavel; Diallo, Mamadou

    2014-01-01

    Nuclear erythroid 2-related factor 2 (Nrf2) is a central regulator of antioxidative response elements-mediated gene expression. It has a significant role in adaptive responses to oxidative stress by interacting with the antioxidant response element, which induces the expression of a variety of downstream targets aimed at cytoprotection. Previous studies suggested oxidative stress and associated damage could represent a common link between different forms of diseases. Oxidative stress has been implicated in various liver diseases, including viral hepatitis, nonalcoholic fatty liver disease/steatohepatitis, alcoholic liver disease and drug-induced liver injury. Nrf2 activation is initiated by oxidative or electrophilic stress, and aids in the detoxification and elimination of potentially harmful exogenous chemicals and their metabolites. The expression of Nrf2 has been observed throughout human tissue, with high expression in detoxification organs, especially the liver. Thus, Nrf2 may serve as a major regulator of several cellular defense associated pathways by which hepatic cells combat oxidative stress. We review the relevant literature concerning the crucial role of Nrf2 and its signaling pathways against oxidative stress to protect hepatic cell from oxidative damage during development of common chronic liver diseases. We also review the use of Nrf2 as a therapeutic target to prevent and treat liver diseases. PMID:25278702

  5. 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. PMID:27093399

  6. Nrf2 as a Chemopreventive Target in Colorectal Cancer

    PubMed Central

    Saw, Constance Lay Lay; Kong, Tony Ah-Ng

    2012-01-01

    Introduction Numerous epidemiological studies have linked consumption of cruciferous vegetables to a reduced risk of colorectal cancer (CRC) in individuals. It is currently well accepted that chronic inflammation is a contributing factor in 15-20% malignancies including CRC. Many chemopreventive compounds are effective in preclinical systems and many on-going clinical trials are showing promising findings. Many of these compounds could activate the antioxidant responsive element (ARE), a critical regulatory element for phase II protective/detoxification and anti-oxidative stress enzymes mediated by nuclear factor-erythroid 2-related factor 2 (Nrf2). Recently, Nrf2 has emerged as a novel target for the prevention of CRC. Areas covered A full literature search was performed using PubMed with the key words ‘ARE, Nrf2, colon, colorectal cancer, chemoprevention, cancer prevention’, and all relevant publications are included. Expert opinion The use of Nrf2 knockout mice has provided key insights into the toxicological and chemopreventive importance of this pathway. Mounting evidence has revealed that Nrf2 is a critical regulator of inflammation as well, a major driving force for CRC progression and formation. Targeting the Nrf2/ARE pathway may present a novel therapeutic approach for the treatment of not only colorectal inflammatory diseases but the frequent subsequent development of CRC as well. PMID:21261563

  7. 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. PMID:24500986

  8. 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. PMID:25975984

  9. 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

  10. 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

  11. Essential role of PH domain and leucine-rich repeat protein phosphatase 2 in Nrf2 suppression via modulation of Akt/GSK3β/Fyn kinase axis during oxidative hepatocellular toxicity.

    PubMed

    Rizvi, F; Shukla, S; Kakkar, P

    2014-01-01

    Instances of sustained oxidative activity have been shown to involve dysregulation of Nrf2-mediated transcriptional induction; however, mechanisms warranting Nrf2-repression remain unclear. In this study, using primary rat hepatocytes, we have attempted to identify factors that may negatively influence Nrf2 survival pathway. Though studies indicate a conspicuous association between Akt and Nrf2, a confirmatory link between the two is unaddressed. On inhibiting PI3K/Akt pathway, we observed compromised activities of antioxidant and detoxification enzymes culminating in oxidative cytotoxicity. This was accompanied by reduced nuclear retention of Nrf2 and its ARE binding affinity, increased Nrf2 ubiquitination and concurrent decline in its downstream targets. Moreover, Akt inhibition enhanced nuclear translocation as well as phosphorylation of Fyn kinase, an enzyme linked to Nrf2 degradation, by relieving GSK3β from phosphorylation-mediated repression. The involvement of Akt and Fyn kinase in influencing Nrf2 signaling was further confirmed in oxidatively stressed hepatocytes by using tert-butyl hydroperoxide (tBHP). tBHP-induced decrease in Nrf2 levels was associated with enhanced Fyn kinase phosphorylation, Fyn kinase nuclear translocation and decreased levels of phosphorylated GSK3β(Ser9) in a time-dependent manner. Interestingly, tBHP induced site-specific deactivation of Akt as only Akt(Ser473) phosphorylation was observed to be affected. Further, protein expression as well as nuclear localization of PHLPP2, a phosphatase specific for Akt(Ser473), was found to be significantly enhanced in tBHP-stressed hepatocytes. Silencing of PHLPP2 not only resulted in considerable restoration of Nrf2 signaling, enhanced Nrf2-ARE binding and reduced Nrf2 ubiquitination but also significantly suppressed tBHP-induced ROS generation and alterations in mitochondrial permeability. We infer that cellular PHLPP2 levels may aggravate oxidative toxicity by suppressing Nrf2/ARE

  12. Exercise, Nrf2 and Antioxidant Signaling in Cardiac Aging.

    PubMed

    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

  13. 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

  14. Nrf2 transcriptional derepression from Keap1 by dietary polyphenols.

    PubMed

    Bayele, Henry K; Debnam, Edward S; Srai, Kaila S

    2016-01-15

    The liver expresses batteries of cytoprotective genes that confer cellular resistance to oxidative stress and xenobiotic toxins, and protection against cancer and other stress-related diseases. These genes are mainly regulated by Nrf2, making this transcription factor a target for small molecule discovery to treat such diseases. In this report, we identified dietary polyphenolic antioxidants that not only activated these genes but also relieved Nrf2 repression by Keap1, a Cul3-dependent ubiquitin ligase adaptor protein that mediates its degradation. Analysis of postprandial liver RNA revealed a marked activation of both genes by all test polyphenols compared with controls. Nrf2 inhibition by RNA interference reduced polyphenol effects on its target gene expression. Our data suggest that polyphenols may induce cellular defense genes by derepressing Nrf2 inhibition by Keap1. We posit that this ability to derepress Nrf2 and reactivate its target genes may underlie the protection conferred by polyphenols against oxidative stress-related diseases. PMID:26655811

  15. Salvianolic acid A protects RPE cells against oxidative stress through activation of Nrf2/HO-1 signaling.

    PubMed

    Zhang, Hui; Liu, Yuan-yuan; Jiang, Qin; Li, Ke-ran; Zhao, Yu-xia; Cao, Cong; Yao, Jin

    2014-04-01

    Reactive oxygen species (ROS) impair the physiological functions of retinal pigment epithelial (RPE) cells, which is known as one major cause of age-related macular degeneration. Salvianolic acid A (Sal A) is the main effective aqueous extract of Salvia miltiorrhiza. The aim of this study was to test the potential role of Sal A against oxidative stress in cultured RPE cells and to investigate the underlying mechanistic signaling pathways. We observed that Sal A significantly inhibited hydrogen peroxide (H2O2)-induced primary and transformed RPE cell death and apoptosis. H2O2-stimulated mitogen-activated protein kinase activation, ROS production, and subsequent proapoptotic AMP-activated protein kinase activation were largely inhibited by Sal A. Further, Sal A stimulation resulted in a fast and dramatic activation of Akt/mammalian target of rapamycin complex 1 (mTORC1) signaling, followed by phosphorylation, accumulation, and nuclear translocation of the NF-E2-related factor 2 (Nrf2), along with increased expression of the antioxidant-response element-dependent gene heme oxygenase-1 (HO-1). Both Nrf2 and HO-1 were required for Sal A-mediated cytoprotective effect, as Nrf2/HO-1 inhibition abolished Sal A-induced beneficial effects against H2O2. Meanwhile, the PI3K/Akt/mTORC1 chemical inhibitors not only suppressed Sal A-induced Nrf2/HO-1 activation, but also eliminated its cytoprotective effect in RPE cells. These observations suggest that Sal A activates the Nrf2/HO-1 axis in RPE cells and protects against oxidative stress via activation of Akt/mTORC1 signaling. PMID:24486344

  16. Src Subfamily Kinases Regulate Nuclear Export and Degradation of Transcription Factor Nrf2 to Switch Off Nrf2-mediated Antioxidant Activation of Cytoprotective Gene Expression*

    PubMed Central

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

    2011-01-01

    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. PMID:21690096

  17. 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

  18. 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

  19. 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.

  20. 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

  1. Nrf2--A regulator of keratinocyte redox signaling.

    PubMed

    Schäfer, Matthias; Werner, Sabine

    2015-11-01

    The skin is frequently exposed to environmental challenges, such as UV irradiation, toxic chemicals, and mechanical wounding. These insults cause an increase in the levels of reactive oxygen species, resulting in oxidative stress and concomitant inflammation, skin aging, and even cancer development. Therefore, an efficient antioxidant defense strategy is of major importance in this tissue. Since the Nrf2 transcription factor regulates a battery of genes involved in the defense against reactive oxygen species and in compound metabolism, it plays a key role in skin homeostasis, repair, and disease. In this review we summarize current knowledge on the expression and function of Nrf2 in normal skin and its role in the acute and chronic UV response as well as in the pathogenesis of epithelial skin cancer and of different inflammatory skin diseases. Finally, we discuss the potential of Nrf2-activating compounds for skin protection under stress conditions and for the treatment of major human skin disorders. PMID:25912479

  2. 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

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

    PubMed

    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

    2016-04-19

    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. The emerging role of the Nrf2–Keap1 signaling pathway in cancer

    PubMed Central

    Jaramillo, Melba C.; Zhang, Donna D.

    2013-01-01

    The Nrf2 (nuclear factor erythroid 2 [NF-E2]-related factor 2 [Nrf2])–Keap1 (Kelch-like erythroid cell-derived protein with CNC homology [ECH]-associated protein 1) signaling pathway is one of the most important cell defense and survival pathways. Nrf2 can protect cells and tissues from a variety of toxicants and carcinogens by increasing the expression of a number of cytoprotective genes. As a result, several Nrf2 activators are currently being tested as chemopreventive compounds in clinical trials. Just as Nrf2 protects normal cells, studies have shown that Nrf2 may also protect cancer cells from chemotherapeutic agents and facilitate cancer progression. Nrf2 is aberrantly accumulated in many types of cancer, and its expression is associated with a poor prognosis in patients. In addition, Nrf2 expression is induced during the course of drug resistance. Collectively, these studies suggest that Nrf2 contributes to both intrinsic and acquired chemoresistance. This discovery has opened up a broad spectrum of research geared toward a better understanding of the role of Nrf2 in cancer. This review provides an overview of (1) the Nrf2–Keap1 signaling pathway, (2) the dual role of Nrf2 in cancer, (3) the molecular basis of Nrf2 activation in cancer cells, and (4) the challenges in the development of Nrf2-based drugs for chemoprevention and chemotherapy. PMID:24142871

  5. The role of the catecholic and the electrophilic moieties of caffeic acid in Nrf2/Keap1 pathway activation in ovarian carcinoma cell lines

    PubMed Central

    Sirota, R.; Gibson, D.; Kohen, R.

    2014-01-01

    In recent years, numerous studies have demonstrated the health benefits of polyphenols. A major portion of polyphenols in western diet are derived from coffee, which is one of the most consumed beverages in the world. It has been shown that many polyphenols gain their beneficial properties (e.g. cancer prevention) through the activation of the Nrf2/Keap1 pathway as well as their direct antioxidant activity. However, activation of Nrf2 in cancer cells might lead to resistance towards therapy through induction of phase II enzymes. In the present work we hypothesize that caffeic acid (CA), a coffee polyphenol, might act as an electrophile in addition to its nucleophilic properties and is capable of inducing the Nrf2/EpRE pathway in cancer cells. The results indicate that CA induces Nrf2 translocation into the nucleus and consequently its transcription. It has been demonstrated that generated hydrogen peroxide is involved in the induction process. It has also been found that this process is induced predominantly via the double bond in CA (Michael acceptor). However, surprisingly the presence of both nucleophilic and electrophilic moieties in CA resulted in a synergetic activation of Nrf2 and phase II enzymes. We also found that CA possesses a dual activity, although inducing GSTP1 and GSR, it inhibiting their enzymatic activity. In conclusion, the mechanism of induction of Nrf2 pathway and phase II enzymes by CA has been elucidated. The electrophilic moiety in CA is essential for the oxidation of the Keap1 protein. It should be noted that while the nucleophilic moiety (the catechol/quinone moiety) can provide scavenging ability, it cannot contribute directly to Nrf2 induction. It was found that this process may be induced by H2O2 produced by the catechol group. On the whole, it appears that CA might play a major role in the cancer cells by enhancing their resistance to treatment. PMID:25498967

  6. Expression of xCT and activity of system xc(-) are regulated by NRF2 in human breast cancer cells in response to oxidative stress.

    PubMed

    Habib, Eric; Linher-Melville, Katja; Lin, Han-Xin; Singh, Gurmit

    2015-08-01

    Cancer cells adapt to high levels of oxidative stress in order to survive and proliferate by activating key transcription factors. One such master regulator, the redox sensitive transcription factor NF E2 Related Factor 2 (NRF2), controls the expression of cellular defense genes including those encoding intracellular redox-balancing proteins involved in glutathione (GSH) synthesis. Under basal conditions, Kelch-like ECH-associated protein 1 (KEAP1) targets NRF2 for ubiquitination. In response to oxidative stress, NRF2 dissociates from KEAP1, entering the nucleus and binding to the antioxidant response element (ARE) in the promoter of its target genes. Elevated reactive oxygen species (ROS) production may deplete GSH levels within cancer cells. System xc(-), an antiporter that exports glutamate while importing cystine to be converted into cysteine for GSH synthesis, is upregulated in cancer cells in response to oxidative stress. Here, we provided evidence that the expression of xCT, the light chain subunit of system xc(-), is regulated by NRF2 in representative human breast cancer cells. Hydrogen peroxide (H2O2) treatment increased nuclear translocation of NRF2, also increasing levels of xCT mRNA and protein and extracellular glutamate release. Overexpression of NRF2 up-regulated the activity of the xCT promoter, which contains a proximal ARE. In contrast, overexpression of KEAP1 repressed promoter activity and decreased xCT protein levels, while siRNA knockdown of KEAP1 up-regulated xCT protein levels and transporter activity. These results demonstrate the importance of the KEAP1/NRF2 pathway in balancing oxidative stress in breast cancer cells through system xc(-). We have previously shown that xCT is upregulated in various cancer cell lines under oxidative stress. In the current investigation, we focused on MCF-7 cells as a model for mechanistic studies. PMID:25827424

  7. 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

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

    PubMed

    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

  9. Oxidised LDL up-regulate CD36 expression by the Nrf2 pathway in 3T3-L1 preadipocytes.

    PubMed

    D'Archivio, Massimo; Scazzocchio, Beatrice; Filesi, Carmela; Varì, Rosaria; Maggiorella, Maria Teresa; Sernicola, Leonardo; Santangelo, Carmela; Giovannini, Claudio; Masella, Roberta

    2008-06-25

    The effect of oxLDL on CD36 expression has been assessed in preadipocytes induced to differentiate. Novel evidence is provided that oxLDL induce a peroxisome proliferator-activated receptor gamma-independent CD36 overexpression, by up-regulating nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2). The nuclear translocation of Nrf2 appeared to depend on PKC pathway activation. In adipocytes, the CD36 up-regulation may indicate a compensation mechanism to meet the demand of excess oxLDL and oxidised lipids in blood, reducing the risk of atherogenesis. Besides strengthening the hypothesis that oxLDL can contribute to the onset of insulin-resistance, data herein presented highlight the significance of oxLDL-induced CD36 overexpression within the cellular defence response. PMID:18514070

  10. Lipoxin A4-Induced Heme Oxygenase-1 Protects Cardiomyocytes against Hypoxia/Reoxygenation Injury via p38 MAPK Activation and Nrf2/ARE Complex

    PubMed Central

    Chen, Xiao-Qing; Wu, Sheng-Hua; Zhou, Yu; Tang, Yan-Rong

    2013-01-01

    Objective To investigate whether lipoxin A4 (LXA4) increases expression of heme oxygenase-1(HO-1) in cardiomyocytes, whether LXA4-induced HO-1 protects cardiomyocytes against hypoxia/reoxygenation (H/R) injury, and what are the mechanisms involved in the LXA4-induced HO-1 induction. Methods Rat cardiomyocytes were exposed to H/R injury with or without preincubation with LXA4 or HO-1 inhibitor ZnPP-IX or various signal molecule inhibitors. Expressions of HO-1 protein and mRNA were analyzed by using Western blot and RT-PCR respectively. Activity of nuclear factor E2-related factor 2 (Nrf2) binding to the HO-1 E1 enhancer was assessed by chromatin immunoprecipitation. Nrf2 binding to the HO-1 antioxidant responsive element (ARE) were measured by using electrophoretic mobility shift assay. Results Pretreatment of the cells undergoing H/R lesion with LXA4 significantly reduced the lactate dehydrogenase and creatine kinase productions, increased the cell viability, and increased the expressions of HO-1 protein and mRNA and HO-1 promoter activity. HO-1 inhibition abolished the protective role of LXA4 on the cells undergoing H/R lesion. LXA4 increased p38 mitogen-activated protein kinase (p38 MAPK) activation, nuclear translocation of Nrf2, Nrf2 binding to the HO-1 ARE and E1 enhancer in cardiomyocytes with or without H/R exposure. Conclusion The protection role of LXA4 against H/R injury of cardiomyocytes is related to upregulation of HO-1, via activation of p38 MAPK pathway and nuclear translocation of Nrf2 and Nrf2 binding to the HO-1 ARE and E1 enhancer, but not via activation of phosphatidyinositol-3-kinase or extracellular signal-regulated kinase pathway. PMID:23826208

  11. Carbon monoxide mediates heme oxygenase 1 induction via Nrf2 activation in hepatoma cells

    SciTech Connect

    Lee, Bok-Soo; Heo, JungHee; Kim, Yong-Man; Shim, Sang Moo; Pae, Hyun-Ock; Kim, Young-Myeong; Chung, Hun-Taeg . E-mail: htchung@wonkwang.ac.kr

    2006-05-12

    Carbon monoxide (CO) and nitric oxide (NO) are two gas molecules which have cytoprotective functions against oxidative stress and inflammatory responses in many cell types. Currently, it is known that NO produced by nitric oxide synthase (NOS) induces heme oxygenase 1 (HO1) expression and CO produced by the HO1 inhibits inducible NOS expression. Here, we first show CO-mediated HO1 induction and its possible mechanism in human hepatocytes. Exposure of HepG2 cells or primary hepatocytes to CO resulted in dramatic induction of HO1 in dose- and time-dependent manner. The CO-mediated HO1 induction was abolished by MAP kinase inhibitors (MAPKs) but not affected by inhibitors of PI3 kinase or NF-{kappa}B. In addition, CO induced the nuclear translocation and accumulation of Nrf2, which suppressed by MAPKs inhibitors. Taken together, we suggest that CO induces Nrf2 activation via MAPKs signaling pathways, thereby resulting in HO1 expression in HepG2 cells.

  12. 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

  13. Trafficking of the transcription factor Nrf2 to promyelocytic leukemia-nuclear bodies: implications for degradation of NRF2 in the nucleus.

    PubMed

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

    2013-05-17

    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

  14. The Nrf2-ARE pathway: a valuable therapeutic target for the treatment of neurodegenerative diseases

    PubMed Central

    Joshi, Gururaj; Johnson, Jeffrey A.

    2013-01-01

    Modulation of NF-E2 related factor 2 (Nrf2) has been shown in several neurodegenerative disorders. The overexpression of Nrf2 has become a potential therapeutic avenue for various neurodegenerative disorders such as Parkinson, Amyotrophic lateral sclerosis, and Alzheimer’s disease. The expression of phase II detoxification enzymes is governed by the cis-acting regulatory element known as antioxidant response element (ARE). The transcription factor Nrf2 binds to ARE thereby transcribing multitude of antioxidant genes. Keap1, a culin 3-based E3 ligase that targets Nrf2 for degradation, sequesters Nrf2 in cytoplasm. Disruption of Keap1-Nrf2 interaction or genetic overexpression of Nrf2 can increase the endogenous antioxidant capacity of the brain thereby rendering protection against oxidative stress in neurodegenerative disorders. This review primarily focuses on targeted Nrf2 overexpression as a promising therapeutic strategy for the treatment of neurodegenerative disorders. PMID:22742419

  15. S-Propargyl-cysteine Exerts a Novel Protective Effect on Methionine and Choline Deficient Diet-Induced Fatty Liver via Akt/Nrf2/HO-1 Pathway

    PubMed Central

    Li, Wenwen; Ma, Fenfen; Zhang, Laiyin; Huang, Yong; Li, Xinghui; Zhang, Aijie; Hou, Cuilan; Zhu, Yichun; Zhu, YiZhun

    2016-01-01

    This study investigated the antioxidative effect of S-propargyl-cysteine (SPRC) on nonalcoholic fatty liver (NAFLD) by treating mice fed a methionine and choline deficient (MCD) diet with SPRC for four weeks. We found that SPRC significantly reduced hepatic reactive oxygen species (ROS) and methane dicarboxylic aldehyde (MDA) levels. Moreover, SPRC also increased the superoxide dismutase (SOD) activity. By Western blot, we found that this protective effect of SPRC was importantly attributed to the regulated hepatic antioxidant-related proteins, including protein kinase B (Akt), heme oxygenase-1 (HO-1), nuclear factor erythroid 2-related factor 2 (Nrf2), and cystathionine γ-lyase (CSE, an enzyme that synthesizes hydrogen sulfide). Next, we examined the detailed molecular mechanism of the SPRC protective effect using oleic acid- (OA-) induced HepG2 cells. The results showed that SPRC significantly decreased intracellular ROS and MDA levels in OA-induced HepG2 cells by upregulating the phosphorylation of Akt, the expression of HO-1 and CSE, and the translocation of Nrf2. SPRC-induced HO-1 expression and Nrf2 translocation were abolished by the phosphoinositide 3-kinase (PI3K) inhibitor LY294002. Moreover, the antioxidative effect of SPRC was abolished by CSE inhibitor DL-propargylglycine (PAG) and HO-1 siRNA. Therefore, these results proved that SPRC produced an antioxidative effect on NAFLD through the PI3K/Akt/Nrf2/HO-1 signaling pathway. PMID:27313828

  16. S-Propargyl-cysteine Exerts a Novel Protective Effect on Methionine and Choline Deficient Diet-Induced Fatty Liver via Akt/Nrf2/HO-1 Pathway.

    PubMed

    Li, Wenwen; Ma, Fenfen; Zhang, Laiyin; Huang, Yong; Li, Xinghui; Zhang, Aijie; Hou, Cuilan; Zhu, Yichun; Zhu, YiZhun

    2016-01-01

    This study investigated the antioxidative effect of S-propargyl-cysteine (SPRC) on nonalcoholic fatty liver (NAFLD) by treating mice fed a methionine and choline deficient (MCD) diet with SPRC for four weeks. We found that SPRC significantly reduced hepatic reactive oxygen species (ROS) and methane dicarboxylic aldehyde (MDA) levels. Moreover, SPRC also increased the superoxide dismutase (SOD) activity. By Western blot, we found that this protective effect of SPRC was importantly attributed to the regulated hepatic antioxidant-related proteins, including protein kinase B (Akt), heme oxygenase-1 (HO-1), nuclear factor erythroid 2-related factor 2 (Nrf2), and cystathionine γ-lyase (CSE, an enzyme that synthesizes hydrogen sulfide). Next, we examined the detailed molecular mechanism of the SPRC protective effect using oleic acid- (OA-) induced HepG2 cells. The results showed that SPRC significantly decreased intracellular ROS and MDA levels in OA-induced HepG2 cells by upregulating the phosphorylation of Akt, the expression of HO-1 and CSE, and the translocation of Nrf2. SPRC-induced HO-1 expression and Nrf2 translocation were abolished by the phosphoinositide 3-kinase (PI3K) inhibitor LY294002. Moreover, the antioxidative effect of SPRC was abolished by CSE inhibitor DL-propargylglycine (PAG) and HO-1 siRNA. Therefore, these results proved that SPRC produced an antioxidative effect on NAFLD through the PI3K/Akt/Nrf2/HO-1 signaling pathway. PMID:27313828

  17. 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

  18. Induction of the Nrf2-driven antioxidant response confers neuroprotection during mitochondrial stress in vivo.

    PubMed

    Shih, Andy Y; Imbeault, Sophie; Barakauskas, Vilte; Erb, Heidi; Jiang, Lei; Li, Ping; Murphy, Timothy H

    2005-06-17

    NF-E2 related factor (Nrf2) controls a pleiotropic cellular defense, where multiple antioxidant/detoxification pathways are up-regulated in unison. Although small molecule inducers of Nrf2 activity have been reported to protect neurons in vitro, whether similar pathways can be accessed in vivo is not known. We have investigated whether in vivo toxicity of the mitochondrial complex II inhibitor 3-nitropropionic acid (3-NP) can be attenuated by constitutive and inducible Nrf2 activity. The absence of Nrf2 function in Nrf2(-/-) mice resulted in 3-NP hypersensitivity that became apparent with time and increasing dose, causing motor deficits and striatal lesions on a more rapid time scale than identically treated Nrf2(+/+) and Nrf2(+/-) controls. Striatal succinate dehydrogenase activity, the target of 3-NP, was inhibited to the same extent in all genotypes by a single acute dose of 3-NP, suggesting that brain concentrations of 3-NP were similar. Dietary supplementation with the Nrf2 inducer tert-butylhydroquinone attenuated 3-NP toxicity in Nrf2(+/-) mice, but not Nrf2(-/-), confirming the Nrf2-specific action of the inducer in vivo. Increased Nrf2 activity alone was sufficient to protect animals from 3-NP toxicity because intrastriatal adenovirus-mediated Nrf2 overexpression significantly reduced lesion size compared with green fluorescent protein overexpressing controls. In cultured astrocytes, 3-NP was found to increase Nrf2 activity leading to antioxidant response element-dependent gene expression providing a potential mechanism for the increased sensitivity of Nrf2(-/-) animals to 3-NP toxicity in vivo. We conclude that Nrf2 may underlie a feedback system limiting oxidative load during chronic metabolic stress. PMID:15840590

  19. 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

  20. Regulatory Nexus of Synthesis and Degradation Deciphers Cellular Nrf2 Expression Levels

    PubMed Central

    Suzuki, Takafumi; Shibata, Tatsuhiro; Takaya, Kai; Shiraishi, Kouya; Kohno, Takashi; Kunitoh, Hideo; Tsuta, Koji; Furuta, Koh; Goto, Koichi; Hosoda, Fumie; Sakamoto, Hiromi; Motohashi, Hozumi

    2013-01-01

    Transcription factor Nrf2 (NF-E2-related factor 2) is essential for oxidative and electrophilic stress responses. While it has been well characterized that Nrf2 activity is tightly regulated at the protein level through proteasomal degradation via Keap1 (Kelch-like ECH-associated protein 1)-mediated ubiquitination, not much attention has been paid to the supply side of Nrf2, especially regulation of Nrf2 gene transcription. Here we report that manipulation of Nrf2 transcription is effective in changing the final Nrf2 protein level and activity of cellular defense against oxidative stress even in the presence of Keap1 and under efficient Nrf2 degradation, determined using genetically engineered mouse models. In excellent agreement with this finding, we found that minor A/A homozygotes of a single nucleotide polymorphism (SNP) in the human NRF2 upstream promoter region (rs6721961) exhibited significantly diminished NRF2 gene expression and, consequently, an increased risk of lung cancer, especially those who had ever smoked. Our results support the notion that in addition to control over proteasomal degradation and derepression from degradation/repression, the transcriptional level of the Nrf2 gene acts as another important regulatory point to define cellular Nrf2 levels. These results thus verify the critical importance of human SNPs that influence the levels of transcription of the NRF2 gene for future personalized medicine. PMID:23572560

  1. Regulatory nexus of synthesis and degradation deciphers cellular Nrf2 expression levels.

    PubMed

    Suzuki, Takafumi; Shibata, Tatsuhiro; Takaya, Kai; Shiraishi, Kouya; Kohno, Takashi; Kunitoh, Hideo; Tsuta, Koji; Furuta, Koh; Goto, Koichi; Hosoda, Fumie; Sakamoto, Hiromi; Motohashi, Hozumi; Yamamoto, Masayuki

    2013-06-01

    Transcription factor Nrf2 (NF-E2-related factor 2) is essential for oxidative and electrophilic stress responses. While it has been well characterized that Nrf2 activity is tightly regulated at the protein level through proteasomal degradation via Keap1 (Kelch-like ECH-associated protein 1)-mediated ubiquitination, not much attention has been paid to the supply side of Nrf2, especially regulation of Nrf2 gene transcription. Here we report that manipulation of Nrf2 transcription is effective in changing the final Nrf2 protein level and activity of cellular defense against oxidative stress even in the presence of Keap1 and under efficient Nrf2 degradation, determined using genetically engineered mouse models. In excellent agreement with this finding, we found that minor A/A homozygotes of a single nucleotide polymorphism (SNP) in the human NRF2 upstream promoter region (rs6721961) exhibited significantly diminished NRF2 gene expression and, consequently, an increased risk of lung cancer, especially those who had ever smoked. Our results support the notion that in addition to control over proteasomal degradation and derepression from degradation/repression, the transcriptional level of the Nrf2 gene acts as another important regulatory point to define cellular Nrf2 levels. These results thus verify the critical importance of human SNPs that influence the levels of transcription of the NRF2 gene for future personalized medicine. PMID:23572560

  2. Nrf2 null enhances UVB-induced skin inflammation and extracellular matrix damages.

    PubMed

    Saw, Constance Lay Lay; Yang, Anne Yuqing; Huang, Mou-Tuan; Liu, Yue; Lee, Jong Hun; Khor, Tin Oo; Su, Zheng-Yuan; Shu, Limin; Lu, Yaoping; Conney, Allan H; Kong, Ah-Ng Tony

    2014-01-01

    Nrf2 plays a critical role in defending against oxidative stress and inflammation. We previously reported that Nrf2 confers protection against ultraviolet-B (UVB)-induced inflammation, sunburn reaction, and is involved in sulforaphane-mediated photo-protective effects in the skin. In this study, we aimed to demonstrate the protective role of Nrf2 against inflammation-mediated extracellular matrix (ECM) damage induced by UVB irradiation. Ear biopsy weights were significantly increased in both Nrf2 wild-type (Nrf2 WT) and knockout (Nrf2 KO) mice one week after UVB irradiation. However, these weights increased more significantly in KO mice compared to WT mice, suggesting a greater inflammatory response in KO mice. In addition, we analyzed the protein expression of numerous markers, including macrophage inflammatory protein-2 (MIP-2), pro-matrix metalloproteinase-9 (MMP-9), and p53. p53, a regulator of DNA repair, was overexpressed in Nrf2 KO mice, indicating that the absence of Nrf2 led to more sustained DNA damage. There was also more substantial ECM degradation and increased inflammation in UVB-irradiated Nrf2 KO mice compared to UVB-irradiated WT mice. Furthermore, the protective effects of Nrf2 in response to UVB irradiation were mediated by increased HO-1 protein expression. Collectively, our results show that Nrf2 plays a key role in protecting against UVB irradiation and that the photo-protective effect of Nrf2 is closely related to the inhibition of ECM degradation and inflammation. PMID:25228981

  3. Hydrolyzed Methylhesperidin Induces Antioxidant Enzyme Expression via the Nrf2-ARE Pathway in Normal Human Epidermal Keratinocytes.

    PubMed

    Kuwano, Tetsuya; Watanabe, Manabu; Kagawa, Daiji; Murase, Takatoshi

    2015-09-16

    Methylhesperidin (MHES) is a mixture of methylated derivatives of the citrus flavonoid hesperidin and is used as a food or pharmaceutical additive. Dietary MHES could be hydrolyzed by gut microflora to give aglycons. Therefore, we prepared hydrolyzed methylhesperidin (h-MHES) and assessed its pharmacological activity in human epidermal keratinocytes. h-MHES promoted nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear translocation and the expression of cytoprotective genes (e.g., heme oxygenase-1 (HO-1) and glutamate cysteine ligase catalytic subunit (GCLC)). h-MHES also increased intracellular glutathione levels and reduced UVB-induced reactive oxygen species. Moreover, h-MHES increased phosphorylation of p38 mitogen-activated protein kinase (MAPK), and a p38 MAPK inhibitor significantly attenuated h-MHES-induced HO-1 and GCLC expression. Furthermore, when we purified the components of h-MHES, we identified two methoxy-chalcones as novel Nrf2 activators. Our study demonstrates that h-MHES can induce cytoprotective gene expression and reduce oxidative stress via the Nrf2-ARE pathway in keratinocytes, suggesting that MHES may contribute to the suppression of UVB-induced skin damage in vivo. PMID:26313892

  4. eckol enhances heme oxygenase-1 expression through activation of Nrf2/JNK pathway in HepG2 cells.

    PubMed

    Jun, Young-Jin; Lee, Minsup; Shin, Taisun; Yoon, Nayoung; Kim, Ji-Hoe; Kim, Hyeung-Rak

    2014-01-01

    Eckol isolated from Ecklonia stolonifera was previously reported to exhibit cytoprotective activity with its intrinsic antioxidant activity in in vitro studies. In this study, we characterized the mechanism underlying the eckol-mediated the expression of heme oxygenase-1 (HO-1). Eckol suppressed the production of intracellular reactive oxygen species and increased glutathione level in HepG2 cells. Eckol treatment enhanced the expression of HO-1 at the both level of protein and mRNA in HepG2 cells. Enhanced expression of HO-1 by eckol was presumed to be the activation of the nuclear factor erythroid-derived 2-like 2 (Nrf2) demonstrated by its nuclear translocation and increased transcriptional activity. c-Jun NH2-terminal kinases (JNKs) and PI3K/Akt contributed to Nrf2-mediated HO-1 expression. These results demonstrate that the eckol-mediated expression of HO-1 in HepG2 cells is regulated by Nrf2 activation via JNK and PI3K/Akt signaling pathways, suggesting that eckol may be used as a natural antioxidant and cytoprotective agent. PMID:25268719

  5. The Keap1-Nrf2-antioxidant response element pathway: a review of its regulation by melatonin and the proteasome.

    PubMed

    Vriend, Jerry; Reiter, Russel J

    2015-02-01

    Both melatonin and proteasome inhibitors upregulate antioxidant enzymes including superoxide dismutase (SOD), glutathione peroxidase (GP), hemoxygenase 1 (HO-1), and NADPH:quinone oxidoreductase (NQO1). Recent evidence suggests that the antioxidant action of both melatonin and proteasome inhibitors involves the Keap1-ARE (Keap1 antioxidant response element) pathway via the upregulation of Nrf2. Melatonin and proteasome inhibitors suppress the degradation of Nrf2 and also enhance its nuclear translocation. In the nucleus Nrf2, together with a cofactor, stimulates the transcription of antioxidant enzymes and detoxifying enzymes. The ligase (E3) complex (Keap1-Cul3-Rbx1) responsible for ubiquitinating Nrf2, prior to proteasomal degradation, also ubiquitinates IkB kinase and the antiapoptotic factor Bcl-2, and possibly additional proteins. In various systems, NF-κB, which is inhibited by IkBα, is downregulated by proteasome inhibitors as well as by melatonin. Similarly in leukemic cells, Bcl-2 is down-regulated by the proteasome inhibitor, bortezomib, and also by melatonin. Thus melatonin administration modulates the activity of three separate substrates of the Keap1-Cul3-Rbx1 ubiquitin ligase. These facts could be accounted for by the hypothesis that melatonin interacts with the ubiquitin ligase complex or, more likely, by the hypothesis that melatonin acts as a proteasome inhibitor. A recent study documented that melatonin acts as a proteasome inhibitor in cancer cells as well as inhibiting chymotrypsin-like activity in cell-free systems of these cells. Further studies, however, are needed to clarify the interaction of melatonin and the ubiquitin-proteasome system as they relate to oxidative stress. PMID:25528518

  6. Frataxin Deficiency Leads to Defects in Expression of Antioxidants and Nrf2 Expression in Dorsal Root Ganglia of the Friedreich's Ataxia YG8R Mouse Model

    PubMed Central

    Shan, Yuxi; Schoenfeld, Robert A.; Hayashi, Genki; Napoli, Eleonora; Akiyama, Tasuku; Iodi Carstens, Mirela; Carstens, Earl E.; Pook, Mark A.

    2013-01-01

    Abstract Aims: Oxidative stress is thought to be involved in Friedreich's ataxia (FRDA), yet it has not been demonstrated in the target neurons that are first to degenerate. Using the YG8R mouse model of FRDA, microarray and neuritic growth experiments were carried out in the dorsal root ganglion (DRG), the primary site of neurodegeneration in this disease. Results: YG8R hemizygous mice exhibited defects in movement, and DRG neurites had growth defects. Microarray of DRG tissue identified decreased transcripts encoding the antioxidants, including peroxiredoxins, glutaredoxins, and glutathione S-transferase, and these were confirmed by immunoblots and quantitative real-time PCR. Because the decreased gene transcripts are the known targets of the antioxidant transcription factor nuclear factor-E2-related factor-2 (Nrf2), Nrf2 expression was measured; it was significantly decreased at the transcript and protein level in both the DRG and the cerebella of the YG8R hemizygous mouse; further, frataxin expression was significantly correlated with Nrf2 expression. Functionally, in YG8R hemizygous DRG, the total glutathione levels were reduced and explanted cells were more sensitive to the thioredoxin reductase (TxnRD) inhibitor auranofin, a thiol oxidant. In cell models of FRDA, including Schwann and the DRG, frataxin deficiency caused a decreased expression of the Nrf2 protein level in the nucleus, but not a defect in its translocation from the cytosol. Further, frataxin-deficient cells had decreased enzyme activity and expression of TxnRD, which is regulated by Nrf2, and were sensitive the TxnRD inhibitor auranofin. Innovation and Conclusion: These results support a mechanistic hypothesis in which frataxin deficiency decreases Nrf2 expression in vivo, causing the sensitivity to oxidative stress in target tissues the DRG and the cerebella, which contributes to the process of neurodegeneration. Antioxid. Redox Signal. 19, 1481–1493. PMID:23350650

  7. 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.

  8. 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. PMID:27016073

  9. Recurrent Loss of NFE2L2 Exon 2 Is a Mechanism for Nrf2 Pathway Activation in Human Cancers.

    PubMed

    Goldstein, Leonard D; Lee, James; Gnad, Florian; Klijn, Christiaan; Schaub, Annalisa; Reeder, Jens; Daemen, Anneleen; Bakalarski, Corey E; Holcomb, Thomas; Shames, David S; Hartmaier, Ryan J; Chmielecki, Juliann; Seshagiri, Somasekar; Gentleman, Robert; Stokoe, David

    2016-09-01

    The Nrf2 pathway is frequently activated in human cancers through mutations in Nrf2 or its negative regulator KEAP1. Using a cell-line-derived gene signature for Nrf2 pathway activation, we found that some tumors show high Nrf2 activity in the absence of known mutations in the pathway. An analysis of splice variants in oncogenes revealed that such tumors express abnormal transcript variants from the NFE2L2 gene (encoding Nrf2) that lack exon 2, or exons 2 and 3, and encode Nrf2 protein isoforms missing the KEAP1 interaction domain. The Nrf2 alterations result in the loss of interaction with KEAP1, Nrf2 stabilization, induction of a Nrf2 transcriptional response, and Nrf2 pathway dependence. In all analyzed cases, transcript variants were the result of heterozygous genomic microdeletions. Thus, we identify an alternative mechanism for Nrf2 pathway activation in human tumors and elucidate its functional consequences. PMID:27568559

  10. The Keap1-Nrf2 pathway: Mechanisms of activation and dysregulation in cancer☆

    PubMed Central

    Kansanen, Emilia; Kuosmanen, Suvi M.; Leinonen, Hanna; Levonen, Anna-Liisa

    2013-01-01

    The Keap1-Nrf2 pathway is the major regulator of cytoprotective responses to oxidative and electrophilic stress. Although cell signaling pathways triggered by the transcription factor Nrf2 prevent cancer initiation and progression in normal and premalignant tissues, in fully malignant cells Nrf2 activity provides growth advantage by increasing cancer chemoresistance and enhancing tumor cell growth. In this graphical review, we provide an overview of the Keap1-Nrf2 pathway and its dysregulation in cancer cells. We also briefly summarize the consequences of constitutive Nrf2 activation in cancer cells and how this can be exploited in cancer gene therapy. PMID:24024136

  11. Genetic or Pharmacologic Amplification of Nrf2 Signaling Inhibits Acute Inflammatory Liver Injury in Mice

    PubMed Central

    Osburn, William O.; Yates, Melinda S.; Dolan, Patrick D.; Liby, Karen T.; Sporn, Michael B.; Taguchi, Keiko; Yamamoto, Masayuki; Kensler, Thomas W.

    2008-01-01

    Oxidative stress-mediated destruction of normal parenchymal cells during hepatic inflammatory responses contributes to the pathogenesis of immune-mediated hepatitis and is implicated in the progression of acute inflammatory liver injury to chronic inflammatory liver disease. The transcription factor NF-E2-related factor 2 (Nrf2) regulates the expression of a battery of antioxidative enzymes and Nrf2 signaling can be activated by small-molecule drugs that disrupt Keap1-mediated repression of Nrf2 signaling. Therefore, genetic and pharmacologic approaches were used to activate Nrf2 signaling to assess protection against inflammatory liver injury. Profound increases in ind of cell death were observed in both Nrf2 wild-type (Nrf2-WT) mice and Nrf2-disrupted (Nrf2-KO) mice 24-hr following intravenous injection of concanavalin A (12.5 mg/kg, ConA), a model for T cell-mediated acute inflammatory liver injury. However, hepatocyte-specific conditional Keap1 null (Alb-Cre:Keap1flox/−, cKeap1-KO) mice with constitutively enhanced expression of Nrf2-regulated antioxidative genes as well as Nrf2-WT mice but not Nrf2-KO mice pretreated with three daily doses of a triterpenoid that potently activates Nrf2 (30 µmole/kg, CDDO-Im) were highly resistant to ConA-mediated inflammatory liver injury. CDDO-Im pretreatment of both Nrf2-WT and Nrf2-KO mice resulted in equivalent suppression of serum pro-inflammatory soluble proteins suggesting that the hepatoprotection afforded by CDDO-Im pretreatment of Nrf2-WT mice but not Nrf2-KO mice was not due to suppression of systemic pro-inflammatory signaling, but instead was due to activation of Nrf2 signaling in the liver. Enhanced hepatic expression of Nrf2-regulated antioxidative genes inhibited inflammation-mediated oxidative stress, thereby preventing hepatocyte necrosis. Attenuation of hepatocyte death in cKeap1-KO mice and CDDO-Im pretreated Nrf2-WT mice resulted in decreased late-phase pro-inflammatory gene expression in the liver

  12. 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

  13. Nrf2 suppresses macrophage inflammatory response by blocking proinflammatory cytokine transcription

    PubMed Central

    Kobayashi, Eri H.; Suzuki, Takafumi; Funayama, Ryo; Nagashima, Takeshi; Hayashi, Makiko; Sekine, Hiroki; Tanaka, Nobuyuki; Moriguchi, Takashi; Motohashi, Hozumi; Nakayama, Keiko; Yamamoto, Masayuki

    2016-01-01

    Nrf2 (NF-E2-related factor-2) transcription factor regulates oxidative/xenobiotic stress response and also represses inflammation. However, the mechanisms how Nrf2 alleviates inflammation are still unclear. Here, we demonstrate that Nrf2 interferes with lipopolysaccharide-induced transcriptional upregulation of proinflammatory cytokines, including IL-6 and IL-1β. Chromatin immunoprecipitation (ChIP)-seq and ChIP-qPCR analyses revealed that Nrf2 binds to the proximity of these genes in macrophages and inhibits RNA Pol II recruitment. Further, we found that Nrf2-mediated inhibition is independent of the Nrf2-binding motif and reactive oxygen species level. Murine inflammatory models further demonstrated that Nrf2 interferes with IL6 induction and inflammatory phenotypes in vivo. Thus, contrary to the widely accepted view that Nrf2 suppresses inflammation through redox control, we demonstrate here that Nrf2 opposes transcriptional upregulation of proinflammatory cytokine genes. This study identifies Nrf2 as the upstream regulator of cytokine production and establishes a molecular basis for an Nrf2-mediated anti-inflammation approach. PMID:27211851

  14. Innate immunity against bacterial infection following hyperoxia exposure is impaired in Nrf2-deficient mice

    PubMed Central

    Reddy, Narsa M.; Suryanarayana, Vegiraju; Kalvakolanu, Dhananjaya V; Yamamoto, Masayuki; Kensler, Thomas W.; Hassoun, Paul M.; Kleeberger, Steven R.; Reddy, Sekhar P.

    2010-01-01

    Summary Oxygen supplementation is used as therapy to support critically ill patients with severe respiratory impairment. Although hyperoxia has been shown to enhance the lung susceptibility to subsequent bacterial infection, the mechanisms underlying enhanced susceptibility remain enigmatic. We have reported that disruption of Nrf2, a master transcription regulator of various stress response pathways, enhances susceptibility to hyperoxia-induced acute lung injury (ALI) in mice, and have also demonstrated an association between a polymorphism in the NRF2 promoter and increased susceptibility to ALI. In this study, we show that Nrf2-deficient (Nrf2−/−) but not wild-type (Nrf2+/+) mice exposed to sub-lethal hyperoxia succumbed to death during recovery after P. aeruginosa infection. Nrf2-deficiency caused persistent bacterial pulmonary burden and enhanced levels of inflammatory cell infiltration as well as edema. Alveolar macrophages isolated from Nrf2−/− mice exposed to hyperoxia displayed persistent oxidative stress and inflammatory cytokine expression concomitant with diminished levels of antioxidant enzymes, such as Gclc, required for GSH biosynthesis. In vitro exposure of Nrf2−/− macrophages to hyperoxia strongly diminished their antibacterial activity and enhanced inflammatory cytokine expression compared to Nrf2+/+ cells. However, GSH supplementation during hyperoxic insult restored the ability of Nrf2−/− cells to mount antibacterial response and suppressed cytokine expression. Thus, loss of Nrf2 impairs lung innate immunity and promotes susceptibility to bacterial infection after hyperoxia exposure, ultimately leading to death of the host. PMID:19734219

  15. 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. PMID:27306194

  16. 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. PMID:25986586

  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. 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

  19. Association of Nrf2 Polymorphism Haplotypes with Acute Lung Injury Phenotypes in Inbred Strains of Mice

    PubMed Central

    Jedlicka, Anne E.; Gladwell, Wesley; Marzec, Jacqui; McCaw, Zackary R.; Bienstock, Rachelle J.; Kleeberger, Steven R.

    2015-01-01

    Abstract Aims: Nrf2 is a master transcription factor for antioxidant response element (ARE)-mediated cytoprotective gene induction. A protective role for pulmonary Nrf2 was determined in model oxidative disorders, including hyperoxia-induced acute lung injury (ALI). To obtain additional insights into the function and genetic regulation of Nrf2, we assessed functional single nucleotide polymorphisms (SNPs) of Nrf2 in inbred mouse strains and tested whether sequence variation is associated with hyperoxia susceptibility. Results: Nrf2 SNPs were compiled from publicly available databases and by re-sequencing DNA from inbred strains. Hierarchical clustering of Nrf2 SNPs categorized the strains into three major haplotypes. Hyperoxia susceptibility was greater in haplotypes 2 and 3 strains than in haplotype 1 strains. A promoter SNP −103 T/C adding an Sp1 binding site in haplotype 2 diminished promoter activation basally and under hyperoxia. Haplotype 3 mice bearing nonsynonymous coding SNPs located in (1862 A/T, His543Gln) and adjacent to (1417 T/C, Thr395Ile) the Neh1 domain showed suppressed nuclear transactivation of pulmonary Nrf2 relative to other strains, and overexpression of haplotype 3 Nrf2 showed lower ARE responsiveness than overexpression of haplotype 1 Nrf2 in airway cells. Importantly, we found a significant correlation of Nrf2 haplotypes and hyperoxic lung injury phenotypes. Innovation and Conclusion: The results indicate significant influence of Nrf2 polymorphisms and haplotypes on gene function and hyperoxia susceptibility. Our findings further support Nrf2 as a genetic determinant in ALI pathogenesis and provide useful tools for investigators who use mouse strains classified by Nrf2 haplotypes to elucidate the role for Nrf2 in oxidative disorders. Antioxid. Redox Signal. 22, 325–338. PMID:25268541

  20. Nrf2 Deficiency Improves Glucose Tolerance in Mice Fed a High-Fat Diet

    PubMed Central

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

    2012-01-01

    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. PMID:23017736

  1. The Cytoprotective Effect of Hyperoside against Oxidative Stress Is Mediated by the Nrf2-ARE Signaling Pathway through GSK-3β Inactivation

    PubMed Central

    Xing, Hai-Yan; Cai, Yong-Qing; Wang, Xian-Feng; Wang, Lin-Li; Li, Pan; Wang, Guan-Ying; Chen, Jian-Hong

    2015-01-01

    Glycogen synthase kinase-3β (GSK-3β) acts as a negative regulator of NF-E2 related factor 2 (Nrf2) by inducing Nrf2 degradation and nuclear export. Our previous study demonstrated that the flavonoid hyperoside elicits cytoprotection against oxidative stress by activating the Keap1-Nrf2-ARE signaling pathway, thus increasing the expression of antioxidant enzymes, such as heme oxygenase-1 (HO-1), superoxide dismutase (SOD) and catalase. However, the role of GSK-3β in hyperoside-mediated Nrf2 activation is unclear. Here, we demonstrate that in a normal human hepatocyte cell line, (L02), hyperoside is capable of inducing the phosphorylation of GSK-3β at Ser9 without affecting the protein levels of GSK-3β and its phosphorylation at Thr390. Lithium chloride (LiCl) and short interfering RNA (siRNA)-mediated inhibition of GSK-3β significantly enhanced the ability of hyperoside to protect L02 liver cells from H2O2-induced oxidative damage, leading to increased cell survival shown by the maintenance of cell membrane integrity and elevated levels of glutathione (GSH), one of the endogenous antioxidant biomarkers. Further study showed that LiCl and siRNA-mediated inhibition of GSK-3β increased hyperoside-induced HO-1 expression, and the effect was dependent upon enhanced Nrf2 nuclear translocation and gene expression. These activities were followed by ARE-mediated transcriptional activation in the presence of hyperoside, which was abolished by the transfection of the cells with Nrf2 siRNA. Furthermore, the siRNA-mediated inhibition of Keap1 also enhanced hyperoside-induced Nrf2 nuclear accumulation and HO-1 expression, which was relatively smaller than the effects obtained from GSK-3β siRNA administration. Moreover, Keap1 siRNA administration alone had no significant effect on the phosphorylation and protein expression of GSK-3β. Collectively, our data provide evidence that hyperoside attenuates H2O2 -induced L02 cell damage by activating the Nrf2-ARE signaling

  2. Activation of NRF2 Signaling in HEK293 Cells by a First-In-Class Direct KEAP1-NRF2 Inhibitor

    PubMed Central

    Wen, Xia; Thorne, Gabriell; Hu, Longqin; Joy, Melanie S.; Aleksunes, Lauren M.

    2015-01-01

    Under basal conditions, the antioxidant transcription factor NRF2 is bound to the KEAP1 protein and targeted for proteasomal degradation in the cytoplasm. In response to cellular injury or chemical treatment, NRF2 dissociates from KEAP1 and activates the transcription of protective genes and defends against injury. LH601A is a first-in-class direct inhibitor of the KEAP1-NRF2 protein-protein interaction. The purpose of this study was to determine whether LH601A activates NRF2 signaling in human kidney cells. HEK293 cells were treated with LH601A or the indirect NRF2 activator, sulforaphane (SFN) for 6 or 16 h. SFN and LH601A up-regulated NRF2 target genes HO-1 (2- to 7-fold), TRX1 (2-fold) and NQO1 mRNAs (2-fold). Both compounds also elevated HO-1 and TRX1 protein expression. Since NRF2 activation can protect tissues from injury, LH601A, a direct inhibitor of the KEAP1-NRF2 interaction may be used to defend against kidney injury and/or diseases. PMID:25683455

  3. LAPTM4B is associated with poor prognosis in NSCLC and promotes the NRF2-mediated stress response pathway in lung cancer cells.

    PubMed

    Maki, Yuho; Fujimoto, Junya; Lang, Wenhua; Xu, Li; Behrens, Carmen; Wistuba, Ignacio I; Kadara, Humam

    2015-01-01

    We recently demonstrated that lysosomal protein transmembrane 4 beta (LAPTM4B) is elevated in non-small cell lung cancers (NSCLCs) and in the surrounding premalignant airway field of cancerization. In the present study, we sought to begin to understand the relevance of LAPTM4B expression and signaling to NSCLC pathogenesis. In situ hybridization analysis of LAPTM4B transcript in tissue microarrays comprised of 368 NSCLCs demonstrated that LAPTM4B expression was significantly increased in smoker compared to non-smoker lung adenocarcinoma tumors (P < 0.001) and was significantly associated with poor overall survival (P < 0.05) in adenocarcinoma patients. Knockdown of LAPTM4B expression inhibited cell growth, induced cellular apoptosis and decreased cellular autophagy in serum starved lung cancer cells. Expression profiling coupled with pathways analysis revealed decreased activation of the nuclear factor erythroid 2-like 2 (NRF2) stress response pathway following LAPTM4B knockdown. Further analysis demonstrated that LAPTM4B augmented the expression and nuclear translocation of the NRF2 transcription factor following serum deprivation as well as increased the expression of NRF2 target genes such as heme oxygenase 1/HMOX1). Our study points to the relevance of LAPTM4B expression to NSCLC pathogenesis as well as to the probable role of LAPTM4B/NRF2 signaling in promoting lung cancer cell survival. PMID:26343532

  4. LAPTM4B is associated with poor prognosis in NSCLC and promotes the NRF2-mediated stress response pathway in lung cancer cells

    PubMed Central

    Maki, Yuho; Fujimoto, Junya; Lang, Wenhua; Xu, Li; Behrens, Carmen; Wistuba, Ignacio I.; Kadara, Humam

    2015-01-01

    We recently demonstrated that lysosomal protein transmembrane 4 beta (LAPTM4B) is elevated in non-small cell lung cancers (NSCLCs) and in the surrounding premalignant airway field of cancerization. In the present study, we sought to begin to understand the relevance of LAPTM4B expression and signaling to NSCLC pathogenesis. In situ hybridization analysis of LAPTM4B transcript in tissue microarrays comprised of 368 NSCLCs demonstrated that LAPTM4B expression was significantly increased in smoker compared to non-smoker lung adenocarcinoma tumors (P < 0.001) and was significantly associated with poor overall survival (P < 0.05) in adenocarcinoma patients. Knockdown of LAPTM4B expression inhibited cell growth, induced cellular apoptosis and decreased cellular autophagy in serum starved lung cancer cells. Expression profiling coupled with pathways analysis revealed decreased activation of the nuclear factor erythroid 2-like 2 (NRF2) stress response pathway following LAPTM4B knockdown. Further analysis demonstrated that LAPTM4B augmented the expression and nuclear translocation of the NRF2 transcription factor following serum deprivation as well as increased the expression of NRF2 target genes such as heme oxygenase 1/HMOX1). Our study points to the relevance of LAPTM4B expression to NSCLC pathogenesis as well as to the probable role of LAPTM4B/NRF2 signaling in promoting lung cancer cell survival. PMID:26343532

  5. 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.

  6. Vitamin E prevents NRF2-suppression by allergen in asthmatic alveolar macrophages in vivo

    PubMed Central

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

    2011-01-01

    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 of 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 effect 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. PMID:21605660

  7. 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. PMID:27192495

  8. 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

  9. Heme activates the heme oxygenase-1 gene in renal epithelial cells by stabilizing Nrf2.

    PubMed

    Alam, Jawed; Killeen, Erin; Gong, Pengfei; Naquin, Ryan; Hu, Bin; Stewart, Daniel; Ingelfinger, Julie R; Nath, Karl A

    2003-04-01

    The mechanism of heme oxygenase-1 gene (ho-1) activation by heme in immortalized rat proximal tubular epithelial cells was examined. Analysis of the ho-1 promoter identified the heme-responsive sequences as the stress-response element (StRE), multiple copies of which are present in two enhancer regions, E1 and E2. Electrophoretic mobility shift assays identified Nrf2, MafG, ATF3, and Jun and Fos family members as StRE-binding proteins; binding of Nrf2, MafG, and ATF3 was increased in response to heme. Dominant-negative mutants of Nrf2 and Maf, but not of c-Fos and c-Jun, inhibited basal and heme-induced expression of an E1-controlled luciferase gene. Heme did not affect the transcription activity of Nrf2, dimerization between Nrf2 and MafG, or the level of MafG, but did stimulate expression of Nrf2. Heme did not influence the level of Nrf2 mRNA but increased the half-life of Nrf2 protein from approximately 10 min to nearly 110 min. These results indicate that heme promotes stabilization of Nrf2, leading to accumulation of Nrf2. MafG dimers that bind to StREs to activate the ho-1 gene. PMID:12453873

  10. 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. PMID:24500083

  11. Green Tea Polyphenol (−)-Epigallocatechin-3-Gallate Restores Nrf2 Activity and Ameliorates Crescentic Glomerulonephritis

    PubMed Central

    Zhou, Jason K.; Peng, Ai; Vaziri, Nosratola D.; Mohan, Chandra; Xu, Yan; Zhou, Xin J.

    2015-01-01

    Crescentic glomerulonephritis (GN) is the most severe form of GN and is associated with significant morbidity and mortality despite aggressive immunotherapy with steroids, cytotoxic drugs, and plasmapheresis. We examined the therapeutic efficacy of the green tea polyphenol (−)-epigallocatechin-3-gallate (EGCG, 50 mg/kg BW/day x3weeks), a potent anti-inflammatory and anti-oxidant agent, on experimental crescentic GN induced in 129/svJ mice by administration of rabbit anti-mouse glomerular basement membrane sera. Routine histology and key molecules involved in inflammatory and redox signaling were studied. EGCG treatment significantly reduced mortality, decreased proteinuria and serum creatinine, and markedly improved renal histology when compared with vehicle-treated mice. The improvements in renal function and histology were accompanied by the restoration of Nrf2 signaling (which was impaired in vehicle-treated mice) as shown by increased nuclear translocation of Nrf2 and cytoplasmic glutamate cysteine ligase catalytic subunit, glutamate cysteine ligase modifier subunit, and glutathione peroxidase. EGCG-treated mice also showed reduction in p-Akt, p-JNK, p-ERK1/2 and p-P38 as well as restoration of PPARγ and SIRT1 levels. Lower dose of EGCG (25 mg/kg BW/day x2 weeks) treatment also significantly decreased proteinuria and serum creatinine, and markedly improved renal histology when compared with vehicle-treated mice. Thus, our data illustrate the efficacy of EGCG in reversing the progression of crescentic GN in mice by targeting multiple signaling and inflammatory pathways as well as countering oxidative stress. PMID:25785827

  12. 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

  13. 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

  14. Allergic skin inflammation induced by chemical sensitizers is controlled by the transcription factor Nrf2.

    PubMed

    El Ali, Zeina; Gerbeix, Cédric; Hemon, Patrice; Esser, Philipp R; Martin, Stefan F; Pallardy, Marc; Kerdine-Römer, Saadia

    2013-07-01

    Allergic contact dermatitis (ACD) is induced by low-molecular weight electrophilic chemicals and metal ions. Chemical contact sensitizers trigger reactive oxygen species production and provoke electrophilic stress, leading to the accumulation of the transcription factor nuclear-related factor 2 (Nrf2) in innate immune cell types. The objective of this work was to identify the role of Nrf2 in the regulation of ACD. We used the local lymph node assay (LLNA) and the mouse ear swelling test (MEST) to study the role of Nrf2 in both the sensitization and elicitation phase in nrf2 knockout (nrf2(-/-)) and wild-type (nrf2(+/+)) mice. Five chemicals were used: two compounds known to react with cysteine residues, 2,4-dinitrochlorobenzene (DNCB) and cinnamaldehyde (CinA); one sensitizer known to exhibit mixed reactivity to cysteine and lysine residues, isophorone diisocyanate; and one reacting specifically with lysine residues, trimellitic anhydride and croton oil, a well-known irritant. In the MEST assay, DNCB (1 and 2%) induced a significant increase in ear thickness in nrf2(-/-) compared with nrf2(+/+) mice, suggesting a role for Nrf2 in the control of the inflammatory process. When DNCB was used at 0.25 and 0.5% or when mice were treated with CinA, inflammation was found only in nrf2(-/-) mice. In the LLNA, all chemical sensitizers induced an increase of lymphocyte proliferation in nrf2(-/-) compared with nrf2(+/+) mice for the same chemical concentration. These results reveal an important role for Nrf2 in controlling ACD and lymphocyte proliferation in response to sensitizers. PMID:23564646

  15. Poly(ADP-ribose) polymerase-1 modulates Nrf2-dependent transcription.

    PubMed

    Wu, Tongde; Wang, Xiao-Jun; Tian, Wang; Jaramillo, Melba C; Lau, Alexandria; Zhang, Donna D

    2014-02-01

    The basic leucine zipper transcription factor Nrf2 has emerged as a master regulator of intracellular redox homeostasis by controlling the expression of a battery of redox-balancing antioxidants and phase II detoxification enzymes. Under oxidative stress conditions, Nrf2 is induced at the protein level through redox-sensitive modifications on critical cysteine residues in Keap1, a component of an E3 ubiquitin ligase complex that targets Nrf2 for proteasomal degradation. Poly(ADP-ribose) polymerase-1 (PARP-1) is historically known to function in DNA damage detection and repair; however, recently PARP-1 has been shown to play an important role in other biochemical activities, such as DNA methylation and imprinting, insulator activity, chromosome organization, and transcriptional regulation. The exact role of PARP-1 in transcription modulation and the underlying mechanisms remain poorly defined. In this study, we report that PARP-1 forms complexes with the antioxidant response element (ARE) within the promoter region of Nrf2 target genes and upregulates the transcriptional activity of Nrf2. Interestingly, PARP-1 neither physically interacts with Nrf2 nor promotes the expression of Nrf2. In addition, PARP-1 does not target Nrf2 for poly(ADP-ribosyl)ation. Instead, PARP-1 interacts directly with small Maf proteins and the ARE of Nrf2 target genes, which augments ARE-specific DNA-binding of Nrf2 and enhances the transcription of Nrf2 target genes. Collectively, these results suggest that PARP-1 serves as a transcriptional coactivator, upregulating the transcriptional activity of Nrf2 by enhancing the interaction among Nrf2, MafG, and the ARE. PMID:24140708

  16. 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.

  17. 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

  18. 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

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

    2016-05-01

    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

  19. 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

  20. 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

  1. 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.

  2. 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.

  3. Nrf2 Expressions Correlate with WHO Grades in Gliomas and Meningiomas

    PubMed Central

    Tsai, Wen-Chiuan; Hueng, Dueng-Yuan; Lin, Chii-Ruey; Yang, Thomas C. K.; Gao, Hong-Wei

    2016-01-01

    Background: Nuclear factor erythroid 2-related factor 2 (NFE2L2, also known as Nrf2) is associated with cellular progression and chemotherapeutic resistance in some human cancers. We tested the relationship between Nrf2 expression and survival of patients with primary brain tumors (PBTs). Methods: In order to realize Nrf2 protein expression in gliomas, Western blot analysis was performed in normal brain tissue and U87MG, LN229, GBM8401 and U118MG glioma cell lines protein lysates. Then, U87MG, LN229, and GBM8401 mRNA were applied to performed quantitative RT-PCR for detect Nrf2 gene expression in glioma cell lines. At last, immunohistochemical analysis was used to determine the expression of Nrf2 in samples from 178 PBTs and 10 non-neoplastic brain tissues. Results: In these included in vitro studies, both Nrf2 protein and mRNA expression in all human glioma cell lines were higher than normal brain tissue. Similarly, on the viewpoint of immunohistochemistry, Nrf2 expression in gliomas were positively correlated with World Health Organization (WHO) grades. Additionally, compared with the expression of Nrf2 in non-neoplastic brain tissue, expression in meningiomas was of a stronger intensity and was present in a higher percentage of cells. Furthermore, scores were significantly higher in WHO grade II than in WHO grade I meningiomas. Finally, overall survival tended to be shorter in patients whose PBTs had higher expression of Nrf2, although the correlation was not statistically significant. Conclusions: Nrf2 overexpression positively correlated with WHO grade in gliomas and meningiomas. On the other hand, Nrf2 immunohistochemical stain could help pathologists to differentiate atypical meningiomas from benign tumors. Therefore, Nrf2 expression may be a useful biomarker to predict WHO grade and cellular behavior of PBTs. PMID:27187376

  4. Mutant p53 confers chemoresistance in non-small cell lung cancer by upregulating Nrf2

    PubMed Central

    Wang, Yao-Chen; He, Tsung-Ying; Lee, Ming-Ching; Yeh, Sauh-Der; Chen, Chih-Yi; Lee, Huei

    2015-01-01

    Nrf2 is a key transcription factor for genes coding for antioxidants, detoxification enzymes, and multiple drug resistance and it also confers resistance to anticancer drugs. Here, we hypothesized that mutant p53 could upregulate Nrf2 expression at the transcriptional level, thereby conferring cisplatin resistance in non-small cell lung cancer (NSCLC). Luciferase reporter assays and real-time PCR analysis indicated that the Nrf2 promoter activity and its mRNA levels were markedly suppressed by wild-type p53, but not by mutant p53. Chromatin immunoprecipitation (ChIP) further confirmed that wild-type p53 binds at the p53 putative binding site to block Sp1 binding to the Nrf2 promoter and consequently to suppress the Nrf2 promoter activity. The MTT assay indicated that an increase in Nrf2 expression by mutant p53 is responsible for cisplatin resistance. Among the Nrf2 downstream genes, Bcl-2 and Bcl-xL contribute more strongly to Nrf2-mediated cisplatin resistance when compared with heme oxygenase 1 (HO-1). Cox regression analysis showed that patients with high-Nrf2, high-Bcl-2, high-Bcl-xL mRNA tumors were more commonly occurred unfavorable response to cisplatin-based chemotherapy than their counterparts. The prognostic significance of Nrf2 mRNA levels on OS and RFS was also observed in patients who have received cisplatin-based chemotherapy, particularly in p53-mutant patients. Collectively, mutant p53 may confer cisplatin resistance via upregulation of Nrf2 expression, and Nrf2 mRNA level may predict chemotherapeutic response and outcomes in NSCLC. PMID:26497680

  5. 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.

  6. High levels of Nrf2 determine chemoresistance in type II endometrial cancer

    PubMed Central

    Jiang, Tao; Chen, Ning; Zhao, Fei; Wang, Xiao-Jun; Kong, Beihua; Zheng, Wenxin; Zhang, Donna D.

    2010-01-01

    Type II endometrial cancer, which mainly presents as serous and clear cell types, has proved to be the most malignant and recurrent carcinoma among various female genital malignancies. The transcription factor, Nrf2, was first described as having chemopreventive activity. Activation of the Nrf2-mediated cellular defense response protects cells against the toxic and carcinogenic effects of environmental insults by upregulating an array of genes that detoxify reactive oxygen species (ROS) and restore cellular redox homeostasis. However, the cancer-promoting role of Nrf2 has recently been revealed. Nrf2 is constitutively upregulated in several types of human cancer tissues and cancer cell lines. Furthermore, inhibition of Nrf2 expression sensitizes cancer cells to chemotherapeutic drugs. In this study, the constitutive level of Nrf2 was compared in different types of human endometrial tumors. It was found that Nrf2 was highly expressed in endometrial serous carcinoma (ESC), whereas complex hyperplasia (CH) and endometrial endometrioid carcinoma (EEC) had no or marginal expression of Nrf2. Likewise, the ESC derived SPEC-2 cell line had a higher level of Nrf2 expression and was more resistant to the toxic effects of cisplatin and paclitaxel than that of the Ishikawa cell line, which was generated from EEC. Silencing of Nrf2 rendered SPEC-2 cells more susceptible to chemotherapeutic drugs while it had a limited effect on Ishikawa cells. Inhibition of Nrf2 expression by overexpressing Keap1 sensitized SPEC-2 cells or SPEC-2-derived xenografts to chemotherapeutic treatments using both cell culture and SCID mouse models. Collectively, we provide a molecular basis for the use of Nrf2 inhibitors to increase the efficacy of chemotherapeutic drugs and to combat chemoresistance, the biggest obstacle in chemotherapy. PMID:20530669

  7. 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. PMID:23843400

  8. The flavonoid, eriodictyol, induces long-term protection in ARPE-19 cells through its effects on Nrf2 activation and phase II gene expression

    PubMed Central

    Johnson, Jennifer; Maher, Pamela

    2009-01-01

    Purpose Eriodictyol, a flavonoid found in citrus fruits, is among the most potent compounds reported to protect human RPE cells from oxidative stress-induced cell death. In the present study, we determined whether eriodictyol-induced phase II protein expression further enhances the resistance of human ARPE-19 cells to oxidative stress. Methods We analyzed the ability of eriodictyol to activate Nrf2 and induce the phase II proteins, heme-oxygenase (HO-1), NAD(P)H: quinone oxidoreductase 1 (NQO-1), and the cellular antioxidant glutathione, (GSH). We performed cytoprotection assays in ARPE-19 cells that were overexpressing HO-1 or NQO-1. We compared cell survival after short-term and long-term eriodictyol treatment and tested the mechanism of protection using a dominant negative Nrf2 and an shRNA specific for HO-1. Results We demonstrate that eriodictyol induces the nuclear translocation of Nrf2, enhances the expression of HO-1 and NQO-1, and increases the levels of intracellular glutathione. We show that ARPE-19 cells that overexpress HO-1 or NQO-1 are more resistant to oxidative stress-induced cell death than control cells. We demonstrate that eriodictyol induces long-term protection that is significantly greater than its short-term protection, and this effect is correlated temporally with both the activation of Nrf2 and the induction of phase II enzymes. We demonstrate that this effect can be blocked with the use of a dominant negative to Nrf2 and an shRNA specific to HO-1. Conclusions These findings indicate the greatest benefit from eriodictyol may be its ability to regulate gene expression and enhance multiple cellular defenses to oxidative injury. PMID:19117929

  9. 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

  10. 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

  11. Increased cell migration and plasticity in Nrf2-deficient cancer cell lines.

    PubMed

    Rachakonda, G; Sekhar, K R; Jowhar, D; Samson, P C; Wikswo, J P; Beauchamp, R D; Datta, P K; Freeman, M L

    2010-06-24

    Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) expression is deregulated in many cancers. Genetic and biochemical approaches coupled with functional assays in cultured cells were used to explore the consequences of Nrf2 repression. Nrf2 suppression by Keap1-directed ubiquitylation or the expression of independent short hairpin RNA (shRNA)/siRNA sequences enhanced cellular levels of reactive oxygen species, Smad-dependent tumor cell motility and growth in soft agar. Loss of Nrf2 was accompanied by concomitant Smad linker region/C-terminus phosphorylation, induction of the E-cadherin transcriptional repressor Slug and suppression of the cell-cell adhesion protein E-cadherin. Ectopic expression of the wildtype but not dominant-negative Nrf2 suppressed the activity of a synthetic transforming growth factor-beta1-responsive CAGA-directed luciferase reporter. shRNA knock-down of Nrf2 enhanced the activity of the synthetic CAGA reporter, as well as the expression of the endogenous Smad target gene plasminogen activator inhibitor-1. Finally, we found that Nrf2/Smad3/Smad4 formed an immunoprecipitable nuclear complex. Thus, loss of Nrf2 increased R-Smad phosphorylation and R-Smad signaling, supporting the hypothesis that loss of Nrf2 in an oncogenic context-dependent manner can enhance cellular plasticity and motility, in part by using transforming growth factor-beta/Smad signaling. PMID:20440267

  12. 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

  13. BRCA1 interacts with Nrf2 to regulate antioxidant signaling and cell survival

    PubMed Central

    Gorrini, Chiara; Baniasadi, Pegah S.; Harris, Isaac S.; Silvester, Jennifer; Inoue, Satoshi; Snow, Bryan; Joshi, Purna A.; Wakeham, Andrew; Molyneux, Sam D.; Martin, Bernard; Bouwman, Peter; Cescon, David W.; Elia, Andrew J.; Winterton-Perks, Zoe; Cruickshank, Jennifer; Brenner, Dirk; Tseng, Alan; Musgrave, Melinda; Berman, Hal K.; Khokha, Rama; Jonkers, Jos

    2013-01-01

    Oxidative stress plays an important role in cancer development and treatment. Recent data implicate the tumor suppressor BRCA1 in regulating oxidative stress, but the molecular mechanism and the impact in BRCA1-associated tumorigenesis remain unclear. Here, we show that BRCA1 regulates Nrf2-dependent antioxidant signaling by physically interacting with Nrf2 and promoting its stability and activation. BRCA1-deficient mouse primary mammary epithelial cells show low expression of Nrf2-regulated antioxidant enzymes and accumulate reactive oxygen species (ROS) that impair survival in vivo. Increased Nrf2 activation rescues survival and ROS levels in BRCA1-null cells. Interestingly, 53BP1 inactivation, which has been shown to alleviate several defects associated with BRCA1 loss, rescues survival of BRCA1-null cells without restoring ROS levels. We demonstrate that estrogen treatment partially restores Nrf2 levels in the absence of BRCA1. Our data suggest that Nrf2-regulated antioxidant response plays a crucial role in controlling survival downstream of BRCA1 loss. The ability of estrogen to induce Nrf2 posits an involvement of an estrogen-Nrf2 connection in BRCA1 tumor suppression. Lastly, BRCA1-mutated tumors retain a defective antioxidant response that increases the sensitivity to oxidative stress. In conclusion, the role of BRCA1 in regulating Nrf2 activity suggests important implications for both the etiology and treatment of BRCA1-related cancers. PMID:23857982

  14. Sulforaphane Protects against Cardiovascular Disease via Nrf2 Activation

    PubMed Central

    Bai, Yang; Wang, Xiaolu; Zhao, Song; Ma, Chunye; Cui, Jiuwei; Zheng, Yang

    2015-01-01

    Cardiovascular disease (CVD) causes an unparalleled proportion of the global burden of disease and will remain the main cause of mortality for the near future. Oxidative stress plays a major role in the pathophysiology of cardiac disorders. Several studies have highlighted the cardinal role played by the overproduction of reactive oxygen or nitrogen species in the pathogenesis of ischemic myocardial damage and consequent cardiac dysfunction. Isothiocyanates (ITC) are sulfur-containing compounds that are broadly distributed among cruciferous vegetables. Sulforaphane (SFN) is an ITC shown to possess anticancer activities by both in vivo and epidemiological studies. Recent data have indicated that the beneficial effects of SFN in CVD are due to its antioxidant and anti-inflammatory properties. SFN activates NF-E2-related factor 2 (Nrf2), a basic leucine zipper transcription factor that serves as a defense mechanism against oxidative stress and electrophilic toxicants by inducing more than a hundred cytoprotective proteins, including antioxidants and phase II detoxifying enzymes. This review will summarize the evidence from clinical studies and animal experiments relating to the potential mechanisms by which SFN modulates Nrf2 activation and protects against CVD. PMID:26583056

  15. 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.

  16. 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. PMID:24361900

  17. Seaweed extracts and unsaturated fatty acid constituents from the green alga Ulva lactuca as activators of the cytoprotective Nrf2–ARE pathway

    PubMed Central

    Wang, Rui; Paul, Valerie J.; Luesch, Hendrik

    2013-01-01

    with 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. PMID:23291594

  18. 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. PMID:23291594

  19. 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

    2013-01-01

    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

  20. Dimethyl fumarate and monoethyl fumarate exhibit differential effects on KEAP1, NRF2 activation, and glutathione depletion in vitro.

    PubMed

    Brennan, Melanie S; Matos, Maria F; Li, Bing; Hronowski, Xiaoping; Gao, Benbo; Juhasz, Peter; Rhodes, Kenneth J; Scannevin, Robert H

    2015-01-01

    Delayed-release dimethyl fumarate (also known as gastro-resistant dimethyl fumarate), an oral therapeutic containing dimethyl fumarate (DMF) as the active ingredient, is currently approved for the treatment of relapsing multiple sclerosis. DMF is also a component in a distinct mixture product with 3 different salts of monoethyl fumarate (MEF), which is marketed for the treatment of psoriasis. Previous studies have provided insight into the pharmacologic properties of DMF, including modulation of kelch-like ECH-associated protein 1 (KEAP1), activation of the nuclear factor (erythroid-derived 2)-like 2 (NRF2) pathway, and glutathione (GSH) modulation; however, those of MEF remain largely unexplored. Therefore, the aim of this study was to evaluate the in vitro effects of DMF and MEF on KEAP1 modification, activation of the NRF2 pathway, and GSH conjugation. Using mass spectrometry, DMF treatment resulted in a robust modification of specific cysteine residues on KEAP1. In comparison, the overall degree of KEAP1 modification following MEF treatment was significantly less or undetectable. Consistent with KEAP1 cysteine modification, DMF treatment resulted in nuclear translocation of NRF2 and a robust transcriptional response in treated cells, as did MEF; however, the responses to MEF were of a lower magnitude or distinct compared to DMF. DMF was also shown to produce an acute concentration-dependent depletion of GSH; however, GSH levels eventually recovered and rose above baseline by 24 hours. In contrast, MEF did not cause acute reductions in GSH, but did produce an increase by 24 hours. Overall, these studies demonstrate that DMF and MEF are both pharmacologically active, but have differing degrees of activity as well as unique actions. These differences would be expected to result in divergent effects on downstream biology. PMID:25793262

  1. Nrf2 Knockout Attenuates the Anti-Inflammatory Effects of Phenethyl Isothiocyanate and Curcumin

    PubMed Central

    2015-01-01

    The role of phytochemicals in preventive and therapeutic medicine is a major area of scientific research. Several studies have illustrated the mechanistic roles of phytochemicals in Nrf2 transcriptional activation. The present study aims to examine the importance of the transcription factor Nrf2 by treating peritoneal macrophages from Nrf2+/+ and Nrf2–/– mice ex vivo with phenethyl isothiocyanate (PEITC) and curcumin (CUR). The peritoneal macrophages were pretreated with the drugs and challenged with lipopolysaccharides (LPSs) alone and in combination with PEITC or CUR to assess their anti-inflammatory and antioxidative effects based on gene and protein expression in the treated cells. LPS treatment resulted in an increase in the expression of inflammatory markers such as cycloxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in both Nrf2+/+ and Nrf2–/– macrophages, detected by quantitative polymerase chain reaction (qPCR). Nrf2+/+ macrophages treated with PEITC and CUR exhibited a significant decrease in the expression of these anti-inflammatory genes along with an increase in the expression of hemeoxygenase-1 (HO-1), which is an antioxidative stress gene downstream of the Nrf2 transcription factor battery. Although there was no significant decrease in the expression of the anti-inflammatory genes or an increase in HO-1 expression in Nrf2–/– macrophages treated with either PEITC or CUR, there was a significant decrease in the protein expression of COX-2 and an increase in the expression of HO-1 in Nrf2+/+ macrophages treated with PEITC compared to that with CUR treatment. No significant changes were observed in the macrophages from knockout animals. Additionally, there was a significant decrease in LPS-induced IL-6 and TNF-α production following PEITC treatment compared with that following CUR in Nrf2+/+ macrophages, whereas no change was observed in the macrophages from knockout

  2. 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

  3. Cytoprotective Role of Nrf2 in Electrical Pulse Stimulated C2C12 Myotube

    PubMed Central

    Horie, Masaki; Warabi, Eiji; Komine, Shoichi; Oh, Sechang; Shoda, Junichi

    2015-01-01

    Regular physical exercise is central to a healthy lifestyle. However, exercise-related muscle contraction can induce reactive oxygen species and reactive nitrogen species (ROS/RNS) production in skeletal muscle. The nuclear factor-E2-related factor-2 (Nrf2) transcription factor is a cellular sensor for oxidative stress. Regulation of nuclear Nrf2 signaling regulates antioxidant responses and protects organ structure and function. However, the role of Nrf2 in exercise- or contraction-induced ROS/RNS production in skeletal muscle is not clear. In this study, using differentiated C2C12 cells and electrical pulse stimulation (EPS) of muscle contraction, we explored whether Nrf2 plays a role in the skeletal muscle response to muscle contraction-induced ROS/RNS. We found that EPS (40 V, 1 Hz, 2 ms) stimulated ROS/RNS accumulation and Nrf2 activation. We also showed that expression of NQO1, HO-1 and GCLM increased after EPS-induced muscle contraction and was remarkably suppressed in cells with Nrf2 knockdown. We also found that the antioxidant N-acetylcysteine (NAC) significantly attenuated Nrf2 activation after EPS, whereas the nitric oxide synthetase inhibitor Nω-nitro-L-arginine methyl ester (L-NAME) did not. Furthermore, Nrf2 knockdown after EPS markedly decreased ROS/RNS redox potential and cell viability and increased expression of the apoptosis marker Annexin V in C2C12 myotubes. These results indicate that Nrf2 activation and expression of Nrf2 regulated-genes protected muscle against the increased ROS caused by EPS-induced muscle contraction. Thus, our findings suggest that Nrf2 may be a key factor for preservation of muscle function during muscle contraction. PMID:26658309

  4. Molecular Basis of Electrophilic and Oxidative Defense: Promises and Perils of Nrf2

    PubMed Central

    Ma, Qiang; He, Xiaoqing

    2015-01-01

    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

  5. 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.

  6. Epigenetic DNA methylation of antioxidative stress regulator NRF2 in human prostate cancer.

    PubMed

    Khor, Tin Oo; Fuentes, Francisco; Shu, Limin; Paredes-Gonzalez, Ximena; Yang, Anne Yuqing; Liu, Yue; Smiraglia, Dominic J; Yegnasubramanian, Srinivasan; Nelson, William G; Kong, Ah-Ng Tony

    2014-12-01

    Epigenetic control of NRF2, a master regulator of many critical antioxidative stress defense genes in human prostate cancer (CaP), is unknown. Our previous animal study found decreased Nrf2 expression through promoter CpG methylation/histone modifications during prostate cancer progression in TRAMP mice. In this study, we evaluated CpG methylation of human NRF2 promoter in 27 clinical prostate cancer samples and in LNCaP cells using MAQMA analysis and bisulfite genomic DNA sequencing. Prostate cancer tissue microarray (TMA) containing normal and prostate cancer tissues was studied by immunohistochemistry. Luciferase reporter assay using specific human NRF2 DNA promoter segments and chromatin immunoprecipitation (ChIP) assay against histone modifying proteins were performed in LNCaP cells. Three specific CpG sites in the NRF2 promoter were found to be hypermethylated in clinical prostate cancer samples (BPHNRF2 staining in human prostate cancer TMA showed a decreasing trend for both intensity and percentage of positive cells from normal tissues to advanced-stage prostate cancer (Gleason score from 3-9). Reporter assays in the LNCaP cells containing these three CpG sites showed methylation-inhibited transcriptional activity of the NRF2 promoter. LNCaP cells treated with 5-aza/TSA restored the expression of NRF2 and NRF2 downstream target genes, decreased expression levels of DNMT and HDAC proteins, and ChIP assays showed increased RNA Pol II and H3Ac with a concomitant decrease in H3K9me3, MBD2, and MeCP2 at CpG sites of human NRF2 promoter. Taken together, these findings suggest that epigenetic modification may contribute to the regulation of transcription activity of NRF2, which could be used as prevention and treatment target of human prostate cancer. PMID:25266896

  7. NF-κB and Nrf2 signaling pathways contribute to wogonin-mediated inhibition of inflammation-associated colorectal carcinogenesis

    PubMed Central

    Yao, J; Zhao, L; Zhao, Q; Zhao, Y; Sun, Y; Zhang, Y; Miao, H; You, Q-D; Hu, R; Guo, Q-L

    2014-01-01

    The transcriptional factors nuclear factor-κB (NF-κB) and NF-E2-related factor 2 (Nrf2) have been recently reported to have critical roles in protecting various tissues against inflammation and colitis-associated colorectal cancer (aberrant crypt foci). Our previous studies showed that wogonin (5,7-dihydroxy-8-methoxyflavone) possessed anti-neoplastic and anti-inflammatory activities. The present study extended these important earlier findings by exploring the effect of wogonin on the initiation and development of colitis-associated cancer. Wogonin lowered tumor incidence and inhibited the development of colorectal adenomas in azoxymethane- or dextran sulfate sodium-induced mice. We found that wogonin significantly decreased the secretion and expression of IL-6 and IL-1β, reduced cell proliferation and nuclear expression of NF-κB in adenomas and surrounding tissues and promoted Nrf2 nuclear translocation in surrounding tissues, although overexpressed Nrf2 in tumor tissues was independent of wogonin administration. Furthermore, wogonin inhibited the interaction between human monocytic THP-1 cells and human colon cancer HCT116 cells, and significantly downregulated lipopolysaccharide-induced secretion of prototypical pro-inflammatory cytokines IL-6 and IL-1β in THP-1 cells. Further mechanism research revealed that wogonin inhibited the nuclear translocation of NF-κB and phosphorylation of IκB and IKKα/β, and promoted Nrf2 signaling pathway in HCT116 cells and THP-1 cells. Taken together, the present results indicated that wogonin effectively suppressed inflammation-associated colon carcinogenesis and cancer development, suggesting its potential as a chemopreventive agent against colitis-associated colon cancer. PMID:24901054

  8. 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

  9. 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. PMID:26492067

  10. 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. PMID:26371321

  11. The Keap1-Nrf2 System Prevents Onset of Diabetes Mellitus

    PubMed Central

    Uruno, Akira; Furusawa, Yuki; Yagishita, Yoko; Fukutomi, Toshiaki; Muramatsu, Hiroyuki; Negishi, Takaaki; Sugawara, Akira; Kensler, Thomas W.

    2013-01-01

    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. PMID:23716596

  12. 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

  13. 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...

  14. 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

  15. Nrf2 is the key to chemotherapy resistance in MCF7 breast cancer cells under hypoxia

    PubMed Central

    Syu, Jhih-Pu; Chi, Jen-Tsan; Kung, Hsiu-Ni

    2016-01-01

    Hypoxia leads to reactive oxygen species (ROS) imbalance, which is proposed to associate with drug resistance and oncogenesis. Inhibition of enzymes of antioxidant balancing system in tumor cells was shown to reduce chemoresistance under hypoxia. However, the underlying mechanism remains unknown. The key regulator of antioxidant balancing system is nuclear factor erythroid 2-related factor 2 (NFE2L2, Nrf2). In this study, we showed that hypoxia induced ROS production and increased the Nrf2 activity. Nrf2 activation increased levels of its downstream target antioxidant enzymes, including GCLC and GCLM. The Nrf2-overexpressing also confers chemo-resistant MCF7 cells under normoxia. The in vivo mouse model also demonstrated that the chemical inhibition of Nrf2 can increase cisplatin (CDDP) cytotoxicity. Together, these results showed that Nrf2 serves as a key regulator in chemotherapeutic resistance under hypoxia through ROS-Nrf2-GCLC-GSH pathway. Therefore, targeting Nrf2 can be a potential treatment for hypoxia-induced drug resistance in breast cancer cells. PMID:26894974

  16. Novel Chalcone Derivatives as Potent Nrf2 Activators in Mice and Human Lung Epithelial Cells

    PubMed Central

    Kumar, Vineet; Kumar, Sarvesh; Hassan, Mohammad; Wu, Hailong; Thimmulappa, Rajesh K.; Kumar, Amit; Sharma, Sunil K.; Parmar, Virinder S.; Biswal, Shyam; Malhotra, Sanjay V.

    2011-01-01

    Nrf2-mediated activation of antioxidant response element is a central part of molecular mechanisms governing the protective function of phase II detoxification and antioxidant enzymes against carcinogenesis, oxidative stress and inflammation. Nrf2 is sequestered in the cytoplasm by its repressor, Keap1. We have designed and synthesized novel chalcone derivatives as Nrf2 activators. The potency of these compounds was measured by the expression of Nrf2 dependent antioxidant genes, GCLM, NQO1 and HO1, in human lung epithelial cells; while the cytotoxicity was analyzed using MTT assay. In vivo potency of identified lead compounds to activate Nrf2 was evaluated using mouse model. Our studies showed 2-trifluoromethyl-2’-methoxychalone (2b) to be a potent activator of Nrf2, both, in vitro and in mice. Additional experiments showed that the activation of Nrf2 by this compound is independent of reactive oxygen species or redox changes. We have discussed a quantitative structure-activity relationship and proposed a possible mechanism of Nrf2 activation. PMID:21539383

  17. Nrf2 protects against As(III)-induced damage in mouse liver and bladder

    PubMed Central

    Jiang, Tao; Huang, Zheping; Chan, Jefferson Y.; Zhang, Donna D.

    2009-01-01

    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−/− mice displayed more severe pathological changes in the liver and bladder, compared to Nrf2+/+ mice. Furthermore, Nrf2−/− 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. PMID:19538980

  18. 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

  19. 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

  20. Glucose availability is a decisive factor for Nrf2-mediated gene expression.

    PubMed

    Heiss, Elke H; Schachner, Daniel; Zimmermann, Kristin; Dirsch, Verena M

    2013-01-01

    Activation of the transcription factor Nrf2 (nuclear factor-erythroid 2-related factor 2) is one of the major cellular defense lines against oxidative and xenobiotic stress, but also influences genes involved in lipid and glucose metabolism. It is unresolved whether the cytoprotective and metabolic responses mediated by Nrf2 are connected or separable events in non-malignant cells. In this study we show that activation of Nrf2, either by the small molecule sulforaphane or knockout of the Nrf2 inhibitor Keap1, leads to increased cellular glucose uptake and increased glucose addiction in fibroblasts. Upon Nrf2 activation glucose is preferentially metabolized through the pentose phosphate pathway with increased production of NADPH. Interference with the supply of glucose or the pentose phosphate pathway and NADPH generation not only hampers Nrf2-mediated detoxification of reactive oxygen species on the enzyme level but also Nrf2-initiated expression of antioxidant defense proteins, such as glutathione reductase and heme-oxygenase1. We conclude that the Nrf2-dependent protection against oxidative stress relies on an intact pentose phosphate pathway and that there is crosstalk between metabolism and detoxification already at the level of gene expression in mammalian cells. PMID:24024172

  1. Glucose availability is a decisive factor for Nrf2-mediated gene expression☆

    PubMed Central

    Heiss, Elke H.; Schachner, Daniel; Zimmermann, Kristin; Dirsch, Verena M.

    2013-01-01

    Activation of the transcription factor Nrf2 (nuclear factor-erythroid 2-related factor 2) is one of the major cellular defense lines against oxidative and xenobiotic stress, but also influences genes involved in lipid and glucose metabolism. It is unresolved whether the cytoprotective and metabolic responses mediated by Nrf2 are connected or separable events in non-malignant cells. In this study we show that activation of Nrf2, either by the small molecule sulforaphane or knockout of the Nrf2 inhibitor Keap1, leads to increased cellular glucose uptake and increased glucose addiction in fibroblasts. Upon Nrf2 activation glucose is preferentially metabolized through the pentose phosphate pathway with increased production of NADPH. Interference with the supply of glucose or the pentose phosphate pathway and NADPH generation not only hampers Nrf2-mediated detoxification of reactive oxygen species on the enzyme level but also Nrf2-initiated expression of antioxidant defense proteins, such as glutathione reductase and heme-oxygenase1. We conclude that the Nrf2-dependent protection against oxidative stress relies on an intact pentose phosphate pathway and that there is crosstalk between metabolism and detoxification already at the level of gene expression in mammalian cells. PMID:24024172

  2. Nrf2 sensitizes prostate cancer cells to radiation via decreasing basal ROS levels.

    PubMed

    Liu, Min; Yao, Xu-Dong; Li, Wei; Geng, Jiang; Yan, Yang; Che, Jian-Ping; Xu, Yun-Fei; Zheng, Jun-Hua

    2015-01-01

    Androgen deprivation therapy (ADT) was reported to lower basal ROS level in prostate cancer (PCa) and to sensitize PCa to radiation. We aimed to seek for the underlying molecular mechanism and to develop novel additive treatments to ADT in this regard. We simulated human androgen milieu in vitro and tested the ROS level in PCa cells undergoing ADT. We also tested the Nrf2 level in PCa cells with or without ADT. Genetic and pharmaceutical upregulation of Nrf2 was applied in vitro and in vivo in transgenic adenocarcinoma of the mouse prostate (TRAMP) mice with or without castration to investigate whether Nrf2 overexpression supplemented the effect of ADT in PCa. We first discovered that androgen deprivation increased basal ROS level in PCa cells with AR expression. We then found that genetic Nrf2 upregulation lowered basal ROS similar to ADT. Also, SFN sensitized PCa cell to radiation via upregulation of Nrf2. We then found that Nrf2 level in control TRAMP groups was lower than castration or SFN groups. The SFN treated TRAMP mice showed similar level of Nrf2 to castration. Genetic and pharmaceutical upregulation of Nrf2 lowered the ROS in PCa cells and sensitized PCa cells to radiation similar to ADT, implicating possible administration of SFN in place of ADT for PCa patients requiring radiotherapy. PMID:25728635

  3. Hrd1 suppresses Nrf2-mediated cellular protection during liver cirrhosis

    PubMed Central

    Wu, Tongde; Zhao, Fei; Gao, Beixue; Tan, Can; Yagishita, Naoko; Nakajima, Toshihiro; Wong, Pak K.; Chapman, Eli; Fang, Deyu; Zhang, Donna D.

    2014-01-01

    Increased endoplasmic reticulum (ER) stress and reactive oxygen species (ROS) are the salient features of end-stage liver diseases. Using liver tissues from liver cirrhosis patients, we observed up-regulation of the XBP1–Hrd1 arm of the ER stress response pathway and down-regulation of the Nrf2-mediated antioxidant response pathway. We further confirmed this negative regulation of Nrf2 by Hrd1 using Hrd1 conditional knockout mice. Down-regulation of Nrf2 was a surprising result, since the high levels of ROS should have inactivated Keap1, the primary ubiquitin ligase regulating Nrf2 levels. Here, we identified Hrd1 as a novel E3 ubiquitin ligase responsible for compromised Nrf2 response during liver cirrhosis. In cirrhotic livers, activation of the XBP1–Hrd1 arm of ER stress transcriptionally up-regulated Hrd1, resulting in enhanced Nrf2 ubiquitylation and degradation and attenuation of the Nrf2 signaling pathway. Our study reveals not only the convergence of ER and oxidative stress response pathways but also the pathological importance of this cross-talk in liver cirrhosis. Finally, we showed the therapeutic importance of targeting Hrd1, rather than Keap1, to prevent Nrf2 loss and suppress liver cirrhosis. PMID:24636985

  4. 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. PMID:27185859

  5. The dual roles of NRF2 in tumor prevention and progression: possible implications in cancer treatment

    PubMed Central

    Moon, Eui Jung; Giaccia, Amato

    2015-01-01

    The Cap’N’Collar (CNC) family serves as cellular sensors of oxidative and electrophilic stresses and shares structural similarities including basic leucine zipper (bZIP) and CNC domains,. They form heterodimers with small MAF proteins to regulate antioxidant and phase II enzymes through antioxidant response element (ARE)-mediated transactivation. Among the CNC family members, NRF2 is required for systemic protection against redox-mediated injury and carcinogenesis. On the other hand, NRF2 is activated by oncogenic pathways, metabolism, and hypoxia. Constitutive NRF2 activation is observed in a variety of human cancers and it is highly correlated with tumor progression and aggressiveness. In this review, we will discuss how NRF2 plays dual roles in cancer prevention and progression depending on the cellular context and environment. Therefore, a better understanding of NRF2 will be necessary to exploit this complex network of balancing antioxidant pathways to inhibit tumor progression. PMID:25458917

  6. 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.

  7. Effects of that ATRA inhibits Nrf2-ARE pathway on glial cells activation after intracerebral hemorrhage

    PubMed Central

    Yin, Xiao-Ping; Zhou, Jun; Wu, Dan; Chen, Zhi-Ying; Bao, Bing

    2015-01-01

    Previous studies indicate that the Nrf2-ARE signaling pathway plays a neruo-protective role in glia cell, however, the mechanism was also elusive. This study aims to explore the inhibitive function of all-trans-retinoic (ATRA) on Nrf2-ARE pathway in intracerebral hemorrhage (ICH), and investigate the mechanism. In this study, the femoral artery injection method was employed to establish ICH model. The model rats were randomly divided into four groups, including Sham group, ICH group, ATRA group and DMSO group. The neurological scores were evaluated for the four groups at different time points. Hematoxylin-Eosin staining was used to stain the CD11b positive glia cells. Double immunofluorescence staining method was utilized to observe the co-expression of HO-1, NF-κB, Nrf2 and TNF-α and CD11b marker in glia cells. Western blot assay was used to detect the Nrf2 protein (total and binding Nrf2), HO-1, NF-κB and TNF-α proteins in every group. The results indicated that neurologiclal scores were significantly decreased in ATRA group compared to ICH gorup (P < 0.05). The glia cells were significantly activated and accumulated in ICH rats. ATRA significantly decreased co-expression of Nrf2, HO-1 and CD11b, and increased co-expression of NF-κB, TNF-α and CD11b of glia cells. ATRA significantly decreased total Nrf2 expression and increased binding Nrf2 expression in ATRA group compared to ICH group (P < 0.05). ATRA decreased anti-oxygen protein Nrf2 and HO-1, and increases inflammatory factors NF-κB and TNF-α. In conclusion, the application of ATRA could inhibit the neuro-protective function effectively by blocking the Nrf2-ARE pathway in glia cells. PMID:26617752

  8. Nrf2-deficiency creates a responsive microenvironment for metastasis to the lung.

    PubMed

    Satoh, Hironori; Moriguchi, Takashi; Taguchi, Keiko; Takai, Jun; Maher, Jonathan M; Suzuki, Takafumi; Winnard, Paul T; Raman, Venu; Ebina, Masahito; Nukiwa, Toshihiro; Yamamoto, Masayuki

    2010-10-01

    The Nrf2 transcription factor is crucial for regulating the cellular defense against various carcinogens. However, relationship between host Nrf2 and cancer metastasis remains unexplored. To address this issue, we examined susceptibility of Nrf2-deficient mice to pulmonary cancer metastasis following implantation of the mouse Lewis lung carcinoma (3LL) cell line. Nrf2-deficient mice reproducibly exhibited a higher number of pulmonary metastatic nodules than wild-type mice did. The lung and bone marrow (BM) of cancer-bearing Nrf2-deficient mice contained increased numbers of inflammatory cells, including myeloid-derived suppressor cells (MDSCs), a potent population of immunosuppressive cells. MDSCs can attenuate CD8(+) T-cell immunity through modification of the T-cell receptor complex exploiting reactive oxygen species (ROS). MDSCs of Nrf2-deficient mice retained elevated levels of ROS relative to wild-type mice. BM transplantation experiments revealed functional disturbance in the hematopoietic and immune systems of Nrf2-deficient mice. Wild-type recipient mice with Nrf2-deficient BM cells showed increased levels of lung metastasis after cancer cell inoculation. These mice exhibited high-level accumulation of ROS in MDSCs, which showed very good coincidence to the decrease of splenic CD8(+) T-cells. In contrast, Keap1-knockdown mutant mice harboring high-level Nrf2 expression displayed increased resistance against the cancer cell metastasis to the lung, accompanied by a decrease in ROS in the MDSCs fraction. Our results thus reveal a novel function for Nrf2 in the prevention of cancer metastasis, presumably by its ability to preserve the redox balance in the hematopoietic and immune systems. PMID:20513672

  9. 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

  10. 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 Central

    Mandal, Animesh; Bishayee, Anupam

    2015-01-01

    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. pofrtulacastrum 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. PMID:25622256

  11. 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

    2015-01-01

    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. PMID:25622256

  12. Monascin attenuates oxidative stress-mediated lung inflammation via peroxisome proliferator-activated receptor-gamma (PPAR-γ) and nuclear factor-erythroid 2 related factor 2 (Nrf-2) modulation.

    PubMed

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

    2014-06-11

    We speculated that peroxisome proliferator-activated receptor (PPAR)-γ agonists may modulate the oxidative stress pathway to ameliorate the development of airway inflammation. The effect of Monascus-fermented metabolite monascin (MS) and rosiglitazone (Rosi) on oxidative stress-induced lung inflammation was evaluated. Luciferase assay and DNA binding activity assay were used to point out that MS may be a novel PPAR-γ agonist and nuclear factor-erythroid 2 related factor 2 (Nrf-2) activator. We used hydrogen peroxide (H2O2) to induce inflammation in lung epithelial cells. MS and Rosi prevented H2O2-induced ROS generation in A549 epithelial cells through PPAR-γ translocation, avoiding inflammatory mediator expression via inhibiting nuclear factor (NF)-κB translocation. The regulatory ability of MS was abolished by siRNA against PPAR-γ. MS also elevated antioxidant enzyme expression via Nrf-2 activation. Both PPAR-γ and Nrf-2 might have benefits against lung inflammation. MS regulated PPAR-γ and Nrf-2 to improve lung oxidative inflammation. PMID:24865672

  13. 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

  14. Coenzyme Q0 regulates NFκB/AP-1 activation and enhances Nrf2 stabilization in attenuation of LPS-induced inflammation and redox imbalance: Evidence from in vitro and in vivo studies.

    PubMed

    Yang, Hsin-Ling; Lin, Ming-Wei; Korivi, Mallikarjuna; Wu, Jia-Jiuan; Liao, Chun-Huei; Chang, Chia-Ting; Liao, Jiunn-Wang; Hseu, You-Cheng

    2016-02-01

    Coenzyme Q (CoQ) analogs with variable number of isoprenoid units have been demonstrated as anti-inflammatory and antioxidant/pro-oxidant molecules. In this study we used CoQ0 (2,3-dimethoxy-5-methyl-1,4-benzoquinone, zero isoprenoid side-chains), a novel quinone derivative, and investigated its molecular actions against LPS-induced inflammation and redox imbalance in murine RAW264.7 macrophages and mice. In LPS-stimulated macrophages, non-cytotoxic concentrations of CoQ0 (2.5-10 μM) inhibited iNOS/COX-2 protein expressions with subsequent reductions of NO, PGE2, TNF-α and IL-1β secretions. This inhibition was reasoned by suppression of NFκB (p65) activation, and inhibition of AP-1 (c-Jun., c-Fos, ATF2) translocation. Our findings indicated that IKKα-mediated I-κB degradation and MAPK-signaling are involved in regulation of NFκB/AP-1 activation. Furthermore, CoQ0 triggered HO-1 and NQO-1 genes through increased Nrf2 nuclear translocation and Nrf2/ARE-signaling. This phenomenon was confirmed by diminished CoQ0 protective effects in Nrf2 knockdown cells, where LPS-induced NO, PGE2, TNF-α and IL-1β productions remained high. Molecular evidence revealed that CoQ0 enhanced Nrf2 steady-state level at both transcriptional and translational levels. CoQ0-induced Nrf2 activation appears to be regulated by ROS-JNK-signaling cascades, as evidenced by suppressed Nrf2 activation upon treatment with pharmacological inhibitors of ROS (N-acetylcysteine) and JNK (SP600125). Besides, oral administration of CoQ0 (5 mg/kg) suppressed LPS-induced (1 mg/kg) induction of iNOS/COX-2 and TNF-α/IL-1β through tight regulation of NFκB/Nrf2 signaling in mice liver and spleen. Our findings conclude that pharmacological actions of CoQ0 are mediated via inhibition of NFκB/AP-1 activation and induction of Nrf2/ARE-signaling. Owing to its potent anti-inflammatory and antioxidant properties, CoQ0 could be a promising candidate to treat inflammatory disorders. PMID:26548719

  15. 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. PMID:26291957

  16. DMF, but not other fumarates, inhibits NF-κB activity in vitro in an Nrf2-independent manner.

    PubMed

    Gillard, Geoffrey O; Collette, Brian; Anderson, John; Chao, Jianhua; Scannevin, Robert H; Huss, David J; Fontenot, Jason D

    2015-06-15

    Fumarate-containing pharmaceuticals are potent therapeutic agents that influence multiple cellular pathways. Despite proven clinical efficacy, there is a significant lack of data that directly defines the molecular mechanisms of action of related, yet distinct fumarate compounds. We systematically compared the impact of dimethyl fumarate (DMF), monomethyl fumarate (MMF) and a mixture of monoethyl fumarate salts (Ca(++), Mg(++), Zn(++); MEF) on defined cellular responses. We demonstrate that DMF inhibited NF-κB-driven cytokine production and nuclear translocation of p65 and p52 in an Nrf2-independent manner. Equivalent doses of MMF and MEF did not affect NF-κB signaling. These results highlight a key difference in the biological impact of related, yet distinct fumarate compounds. PMID:26004161

  17. The effect of resveratrol and its methylthio-derivatives on the Nrf2-ARE pathway in mouse epidermis and HaCaT keratinocytes.

    PubMed

    Krajka-Kuźniak, Violetta; Szaefer, Hanna; Stefański, Tomasz; Sobiak, Stanisław; Cichocki, Michał; Baer-Dubowska, Wanda

    2014-09-01

    Resveratrol is the most extensively studied stilbene derivative. We previously showed that methylthiostilbenes were more effective inhibitors of CYP1A1 and 1B1 activity than resveratrol. In this study, we investigated whether resveratrol and its methylthio-substituted derivatives, i.e. 3-M-4'-MTS (S2), 3,5-DM-4'-MTS (S5) and 3,4,5-TM-4'-MTS (S7) could activate Nrf2 signaling in the mouse epidermis and in human keratinocytes. Western blot analysis showed translocation of Nrf2 from the cytosol to the nucleus in both models. All of the tested stilbenes increased GST activity, but resveratrol was the most effective inducer. Moreover, only resveratrol increased the protein level of GSTP in the mouse epidermis. GSTM was enhanced in HaCaT cells after the treatment with derivatives S2 and S5. The same effect was observed for GSTP in the case of compound S2. Resveratrol and its derivatives reduced the NQO2 protein level in HaCaT cells. Thus, it is possible that increased expression of GSTP or GSTM and GST activity was linked with NQO2 inhibition in these cells. The results of this study indicate that resveratrol and its methylthioderivatives activate Nrf2 not only in the mouse epidermis, but also in human keratinocytes. Upregulating GST isozymes might be particularly important for deactivating chemical carcinogens, such as PAH. PMID:25169438

  18. Role of Nrf2 in retinal vascular development and the vaso-obliterative phase of oxygen-induced retinopathy

    PubMed Central

    Uno, Koichi; Prow, Tarl W.; Bhutto, Imran A.; Yerrapureddy, Adi; McLeod, D. Scott; Yamamoto, Masayuki; Reddy, Sekhar P.; Lutty, Gerard A.

    2016-01-01

    In the initial stage of retinopathy of prematurity (ROP), hyperoxia causes retinal blood vessel obliteration. This is thought to occur in part through oxidative stress-induced apoptosis of endothelial cells. This study was designed to determine what role NF-E2-related factor 2 (Nrf2) plays in this process. Nrf2 is a transcription factor of the anti-oxidant response element that, if induced, may protect the retina from hyperoxia-induced oxidative stress. Nrf2 knockout mice (Nrf2−/−), Nrf2 wild type control mice (Nrf2 +/+), and C57BL/6 mice were exposed to hyperoxia (75% O2) or normoxia from P7 through P12. Mice were sacrificed on P9 and P12 and the retinas were stained with GSA lectin-Cy3 to visualize retinal blood vessels. Hyperoxia exposed retinas were flat mounted and photographed, then the size of the avascular areas was determined. Additionally, retinas were cryopreserved after lectin staining and area analysis and then sectioned. Secondary or deep capillaries were then hand-counted in sections. In hyperoxia-treated mice, the avascular areas in Nrf2−/− P9 mice were significantly larger than those in Nrf2+/+ P9 mice (P = 0.01). However, there was no significant difference between Nrf2−/− and Nrf2+/+ mice at P12. Avascular areas at P12 were significantly smaller than that at P9 in Nrf2−/−, Nrf2+/+, and C57BL/6 mice (P = 0.0011, P = 0.009, and P = 0.001 respectively). The numbers of deep or secondary capillaries in air-reared Nrf2−/− mice were significantly decreased, when compared to Nrf2+/+ mice at P9 (P = 0.0082). On the other hand, there was no significant difference in deep capillary formation between air-reared Nrf2−/− and Nrf2+/+ mice at P12. Akt signaling activates Nrf2 and Akt was localized to retinal blood vessels in all animals and was increased in Nrf2+/+ and Nrf2−/− mice exposed to hyperoxia as compared to normoxia mice. Interestingly, during normal development this protection by Nrf2 occurs in a specific window of time

  19. Autocrine and Paracrine Regulation of Keratinocyte Proliferation through a Novel Nrf2-IL-36γ Pathway.

    PubMed

    Kurinna, Svitlana; Muzumdar, Sukalp; Köhler, Ulrike Anne; Kockmann, Tobias; Auf dem Keller, Ulrich; Schäfer, Matthias; Werner, Sabine

    2016-06-01

    The Nrf2 transcription factor is well known for its cytoprotective functions through regulation of genes involved in the detoxification of reactive oxygen species or toxic compounds. Therefore, activation of Nrf2 is a promising strategy for the protection of tissues from various types of insults and for cancer prevention. However, recent studies revealed a proinflammatory activity of activated Nrf2 and a stimulating effect on epithelial cell proliferation, but the underlying mechanisms of action and the responsible target genes are largely unknown. Using a combination of gene expression profiling, chromatin immunoprecipitation, and targeted proteomics via selected reaction monitoring, we show that the gene encoding the proinflammatory cytokine IL-36γ is a novel direct target of Nrf2 in keratinocytes and hepatocytes in vitro and in vivo. As a consequence, upregulation of IL-36γ expression occurred upon genetic or pharmacological activation of Nrf2 in the epidermis and in the normal and regenerating liver. Functional in vitro studies demonstrate that IL-36γ directly stimulates proliferation of keratinocytes. In particular, it induces expression of keratinocyte mitogens in fibroblasts, suggesting that the Nrf2-IL-36γ axis promotes keratinocyte proliferation through a double paracrine loop. These results provide mechanistic insight into Nrf2 action in the control of inflammation and cell proliferation through regulation of a proinflammatory cytokine with a key function in various inflammatory diseases. PMID:27183581

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

    PubMed

    Chartoumpekis, Dionysios V; Wakabayashi, Nobunao; Kensler, Thomas W

    2015-08-01

    Cancer cells adapt their metabolism to their increased needs for energy and substrates for protein, lipid and nucleic acid synthesis. Nuclear erythroid factor 2-like 2 (Nrf2) pathway is usually activated in cancers and has been suggested to promote cancer cell survival mainly by inducing a large battery of cytoprotective genes. This mini review focuses on metabolic pathways, beyond cytoprotection, which can be directly or indirectly regulated by Nrf2 in cancer cells to affect their survival. The pentose phosphate pathway (PPP) is enhanced by Nrf2 in cancers and aids their growth. PPP has also been found to be up-regulated in non-cancer tissues and other pathways, such as de novo lipogenesis, have been found to be repressed after activation of the Nrf2 pathway. The importance of these Nrf2-regulated metabolic pathways in cancer compared with non-cancer state remains to be determined. Last but not least, the importance of context about Nrf2 and cancer is highlighted as the Nrf2 pathway may be activated in cancers but its pharmacological activators are useful in chemoprevention. PMID:26551705

  1. 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. PMID:26936880

  2. Activation of Nrf2 in keratinocytes causes chloracne (MADISH)-like skin disease in mice.

    PubMed

    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

    2014-04-01

    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

  3. Nrf2 Activation Promotes Keratinocyte Survival during Early Skin Carcinogenesis via Metabolic Alterations.

    PubMed

    Rolfs, Frank; Huber, Marcel; Kuehne, Andreas; Kramer, Stefan; Haertel, Eric; Muzumdar, Sukalp; Wagner, Johanna; Tanner, Yasmine; Böhm, Friederike; Smola, Sigrun; Zamboni, Nicola; Levesque, Mitchell P; Dummer, Reinhard; Beer, Hans-Dietmar; Hohl, Daniel; Werner, Sabine; Schäfer, Matthias

    2015-11-15

    Pharmacologic activation of the transcription factor NRF2 has been suggested to offer a strategy for cancer prevention. In this study, we present evidence from murine tumorigenesis experiments suggesting there may be limitations to this possibility, based on tumorigenic effects of Nrf2 in murine keratinocytes that have not been described previously. In this setting, Nrf2 expression conferred metabolic alterations in keratinocytes that were protumorigenic in nature, affecting enzymes involved in glutathione biosynthesis or in the oxidative pentose phosphate pathway and other NADPH-producing enzymes. Under stress conditions, coordinate increases in NADPH, purine, and glutathione levels promoted the survival of keratinocytes harboring oncogenic mutations, thereby promoting tumor development. The protumorigenic activity of Nrf2 in keratinocytes was particularly significant in a mouse model of skin tumorigenesis that did not rely upon chemical carcinogenesis. In exploring the clinical relevance of our findings, we confirm that NRF2 and protumorigenic NRF2 target genes were activated in some actinic keratoses, the major precancerous lesion in human skin. Overall, our results reveal an unexpected tumor-promoting activity of activated NRF2 during early phases of skin tumorigenesis. PMID:26530903

  4. 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

  5. [Recent advances in the study of Nrf2 and inflammatory respiratory diseases].

    PubMed

    Xie, Jian-lin; Lin, Ming-bao; Hou, Qi

    2015-09-01

    Nuclear factor-erythroid 2 related factor 2 (Nrf2) is an ubiquitous and important transcription factor. It regulates antioxidant response elements (AREs)-mediated expression of antioxidant enzyme and cytoprotective proteins. A large body of research showed that Nrf2-Keap1 (Kelch-like ECH-associated protein 1, Keap 1)-ARE signaling pathway is involved in the endogenous antioxidant defense mechanisms. Nrf2 increases the expression of a number of cytoprotective genes, protects cells and tissues from the injury of a variety of toxicants and carcinogens. As a result, Nrf2 enhances the expression of glutathione and antioxidants such as superoxide dismutase and glutathione S-transferase, and subsequently scavenging free radicals. Air pollution especially from PM2.5 particles, is associated with an increasing morbidity of inflammatory pulmonary diseases and their deterioration. More and more studies demonstrated that Nrf2 was a novel signaling molecule in the modulation of inflammatory responses in these inflammatory respiratory diseases, such as asthma, acute lung injury (ALI) and COPD. Therefore, Nrf2 targeting might be a therapeutic target, which will provide clinical benefit by reducing both oxidative stress and inflammation in asthma, acute lung injury (ALI) and COPD. This review focused on the relationship between Nrf2 and inflammatory respiratory diseases and oxidative stress. PMID:26757542

  6. 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. PMID:27037625

  7. Effects of Nrf2 Deficiency on Bone Microarchitecture in an Experimental Model of Osteoporosis

    PubMed Central

    Ibáñez, Lidia; Ferrándiz, María Luisa; Brines, Rita; Alcaraz, Maria José

    2014-01-01

    Objective. Redox imbalance contributes to bone fragility. We have evaluated the in vivo role of nuclear factor erythroid derived 2-related factor-2 (Nrf2), an important regulator of cellular responses to oxidative stress, in bone metabolism using a model of postmenopausal osteoporosis. Methods. Ovariectomy was performed in both wild-type and mice deficient in Nrf2 (Nrf2−/−). Bone microarchitecture was analyzed by μCT. Serum markers of bone metabolism were also measured. Reactive oxygen species production was determined using dihydrorhodamine 123. Results. Sham-operated or ovariectomized Nrf2−/− mice exhibit a loss in trabecular bone mineral density in femur, accompanied by a reduction in cortical area in vertebrae. Nrf2 deficiency tended to increase osteoblastic markers and significantly enhanced osteoclastic markers in sham-operated animals indicating an increased bone turnover with a main effect on bone resorption. We have also shown an increased production of oxidative stress in bone marrow-derived cells from sham-operated or ovariectomized Nrf2−/− mice and a higher responsiveness of bone marrow-derived cells to osteoclastogenic stimuli in vitro. Conclusion. We have demonstrated in vivo a key role of Nrf2 in the maintenance of bone microarchitecture. PMID:25120886

  8. Nrf2 the rescue: Effects of the antioxidative/electrophilic response on the liver

    SciTech Connect

    Klaassen, Curtis D.; Reisman, Scott A.

    2010-04-01

    Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that positively regulates the basal and inducible expression of a large battery of cytoprotective genes. These gene products include proteins that catalyze reduction reactions (NAD(P)H:quinone oxidoreductase 1, Nqo1), conjugation reactions (glutathione-S-transferases, Gsts and UDP-glucuronosyltransferases, Ugts), as well as the efflux of potentially toxic xenobiotics and xenobiotic conjugates (multidrug resistance-associated proteins, Mrps). The significance of Nrf2 in the liver has been established, as livers of Nrf2-null mice are more susceptible to various oxidative/electrophilic stress-induced pathologies than wild-type mice. In contrast, both pharmacological and genetic models of hepatic Nrf2 activation are protective against oxidative/electrophilic stress. Furthermore, because certain Nrf2-target genes in the liver could affect the distribution, metabolism, and excretion of xenobiotics, the effects of Nrf2 on the kinetics of drugs and other xenobiotics should also be considered, with a special emphasis on metabolism and excretion. Therefore, this review highlights the research that has contributed to the understanding of the importance of Nrf2 in toxicodynamics and toxicokinetics, especially that which pertains to the liver.

  9. Cancer Cell Growth Is Differentially Affected by Constitutive Activation of NRF2 by KEAP1 Deletion and Pharmacological Activation of NRF2 by the Synthetic Triterpenoid, RTA 405

    PubMed Central

    Probst, Brandon L.; McCauley, Lyndsey; Trevino, Isaac; Wigley, W. Christian; Ferguson, Deborah A.

    2015-01-01

    Synthetic triterpenoids are antioxidant inflammation modulators (AIMs) that exhibit broad anticancer activity. AIMs bind to KEAP1 and inhibit its ability to promote NRF2 degradation. As a result, NRF2 increases transcription of genes that restore redox balance and reduce inflammation. AIMs inhibit tumor growth and metastasis by increasing NRF2 activity in the tumor microenvironment and by modulating the activity of oncogenic signaling pathways, including NF-κB, in tumor cells. Accumulating evidence suggests that KEAP1 loss or mutation—which results in high levels of sustained NRF2 activity—may promote cancer growth and increase chemoresistance. Loss of KEAP1 also increases the levels of other oncogenic proteins, including IKKβ and BCL2. The apparent survival advantage provided to some tumor cells by loss of functional KEAP1 raises the question of whether pharmacological inhibition of KEAP1 could promote tumor growth. To address this issue, we characterized the basal levels of KEAP1 and NRF2 in a panel of human tumor cell lines and profiled the activity of an AIM, RTA 405. We found that in tumor cell lines with low or mutant KEAP1, and in Keap1-/- murine embryonic fibroblasts, multiple KEAP1 targets including NRF2, IKKβ, and BCL2 were elevated. Keap1-/- murine embryonic fibroblasts also had higher rates of proliferation and colony formation than their wild-type counterparts. In cells with functional KEAP1, RTA 405 increased NRF2 levels, but not IKKβ or BCL2 levels, and did not increase cell proliferation or survival. Moreover, RTA 405 inhibited growth at similar concentrations in cells with different basal NRF2 activity levels and in cells with wild-type or mutant KRAS. Finally, pre-treatment with RTA 405 did not protect tumor cells from doxorubicin- or cisplatin-mediated growth inhibition. Collectively, these data demonstrate that pharmacological activation of NRF2 by AIMs is distinct from genetic activation and does not provide a growth or survival

  10. Nrf2 activation protects against solar-simulated ultraviolet radiation in mice and humans

    PubMed Central

    Knatko, Elena V.; Ibbotson, Sally H.; Zhang, Ying; Higgins, Maureen; Fahey, Jed W.; Talalay, Paul; Dawe, Robert S.; Ferguson, James; Huang, Jeffrey T.-J.; Clarke, Rosemary; Zheng, Suqing; Saito, Akira; Kalra, Sukirti; Benedict, Andrea L.; Honda, Tadashi; Proby, Charlotte M.; Dinkova-Kostova, Albena T.

    2015-01-01

    The transcription factor Nrf2 determines the ability to adapt and survive under conditions of electrophilic, oxidative and inflammatory stress by regulating the expression of elaborate networks comprising nearly 500 genes encoding proteins with versatile cytoprotective functions. In mice, disruption of Nrf2 increases susceptibility to carcinogens and accelerates disease pathogenesis. Paradoxically, Nrf2 is upregulated in established human tumors, but whether this upregulation drives carcinogenesis is not known. Here we show that the incidence, multiplicity and burden of solar-simulated UV radiation-mediated cutaneous tumors that form in SKH-1 hairless mice in which Nrf2 is genetically constitutively activated, are lower than those that arise in their wild-type counterparts. Pharmacological Nrf2 activation by topical bi-weekly applications of small (40 nmol) quantities of the potent bis(cyano enone) inducer TBE-31 has a similar protective effect against solar-simulated UV radiation in animals receiving long-term treatment with the immunosuppressive agent azathioprine. Genetic or pharmacological Nrf2 activation lowers the expression of the pro-inflammatory factors interleukin (IL)-6 and IL-1β, and cyclooxygenase (COX)-2 after acute exposure of mice to UV radiation. In healthy human subjects, topical applications of extracts delivering the Nrf2 activator sulforaphane, reduced the degree of solar-simulated UV radiation-induced skin erythema, a quantifiable surrogate end-point for cutaneous damage and skin cancer risk. Collectively, these data show that Nrf2 is not a driver for tumorigenesis even upon exposure to a very potent and complete carcinogen, and strongly suggest that the frequent activation of Nrf2 in established human tumors is a marker of metabolic adaptation. PMID:25804610

  11. Nrf2 is required for normal postnatal bone acquisition in mice

    PubMed Central

    Kim, Jung-Hyun; Singhal, Vandana; Biswal, Shyam; Thimmulappa, Rajesh K; DiGirolamo, Douglas J

    2014-01-01

    A large body of literature suggests that bone metabolism is susceptible to the ill effects of reactive species that accumulate in the body and cause cellular dysfunction. One of the body’s front lines in defense against such damage is the transcription factor, Nrf2. This transcription factor regulates a plethora of antioxidant and cellular defense pathways to protect cells from such damage. Despite the breadth of knowledge of both the function of Nrf2 and the effects of reactive species in bone metabolism, the direct role of Nrf2 in skeletal biology has yet to be thoroughly examined. Thus, in the current study, we have examined the role of Nrf2 in postnatal bone metabolism in mice. Mice lacking Nrf2 (Nrf2−/−) exhibited a marked deficit in postnatal bone acquisition, which was most severe at 3 weeks of age when osteoblast numbers were 12-fold less than observed in control animals. While primary osteoblasts from Nrf2−/− mice functioned normally in vitro, the colony forming capacity of bone marrow stromal cells (BMSCs) from these mice was significantly reduced compared to controls. This defect could be rescued through treatment with the radical scavenger N-acetyl cysteine (NAC), suggesting that increased reactive species stress might impair early osteoblastogenesis in BMSCs and lead to the failure of bone acquisition observed in Nrf2−/− animals. Taken together, these studies suggest Nrf2 represents a key pathway in regulating bone metabolism, which may provide future therapeutic targets to treat osteoporosis. PMID:26273528

  12. Activation of Nrf2-mediated oxidative stress response in macrophages by hypochlorous acid

    SciTech Connect

    Pi Jingbo Zhang Qiang; Woods, Courtney G.; Wong, Victoria; Collins, Sheila; Andersen, Melvin E.

    2008-02-01

    Hypochlorous acid (HOCl), a potent oxidant generated when chlorine gas reacts with water, is important in the pathogenesis of many disorders. Transcription factor Nrf2-mediated antioxidant response represents a critical cellular defense mechanism that serves to maintain intracellular redox homeostasis and limit oxidative damage. In the present study, the effect of HOCl on Nrf2 activation was investigated in macrophages, one of the target cells of chlorine gas exposure. Exposure of RAW 264.7 macrophages to HOCl resulted in increased protein levels of Nrf2 in nuclear extractions, as well as a time- and dose-dependent increase in the expression of Nrf2 target genes, including heme oxygenase-1, NAD(P)H:quinone oxidoreductase 1 (NQO-1), glutamate cysteine ligase catalytic subunit (GCLC), and glutathione synthetase (GS). Additionally, intracellular glutathione (GSH), which is the prime scavenger for HOCl in cells, decreased within the first hour of HOCl exposure. The decline was followed by a GSH rebound that surpassed the initial basal levels by up to 4-fold. This reversal in GSH levels closely correlated with the gene expression profile of GCLC and GS. To study the mechanisms of Nrf2 activation in response to HOCl exposure, we examined the effects of several antioxidants on Nrf2-mediated response. Pretreatment with cell-permeable catalase, N-acetyl-L-cysteine or GSH-monoethyl ester markedly reduced expression of NQO-1 and GCLC under HOCl challenge conditions, suggesting intracellular ROS-scavenging capacity affects HOCl-induced Nrf2 activation. Importantly, pre-activation of Nrf2 with low concentrations of pro-oxidants protected the cells against HOCl-induced cell damage. Taken together, we provide direct evidence that HOCl activates Nrf2-mediated antioxidant response, which protects cells from oxidative damage.

  13. Upregulation of phase II enzymes through phytochemical activation of Nrf2 protects cardiomyocytes against oxidant stress.

    PubMed

    Reuland, Danielle J; Khademi, Shadi; Castle, Christopher J; Irwin, David C; McCord, Joe M; Miller, Benjamin F; Hamilton, Karyn L

    2013-03-01

    Increased production of reactive oxygen species has been implicated in the pathogenesis of cardiovascular disease (CVD), and enhanced endogenous antioxidants have been proposed as a mechanism for regulating redox balance. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a transcriptional regulator of phase II antioxidant enzymes, and activation of Nrf2 has been suggested to be an important step in attenuating oxidative stress associated with CVD. A well-defined combination of five widely studied medicinal plants derived from botanical sources (Bacopa monniera, Silybum marianum (milk thistle), Withania somnifera (Ashwagandha), Camellia sinensis (green tea), and Curcuma longa (turmeric)) has been shown to activate Nrf2 and induce phase II enzymes through the antioxidant response element. The purpose of these experiments was to determine if treatment of cardiomyocytes with this phytochemical composition, marketed as Protandim, activates Nrf2, induces phase II detoxification enzymes, and protects cardiomyocytes from oxidant-induced apoptosis in a Nrf2-dependent manner. In cultured HL-1 cardiomyocytes, phytochemical treatment was associated with nuclear accumulation of Nrf2, significant induction of phase II enzymes, and concomitant protection against hydrogen peroxide-induced apoptosis. The protection against oxidant stress was abolished when Nrf2 was silenced by shRNA, suggesting that our phytochemical treatment worked through the Nrf2 pathway. Interestingly, phytochemical treatment was found to be a more robust activator of Nrf2 than oxidant treatment, supporting the use of the phytochemicals as a potential treatment to increase antioxidant defenses and protect heart cells against an oxidative challenge. PMID:23201694

  14. Nrf2 in ischemic neurons promotes retinal vascular regeneration through regulation of semaphorin 6A

    PubMed Central

    Wei, Yanhong; Gong, Junsong; Xu, Zhenhua; Thimmulappa, Rajesh K.; Mitchell, Katherine L.; Welsbie, Derek S.; Biswal, Shyam; Duh, Elia J.

    2015-01-01

    Delayed revascularization of ischemic neural tissue is a major impediment to preservation of function in central nervous system (CNS) diseases including stroke and ischemic retinopathies. Therapeutic strategies allowing rapid revascularization are greatly needed to reduce ischemia-induced cellular damage and suppress harmful pathologic neovascularization. However, key mechanisms governing vascular recovery in ischemic CNS, including regulatory molecules governing the transition from tissue injury to tissue repair, are largely unknown. NF-E2-related factor 2 (Nrf2) is a major stress-response transcription factor well known for its cell-intrinsic cytoprotective function. However, its role in cell–cell crosstalk is less appreciated. Here we report that Nrf2 is highly activated in ischemic retina and promotes revascularization by modulating neurons in their paracrine regulation of endothelial cells. Global Nrf2 deficiency strongly suppresses retinal revascularization and increases pathologic neovascularization in a mouse model of ischemic retinopathy. Conditional knockout studies demonstrate a major role for neuronal Nrf2 in vascular regrowth into avascular retina. Deletion of neuronal Nrf2 results in semaphorin 6A (Sema6A) induction in hypoxic/ischemic retinal ganglion cells in a hypoxia-inducible factor-1 alpha (HIF-1α)-dependent fashion. Sema6A expression increases in avascular inner retina and colocalizes with Nrf2 in human fetal eyes. Extracellular Sema6A leads to dose-dependent suppression of the migratory phenotype of endothelial cells through activation of Notch signaling. Lentiviral-mediated delivery of Sema6A small hairpin RNA (shRNA) abrogates the defective retinal revascularization in Nrf2-deficient mice. Importantly, pharmacologic Nrf2 activation promotes reparative angiogenesis and suppresses pathologic neovascularization. Our findings reveal a unique function of Nrf2 in reprogramming ischemic tissue toward neurovascular repair via Sema6A regulation

  15. Interplay between cytosolic disulfide reductase systems and the Nrf2/Keap1 pathway

    PubMed Central

    Schmidt, Edward E.

    2015-01-01

    NADPH transfers reducing power from bioenergetic pathways to thioredoxin reductase-1 (TrxR1) and glutathione reductase (GR) to support essential reductive systems. Surprisingly, it was recently shown that mouse livers lacking both TrxR1 and GR (‘TR/GR-null’) can sustain redox (reduction-oxidation) homoeostasis using a previously unrecognized NADPH-independent source of reducing power fuelled by dietary methionine. The NADPH-dependent systems are robustly redundant in liver, such that disruption of either TrxR1 or GR alone does not cause oxidative stress. However, disruption of TrxR1 induces transcription factor Nrf2 (nuclear factor erythroid-derived 2-like-2) whereas disruption of GR does not. This suggests the Nrf2 pathway responds directly to the status of the thioredoxin-1 (Trx1) system. The proximal regulator of Nrf2 is Keap1 (Kelch-like ECH-associated protein-1), a cysteine (Cys)-rich protein that normally interacts transiently with Nrf2, targeting it for degradation. During oxidative stress, this interaction is stabilized, preventing degradation of newly synthesized Nrf2, thereby allowing Nrf2 accumulation. Within the Trx1 system, TrxR1 and peroxiredoxins (Prxs) contain some of the most reactive nucleophilic residues in the cell, making them likely targets for oxidants or electrophiles. We propose that Keap1 activity and therefore Nrf2 is regulated by interactions of Trx1 system enzymes with oxidants. In TR/GR-null livers, Nrf2 activity is further induced, revealing that TrxR-independent systems also repress Nrf2 and these might be induced by more extreme challenges. PMID:26551704

  16. The clinical potential of influencing Nrf2 signaling in degenerative and immunological disorders

    PubMed Central

    Gao, Bifeng; Doan, An; Hybertson, Brooks M

    2014-01-01

    Nuclear factor (erythroid-derived 2)-like 2 (Nrf2; encoded in humans by the NFE2L2 gene) is a transcription factor that regulates the gene expression of a wide variety of cytoprotective phase II detoxification and antioxidant enzymes through a promoter sequence known as the antioxidant-responsive element (ARE). The ARE is a promoter element found in many cytoprotective genes; therefore, Nrf2 plays a pivotal role in the ARE-driven cellular defense system against environmental stresses. Agents that target the ARE/Nrf2 pathway have been tested in a wide variety of disorders, with at least one new Nrf2-activating drug now approved by the US Food and Drug Administration. Examination of in vitro and in vivo experimental results, and taking into account recent human clinical trial results, has led to an opinion that Nrf2-activating strategies – which can include drugs, foods, dietary supplements, and exercise – are likely best targeted at disease prevention, disease recurrence prevention, or slowing of disease progression in early stage illnesses; they may also be useful as an interventional strategy. However, this rubric may be viewed even more conservatively in the pathophysiology of cancer. The activation of the Nrf2 pathway has been widely accepted as offering chemoprevention benefit, but it may be unhelpful or even harmful in the setting of established cancers. For example, Nrf2 activation might interfere with chemotherapies or radiotherapies or otherwise give tumor cells additional growth and survival advantages, unless they already possess mutations that fully activate their Nrf2 pathway constitutively. With all this in mind, the ARE/Nrf2 pathway remains of great interest as a possible target for the pharmacological control of degenerative and immunological diseases, both by activation and by inhibition, and its regulation remains a promising biological target for the development of new therapies. PMID:24520207

  17. Nrf2 Protein Up-regulates Antiapoptotic Protein Bcl-2 and Prevents Cellular Apoptosis*

    PubMed Central

    Niture, Suryakant K.; Jaiswal, Anil K.

    2012-01-01

    Nuclear transcription factor Nrf2 regulates the expression and coordinated induction of a battery of genes encoding cytoprotective and drug transporter proteins in response to chemical and radiation stress. This leads to reduced apoptosis, enhanced cell survival, and increased drug resistance. In this study, we investigated the role of Nrf2 in up-regulation of antiapoptotic protein Bcl-2 and its contribution to stress-induced apoptosis and cell survival. Exposure of mouse hepatoma (Hepa-1) and human hepatoblastoma (HepG2) cells to antioxidant tert-butylhydroquinone led to induction of Bcl-2. Mutagenesis and transfection assays identified an antioxidant response element between nucleotides −3148 and −3140 on the reverse strand of the Bcl-2 gene promoter that was essential for activation of Bcl-2 gene expression. Band/supershift and ChIP assays demonstrated binding of Nrf2 to Bcl-2 antioxidant response element. Alterations in Nrf2 led to altered Bcl-2 induction and cellular apoptosis. Moreover, dysfunctional/mutant inhibitor of Nrf2 (INrf2) in human lung cancer cells failed to degrade Nrf2, resulting in an increased Bcl-2 level and decreased etoposide- and UV/γ radiation-mediated DNA fragmentation. In addition, siRNA-mediated down-regulation of Nrf2 also led to decreased apoptosis and increased cell survival. Furthermore, the specific knockdown of Bcl-2 in Nrf2-activated tumor cells led to increased etoposide-induced apoptosis and decreased cell survival and growth/proliferation. These data provide the first evidence of Nrf2 in control of Bcl-2 expression and apoptotic cell death with implications in antioxidant protection, survival of cancer cells, and drug resistance. PMID:22275372

  18. Association between plasma selenium level and NRF2 target genes expression in humans.

    PubMed

    Reszka, Edyta; Wieczorek, Edyta; Jablonska, Ewa; Janasik, Beata; Fendler, Wojciech; Wasowicz, Wojciech

    2015-04-01

    Animal studies in rodent and in vitro studies indicate compensatory role of nuclear factor (erythroid-derived 2)-like (Nrf2) and Nrf2-regulated antioxidant and phase II biotransformation enzymes for the dietary selenium (Se) deficiency or for the loss of selenoproteins. To explore associations between plasma Se level and NRF2-regulated cytoprotective genes expression, an observational study was conducted in a population of 96 healthy non-smoking men living in Central Poland aged 18-83 years with relatively low plasma Se level. NRF2, KEAP2, CAT, EPHX1, GCLC, GCLM, GPX2, GSR, GSTA1, GSTM1, GSTP1, GSTT1, HMOX1, NQO1, PRDX1, SOD1, SOD2, TXNRD1 transcript levels in peripheral blood leukocytes and polymorphism of NRF2-617C/A (rs6721961) in blood genomic DNA were determined by means of quantitative real-time PCR. Mean plasma Se level was found to be 51.10±15.25μg/L (range 23.86-96.18μg/L). NRF2 mRNA level was positively correlated with expression of investigated NRF2-target genes. The multivariate linear regression adjusting for selenium status showed that plasma Se level was significantly inversely associated only with expression of GSTP1 (β-coef.=-0.270, p=0.009), PRDXR1 (β-coef.=-0.245, p=0.017) and SOD2 with an inverse trend toward significance (β-coef.=-0.186, p=0.074), but without an effect of NRF2 gene variants. NRF2 expression was inversely associated with age (r=-0.23, p=0.03) and body mass index (r=-0.29, p<0.001). The findings may suggest a possible link between plasma Se level and cytoprotective response at gene level in humans. PMID:25524402

  19. Aldosterone Activates Transcription Factor Nrf2 in Kidney Cells Both In Vitro and In Vivo

    PubMed Central

    Oteiza, Patricia I.; Link, Samuel; Hey, Valentin; Stopper, Helga; Schupp, Nicole

    2014-01-01

    Abstract Aims: An increased kidney cancer risk was found in hypertensive patients, who frequently exhibit hyperaldosteronism, known to contribute to kidney injury, with oxidative stress playing an important role. The capacity of kidney cells to up-regulate transcription factor nuclear factor-erythroid-2-related factor 2 (Nrf2), a key regulator of the cellular antioxidative defense, as a prevention of aldosterone-induced oxidative damage was investigated both in vitro and in vivo. Results: Aldosterone activated Nrf2 and increased the expression of enzymes involved in glutathione (GSH) synthesis and detoxification. This activation depended on the mineralocorticoid receptor (MR) and oxidative stress. In vitro, Nrf2 activation, GSH amounts, and target gene levels decreased after 24 h, while oxidant levels remained high. Nrf2 activation could not protect cells against oxidative DNA damage, as aldosterone-induced double-strand breaks and 7,8-dihydro-8-oxo-guanine (8-oxodG) lesions steadily rose. The Nrf2 activator sulforaphane enhanced the Nrf2 response both in vitro and in vivo, thereby preventing aldosterone-induced DNA damage. In vivo, Nrf2 activation further had beneficial effects on the aldosterone-caused blood pressure increase and loss of kidney function. Innovation: This is the first study showing the activation of Nrf2 by aldosterone. Moreover, the results identify sulforaphane as a substance that is capable of preventing aldosterone-induced damage both in vivo and in vitro. Conclusion: Aldosterone-induced Nrf2 adaptive response cannot neutralize oxidative actions of chronically increased aldosterone, which, therefore could be causally involved in the increased cancer incidence of hypertensive individuals. Enhancing the cellular antioxidative defense with sulforaphane might exhibit beneficial effects. Antioxid. Redox Signal. 21, 2126–2142. PMID:24512358

  20. Notoginsenoside R1-mediated neuroprotection involves estrogen receptor-dependent crosstalk between Akt and ERK1/2 pathways: a novel mechanism of Nrf2/ARE signaling activation.

    PubMed

    Meng, X; Sun, G; Ye, J; Xu, H; Wang, H; Sun, X

    2014-04-01

    Notoginsenoside R1 (NGR1), a novel phytoestrogen isolated from Panax notoginseng, has antioxidant and anti-apoptotic properties. Oxidative stress plays a pivotal role in neurodegenerative diseases. To mimic oxidative stress in neurons and explore the neuroprotection of NGR1, H₂O₂-induced neurotoxicity in NGF-induced differentiation of PC12 cells was used. In this study, NGR1 preconditioning provided neuroprotective effects via suppressing H₂O₂-induced the intracellular ROS accumulation, the increase in the product of lipid peroxidation (MDA), protein oxidation (protein carbonyl), and DNA fragmentation (8-OHdG), and mitochondrial membrane depolarization as well as caspase-3 activation. Moreover, NGR1 treatment alone potently increased the nuclear translocation of Nrf2, augmented ARE enhancer activity, and upregulated the expression and activity of phase II antioxidant enzymes including HO-1, NQO-1, and γ-GCSc. NGR1 could also increase the ERE activity and activate Akt and ERK1/2 pathways. NGR1-mediated activation of Nrf2/ARE signaling and neuroprotection were abolished by genetic silencing of Nrf2 using siRNA or the pharmacological blockade of estrogen receptors using ICI-182780, and partially inhibited by Akt siRNA or ERK siRNA transfection. In addition, the phosphorylation of ERK1/2 mediated by NGR1 was markedly inhibited in PC12 cells transfected with Akt siRNA. On the contrary, ERK1/2 siRNA transfection hardly had any effect on the phosphorylation of Akt mediated by NGR1. NGR1-mediated activation of Akt and ERK1/2 pathways was blocked by ICI-182780. In conclusion, NGR1 provided neuroprotection via inducing an estrogen receptor-dependent crosstalk between Akt and ERK1/2 pathways, subsequently activating Nrf2/ARE signaling and thereby up-regulating phase II antioxidant enzymes. PMID:24437944

  1. Epigallocatechin-3-Gallate Attenuates Oxidative Stress and Inflammation in Obstructive Nephropathy via NF-κB and Nrf2/HO-1 Signalling Pathway Regulation.

    PubMed

    Wang, Yanqiu; Wang, Bowen; Du, Feng; Su, Xuesong; Sun, Guangping; Zhou, Guangyu; Bian, Xiaohui; Liu, Na

    2015-09-01

    Oxidative stress and inflammation contribute importantly to the pathogenesis of chronic kidney disease (CKD). Epigallocatechin-3-gallate (EGCG), which is the most abundant and most active catechin polyphenol extracted from green tea, has been proved to have many bioactivities. In this study, the renoprotective effect of EGCG was evaluated in a widely used kidney disease model, the unilateral ureteral obstruction (UUO) mice model. After 14 days of EGCG administration, mean arterial blood pressure, body-weight and obstructed kidney weight were measured. Levels of blood urea nitrogen (BUN) and creatinine (CR) and activities of glutamic-pyruvic transaminase (GPT) and lactate dehydrogenase (LDH) in serum were estimated as indicators of renal function. Periodic acid-Schiff (PAS) staining was performed to observe the pathological changes of the obstructed kidney. Antioxidant enzymes and pro-inflammatory cytokine production were estimated to reflect the oxidative stress and inflammatory state in the obstructed kidney. Finally, the main proteins in the NF-κB and Nrf2 signalling pathway and DNA binding activity of NF-κB and Nrf2 were measured to investigate the effect of EGCG on these two pathways. The results demonstrated that EGCG could restore UUO-induced kidney weight loss and renal dysfunction. In addition, UUO-induced oxidative stress and inflammatory responses in the obstructed kidney were also prevented by EGCG. Furthermore, EGCG could induce both NF-κB and Nrf2 nuclear translocation in the UUO kidney and promote heme oxygenase-1 (HO-1) production. These results indicated that the renoprotective effect of EGCG might be through its NF-κB and Nrf2 signalling pathway regulations. PMID:25625183

  2. Cytoplasmic localization of Nrf2 promotes colorectal cancer with more aggressive tumors via upregulation of PSMD4.

    PubMed

    Lin, Po-Lin; Chang, Jinghua Tsai; Wu, De-Wei; Huang, Chi-Chou; Lee, Huei

    2016-06-01

    Differences in subcellular localization of Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) have been associated with poor outcomes in human cancers. However, the prognostic value of subcellular localization of Nrf2 in colorectal cancer and the underlying mechanism in tumor invasion remain unknown. We enrolled tumors from colorectal patients to evaluate Nrf2, NQO1, and HO-1 expression by immunohistochemistry. NQO1 and HO-1 positive tumors showed nearly complete expression of Nrf2 in the nucleus and/or showed partial expression in the nucleus/cytoplasm (nNrf2); however, tumors negative for NQO1 and HO-1 showed almost complete expression of Nrf2 in the cytoplasm and/or partial expression in the nucleus/cytoplasm (cNrf2). Kaplan-Meier and Cox regression analysis indicated poorer overall survival in patients with cNrf2 tumors than with nNrf2 tumors. Cell models provided evidence that cNrf2, rather than nNrf2, was responsible for cell invasion and soft agar growth triggered by activation of the NF-κB/AKT/β-catenin cascade. Mechanistically, cNrf2 persistently increased PSMD4 expression by the HIF1α/β-catenin axis, whereas PSMD4 reciprocally enhanced Nrf2 nuclear export by increasing CRM1 expression through p53 degradation. The mechanistic action of the cell model was further confirmed with a nude mouse animal model in which xenograft tumors induced by cNrf2 were nearly completely suppressed by the proteasomal inhibitor carfilzomib or the β-catenin inhibitor XAV939. We therefore suggest that PSMD4 or β-catenin might be potential targets for suppressing tumor aggressiveness, and consequently, improving outcomes in patients whose tumors express cNrf2. PMID:27033953

  3. Epigenetics Reactivation of Nrf2 in Prostate TRAMP C1 Cells by Curcumin Analogue FN1.

    PubMed

    Li, Wenji; Pung, Doug; Su, Zheng-Yuan; Guo, Yue; Zhang, Chengyue; Yang, Anne Yuqing; Zheng, Xi; Du, Zhi-Yun; Zhang, Kun; Kong, Ah-Ng

    2016-04-18

    It has previously been shown that curcumin can effectively inhibit prostate cancer proliferation and progression in TRAMP mice, potentially acting through the hypomethylation of the Nrf2 gene promoter and hence activation of the Nrf2 pathway to enhance cell antioxidative defense. FN1 is a synthetic curcumin analogue that shows stronger anticancer activity than curcumin in other reports. We aimed to explore the epigenetic modification of FN1 that restores Nrf2 expression in TRAMP-C1 cells. Stably transfected HepG2-C8 cells were used to investigate the effect of FN1 on the Nrf2- antioxidant response element (ARE) pathway. Real-time quantitative PCR and Western blotting were applied to study the influence of FN1 on endogenous Nrf2 and its downstream genes. Bisulfite genomic sequencing (BGS) and methylated DNA immunoprecipitation (MeDIP) were then performed to examine the methylation profile of the Nrf2 promoter. An anchorage-independent colony-formation analysis was conducted to examine the tumor inhibition activity of FN1. Epigenetic modification enzymes, including DNMTs and HDACs, were investigated by Western blotting. The luciferase reporter assay indicated that FN1 was more potent than curcumin in activating the Nrf2-ARE pathway. FN1 increased the expression of Nrf2 and its downstream detoxifying enzymes. FN1 significantly inhibited the colony formation of TRAMP-C1 cells. BGS and MeDIP assays revealed that FN1 treatment (250 nM for 3 days) reduced the percentage of CpG methylation of the Nrf2 promoter. FN1 also downregulated epigenetic modification enzymes. In conclusion, our results suggest that FN1 is a novel anticancer agent for prostate cancer. In the TRAMP-C1 cell line, FN1 can increase the level of Nrf2 and downstream genes via activating the Nrf2-ARE pathway and inhibit the colony formation potentially through the decreased expression of keap1 coupled with CpG demethylation of the Nrf2 promoter. This CpG demethylation effect may come from decreased

  4. Regulation of Cigarette Smoke (CS)-Induced Autophagy by Nrf2.

    PubMed

    Zhu, Lingxiang; Barrett, Erika C; Barret, Erika C; Xu, Yuxue; Liu, Zuguo; Manoharan, Aditya; Chen, Yin

    2013-01-01

    Cigarette smoke (CS) has been reported to induce autophagy in airway epithelial cells. The subsequent autophagic cell death has been proposed to play an important pathogenic role in chronic obstructive pulmonary disease (COPD); however, the underlying molecular mechanism is not entirely clear. Using CS extract (CSE) as a surrogate for CS, we found that it markedly increased the expressions of both LC3B-I and LC3B-II as well as autophagosomes in airway epithelial cells. This is in contrast to the common autophagy inducer (i.e., starvation) that increases LC3B-II but reduces LC3B-I. Further studies indicate that CSE regulated LC3B at transcriptional and post-translational levels. In addition, CSE, but not starvation, activated Nrf2-mediated adaptive response. Increase of cellular Nrf2 by either Nrf2 overexpression or the knockdown of Keap1 (an Nrf2 inhibitor) significantly repressed CSE-induced LC3B-I and II as well as autophagosomes. Supplement of NAC (a GSH precursor) or GSH recapitulated the effect of Nrf2, suggesting the increase of cellular GSH level is responsible for Nrf2 effect on LC3B and autophagosome. Interestingly, neither Nrf2 activation nor GSH supplement could restore the repressed activities of mTOR or its downstream effctor-S6K. Thus, the Nrf2-dependent autophagy-suppression was not due to the re-activation of mTOR-the master repressor of autophagy. To search for the downstream effector of Nrf2 on LC3B and autophagosome, we tested Nrf2-dependent genes (i.e., NQO1 and P62) that are also increased by CSE treatment. We found that P62, but not NQO1, could mimic the effect of Nrf2 activation by repressing LC3B expression. Thus, Nrf2->P62 appears to play an important role in the regulation of CSE-induced LC3B and autophagosome. PMID:23585825

  5. Frugal chemoprevention: targeting Nrf2 with foods rich in sulforaphane.

    PubMed

    Yang, Li; Palliyaguru, Dushani L; Kensler, Thomas W

    2016-02-01

    With the properties of efficacy, safety, tolerability, practicability and low cost, foods containing bioactive phytochemicals are gaining significant attention as elements of chemoprevention strategies against cancer. Sulforaphane [1-isothiocyanato-4-(methylsulfinyl)butane], a naturally occurring isothiocyanate produced by cruciferous vegetables such as broccoli, is found to be a highly promising chemoprevention agent against not only a variety of cancers such as breast, prostate, colon, skin, lung, stomach or bladder, but also cardiovascular disease, neurodegenerative diseases, and diabetes. For reasons of experimental exigency, preclinical studies have focused principally on sulforaphane itself, while clinical studies have relied on broccoli sprout preparations rich in either sulforaphane or its biogenic precursor, glucoraphanin. Substantive subsequent evaluation of sulforaphane pharmacokinetics and pharmacodynamics has been undertaken using either pure compound or food matrices. Sulforaphane affects multiple targets in cells. One key molecular mechanism of action for sulforaphane entails activation of the Nrf2-Keap1 signaling pathway although other actions contribute to the broad spectrum of efficacy in different animal models. This review summarizes the current status of pre-clinical chemoprevention studies with sulforaphane and highlights the progress and challenges for the application of foods rich in sulforaphane and/or glucoraphanin in the arena of clinical chemoprevention. PMID:26970133

  6. Hepatic Gene Expression Profiling in Nrf2 Knockout Mice after Long-Term High-Fat Diet-Induced Obesity

    PubMed Central

    Chartoumpekis, Dionysios V.; Ziros, Panos G.; Zaravinos, Apostolos; Iskrenova, Ralitsa P.; Psyrogiannis, Agathoklis I.; Kyriazopoulou, Venetsana E.; Sykiotis, Gerasimos P.; Habeos, Ioannis G.

    2013-01-01

    Introduction. The transcription factor NFE2-related factor 2 (Nrf2) is a central regulator of antioxidant and detoxification gene expression in response to electrophilic or oxidative stress. Nrf2 has recently been shown to cross-talk with metabolic pathways, and its gene deletion protected mice from high-fat-diet-(HFD-) induced obesity and insulin resistance. This study aimed to identify potential Nrf2-regulated genes of metabolic interest by comparing gene expression profiles of livers of wild-type (WT) versus Nrf2 knockout (Nrf2-KO) mice after a long-term HFD. Methods. WT and Nrf2-KO mice were fed an HFD for 180 days; total RNA was prepared from liver and used for microarray analysis and quantitative real-time RT-PCR (qRT-PCR). Results. The microarray analysis identified 601 genes that were differentially expressed between WT and Nrf2-KO mice after long-term HFD. Selected genes, including ones known to be involved in metabolic regulation, were prioritized for verification by qRT-PCR: Cyp7a1 and Fabp5 were significantly overexpressed in Nrf2-KO mice; in contrast, Car, Cyp2b10, Lipocalin 13, Aquaporin 8, Cbr3, Me1, and Nqo1 were significantly underexpressed in Nrf2-KO mice. Conclusion. Transcriptome profiling after HFD-induced obesity confirms that Nrf2 is implicated in liver metabolic gene networks. The specific genes identified here may provide insights into Nrf2-dependent mechanisms of metabolic regulation. PMID:23710285

  7. NRF2/Long Noncoding RNA ROR Signaling Regulates Mammary Stem Cell Expansion and Protects against Estrogen Genotoxicity*

    PubMed Central

    Zhang, Yongshu; Xia, Jixiang; Li, Qinglin; Yao, Yuan; Eades, Gabriel; Gernapudi, Ramkishore; Duru, Nadire; Kensler, Thomas W.; Zhou, Qun

    2014-01-01

    Long noncoding RNAs (lncRNAs) have emerged as key regulators of gene expression in embryonic stem cell (ESC) self-renewal and differentiation. In ESCs, lncRNAs are regulated at the genetic level via transcription factor binding to lncRNA gene promoters. Here we demonstrate that the key cytoprotective transcription factor NRF2 controls lncRNA expression in mammary stem cells. By profiling lncRNAs in wild-type and NRF2 knockdown mammary stem cells, we demonstrate that the lncRNA ROR, a regulator of embryonic stem cell pluripotency, is overexpressed upon NRF2 knockdown. We performed promoter analyses and examined predicted NRF2 binding elements in the ROR promoter using luciferase reporter constructs of a ROR promoter deletion series. Our studies revealed that NRF2 binds to two specific NRF2 response elements flanking the ROR promoter and that these two NRF2 response elements are equally important to suppress ROR transcription. In addition, we identified associated H3K27me3 chromatin modification and EZH2 binding at the ROR promoter that was dependent on NRF2 binding. We observed that NRF2 knockdown or ROR overexpression leads to increased stem cell self-renewal in mammary stem cells. Furthermore, we demonstrate Nrf2 regulation of the mammary stem cell population in vivo. These observations provide further evidence for the critical role of NRF2 in maintaining normal stem cell subpopulations in mammary epithelium. PMID:25231996

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

    PubMed

    Zhang, Yongshu; Xia, Jixiang; Li, Qinglin; Yao, Yuan; Eades, Gabriel; Gernapudi, Ramkishore; Duru, Nadire; Kensler, Thomas W; Zhou, Qun

    2014-11-01

    Long noncoding RNAs (lncRNAs) have emerged as key regulators of gene expression in embryonic stem cell (ESC) self-renewal and differentiation. In ESCs, lncRNAs are regulated at the genetic level via transcription factor binding to lncRNA gene promoters. Here we demonstrate that the key cytoprotective transcription factor NRF2 controls lncRNA expression in mammary stem cells. By profiling lncRNAs in wild-type and NRF2 knockdown mammary stem cells, we demonstrate that the lncRNA ROR, a regulator of embryonic stem cell pluripotency, is overexpressed upon NRF2 knockdown. We performed promoter analyses and examined predicted NRF2 binding elements in the ROR promoter using luciferase reporter constructs of a ROR promoter deletion series. Our studies revealed that NRF2 binds to two specific NRF2 response elements flanking the ROR promoter and that these two NRF2 response elements are equally important to suppress ROR transcription. In addition, we identified associated H3K27me3 chromatin modification and EZH2 binding at the ROR promoter that was dependent on NRF2 binding. We observed that NRF2 knockdown or ROR overexpression leads to increased stem cell self-renewal in mammary stem cells. Furthermore, we demonstrate Nrf2 regulation of the mammary stem cell population in vivo. These observations provide further evidence for the critical role of NRF2 in maintaining normal stem cell subpopulations in mammary epithelium. PMID:25231996

  9. Identification of novel NRF2-regulated genes by ChIP-Seq: influence on retinoid X receptor alpha

    PubMed Central

    Chorley, Brian N.; Campbell, Michelle R.; Wang, Xuting; Karaca, Mehmet; Sambandan, Deepa; Bangura, Fatu; Xue, Peng; Pi, Jingbo; Kleeberger, Steven R.; Bell, Douglas A.

    2012-01-01

    Cellular oxidative and electrophilic stress triggers a protective response in mammals regulated by NRF2 (nuclear factor (erythroid-derived) 2-like; NFE2L2) binding to deoxyribonucleic acid-regulatory sequences near stress-responsive genes. Studies using Nrf2-deficient mice suggest that hundreds of genes may be regulated by NRF2. To identify human NRF2-regulated genes, we conducted chromatin immunoprecipitation (ChIP)-sequencing experiments in lymphoid cells treated with the dietary isothiocyanate, sulforaphane (SFN) and carried out follow-up biological experiments on candidates. We found 242 high confidence, NRF2-bound genomic regions and 96% of these regions contained NRF2-regulatory sequence motifs. The majority of binding sites were near potential novel members of the NRF2 pathway. Validation of selected candidate genes using parallel ChIP techniques and in NRF2-silenced cell lines indicated that the expression of about two-thirds of the candidates are likely to be directly NRF2-dependent including retinoid X receptor alpha (RXRA). NRF2 regulation of RXRA has implications for response to retinoid treatments and adipogenesis. In mouse, 3T3-L1 cells’ SFN treatment affected Rxra expression early in adipogenesis, and knockdown of Nrf2-delayed Rxra expression, both leading to impaired adipogenesis. PMID:22581777

  10. The Effects of Sequence Variation on Genome-wide NRF2 Binding—New Target Genes and Regulatory SNPs

    PubMed Central

    Kuosmanen, Suvi M.; Viitala, Sari; Laitinen, Tuomo; Peräkylä, Mikael; Pölönen, Petri; Kansanen, Emilia; Leinonen, Hanna; Raju, Suresh; Wienecke-Baldacchino, Anke; Närvänen, Ale; Poso, Antti; Heinäniemi, Merja; Heikkinen, Sami; Levonen, Anna-Liisa

    2016-01-01

    Transcription factor binding specificity is crucial for proper target gene regulation. Motif discovery algorithms identify the main features of the binding patterns, but the accuracy on the lower affinity sites is often poor. Nuclear factor E2-related factor 2 (NRF2) is a ubiquitous redox-activated transcription factor having a key protective role against endogenous and exogenous oxidant and electrophile stress. Herein, we decipher the effects of sequence variation on the DNA binding sequence of NRF2, in order to identify both genome-wide binding sites for NRF2 and disease-associated regulatory SNPs (rSNPs) with drastic effects on NRF2 binding. Interactions between NRF2 and DNA were studied using molecular modelling, and NRF2 chromatin immunoprecipitation-sequence datasets together with protein binding microarray measurements were utilized to study binding sequence variation in detail. The binding model thus generated was used to identify genome-wide binding sites for NRF2, and genomic binding sites with rSNPs that have strong effects on NRF2 binding and reside on active regulatory elements in human cells. As a proof of concept, miR-126–3p and -5p were identified as NRF2 target microRNAs, and a rSNP (rs113067944) residing on NRF2 target gene (Ferritin, light polypeptide, FTL) promoter was experimentally verified to decrease NRF2 binding and result in decreased transcriptional activity. PMID:26826707

  11. Arsenic-mediated activation of the Nrf2-Keap1 antioxidant pathway.

    PubMed

    Lau, Alexandria; Whitman, Samantha A; Jaramillo, Melba C; Zhang, Donna D

    2013-02-01

    Arsenic is present in the environment and has become a worldwide health concern due to its toxicity and carcinogenicity. However, the specific mechanism(s) by which arsenic elicits its toxic effects has yet to be fully elucidated. The transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) has been recognized as the master regulator of a cellular defense mechanism against toxic insults. This review highlights studies demonstrating that arsenic activates the Nrf2-Keap1 antioxidant pathway by a distinct mechanism from that of natural compounds such as sulforaphane (SF) found in broccoli sprouts or tert-butylhyrdoquinone (tBHQ), a natural antioxidant commonly used as a food preservative. Evidence also suggests that arsenic prolongs Nrf2 activation and may mimic constitutive activation of Nrf2, which has been found in several human cancers due to disruption of the Nrf2-Keap1 axis. The current literature strongly suggests that activation of Nrf2 by arsenic potentially contributes to, rather than protects against, arsenic toxicity and carcinogenicity. The mechanism(s) by which known Nrf2 activators, such as the natural chemopreventive compounds SF and lipoic acid, protect against the deleterious effects caused by arsenic will also be discussed. These findings will provide insight to further understand how arsenic promotes a prolonged Nrf2 response, which will lead to the identification of novel molecular markers and development of rational therapies for the prevention or intervention of arsenic-induced diseases. The National Institute of Environmental Health Science (NIEHS) Outstanding New Environmental Scientist (ONES) award has provided the opportunity to review the progress both in the fields of arsenic toxicology and Nrf2 biology. Much of the funding has led to (1) the novel discovery that arsenic activates the Nrf2 pathway by a mechanism different to that of other Nrf2 activators, such as sulforaphane and tert-butylhydroquinone, (2) activation of Nrf

  12. Altered behavioral development in Nrf2 knockout mice following early postnatal exposure to valproic acid

    PubMed Central

    Furnari, Melody A.; Saw, Constance Lay-Lay; Kong, Ah-Ng; Wagner, George C

    2015-01-01

    Early exposure to valproic acid results in autism-like neural and behavioral deficits in humans and other animals through oxidative stress-induced neural damage. In the present study, valproic acid was administered to genetically altered mice lacking the Nrf2 (nuclear factor-erythroid 2 related factor 2) gene on postnatal day 14 (P14). Nrf2 is a transcription factor that induces genes that protect against oxidative stress. It was found that valproic acid-treated Nrf2 knockout mice were less active in open field activity chambers, less successful on the rotorod, and had deficits in learning and memory in the Morris water maze compared to the valproic acid-treated wild type mice. Given these results, it appears that Nrf2 knockout mice were more sensitive to the neural damage caused by valproic acid administered during early development. PMID:25454122

  13. Control of antioxidative response by the tumor suppressor protein PML through regulating Nrf2 activity

    PubMed Central

    Guo, Shuang; Cheng, Xiwen; Lim, Jun-Hee; Liu, Yu; Kao, Hung-Ying

    2014-01-01

    Oxidative stress is a consequence of an imbalance between reactive oxygen species (ROS) production and the ability of the cytoprotective system to detoxify the reactive intermediates. The tumor suppressor promyelocytic leukemia protein (PML) functions as a stress sensor. Loss of PML results in impaired mitochondrial complex II activity, increased ROS, and subsequent activation of nuclear factor erythroid 2–related factor 2 (Nrf2) antioxidative pathway. We also demonstrate that sulforaphane (SFN), an antioxidant, regulates Nrf2 activity by controlling abundance and subcellular distribution of PML and that PML is essential for SFN-mediated ROS increase, Nrf2 activation, antiproliferation, antimigration, and antiangiogenesis. Taking the results together, we have uncovered a novel antioxidative mechanism by which PML regulates cellular oxidant homeostasis by controlling complex II integrity and Nrf2 activity and identified PML as an indispensable mediator of SFN activity. PMID:24943846

  14. Introducing the “TCDD-Inducible AhR-Nrf2 Gene Battery”

    PubMed Central

    Yeager, Ronnie L.; Reisman, Scott A.; Aleksunes, Lauren M.; Klaassen, Curtis D.

    2009-01-01

    2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) induces genes via the transcription factor aryl hydrocarbon receptor (AhR), including Cyp1a1, NAD(P)H:quinone oxidoreductase 1 (Nqo1), UDP-glucuronosyltransferase 1a6 (Ugt1a6), and glutathione S-transferase a1 (Gsta1). These genes are referred to as the “AhR gene battery.” However, Nqo1 is also considered a prototypical target gene of the transcription factor nuclear factor erythroid 2–related factor 2 (Nrf2). In mice, TCDD induction of Nrf2 and Nrf2 target, Nqo1, is dependent on AhR, and thus TCDD induction of drug-processing genes may be routed through an AhR-Nrf2 sequence. There has been speculation that Nrf2 may be involved in the TCDD induction of drug-processing genes; however, the data are not definitive. Therefore, to address whether TCDD induction of Nqo1, Ugts, and Gsts is dependent on Nrf2, we conducted the definitive experiment by administering TCDD (50 μg/kg, ip) to Nrf2-null and wild-type (WT) mice and collecting livers 24 h later to quantify the mRNA of drug-processing genes. TCDD induction of Cyp1a1 and Ugt1a1 was similar in WT and Nrf2-null mice, whereas TCDD induction of Ugt1a5 and 1a9 was blunted in Nrf2-null mice. TCDD induced Nqo1, Ugt1a6, 2b34, 2b35, 2b36, UDP-glucuronic acid–synthesizing gene UDP-glucose dehydrogenase, and Gsta1, m1, m2, m3, m6, p2, t2, and microsomal Gst1 in WT mice but not in Nrf2-null mice. Therefore, the present study demonstrates the novel finding that Nrf2 is required for TCDD induction of classical AhR battery genes Nqo1, Ugt1a6, and Gsta1, as well as most Ugt and Gst isoforms in livers of mice. PMID:19474220

  15. Synthesis, evaluation, and metabolism of novel [6]-shogaol derivatives as potent Nrf2 activators.

    PubMed

    Zhu, Yingdong; Wang, Pei; Zhao, Yantao; Yang, Chun; Clark, Anderson; Leung, TinChung; Chen, Xiaoxin; Sang, Shengmin

    2016-06-01

    Oxidative stress is a central component of many chronic diseases. The Kelch-like ECH-associated protein 1 (Keap1)-nuclear factor erythroid 2 p45-related factor 2 (Nrf2) system is a major regulatory pathway of cytoprotective genes against oxidative and electrophilic stress. Activation of the Nrf2 pathway plays crucial roles in the chemopreventive effects of various inducers. In this study, we developed a novel class of potent Nrf2 activators derived from ginger compound, [6]-shogaol (6S), using the Tg[glutathione S-transferase pi 1 (gstp1):green fluorescent protein (GFP)] transgenic zebrafish model. Investigation of structure-activity relationships of 6S derivatives indicates that the combination of an α,β-unsaturated carbonyl entity and a catechol moiety in one compound enhances the Tg(gstp1:GFP) fluorescence signal in zebrafish embryos. Chemical reaction and in vivo metabolism studies of the four most potent 6S derivatives showed that both α,β-unsaturated carbonyl entity and catechol moiety act as major active groups for conjugation with the sulfhydryl groups of the cysteine residues. In addition, we further demonstrated that 6S derivatives increased the expression of Nrf2 downstream target, heme oxygenase-1, in both a dose- and time-dependent manner. These results suggest that α,β-unsaturated carbonyl entity and catechol moiety of 6S derivatives may react with the cysteine residues of Keap1, disrupting the Keap1-Nrf2 complex, thereby liberating and activating Nrf2. Our findings of natural product-derived Nrf2 activators lead to design options of potent Nrf2 activators for further optimization. PMID:27021962

  16. Dimethyl fumarate treatment induces adaptive and innate immune modulation independent of Nrf2.

    PubMed

    Schulze-Topphoff, Ulf; Varrin-Doyer, Michel; Pekarek, Kara; Spencer, Collin M; Shetty, Aparna; Sagan, Sharon A; Cree, Bruce A C; Sobel, Raymond A; Wipke, Brian T; Steinman, Lawrence; Scannevin, Robert H; Zamvil, Scott S

    2016-04-26

    Dimethyl fumarate (DMF) (BG-12, Tecfidera) is a fumaric acid ester (FAE) that was advanced as a multiple sclerosis (MS) therapy largely for potential neuroprotection as it was recognized that FAEs are capable of activating the antioxidative transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathway. However, DMF treatment in randomized controlled MS trials was associated with marked reductions in relapse rate and development of active brain MRI lesions, measures considered to reflect CNS inflammation. Here, we investigated the antiinflammatory contribution of Nrf2 in DMF treatment of the MS model, experimental autoimmune encephalomyelitis (EAE). C57BL/6 wild-type (WT) and Nrf2-deficient (Nrf2(-/-)) mice were immunized with myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 (p35-55) for EAE induction and treated with oral DMF or vehicle daily. DMF protected WT and Nrf2(-/-) mice equally well from development of clinical and histologic EAE. The beneficial effect of DMF treatment in Nrf2(-/-) and WT mice was accompanied by reduced frequencies of IFN-γ and IL-17-producing CD4(+) cells and induction of antiinflammatory M2 (type II) monocytes. DMF also modulated B-cell MHC II expression and reduced the incidence of clinical disease in a B-cell-dependent model of spontaneous CNS autoimmunity. Our observations that oral DMF treatment promoted immune modulation and provided equal clinical benefit in acute EAE in Nrf2(-/-) and WT mice, suggest that the antiinflammatory activity of DMF in treatment of MS patients may occur through alternative pathways, independent of Nrf2. PMID:27078105

  17. Dimethyl fumarate treatment induces adaptive and innate immune modulation independent of Nrf2

    PubMed Central

    Schulze-Topphoff, Ulf; Varrin-Doyer, Michel; Pekarek, Kara; Spencer, Collin M.; Shetty, Aparna; Sagan, Sharon A.; Cree, Bruce A. C.; Sobel, Raymond A.; Wipke, Brian T.; Steinman, Lawrence; Scannevin, Robert H.; Zamvil, Scott S.

    2016-01-01

    Dimethyl fumarate (DMF) (BG-12, Tecfidera) is a fumaric acid ester (FAE) that was advanced as a multiple sclerosis (MS) therapy largely for potential neuroprotection as it was recognized that FAEs are capable of activating the antioxidative transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathway. However, DMF treatment in randomized controlled MS trials was associated with marked reductions in relapse rate and development of active brain MRI lesions, measures considered to reflect CNS inflammation. Here, we investigated the antiinflammatory contribution of Nrf2 in DMF treatment of the MS model, experimental autoimmune encephalomyelitis (EAE). C57BL/6 wild-type (WT) and Nrf2-deficient (Nrf2−/−) mice were immunized with myelin oligodendrocyte glycoprotein (MOG) peptide 35–55 (p35–55) for EAE induction and treated with oral DMF or vehicle daily. DMF protected WT and Nrf2−/− mice equally well from development of clinical and histologic EAE. The beneficial effect of DMF treatment in Nrf2−/− and WT mice was accompanied by reduced frequencies of IFN-γ and IL-17–producing CD4+ cells and induction of antiinflammatory M2 (type II) monocytes. DMF also modulated B-cell MHC II expression and reduced the incidence of clinical disease in a B-cell–dependent model of spontaneous CNS autoimmunity. Our observations that oral DMF treatment promoted immune modulation and provided equal clinical benefit in acute EAE in Nrf2−/− and WT mice, suggest that the antiinflammatory activity of DMF in treatment of MS patients may occur through alternative pathways, independent of Nrf2. PMID:27078105

  18. UHRF1 regulation of the Keap1-Nrf2 pathway in pancreatic cancer contributes to oncogenesis.

    PubMed

    Abu-Alainin, Wafa; Gana, Thompson; Liloglou, Triantafillos; Olayanju, Adedamola; Barrera, Lawrence N; Ferguson, Robert; Campbell, Fiona; Andrews, Timothy; Goldring, Christopher; Kitteringham, Neil; Park, Brian K; Nedjadi, Taoufik; Schmid, Michael C; Slupsky, Joseph R; Greenhalf, William; Neoptolemos, John P; Costello, Eithne

    2016-02-01

    The cellular defence protein Nrf2 is a mediator of oncogenesis in pancreatic ductal adenocarcinoma (PDAC) and other cancers. However, the control of Nrf2 expression and activity in cancer is not fully understood. We previously reported the absence of Keap1, a pivotal regulator of Nrf2, in ∼70% of PDAC cases. Here we describe a novel mechanism whereby the epigenetic regulator UHRF1 suppresses Keap1 protein levels. UHRF1 expression was observed in 20% (5 of 25) of benign pancreatic ducts compared to 86% (114 of 132) of pancreatic tumours, and an inverse relationship between UHRF1 and Keap1 levels in PDAC tumours (n = 124) was apparent (p = 0.002). We also provide evidence that UHRF1-mediated regulation of the Nrf2 pathway contributes to the aggressive behaviour of PDAC. Depletion of UHRF1 from PDAC cells decreased growth and enhanced apoptosis and cell cycle arrest. UHRF1 depletion also led to reduced levels of Nrf2-regulated downstream proteins and was accompanied by heightened oxidative stress, in the form of lower glutathione levels and increased reactive oxygen species. Concomitant depletion of Keap1 and UHRF1 restored Nrf2 levels and reversed cell cycle arrest and the increase in reactive oxygen species. Mechanistically, depletion of UHRF1 reduced global and tumour suppressor promoter methylation in pancreatic cancer cell lines, and KEAP1 gene promoter methylation was reduced in one of three cell lines examined. Thus, methylation of the KEAP1 gene promoter may contribute to the suppression of Keap1 protein levels by UHRF1, although our data suggest that additional mechanisms need to be explored. Finally, we demonstrate that K-Ras drives UHRF1 expression, establishing a novel link between this oncogene and Nrf2-mediated cellular protection. Since UHRF1 over-expression occurs in other cancers, its ability to regulate the Keap1-Nrf2 pathway may be critically important to the malignant behaviour of these cancers. PMID:26497117

  19. Nrf2 induces cisplatin resistance through activation of autophagy in ovarian carcinoma

    PubMed Central

    Bao, Ling-Jie; Jaramillo, Melba C; Zhang, Zhen-Bo; Zheng, Yun-Xi; Yao, Ming; Zhang, Donna D; Yi, Xiao-Fang

    2014-01-01

    Cisplatin resistance is a major problem affecting ovarian carcinoma treatment. NF-E2-related factor 2 (Nrf2), a nuclear transcription factor, plays an important role in chemotherapy resistance. However, the underlying mechanism by which Nrf2 mediates cisplatin chemoresistance is unclear. Methods: The human ovarian carcinoma cell line, A2780, and its cisplatin-resistant variant, A2780cp were cultivated. Cell viability was determined with WST-8 assay. Western blot was applied to detect the expression of Nrf2, Nrf2 target genes, and autophagy-related proteins. RNA interference was used to knock down target genes. Annexin V and propidium iodide (PI) staining was utilized to quantify apoptosis. The ultrastructural analysis of autophagosomes was performed by transmission electron microscopy (TEM). Results: Nrf2 and its targeting genes, NQO1 and HO-1, are overexpressed in A2780cp cells compared with A2780 cells. Knocking down Nrf2 sensitized A2780cp cells to cisplatin treatment and decreased autophagy-related genes, Atg3, Atg6, Atg12 and p62 in both mRNA and protein levels. Furthermore, we demonstrated that in both cell lines cisplatin could induce the formation of autophagosomes and upregulate the expression of autophagy-related genes Atg3, Atg6 and Atg12. Treatment with an autophagy inhibitor, 3-Methyladenine (3-MA), or beclin 1 siRNA enhanced cisplatin-induced cell death in A2780cp cells, suggesting that inhibition of autophagy renders resistant cells to be more sensitive to cisplatin. Taken together, Nrf2 signaling may regulate cisplatin resistance by activating autophagy. Conclusions: Nrf2-activated autophagy may function as a novel mechanism causing cisplatin-resistance. PMID:24817946

  20. Sulforaphane inhibits multiple inflammasomes through an Nrf2-independent mechanism.

    PubMed

    Greaney, Allison J; Maier, Nolan K; Leppla, Stephen H; Moayeri, Mahtab

    2016-01-01

    The inflammasomes are intracellular complexes that have an important role in cytosolic innate immune sensing and pathogen defense. Inflammasome sensors detect a diversity of intracellular microbial ligands and endogenous danger signals and activate caspase-1, thus initiating maturation and release of the proinflammatory cytokines interleukin-1β and interleukin-18. These events, although crucial to the innate immune response, have also been linked to the pathology of several inflammatory and autoimmune disorders. The natural isothiocyanate sulforaphane, present in broccoli sprouts and available as a dietary supplement, has gained attention for its antioxidant, anti-inflammatory, and chemopreventive properties. We discovered that sulforaphane inhibits caspase-1 autoproteolytic activation and interleukin-1β maturation and secretion downstream of the nucleotide-binding oligomerization domain-like receptor leucine-rich repeat proteins NLRP1 and NLRP3, NLR family apoptosis inhibitory protein 5/NLR family caspase-1 recruitment domain-containing protein 4 (NAIP5/NLRC4), and absent in melanoma 2 (AIM2) inflammasome receptors. Sulforaphane does not inhibit the inflammasome by direct modification of active caspase-1 and its mechanism is not dependent on protein degradation by the proteasome or de novo protein synthesis. Furthermore, sulforaphane-mediated inhibition of the inflammasomes is independent of the transcription factor nuclear factor erythroid-derived 2-like factor 2 (Nrf2) and the antioxidant response-element pathway, to which many of the antioxidant and anti-inflammatory effects of sulforaphane have been attributed. Sulforaphane was also found to inhibit cell recruitment to the peritoneum and interleukin-1β secretion in an in vivo peritonitis model of acute gout and to reverse NLRP1-mediated murine resistance to Bacillus anthracis spore infection. These findings demonstrate that sulforaphane inhibits the inflammasomes through a novel mechanism and contributes to

  1. Disruption of Nrf2 enhances susceptibility to severe airway inflammation and asthma in mice.

    PubMed

    Rangasamy, Tirumalai; Guo, Jia; Mitzner, Wayne A; Roman, Jessica; Singh, Anju; Fryer, Allison D; Yamamoto, Masayuki; Kensler, Thomas W; Tuder, Rubin M; Georas, Steve N; Biswal, Shyam

    2005-07-01

    Oxidative stress has been postulated to play an important role in the pathogenesis of asthma; although a defect in antioxidant responses has been speculated to exacerbate asthma severity, this has been difficult to demonstrate with certainty. Nuclear erythroid 2 p45-related factor 2 (Nrf2) is a redox-sensitive basic leucine zipper transcription factor that is involved in the transcriptional regulation of many antioxidant genes. We show that disruption of the Nrf2 gene leads to severe allergen-driven airway inflammation and hyperresponsiveness in mice. Enhanced asthmatic response as a result of ovalbumin sensitization and challenge in Nrf2-disrupted mice was associated with more pronounced mucus cell hyperplasia and infiltration of eosinophils into the lungs than seen in wild-type littermates. Nrf2 disruption resulted in an increased expression of the T helper type 2 cytokines interleukin (IL)-4 and IL-13 in bronchoalveolar lavage fluid and in splenocytes after allergen challenge. The enhanced severity of the asthmatic response from disruption of the Nrf2 pathway was a result of a lowered antioxidant status of the lungs caused by lower basal expression, as well as marked attenuation, of the transcriptional induction of multiple antioxidant genes. Our studies suggest that the responsiveness of Nrf2-directed antioxidant pathways may act as a major determinant of susceptibility to allergen-mediated asthma. PMID:15998787

  2. Nrf2-dependent and -independent Responses to Nitro-fatty Acids in Human Endothelial Cells

    PubMed Central

    Kansanen, Emilia; Jyrkkänen, Henna-Kaisa; Volger, Oscar L.; Leinonen, Hanna; Kivelä, Annukka M.; Häkkinen, Sanna-Kaisa; Woodcock, Steven R.; Schopfer, Francisco J.; Horrevoets, Anton J.; Ylä-Herttuala, Seppo; Freeman, Bruce A.; Levonen, Anna-Liisa

    2009-01-01

    Electrophilic fatty acid derivatives, including nitrolinoleic acid and nitro-oleic acid (OA-NO2), can mediate anti-inflammatory and pro-survival signaling reactions. The transcription factor Nrf2, activated by electrophilic fatty acids, suppresses redox-sensitive pro-inflammatory gene expression and protects against vascular endothelial oxidative injury. It was therefore postulated that activation of Nrf2 by OA-NO2 accounts in part for its anti-inflammatory actions, motivating the characterization of Nrf2-dependent and -independent effects of OA-NO2 on gene expression using genome-wide transcriptional profiling. Control and Nrf2-small interfering RNA-transfected human endothelial cells were treated with vehicle, oleic acid, or OA-NO2, and differential gene expression profiles were determined. Although OA-NO2 significantly induced the expression of Nrf2-dependent genes, including heme oxygenase-1 and glutamate-cysteine ligase modifier subunit, the majority of OA-NO2-regulated genes were regulated by Nrf2-independent pathways. Moreover, gene set enrichment analysis revealed that the heat shock response is the major pathway activated by OA-NO2, with robust induction of a number of heat shock genes regulated by the heat shock transcription factor. Inasmuch as the heat shock response mediates anti-inflammatory and cytoprotective actions, this mechanism is proposed to contribute to the protective cell signaling functions of nitro-fatty acids and other electrophilic fatty acid derivatives. PMID:19808663

  3. Colloidal carbon stimulation of Kupffer cells triggers Nrf2 activation in the isolated perfused rat liver.

    PubMed

    Núñez, Bárbara; Vargas, Romina; Castillo, Iván; Videla, Luis A

    2012-06-01

    Activation of transcription factor Nrf2 was investigated in the isolated perfused rat liver infused with 0.5 mg of colloidal carbon (CC)/ml for 5-15 min to stimulated Kupffer cell function. Infusion of CC enhanced liver O(2) consumption over basal levels, with a time-dependent increase in CC-induced O(2) uptake, at constant rates of CC phagocytosis by Kupffer cells, as assessed histologically, and adequate viability conditions of the livers, as shown by the marginal (0.34 %) total sinusoidal lactate dehydrogenase (LDH) efflux over intrahepatic LDH activity. Under these conditions, cytosolic protein levels of Nrf2 (Western blot) and inhibitor of Nrf2 Keap1 progressively declined by CC infusion, those of nuclear Nrf2 increased, leading to enhancement in the nuclear/cytosolic Nrf2 ratios. It is concluded that the respiratory burst of CC-stimulated Kupffer cells triggers Nrf2 activation in the perfused liver, a response that may afford cellular protection under pro-oxidant conditions underlying Kupffer cell stimulation. PMID:22461194

  4. NRF2 Oxidative Stress Induced by Heavy Metals is Cell Type Dependent

    PubMed Central

    Simmons, Steven O; Fan, Chun-Yang; Yeoman, Kim; Wakefield, John; Ramabhadran, Ram

    2011-01-01

    Exposure to metallic environmental toxicants has been demonstrated to induce a variety of oxidative stress responses in mammalian cells. The transcription factor Nrf2 is activated in response to oxidative stress and coordinates the expression of antioxidant gene products. In this study, we describe the development of an Nrf2-specific reporter gene assay that can be used to study the oxidative stress response in multiple cell types. Using five different cell lines, the Nrf2-activating potency of twenty metals was assessed across a range of concentrations. While ten of the metals tested (cadmium, cobalt, copper, gold, iron, lead, mercury, silver, sodium arsenite and zinc) stimulated Nrf2-dependent transcriptional activity in at least three of the engineered cell lines, only three (cadmium, copper and sodium arsenite) were active in all five cell lines. A comparison of metal-induced Nrf2 transcriptional activation revealed significant differences in the absolute magnitude of activation as well as the relative potencies between the cell lines tested. However, there was no direct correlation between activity and potency. Taken together, these results show that the capacity to stimulate Nrf2 activity and relative potencies of these test compounds are highly dependent on the cell type tested. Since oxidative stress is thought to be involved in the mode of action of many toxicological studies, this observation may inform the design of paradigms for toxicity testing for toxicant prioritization and characterization. PMID:21643505

  5. Methylation of arginine by PRMT1 regulates Nrf2 transcriptional activity during the antioxidative response.

    PubMed

    Liu, Xin; Li, Hongyuan; Liu, Lingxia; Lu, Yang; Gao, Yanyan; Geng, Pengyu; Li, Xiaoxue; Huang, Baiqu; Zhang, Yu; Lu, Jun

    2016-08-01

    The cap 'n' collar (CNC) family of transcription factors play important roles in resistance of oxidative and electrophilic stresses. Among the CNC family members, NF-E2-related factor 2 (Nrf2) is critical for regulating the antioxidant and phase II enzymes through antioxidant response element (ARE)-mediated transactivation. The activity of Nrf2 is controlled by a variety of post-translational modifications, including phosphorylation, ubiquitination, acetylation and sumoylation. Here we demonstrate that the arginine methyltransferase-1 (PRMT1) methylates Nrf2 protein at a single residue of arginine 437, both in vitro and in vivo. Using the heme oxygenase-1 (HO-1) as a model of phase II enzyme gene, we found that methylation of Nrf2 by PRMT1 led to a moderate increase of its DNA-binding activity and transactivation, which subsequently protected cells against the tBHP-induced glutathione depletion and cell death. Collectively, our results define a novel modification of Nrf2, which operates as a fine-tuning mechanism for the transcriptional activity of Nrf2 under the oxidative stress. PMID:27183873

  6. PGAM5 tethers a ternary complex containing Keap1 and Nrf2 to mitochondria

    SciTech Connect

    Lo, S.-C.; Hannink, Mark

    2008-05-01

    Eukaryote cells balance production of reactive oxygen species (ROS) with levels of anti-oxidant enzyme activity to maintain cellular redox homeostasis. Mitochondria are a major source of ROS, while many anti-oxidant genes are regulated by the Nrf2 transcription factor. Keap1, a redox-regulated substrate adaptor for a cullin-based ubiquitin ligase, targets Nrf2 for proteosome-mediated degradation and represses Nrf2-dependent gene expression. We have previously identified a member of the phosphoglycerate mutase family, PGAM5, as a Keap1-binding protein. In this report, we demonstrate that PGAM5 is targeted to the outer membrane of mitochondria by an N-terminal mitochondrial-localization sequence. Furthermore, we provide evidence that PGAM5 forms a ternary complex containing both Keap1 and Nrf2, in which the dimeric Keap1 protein simultaneously binds both PGAM5 and Nrf2 through their conserved E(S/T)GE motifs. Knockdown of either Keap1 or PGAM5 activates Nrf2-dependent gene expression. We suggest that this ternary complex provides a molecular framework for understanding how nuclear anti-oxidant gene expression is regulated in response to changes in mitochondrial function(s)

  7. Skin-Derived Precursors against UVB-Induced Apoptosis via Bcl-2 and Nrf2 Upregulation

    PubMed Central

    Zhong, Jianqiao

    2016-01-01

    Bcl-2 and Nrf2 are critical factors in protecting cells against UVB-induced apoptosis. Hair-follicle-bulge stem cells could resist ionization through Bcl-2 upregulation. Skin-derived precursors (SKPs) dwelling on the bulge may be against UVB irradiation. Initially, SKPs were isolated and identified. Then, SKPs were exposed to UVB and grew in medium for 24 hours. CCK-8 assay, TUNEL, and Ki67 staining evaluated cells apoptosis/proliferation, while SA-βgal staining evaluated cells senescence and pH2AX immunostaining evaluated DNA damage. Meanwhile, Bcl-2, Nrf2, HO-1, Bax, and Bak expressions were assessed by qRT-PCR and western blot. Two weeks later, floating spheres appeared and were identified as SKPs. After UVB radiation, SKPs maintained spherical colonies and outnumbered unirradiated ones, showing high Ki67 expression and low TUNEL, SA-βgal, and pH2AX expression. Fibroblasts (FBs), however, displayed deformation, senescence, and reduction, with increased TUNEL, SA-βgal, and pH2AX expression. Moreover, Bcl-2 and Nrf2 mRNA expression were significantly higher than Bak and Bax in irradiated SKPs. Conversely, Bcl-2 and Nrf2 mRNA levels greatly decreased compared with Bax and Bak in irradiated FBs. Interestingly, SKPs showed higher protein levels of Bcl-2, Nrf2, and HO-1 than FBs. SKPs exert a beneficial effect on resisting UVB-induced apoptosis, which may be associated with Bcl-2 and Nrf2 upregulation.

  8. NRF2, a Key Regulator of Antioxidants with Two Faces towards Cancer

    PubMed Central

    2016-01-01

    While reactive oxygen species (ROS) is generally considered harmful, a relevant amount of ROS is necessary for a number of cellular functions, including the intracellular signal transduction. In order to deal with an excessive amount of ROS, organisms are equipped with a sufficient amount of antioxidants together with NF-E2-related factor-2 (NRF2), a transcription factor that plays a key role in the protection of organisms against environmental or intracellular stresses. While the NRF2 activity has been generally viewed as beneficial to preserve the integrity of organisms, recent studies have demonstrated that cancer cells hijack the NRF2 activity to survive under the oxidative stress and, therefore, a close check must be kept on the NRF2 activity in cancer. In the present review, we briefly highlight important progresses in understanding the molecular mechanism, structure, and function of KEAP1 and NRF2 interaction. In addition, we provide general perspectives that justify conflicting views on the NRF2 activity in cancer. PMID:27340506

  9. Nrf2 Induces IL-17D to Mediate Tumor and Virus Surveillance.

    PubMed

    Saddawi-Konefka, Robert; Seelige, Ruth; Gross, Emilie T E; Levy, Eric; Searles, Stephen C; Washington, Allen; Santosa, Endi K; Liu, Beichen; O'Sullivan, Timothy E; Harismendy, Olivier; Bui, Jack D

    2016-08-30

    Cells undergoing xenobiotic or oxidative stress activate the transcription factor nuclear factor erythroid-derived 2-like 2 (Nrf2), which initiates an intrinsic "stress surveillance" pathway. We recently found that the cytokine IL-17D effects a form of extrinsic stress surveillance by inducing antitumor immunity, but how IL-17D is regulated remains unknown. Here, we show that Nrf2 induced IL-17D in cancer cell lines. Moreover, both Nrf2 and IL-17D were induced in primary tumors as well as during viral infection in vivo. Expression of IL-17D in tumors and virally infected cells is essential for optimal protection of the host as il17d(-/-) mice experienced a higher incidence of tumors and exacerbated viral infections compared to wild-type (WT) animals. Moreover, activating Nrf2 to induce IL-17D in established tumors led to natural killer cell-dependent tumor regression. These data demonstrate that Nrf2 can initiate both intrinsic and extrinsic stress surveillance pathways and highlight the use of Nrf2 agonists as immune therapies for cancer and infection. PMID:27545889

  10. Sulforaphane prevents pulmonary damage in response to inhaled arsenic by activating the Nrf2-defense response

    SciTech Connect

    Zheng, Yi; Tao, Shasha; Lian, Fangru; Chau, Binh T.; Chen, Jie; Sun, Guifan; Fang, Deyu; Lantz, R. Clark; Zhang, Donna D.

    2012-12-15

    Exposure to arsenic is associated with an increased risk of lung disease. Novel strategies are needed to reduce the adverse health effects associated with arsenic exposure in the lung. Nrf2, a transcription factor that mediates an adaptive cellular defense response, is effective in detoxifying environmental insults and prevents a broad spectrum of diseases induced by environmental exposure to harmful substances. In this report, we tested whether Nrf2 activation protects mice from arsenic-induced toxicity. We used an in vivo arsenic inhalation model that is highly relevant to low environmental human exposure to arsenic-containing dusts. Two-week exposure to arsenic-containing dust resulted in pathological alterations, oxidative DNA damage, and mild apoptotic cell death in the lung; all of which were blocked by sulforaphane (SF) in an Nrf2-dependent manner. Mechanistically, SF-mediated activation of Nrf2 alleviated inflammatory responses by modulating cytokine production. This study provides strong evidence that dietary intervention targeting Nrf2 activation is a feasible approach to reduce adverse health effects associated with arsenic exposure. -- Highlights: ► Exposed to arsenic particles and/or SF have elevated Nrf2 and its target genes. ► Sulforaphane prevents pathological alterations, oxidative damage and cell death. ► Sulforaphane alleviates infiltration of inflammatory cells into the lungs. ► Sulforaphane suppresses arsenic-induced proinflammatory cytokine production.

  11. Expression of LC3, LAMP2, KEAP1 and NRF2 in Salivary Adenoid Cystic Carcinoma.

    PubMed

    Huang, Cong-Fa; Deng, Wei-Wei; Zhang, Lu; Zhang, Wen-Feng; Sun, Zhi-Jun

    2016-01-01

    Salivary Adenoid Cystic Carcinoma (SACC) is a tumor characterized by inevitable local progression and terminal hematogenous metastasis. This study aimed to investigate the expression of LC3, LAMP2, KEAP1 and NRF2 in SACC. Human salivary gland tissue microarray which contains 74 SACC, 12 pleomorphic adenoma and 18 normal salivary gland specimens. High expression of LC3, LAMP2, KEAP1 and NRF2 were found in SACC patients, and LC3, LAMP2, KEAP1 and NRF2 expression were significantly higher in SACC than as compared with pleomorphic adenoma and (or) normal salivary gland. The expression of NRF2 was correlated with pathological type of human SACC (P < 0.05). Moreover, the high-expression of KEAP1 had significant correlations with LC3 (P < 0.001, R = 0.3195), and LAMP2 (P < 0.001, R = 0.3346) and NRF2 (P < 0.05, R = 0.2246) by using the Pearson correlation coefficient test. Our findings demonstrated that up-regulation of LC3, LAMP2, KEAP1 and NRF2 were associated with carcinogenesis and progression of SACC patients, suggesting that they may be useful molecular targets in salivary adenoid cystic carcinoma. PMID:26350055

  12. Role of Nrf2, HO-1 and GSH in Neuroblastoma Cell Resistance to Bortezomib

    PubMed Central

    Furfaro, A. L.; Piras, S.; Domenicotti, C.; Fenoglio, D.; De Luigi, A.; Salmona, M.; Moretta, L.; Marinari, U. M.; Pronzato, M. A.; Traverso, N.; Nitti, M.

    2016-01-01

    The activation of Nrf2 has been demonstrated to play a crucial role in cancer cell resistance to different anticancer therapies. The inhibition of proteasome activity has been proposed as a chemosensitizing therapy but the activation of Nrf2 could reduce its efficacy. Using the highly chemoresistant neuroblastoma cells HTLA-230, here we show that the strong reduction in proteasome activity, obtained by using low concentration of bortezomib (BTZ, 2.5 nM), fails in reducing cell viability. BTZ treatment favours the binding of Nrf2 to the ARE sequences in the promoter regions of target genes such as heme oxygenase 1 (HO-1), the modulatory subunit of γ-glutamylcysteine ligase (GCLM) and the transporter for cysteine (x-CT), enabling their transcription. GSH level is also increased after BTZ treatment. The up-regulation of Nrf2 target genes is responsible for cell resistance since HO-1 silencing and GSH depletion synergistically decrease BTZ-treated cell viability. Moreover, cell exposure to all-trans-Retinoic acid (ATRA, 3 μM) reduces the binding of Nrf2 to the ARE sequences, decreases HO-1 induction and lowers GSH level increasing the efficacy of bortezomib. These data suggest the role of Nrf2, HO-1 and GSH as molecular targets to improve the efficacy of low doses of bortezomib in the treatment of malignant neuroblastoma. PMID:27023064

  13. Activation of the Nrf2 pathway, but decreased {gamma}-glutamylcysteine synthetase heavy subunit chain levels and caspase-3-dependent apoptosis during exposure of primary mouse hepatocytes to diphenylarsinic acid

    SciTech Connect

    Sumi, Daigo; Manji, Aiko; Shinkai, Yasuhiro; Toyama, Takashi; Kumagai, Yoshito

    2007-09-15

    Diphenylarsinic acid (DPAsV) is a degradation product of chemical warfare agents, over which there has been a public outcry in the Kamisu Area of Ibaraki Prefecture in Japan. In this study, we investigated the cytotoxicity of and cellular response to DPAsV in primary mouse hepatocytes. Exposure of the hepatocytes to DPAsV resulted in cell damage accompanied by cellular accumulation of DPAsV in a time-dependent manner. The cell death caused by DPAsV was attributable to apoptosis. DPAsV activated a basic leucine-zipper transcription factor Nrf2 as determined by the nuclear translocation of Nrf2, anti-oxidant response element (ARE)-dependent luciferase activity, and upregulation of downstream gene products. However, {gamma}-glutamylcysteine synthetase heavy subunit chain ({gamma}-GCS{sub H}), which is regulated by Nrf2, underwent cleavage by activated caspase-3 to a 17 kDa fragment, leading to a minimal level of constitutive {gamma}-GCS{sub H} expression 72 h following the exposure (25 {mu}M). Experiments with cycloheximide revealed that the DPAsV-mediated reduction in {gamma}-GCS{sub H} was due to a post-translational modification. The results suggest that DPAsV causes caspase-3-dependent cleavage of {gamma}-GCS{sub H} regardless of Nrf2 activation in primary mouse hepatocytes.

  14. NRF2 Is a Key Target for Prevention of Noise-Induced Hearing Loss by Reducing Oxidative Damage of Cochlea

    PubMed Central

    Honkura, Yohei; Matsuo, Hirotaka; Murakami, Shohei; Sakiyama, Masayuki; Mizutari, Kunio; Shiotani, Akihiro; Yamamoto, Masayuki; Morita, Ichiro; Shinomiya, Nariyoshi; Kawase, Tetsuaki; Katori, Yukio; Motohashi, Hozumi

    2016-01-01

    Noise-induced hearing loss (NIHL) is one of the most common sensorineural hearing deficits. Recent studies have demonstrated that the pathogenesis of NIHL is closely related to ischemia-reperfusion injury of cochlea, which is caused by blood flow decrease and free radical production due to excessive noise. This suggests that protecting the cochlea from oxidative stress is an effective therapeutic approach for NIHL. NRF2 is a transcriptional activator playing an essential role in the defense mechanism against oxidative stress. To clarify the contribution of NRF2 to cochlear protection, we examined Nrf2–/– mice for susceptibility to NIHL. Threshold shifts of the auditory brainstem response at 7 days post-exposure were significantly larger in Nrf2–/– mice than wild-type mice. Treatment with CDDO-Im, a potent NRF2-activating drug, before but not after the noise exposure preserved the integrity of hair cells and improved post-exposure hearing levels in wild-type mice, but not in Nrf2–/– mice. Therefore, NRF2 activation is effective for NIHL prevention. Consistently, a human NRF2 SNP was significantly associated with impaired sensorineural hearing levels in a cohort subjected to occupational noise exposure. Thus, high NRF2 activity is advantageous for cochlear protection from noise-induced injury, and NRF2 is a promising target for NIHL prevention. PMID:26776972

  15. Systemic administration of the apocarotenoid bixin protects skin against solar UV-induced damage through activation of NRF2.

    PubMed

    Tao, Shasha; Park, Sophia L; Rojo de la Vega, Montserrat; Zhang, Donna D; Wondrak, Georg T

    2015-12-01

    Exposure to solar ultraviolet (UV) radiation is a causative factor in skin photodamage and carcinogenesis, and an urgent need exists for improved molecular photoprotective strategies different from (or synergistic with) photon absorption. Recent studies suggest a photoprotective role of cutaneous gene expression orchestrated by the transcription factor NRF2 (nuclear factor-E2-related factor 2). Here we have explored the molecular mechanism underlying carotenoid-based systemic skin photoprotection in SKH-1 mice and provide genetic evidence that photoprotection achieved by the FDA-approved apocarotenoid and food additive bixin depends on NRF2 activation. Bixin activates NRF2 through the critical Cys-151 sensor residue in KEAP1, orchestrating a broad cytoprotective response in cultured human keratinocytes as revealed by antioxidant gene expression array analysis. Following dose optimization studies for cutaneous NRF2 activation by systemic administration of bixin, feasibility of bixin-based suppression of acute cutaneous photodamage from solar UV exposure was investigated in Nrf2(+/+) versus Nrf2(-/-) SKH-1 mice. Systemic administration of bixin suppressed skin photodamage, attenuating epidermal oxidative DNA damage and inflammatory responses in Nrf2(+/+) but not in Nrf2(-/-) mice, confirming the NRF2-dependence of bixin-based cytoprotection. Taken together, these data demonstrate feasibility of achieving NRF2-dependent cutaneous photoprotection by systemic administration of the apocarotenoid bixin, a natural food additive consumed worldwide. PMID:26456052

  16. NRF2 Is a Key Target for Prevention of Noise-Induced Hearing Loss by Reducing Oxidative Damage of Cochlea.

    PubMed

    Honkura, Yohei; Matsuo, Hirotaka; Murakami, Shohei; Sakiyama, Masayuki; Mizutari, Kunio; Shiotani, Akihiro; Yamamoto, Masayuki; Morita, Ichiro; Shinomiya, Nariyoshi; Kawase, Tetsuaki; Katori, Yukio; Motohashi, Hozumi

    2016-01-01

    Noise-induced hearing loss (NIHL) is one of the most common sensorineural hearing deficits. Recent studies have demonstrated that the pathogenesis of NIHL is closely related to ischemia-reperfusion injury of cochlea, which is caused by blood flow decrease and free radical production due to excessive noise. This suggests that protecting the cochlea from oxidative stress is an effective therapeutic approach for NIHL. NRF2 is a transcriptional activator playing an essential role in the defense mechanism against oxidative stress. To clarify the contribution of NRF2 to cochlear protection, we examined Nrf2(-/-) mice for susceptibility to NIHL. Threshold shifts of the auditory brainstem response at 7 days post-exposure were significantly larger in Nrf2(-/-) mice than wild-type mice. Treatment with CDDO-Im, a potent NRF2-activating drug, before but not after the noise exposure preserved the integrity of hair cells and improved post-exposure hearing levels in wild-type mice, but not in Nrf2(-/-) mice. Therefore, NRF2 activation is effective for NIHL prevention. Consistently, a human NRF2 SNP was significantly associated with impaired sensorineural hearing levels in a cohort subjected to occupational noise exposure. Thus, high NRF2 activity is advantageous for cochlear protection from noise-induced injury, and NRF2 is a promising target for NIHL prevention. PMID:26776972

  17. Discovery of potent, novel Nrf2 inducers via quantum modeling, virtual screening and in vitro experimental validation

    PubMed Central

    Williamson, Tracy P.; Amirahmadi, Sara; Joshi, Gururaj; Kaludov, Nikola K.; Martinov, Martin N.; Johnson, Delinda A.; Johnson, Jeffrey A.

    2012-01-01

    Nuclear factor erythroid 2-related factor 2 (Nrf2) is the master transcription factor of the antioxidant response element (ARE) pathway, coordinating the induction of detoxifying and antioxidant enzymes. Nrf2 is normally sequestered in the cytoplasm by Kelch-like ECH associating protein 1 (Keap1). To identify novel small molecules that will disturb Nrf2:Keap1 binding and promote activation of the Nrf2-ARE pathway, we generated a quantum model based on the structures of known Nrf2-ARE activators. We used the quantum model to perform in silico screening on over 18 million commercially available chemicals to identify the structures predicted to activate the Nrf2-ARE pathway based on the quantum model. The top hits were tested in vitro and half of the predicted hits activated the Nrf2-ARE pathway significantly in primary cell culture. In addition, we identified a new family of Nrf2-ARE activating structures that all have comparable activity to tBHQ and protect against oxidative stress and dopaminergic toxins in vitro. The improved ability to identify potent activators of Nrf2 through the combination of in silico and in vitro screening described here improves the speed and cost associated with screening Nrf2-ARE activating compounds for drug development. PMID:22925725

  18. GSK-3β downregulates Nrf2 in cultured cortical neurons and in a rat model of cerebral ischemia-reperfusion

    PubMed Central

    Chen, Xi; Liu, Yuanling; Zhu, Jin; Lei, Shipeng; Dong, Yuan; Li, Lingyu; Jiang, Beibei; Tan, Li; Wu, Jingxian; Yu, Shanshan; Zhao, Yong

    2016-01-01

    The NF-E2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway plays a critical role in protecting against oxidative stress in brain ischemia and reperfusion injury. Glycogen synthase kinase 3β (GSK-3β) may play a critical role in regulating Nrf2 in a Kelch-like ECH-associated protein 1 (Keap1)-independent manner. However, the relationship between GSK-3β and Nrf2 in brain ischemia and reperfusion injury is not clear. In this study, we explored the mechanisms through which GSK-3β regulates Nrf2 and Nrf-2/ARE pathways in vitro and in vivo. We used oxygen and glucose deprivation/reoxygenation (OGD/R) in primary cultured cortical neurons and a middle cerebral artery occlusion-reperfusion (MCAO/R) rat model to mimic ischemic insult. In this study, GSK-3β siRNA and inhibitors (SB216763 and LiCl) were used to inhibit GSK-3β in vitro and in vivo. After inhibiting GSK-3β, expression of total and nuclear Nrf2, Nrf2-ARE binding activity, and expression of Nrf2/ARE pathway-driven genes HO-1 and NQO-1 increased. Overexpression of GSK-3β yielded opposite results. These results suggest that GSK-3β downregulates Nrf2 and the Nrf2/ARE pathway in brain ischemia and reperfusion injury. GSK-3β may be an endogenous antioxidant relevant protein, and may represent a new therapeutic target in treatment of ischemia and reperfusion injury. PMID:26838164

  19. Sustained NRF2 activation in hereditary leiomyomatosis and renal cell cancer (HLRCC) and in hereditary tyrosinemia type 1 (HT1).

    PubMed

    Sandhu, Ivraj Singh; Maksim, Nicholas James; Amouzougan, Eva Alice; Gallion, Bryce Wilson; Raviele, Anthony L J; Ooi, Aikseng

    2015-08-01

    The nuclear erythroid 2-like 2 transcription factor (NRF2), is a major regulator of cellular redox balance. Although NRF2 activation is generally regarded as beneficial to human health, recent studies have identified that sustained NRF2 activation is over-represented in many cancers. This raises the question regarding the role of NRF2 activation in the development and progression of those cancers. This review focuses on the mechanisms and the effects of NRF2 activation in two hereditary cancer predisposition syndromes: hereditary leiomyomatosis and renal cell cancer (HLRCC) and hereditary tyrosinemia type 1 (HT1). Because the cancer initiating mutations in these hereditary syndromes are well defined, they offer a unique opportunity to explore the roles of NRF2 activation in the early stages of carcinogenesis. Over the years, a variety of approaches have been utilized to study the biology of HLRCC and HT1. In HLRCC, in vitro studies have demonstrated the importance of NRF2 activation in sustaining cancer cell proliferation. In the mouse model of HT1 however, NRF2 activation seems to protect cells from malignant transformation. In both HT1 and HLRCC, NRF2 activation promotes the clearance of electrophilic metabolites, enabling cells to survive cancer-initiating mutations. Biological insights gained from the hereditary syndromes' studies may shed light on to the roles of NRF2 activation in sporadic tumours. PMID:26551707

  20. Role of the Nrf2-heme oxygenase-1 pathway in silver nanoparticle-mediated cytotoxicity

    SciTech Connect

    Kang, Su Jin; Ryoo, In-geun; Lee, Young Joon; Kwak, Mi-Kyoung

    2012-01-01

    Silver nanoparticles (nano-Ag) have been widely used in various commercial products including textiles, electronic appliances and biomedical products. However, there remains insufficient information on the potential risk of nano-Ag to human health and environment. In the current study, we have investigated the role of NF-E2-related factor 2 (Nrf2) transcription factor in nano-Ag-induced cytotoxicity. When Nrf2 expression was blocked using interring RNA expression in ovarian carcinoma cell line, nano-Ag treatment showed a substantial decrease in cell viability with concomitant increases in apoptosis and DNA damage compared to the control cells. Target gene analysis revealed that the expression of heme oxygenase-1 (HO-1) was highly elevated by nano-Ag in nonspecific shRNA expressing cells, while Nrf2 knockdown cells (NRF2i) did not increase HO-1 expression. The role of HO-1 in cytoprotection against nano-Ag was reinforced by results using pharmacological inducer of HO-1: cobalt protoporphyrin-mediated HO-1 activation in the NRF2i cells prevented nano-Ag-mediated cell death. Similarly, pharmacological or genetic inhibition of HO-1 in nonspecific control cells exacerbated nano-Ag toxicity. As the upstream signaling mechanism, nano-Ag required the phosphoinositide 3-kinase (PI3K) and p38MAPK signaling cascades for HO-1 induction. The treatment with either PI3K inhibitor or p38MAPK inhibitor suppressed HO-1 induction and intensified nano-Ag-induced cell death. Taken together, these results suggest that Nrf2-dependent HO-1 up-regulation plays a protective role in nano-Ag-induced DNA damage and consequent cell death. In addition, nano-Ag-mediated HO-1 induction is associated with the PI3K and p38MAPK signaling pathways. -- Highlights: ► Role of Nrf2 signaling in silver nanoparticle toxicity. ► Silver nanoparticle toxicity is increased by Nrf2 blockade. ► Nrf2-dependent HO-1 induction protects cells from silver nanoparticle toxicity. ► PI3K and p38MAPK cascades are

  1. Quercetin phospholipid complex significantly protects against oxidative injury in ARPE-19 cells associated with activation of Nrf2 pathway.

    PubMed

    Xu, Xin-Rong; Yu, Hai-Tao; Yang, Yan; Hang, Li; Yang, Xue-Wen; Ding, Shu-Hua

    2016-01-01

    Age-related macular degeneration (AMD) is a major cause of blindness worldwide. Oxidative stress plays a crucial role in the pathogenesis of dry AMD. Quercetin has potent anti-oxidative activities, but poor bioavailability limits its therapeutic application. Herein, we prepared the phospholipid complex of quercetin (quercetin-PC), characterized its structure by differential scanning calorimetry, infrared spectrum and x-ray diffraction. Quercetin-PC had equilibrium solubility of 38.36 and 1351.27μg/ml in water and chloroform, respectively, which was remarkably higher than those of quercetin alone. Then we established hydrogen peroxide (H2O2)-induced oxidative injury model in human ARPE-19 cells to examine the effects of quercetin-PC. Quercetin-PC, stronger than quercetin, promoted cell proliferation, and the proliferation rate was increased to be 78.89% when treated with Quercetin-PC at 400μM. Moreover, quercetin-PC effectively prevented ARPE-19 cells from apoptosis, and the apoptotic rate was reduced to be 3.1% when treated with Quercetin-PC at 200μM. In addition, quercetin-PC at 200μM significantly increased the activities of SOD, CAT and GSH-PX, and reduced the levels of reactive oxygen species and MDA in H2O2-treated ARPE-19 cells, but quercetin at 200μM failed to do so. Molecular examinations revealed that quercetin-PC at 200μM significantly activated Nrf2 nuclear translocation and significantly enhanced the expression of target genes HO-1, NQO-1 and GCL by different folds at both mRNA and protein levels. Our current data collectively indicated that quercetin-PC had stronger protective effects against oxidative-induced damages in ARPE-19 cells, which was associated with activation of Nrf2 pathway and its target genes implicated in antioxidant defense. PMID:26643168

  2. Intrahippocampal injection of a lentiviral vector expressing Nrf2 improves spatial learning in a mouse model of Alzheimer's disease

    PubMed Central

    Kanninen, Katja; Heikkinen, Riikka; Malm, Tarja; Rolova, Taisia; Kuhmonen, Susanna; Leinonen, Hanna; Ylä-Herttuala, Seppo; Tanila, Heikki; Levonen, Anna-Liisa; Koistinaho, Milla; Koistinaho, Jari

    2009-01-01

    The amyloid hypothesis of Alzheimer's disease (AD) postulates that amyloid-β (Aβ) deposition and neurotoxicity play a causative role in AD; oxidative injury is thought to be central in the pathogenesis. An endogenous defense system against oxidative stress is induced by binding of the transcription factor nuclear factor E2-related factor 2 (Nrf2) to the antioxidant response element (ARE) enhancer sequence. The Nrf2-ARE pathway is activated in response to reactive oxygen species to trigger the simultaneous expression of numerous protective enzymes and scavengers. To exploit the Nrf2-ARE pathway therapeutically, we delivered Nrf2 bilaterally into the hippocampus of 9-month-old transgenic AD mice (APP/PS1 mice) using a lentiviral vector encoding human Nrf2. The data indicate that significant reductions in spatial learning deficits of aged APP/PS1 mice in a Morris Water Maze can be achieved by modulating levels of Nrf2 in the brain. Memory improvement in APP/PS1 mice after Nrf2 transduction shifts the balance between soluble and insoluble Aβ toward an insoluble Aβ pool without concomitant change in total brain Aβ burden. Nrf2 gene transfer is associated with a robust reduction in astrocytic but not microglial activation and induction of Nrf2 target gene heme oxygenase 1, indicating overall activation of the Nrf2-ARE pathway in hippocampal neurons 6 months after injection. Results warrant further exploration of the Nrf2-ARE pathway for treatment of AD and suggest that the Nrf2-ARE pathway may represent a potential therapeutic strategy to pursue in AD in humans, particularly in view of the multiple mechanisms by which Nrf2 can exert its protective effects. PMID:19805328

  3. Generation of a New Model Rat: Nrf2 Knockout Rats Are Sensitive to Aflatoxin B1 Toxicity.

    PubMed

    Taguchi, Keiko; Takaku, Misaki; Egner, Patricia A; Morita, Masanobu; Kaneko, Takehito; Mashimo, Tomoji; Kensler, Thomas W; Yamamoto, Masayuki

    2016-07-01

    THE TRANSCRIPTION FACTOR NRF2: (NF-E2-related-factor 2) REGULATES A BATTERY OF ANTIOXIDATIVE STRESS-RESPONSE GENES AND DETOXICATION GENES, AND NRF2 KNOCKOUT LINES OF MICE HAVE BEEN CONTRIBUTING CRITICALLY TO THE CLARIFICATION OF ROLES THAT NRF2 PLAYS FOR CELL PROTECTION HOWEVER, THERE ARE APPARENT LIMITATIONS IN USE OF THE MOUSE MODELS FOR INSTANCE, RATS EXHIBIT MORE SUITABLE FEATURES FOR TOXICOLOGICAL OR PHYSIOLOGICAL EXAMINATIONS THAN MICE IN THIS STUDY, WE GENERATED 2 LINES OF NRF2 KNOCKOUT RATS BY USING A GENOME EDITING TECHNOLOGY; 1 LINE HARBORS A 7-BP DELETION Δ7 AND THE OTHER LINE HARBORS A 1-BP INSERTION +1 IN THE NRF2 GENE IN THE LIVERS OF RATS HOMOZYGOUSLY DELETING THE NRF2 GENE, AN ACTIVATOR OF NRF2 SIGNALING, CDDO-IM, COULD NOT INDUCE EXPRESSION OF REPRESENTATIVE NRF2 TARGET GENES TO EXAMINE ALTERED TOXICOLOGICAL RESPONSE, WE TREATED THE NRF2 KNOCKOUT RATS WITH AFLATOXIN B1 AFB1, A CARCINOGENIC MYCOTOXIN THAT ELICITS GENE MUTATIONS THROUGH BINDING OF ITS METABOLITES TO DNA AND FOR WHICH THE RAT HAS BEEN PROPOSED AS A REASONABLE SURROGATE FOR HUMAN TOXICITY INDEED, IN THE NRF2 KNOCKOUT RAT LIVERS THE ENZYMES OF THE AFB1 DETOXICATION PATHWAY WERE SIGNIFICANTLY DOWNREGULATED SINGLE DOSE ADMINISTRATION OF AFB1 INCREASED HEPATOTOXICITY AND BINDING OF AFB1-N7-GUANINE TO HEPATIC DNA IN NRF2 KNOCKOUT RATS COMPARED WITH WILD-TYPE NRF2 KNOCKOUT RATS REPEATEDLY TREATED WITH AFB1 WERE PRONE TO LETHALITY AND CDDO-IM WAS NO LONGER PROTECTIVE THESE RESULTS DEMONSTRATE THAT NRF2 KNOCKOUT RATS ARE QUITE SENSITIVE TO AFB1 TOXICITIES AND THIS RAT GENOTYPE EMERGES AS A NEW MODEL ANIMAL IN TOXICOLOGY. PMID:27071940

  4. Impact of Nrf2 on UVB-induced skin inflammation/photoprotection and photoprotective effect of sulforaphane.

    PubMed

    Saw, Constance L; Huang, Mou-Tuan; Liu, Yue; Khor, Tin Oo; Conney, Allan H; Kong, Ah-Ng

    2011-06-01

    Ultraviolet (UV) of sunlight is a complete carcinogen that can burn skin, enhance inflammation, and drive skin carcinogenesis. Previously, we have shown that sulforaphane (SFN) inhibited chemically induced skin carcinogenesis via nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and others have shown that broccoli sprout extracts containing high SFN protected against UV-induced skin carcinogenesis in SKH-1 hairless mice. A recent study showed that there was no difference between Nrf2 knockout (Nrf2 KO) and Nrf2 wild-type (WT) BALB/C mice after exposing to high dose of UVB. Since Nrf2 plays critical roles in the anti-oxidative stress/anti-inflammatory responses, it is relevant to assess the role of Nrf2 for photoprotection against UV. In this context, the role of Nrf2 in UVB-induced skin inflammation in Nrf2 WT and Nrf2 KO C57BL/6 mice was studied. A single dose of UVB (300 mJ/cm(2)) resulted in skin inflammation in both WT and Nrf2 KO (-/-) mice (KO mice) at 8 h and 8 d following UVB irradiation. In the WT mice inflammation returned to the basal level to a greater extent when compared to the KO mice. SFN treatment of Nrf2 WT but not Nrf2 KO mice restored the number of sunburn cells back to their basal level by 8 d after UVB irradiation. Additionally, UVB-induced short-term inflammatory biomarkers (interleukin-1β and interleukin-6) were increased in the KO mice and UVB-induced apoptotic cells in the KO mice were significantly higher as compared to that in the WT. Taken together, our results show that functional Nrf2 confers a protective effect against UVB-induced inflammation, sunburn reaction, and SFN-mediated photoprotective effects in the skin. PMID:21557329

  5. The Ties that Undermine.

    PubMed

    Beverley, John

    2016-06-01

    Do biological relations ground responsibilities between biological fathers and their offspring? Few think biological relations ground either necessary or sufficient conditions for responsibility. Nevertheless, many think biological relations ground responsibility at least partially. Various scenarios, such as cases concerning the responsibilities of sperm donors, have been used to argue in favor of biological relations as partially grounding responsibilities. In this article, I seek to undermine the temptation to explain sperm donor scenarios via biological relations by appealing to an overlooked feature of such scenarios. More specifically, I argue that sperm donor scenarios may be better explained by considering the unique abilities of agents involved. Appealing to unique ability does not eliminate the possibility of biological relations providing some explanation for perceived responsibilities on the part of biological fathers. However, since it is unclear exactly why biological relations are supposed to ground responsibility in the first place, and rather clear why unique ability grounds responsibility in those scenarios where it is exhibited, the burden of proof seems shifted to those advocating biological relations as grounds of responsibility to provide an explanation. Since this seems unlikely, I conclude it is best to avoid appealing to biological relations as providing grounds for responsibility. PMID:26456160

  6. Oxidative stress involving changes in Nrf2 and ER stress in early stages of Alzheimer's disease.

    PubMed

    Mota, Sandra I; Costa, Rui O; Ferreira, Ildete L; Santana, Isabel; Caldeira, Gladys L; Padovano, Carmela; Fonseca, Ana C; Baldeiras, Inês; Cunha, Catarina; Letra, Liliana; Oliveira, Catarina R; Pereira, Cláudia M F; Rego, Ana Cristina

    2015-07-01

    Oxidative stress and endoplasmic reticulum (ER) stress have been associated with Alzheimer's disease (AD) progression. In this study we analyzed whether oxidative stress involving changes in Nrf2 and ER stress may constitute early events in AD pathogenesis by using human peripheral blood cells and an AD transgenic mouse model at different disease stages. Increased oxidative stress and increased phosphorylated Nrf2 (p(Ser40)Nrf2) were observed in human peripheral blood mononuclear cells (PBMCs) isolated from individuals with mild cognitive impairment (MCI). Moreover, we observed impaired ER Ca2+ homeostasis and increased ER stress markers in PBMCs from MCI individuals and mild AD patients. Evidence of early oxidative stress defense mechanisms in AD was substantiated by increased p(Ser40)Nrf2 in 3month-old 3xTg-AD male mice PBMCs, and also with increased nuclear Nrf2 levels in brain cortex. However, SOD1 protein levels were decreased in human MCI PBMCs and in 3xTg-AD mice brain cortex; the latter further correlated with reduced SOD1 mRNA levels. Increased ER stress was also detected in the brain cortex of young female and old male 3xTg-AD mice. We demonstrate oxidative stress and early Nrf2 activation in AD human and mouse models, which fails to regulate some of its targets, leading to repressed expression of antioxidant defenses (e.g., SOD-1), and extending to ER stress. Results suggest markers of prodromal AD linked to oxidative stress associated with Nrf2 activation and ER stress that may be followed in human peripheral blood mononuclear cells. PMID:25857617

  7. Regulation of Nrf2 signaling and longevity in naturally long-lived rodents.

    PubMed

    Lewis, Kaitlyn N; Wason, Emily; Edrey, Yael H; Kristan, Deborah M; Nevo, Eviatar; Buffenstein, Rochelle

    2015-03-24

    The preternaturally long-lived naked mole-rat, like other long-lived species and experimental models of extended longevity, is resistant to both endogenous (e.g., reactive oxygen species) and environmental stressors and also resists age-related diseases such as cancer, cardiovascular disease, and neurodegeneration. The mechanisms behind the universal resilience of longer-lived organisms to stress, however, remain elusive. We hypothesize that this resilience is linked to the activity of a highly conserved transcription factor, nuclear factor erythroid 2-related factor (Nrf2). Nrf2 regulates the transcription of several hundred cytoprotective molecules, including antioxidants, detoxicants, and molecular chaperones (heat shock proteins). Nrf2 itself is tightly regulated by mechanisms that either promote its activity or increase its degradation. We used a comparative approach and examined Nrf2-signaling activity in naked mole-rats and nine other rodent species with varying maximum lifespan potential (MLSP). We found that constitutive Nrf2-signaling activity was positively correlated (P = 0.0285) with MLSP and that this activity was also manifested in high levels of downstream gene expression and activity. Surprisingly, we found that species longevity was not linked to the protein levels of Nrf2 itself, but rather showed a significant (P < 0.01) negative relationship with the regulators Kelch-like ECH-Associated Protein 1 (Keap1) and β-transducin repeat-containing protein (βTrCP), which target Nrf2 for degradation. These findings highlight the use of a comparative biology approach for the identification of evolved mechanisms that contribute to health span, aging, and longevity. PMID:25775529

  8. Nrf2 activation diminishes during adipocyte differentiation of ST2 cells.

    PubMed

    Chartoumpekis, Dionysios V; Ziros, Panos G; Sykiotis, Gerasimos P; Zaravinos, Apostolos; Psyrogiannis, Agathoklis I; Kyriazopoulou, Venetsana E; Spandidos, Demetrios A; Habeos, Ioannis G

    2011-11-01

    Adipocyte differentiation (adipogenesis) is a highly controlled process known to be affected, among other factors, by the redox status of the cell. Nrf2 (NFE2-related factor 2) is a transcription factor that orchestrates the expression of a battery of antioxidant and detoxification genes under both basal and stress conditions. The present study investigated the activation of Nrf2 during adipocyte differentiation using as a model the mouse bone marrow-derived ST2 cell line. Treatment of ST2 cells with a differentiation cocktail containing IBMX, indomethacin, hydrocortisone and insulin induced differentiation into adipocytes over 5 days. During adipogenesis, the intracellular glutathione redox potential, which is an indicator of oxidative stress levels, became steadily more oxidized, as shown by real-time measurement in differentiating ST2 cells stably transfected with a redox-sensitive Grx1-roGFP2 fusion protein. The nuclear abundance of Nrf2 was assessed by Western immunoblotting and its DNA binding activity by EMSA (electrophoretic mobility shift assay) performed on nuclear protein extracts prepared every 24 h. The nuclear abundance of Nrf2 continuously decreased during adipogenesis in ST2 cells. Its DNA binding activity reached a nadir during the first two days of differentiation, after which it increased slightly without approaching its initial level. The pattern of Nrf2 DNA binding corresponded to its transcriptional activity as assessed in ST2 cells stably transfected with a reporter construct bearing a Nrf2 bind site upstream of the luciferase gene. In conclusion, the activation of Nrf2 decreased significantly during adipogenesis. The observed changes might lead to increased oxidative stress levels that could facilitate the differentiation process. These findings could shed new light on the pathogenesis of obesity, in which the adipose tissue and oxidative stress play prominent roles. PMID:21805027

  9. Inorganic Arsenic Induces NRF2-Regulated Antioxidant Defenses in Both Cerebral Cortex and Hippocampus in Vivo.

    PubMed

    Zhang, Yang; Duan, Xiaoxu; Li, Jinlong; Zhao, Shuo; Li, Wei; Zhao, Lu; Li, Wei; Nie, Huifang; Sun, Guifang; Li, Bing

    2016-08-01

    Inorganic arsenic is reported to induce the reactive oxygen species-mediated oxidative stress, which is supposed to be one of the main mechanisms of arsenic-related neurological diseases. Nuclear factor erythroid 2-related factor 2 (NRF2), a master regulator of antioxidant defense systems, up-regulates the expression of target genes to fight against oxidative damages caused by harmful substances, including metals. In the present study, mice were used as a model to investigate the oxidative stress levels and the expressions of NRF2-regulated antioxidant substances in both cerebral cortex and hippocampus with 5, 10 and 20 mg/kg NaAsO2 exposure intra-gastrically. Our results showed that acute NaAsO2 treatment resulted in decreased total anti-oxidative capacity (T-AOC) and increased maleic dialdehyde production in the nervous system. We also detected rapidly elevation of NRF2 protein levels by enhancement of Nrf2 transcription, especially at 20 mg/kg NaAsO2 exposure group. In the meantime, mRNA and protein levels of Nrf2 encoding antioxidant enzymes heme oxygenase-1 (HO-1), NAD(P)H: quinine oxidoreductase 1 (NQO1) and glutathione S-transferase (GST) were consistently elevated time- and dose-dependently both in the cerebral cortex and hippocampus. Taken together, the presence study demonstrated the activation of NRF2 pathway, an early antioxidant defensive response, in both cerebral cortex and hippocampus upon inorganic arsenic (iAs) exposure in vivo. A better knowledge on the roles of NRF2 pathway in maintaining cellular redox homeostasis would be helpful for the strategies on improvement of neurotoxicity related to this metalloid. PMID:27165637

  10. Luteolin inhibits the Nrf2 signaling pathway and tumor growth in vivo

    SciTech Connect

    Chian, Song; Thapa, Ruby; Chi, Zhexu; Wang, Xiu Jun; Tang, Xiuwen

    2014-05-16

    Highlights: • Luteolin inhibits the Nrf2 pathway in mouse liver and in xenografted tumors. • Luteolin markedly inhibits the growth of xenograft tumors. • Luteolin enhances the anti-cancer effect of cisplatin in mice in vivo. • Luteolin could serve as an adjuvant in the chemotherapy of NSCLC. - Abstract: Nuclear factor erythroid 2-related factor 2 (Nrf2) is over-expressed in many types of tumor, promotes tumor growth, and confers resistance to anticancer therapy. Hence, Nrf2 is regarded as a novel therapeutic target in cancer. Previously, we reported that luteolin is a strong inhibitor of Nrf2 in vitro. Here, we showed that luteolin reduced the constitutive expression of NAD(P)H quinone oxidoreductase 1 in mouse liver in a time- and dose-dependent manner. Further, luteolin inhibited the expression of antioxidant enzymes and glutathione transferases, decreasing the reduced glutathione in the liver of wild-type mice under both constitutive and butylated hydroxyanisole-induced conditions. In contrast, such distinct responses were not detected in Nrf2{sup −/−} mice. In addition, oral administration of luteolin, either alone or combined with intraperitoneal injection of the cytotoxic drug cisplatin, greatly inhibited the growth of xenograft tumors from non-small-cell lung cancer (NSCLC) cell line A549 cells grown subcutaneously in athymic nude mice. Cell proliferation, the expression of Nrf2, and antioxidant enzymes were all reduced in tumor xenograft tissues. Furthermore, luteolin enhanced the anti-cancer effect of cisplatin. Together, our findings demonstrated that luteolin inhibits the Nrf2 pathway in vivo and can serve as an adjuvant in the chemotherapy of NSCLC.

  11. Accelerated ovarian failure induced by 4-vinyl cyclohexene diepoxide in Nrf2 null mice.

    PubMed

    Hu, Xiaoming; Roberts, Jenny R; Apopa, Patrick L; Kan, Yuet Wai; Ma, Qiang

    2006-02-01

    Genetic and biochemical analyses have uncovered an essential role for nuclear factor erythroid 2-related factor 2 (Nrf2) in regulating phase II xenobiotic metabolism and antioxidant response. Here we show that Nrf2 protects against the ovarian toxicity of 4-vinylcyclohexene diepoxide (VCD) in mice. Nrf2-/- female mice exposed to VCD exhibit an age-dependent decline in reproduction leading to secondary infertility accompanied by hypergonadotropic hypogonadism after 30 weeks of age. VCD is shown to selectively destroy small ovarian follicles, resulting in early depletion of functional follicles. Treatment with VCD induces apoptotic death in cultured cells and in ovarian follicles, suggesting apoptosis as a mechanism of follicle loss. Loss of Nrf2 function blocks the basal and inducible expression of microsomal epoxide hydrolase, a key enzyme in the detoxification of VCD, and increases the oxidative stress in cells that is further exacerbated by VCD. Foxo3a, a repressor in the early stages of follicle activation, displays reduced expression in Nrf2-/- ovaries, causing accelerated growth of follicles in the absence of exposure to exogenous chemicals. Furthermore, Foxo3a is degraded through the 26S proteasome pathway in untreated cells and is induced by VCD via both Nrf2-dependent transcription and protein stabilization. This study demonstrates that Nrf2 serves as an essential sensor and regulator of chemical homeostasis in ovarian cells, protecting the cells from toxic chemicals by controlling metabolic detoxification, reactive oxygen species defense, and Foxo3a expression. In addition, these findings raise the possibility that exposure to environmental or occupational ovotoxicants plays a role in the premature ovarian failure commonly associated with infertility and premature aging in women. PMID:16428448

  12. Oxidative Stress and the Nrf2 Anti-Oxidant Transcription Factor in Age-Related Macular Degeneration.

    PubMed

    Lambros, Mandy L; Plafker, Scott M

    2016-01-01

    Age-related macular degeneration (AMD) is the leading cause of acquired and irreversible blindness among elderly Americans. Most AMD patients have the dry form of the disease (dAMD) for which reliable therapies are lacking. A major obstacle to the development of effective treatments is a deficit in our understanding of what triggers dAMD onset. This is particularly the case with respect to the events that cause retinal pigment epithelial (RPE) cells to transition from a state of health and homeostasis to one of dysfunction and atrophy. These cells provide critical support to the photoreceptors and their atrophy often precipitates photoreceptor death in dAMD. Chronic oxidative stress is a primary driver of age-dependent, RPE atrophy. Sources of this stress have been identified (e.g., cigarette smoke, photooxidized bisretinoids), but we still do not understand how these stressors damage RPE constituents or what age-dependent changes undermine the cytoprotective systems in the RPE. This review focuses on Nrf2, the master antioxidant transcription factor, and its role in the RPE during aging and dAMD onset. PMID:26427395

  13. Arsenic induces NAD(P)H-quinone oxidoreductase I by disrupting the Nrf2 x Keap1 x Cul3 complex and recruiting Nrf2 x Maf to the antioxidant response element enhancer.

    PubMed

    He, Xiaoqing; Chen, Michael G; Lin, Gary X; Ma, Qiang

    2006-08-18

    The ubiquitous toxic metalloid arsenic elicits pleiotropic adverse and adaptive responses in mammalian species. The biological targets of arsenic are largely unknown at present. We analyzed the signaling pathway for induction of detoxification gene NAD(P)H-quinone oxidoreductase (Nqo1) by arsenic. Genetic and biochemical evidence revealed that induction required cap 'n' collar basic leucine zipper transcription factor Nrf2 and the antioxidant response element (ARE) of Nqo1. Arsenic stabilized Nrf2 protein, extending the t(1/2) of Nrf2 from 21 to 200 min by inhibiting the Keap1 x Cul3-dependent ubiquitination and proteasomal turnover of Nrf2. Arsenic markedly inhibited the ubiquitination of Nrf2 but did not disrupt the Nrf2 x Keap1 x Cul3 association in the cytoplasm. In the nucleus, arsenic, but not phenolic antioxidant tert-butylhydroquinone, dissociated Nrf2 from Keap1 and Cul3 followed by dimerization of Nrf2 with a Maf protein (Maf G/Maf K). Chromatin immunoprecipitation demonstrated that Nrf2 and Maf associated with the endogenous Nqo1 ARE enhancer constitutively. Arsenic substantially increased the ARE occupancy by Nrf2 and Maf. In addition, Keap1 was shown to be ubiquitinated in the cytoplasm and deubiquitinated in the nucleus in the presence of arsenic without changing the protein level, implicating nuclear-cytoplasmic recycling of Keap1. Our data reveal that arsenic activates the Nrf2/Keap1 signaling pathway through a distinct mechanism from that by antioxidants and suggest an "on-switch" model of Nqo1 transcription in which the binding of Nrf2 x Maf to ARE controls both the basal and inducible expression of Nqo1. PMID:16785233

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

    PubMed Central

    Kim, Sanghyun; Lee, Hee-Geum; Park, Sin-Aye; Kundu, Joydeb Kumar; Keum, Young-Sam; Cha, Young-Nam; Na, Hye-Kyung; Surh, Young-Joon

    2014-01-01

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

  15. Hydrogen sulfide (H2S) attenuates uranium-induced acute nephrotoxicity through oxidative stress and inflammatory response via Nrf2-NF-κB pathways.

    PubMed

    Zheng, Jifang; Zhao, Tingting; Yuan, Yan; Hu, Nan; Tang, Xiaoqing

    2015-12-01

    As an endogenous gaseous mediator, H2S exerts anti-oxidative, anti-inflammatory and cytoprotective effects in kidneys. This study was designed to investigate the protective effect of H2S against uranium-induced nephrotoxicity in adult SD male rats after in vivo effect of uranium on endogenous H2S formation was explored in kidneys. The levels of endogenous H2S and H2S-producing enzymes (CBS and CSE) were measured in renal homogenates from rats intoxicated by an intraperitoneally (i.p.) injection of uranyl acetate at a single dose of 2.5, 5 or 10 mg/kg. In rats injected i.p. with uranyl acetate (5 mg/kg) or NaHS (an H2S donor, 28 or 56 μmol/kg) alone or in combination, we determined biochemical parameters and histopathological alteration to assess kidney function, examined oxidative stress markers, and investigated Nrf2 and NF-κB pathways in kidney homogenates. The results suggest that uranium intoxication in rats decreased endogenous H2S generation as well as CBS and CSE protein expression. NaHS administration in uranium-intoxicated rats ameliorated the renal biochemical indices and histopathological effects, lowered MDA accumulation, and restored GSH level and anti-oxidative enzymes activities like SOD, CAT, GPx and GST. NaHS treatment in uranium-intoxicated rats activated uranium-inhibited protein expression and nuclear translocation of transcription factor Nrf2, which increased protein expression of downstream target-Nrf2 genes HO-1, NQO-1, GCLC, and TXNRD-1. NaHS administration in uranium-intoxicated rats inhibited uranium-induced nuclear translocation and phosphorylation of transcription factor κB/p65, which decreased protein expression of target-p65 inflammatory genes TNF-α, iNOS, and COX-2. Taken together, these data implicate that H2S can afford protection to rat kidneys against uranium-induced adverse effects through induction of antioxidant defense by activating Nrf2 pathway and reduction of inflammatory response by suppressing NF-κB pathway. PMID

  16. HER2 confers drug resistance of human breast cancer cells through activation of NRF2 by direct interaction.

    PubMed

    Kang, Hyo Jin; Yi, Yong Weon; Hong, Young Bin; Kim, Hee Jeong; Jang, Young-Joo; Seong, Yeon-Sun; Bae, Insoo

    2014-01-01

    Overexpression and/or activation of HER2 confers resistance of cancer cells to chemotherapeutic drugs. NRF2 also gives drug resistance of cancer cells through induction of detoxification and/or drug efflux proteins. Although several upstream effectors of NRF2 overlapped with the downstream molecules of HER2 pathway, no direct link between HER2 and NRF2 has ever been established. Here, we identified that co-expression of a constitutively active HER2 (HER2CA) and NRF2 increased the levels of NRF2 target proteins, HO-1 and MRP5. We also identified HER2CA activated the DNA-binding of NRF2 and the antioxidant response element (ARE)-mediated transcription in an NRF2-dependent manner. In addition, NRF2 and HER2CA cooperatively up-regulated the mRNA expression of various drug-resistant and detoxifying enzymes including GSTA2, GSTP1, CYP3A4, HO-1, MRP1, and MRP5. We also demonstrated that NRF2 binds to HER2 not only in transiently transfected HEK293T cells but also in HER2-amplified breast cancer cells. Functionally, overexpression of HER2CA gave resistance of MCF7 breast cancer cells to either paraquat or doxorubicin. Overexpression of dominant negative NRF2 (DN-NRF2) reduced the HER2CA-induced resistance of MCF7 cells to these agents. Taken together, these results suggest that active HER2 binds and regulates the NRF2-dependent transcriptional activation and induces drug resistance of cancer cells. PMID:25467193

  17. The NRF2 knockout rat: a new animal model to study endothelial dysfunction, oxidant stress, and microvascular rarefaction.

    PubMed

    Priestley, Jessica R C; Kautenburg, Katie E; Casati, Marc C; Endres, Bradley T; Geurts, Aron M; Lombard, Julian H

    2016-02-15

    Nuclear factor (erythroid-derived 2)-like-2 (NRF2) is a master antioxidant and cell protective transcription factor that upregulates antioxidant defenses. In this study we developed a strain of Nrf2 null mutant rats to evaluate the role of reduced NRF2-regulated antioxidant defenses in contributing to endothelial dysfunction and impaired angiogenic responses during salt-induced ANG II suppression. Nrf2(-/-) mutant rats were developed using transcription activator-like effector nuclease technology in the Sprague-Dawley genetic background, and exhibited a 41-bp deletion that included the start codon for Nrf2 and an absence of immunohistochemically detectable NRF2 protein. Expression of mRNA for the NRF2-regulated indicator enzymes heme oxygenase-1, catalase, superoxide dismutase 1, superoxide dismutase 2, and glutathione reductase was significantly lower in livers of Nrf2(-/-) mutant rats fed high salt (HS; 4% NaCl) for 2 wk compared with wild-type controls. Endothelium-dependent dilation to acetylcholine was similar in isolated middle cerebral arteries (MCA) of Nrf2(-/-) mutant rats and wild-type littermates fed low-salt (0.4% NaCl) diet, and was eliminated by short-term (3 days) HS diet in both strains. Low-dose ANG II infusion (100 ng/kg sc) reversed salt-induced endothelial dysfunction in MCA and prevented microvessel rarefaction in wild-type rats fed HS diet, but not in Nrf2(-/-) mutant rats. The results of this study indicate that suppression of NRF2 antioxidant defenses plays an essential role in the development of salt-induced oxidant stress, endothelial dysfunction, and microvessel rarefaction in normotensive rats and emphasize the potential therapeutic benefits of directly upregulating NRF2-mediated antioxidant defenses to ameliorate vascular oxidant stress in humans. PMID:26637559

  18. miRNA Influences in NRF2 Pathway Interactions within Cancer Models

    PubMed Central

    Ayers, Duncan; Baron, Byron; Hunter, Therese

    2015-01-01

    The NRF2 transcription factor (nuclear factor-erythroid 2 p45-related factor 2) has been identified as a key molecular player in orchestrating adaptive cellular interactions following a wide spectrum of cellular stress conditions that could be either extracellular or intracellular. Dysregulation of the NRF2 system is implicated in various disease states, including inflammatory conditions. The NRF2 transcription factor is also known to permit cross talk with several other essential cellular signaling pathways. Recent literature has also elucidated the potential influences of miRNA activity over modulations of the NRF2 signalling network. Consequently, further delving into the knowledge regarding the extent of miRNA-induced epigenetic gene regulatory control on key elements of the NRF2 signalling pathway and its cross talk, particularly within the context of cancer models, can prove to be of high clinical importance. This is so since such miRNAs, once identified and validated, can be potentially exploited as novel drug targets for emerging translational medicine-based therapies. PMID:26345522

  19. Identification of Chromomoric Acid C-I as an Nrf2 Activator in Chromolaena odorata

    PubMed Central

    2014-01-01

    Activation of nuclear factor-erythroid 2-related factor 2 (Nrf2) contributes to several beneficial bioactivities of natural products, including induction of an increased cellular stress resistance and prevention or resolution of inflammation. In this study, the potential of a crude leaf extract of Chromolaena odorata, traditionally used against inflammation and skin lesions, was examined for Nrf2 activation. Guided by an Nrf2-dependent luciferase reporter gene assay, the phytoprostane chromomoric acid C-I (1) was identified as a potent Nrf2 activator from C. odorata with a CD (concentration doubling the response of vehicle-treated cells) of 5.2 μM. When tested at 1–10 μM, 1 was able to induce the endogenous Nrf2 target gene heme oxygenase 1 (HO-1) in fibroblasts. Between 2 and 5 μM, compound 1 induced HO-1 in vascular smooth muscle cells (VSMC) and inhibited their proliferation in a HO-1-dependent manner, without eliciting signs of cytotoxicity. PMID:24476568

  20. Identification of chromomoric acid C-I as an Nrf2 activator in Chromolaena odorata.

    PubMed

    Heiss, Elke H; Tran, Thi Van Anh; Zimmermann, Kristin; Schwaiger, Stefan; Vouk, Corina; Mayerhofer, Barbara; Malainer, Clemens; Atanasov, Atanas G; Stuppner, Hermann; Dirsch, Verena M

    2014-03-28

    Activation of nuclear factor-erythroid 2-related factor 2 (Nrf2) contributes to several beneficial bioactivities of natural products, including induction of an increased cellular stress resistance and prevention or resolution of inflammation. In this study, the potential of a crude leaf extract of Chromolaena odorata, traditionally used against inflammation and skin lesions, was examined for Nrf2 activation. Guided by an Nrf2-dependent luciferase reporter gene assay, the phytoprostane chromomoric acid C-I (1) was identified as a potent Nrf2 activator from C. odorata with a CD (concentration doubling the response of vehicle-treated cells) of 5.2 μM. When tested at 1-10 μM, 1 was able to induce the endogenous Nrf2 target gene heme oxygenase 1 (HO-1) in fibroblasts. Between 2 and 5 μM, compound 1 induced HO-1 in vascular smooth muscle cells (VSMC) and inhibited their proliferation in a HO-1-dependent manner, without eliciting signs of cytotoxicity. PMID:24476568

  1. Nrf2 silencing to inhibit proteolytic defense induced by hyperthermia in HT22 cells

    PubMed Central

    Bozaykut, Perinur; Ozer, Nesrin Kartal; Karademir, Betul

    2016-01-01

    Nrf2 pathway has been known to be protective against cancer progression however recent studies have revealed that the antioxidant activity of Nrf2 contributes to chemotherapy resistance. For many years, hyperthermia has been used as an additional therapy to increase the efficiency of chemotherapy and radiotherapy. Besides the positive effects of hyperthermia during treatment procedure, thermotolerance has been found to develop against heat treatment. Although the involved molecular mechanisms have not been fully clarified, heat shock proteins (HSP) and proteasome activity are known to be involved in the acquisition of thermotolerance. The aim of this study was to investigate the potential beneficial effects of combining hyperthermia with Nrf2 silencing to inhibit molecular mechanisms leading to induction of defense mechanisms in transcription level. Following heat treatment of HT22 cells, HSP70 and the proteasome levels and as well as proteasome activity were found to be elevated in the nucleus. Our results demonstrated that Nrf2 silencing reduced defense mechanisms against heat treatment both in antioxidant and proteolytic manner and Nrf2 may be a potential target for therapeutic approach in order to improve the beneficial effects of hyperthermia in cancer therapy. PMID:26966891

  2. Role of the Keap1/Nrf2 pathway in neurodegenerative diseases.

    PubMed

    Yamazaki, Hiromi; Tanji, Kunikazu; Wakabayashi, Koichi; Matsuura, Shin; Itoh, Ken

    2015-05-01

    As the elderly population increases, a growing number of individuals suffer from age-associated neurodegenerative diseases, such as Alzheimer's disease (AD) and Parkinson's disease (PD). Oxidative stress is considered to play a crucial role in the pathogenesis of age-related diseases. The transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2) is activated by oxidative stress and regulates the expression of a variety of antioxidant enzymes and proteins that exert cytoprotective effects against oxidative stress. Numerous studies have addressed the role of Nrf2 in age-related diseases, including neurodegenerative diseases, using animal or in vitro cell culture models. Here, we introduce the role of oxidative stress in the pathogenesis of neurodegenerative diseases and critically examine the recent findings concerning the role for Nrf2 in the amelioration of AD and PD. Nrf2 not only regulates antioxidant proteins but also regulates the genes associated with autophagy and nerve growth factor signaling. Current research unequivocally demonstrates that the activation of the Nrf2 pathway is a promising novel strategy for the prevention and modification of neurodegenerative diseases. PMID:25707882

  3. Phytoestrogens modulate hepcidin expression by Nrf2: Implications for dietary control of iron absorption

    PubMed Central

    Bayele, Henry K.; Balesaria, Sara; Srai, Surjit K.S.

    2015-01-01

    Hepcidin is a liver-derived antimicrobial peptide that regulates iron absorption and is also an integral part of the acute phase response. In a previous report, we found evidence that this peptide could also be induced by toxic heavy metals and xenobiotics, thus broadening its teleological role as a defensin. However it remained unclear how its sensing of disparate biotic and abiotic stressors might be integrated at the transcriptional level. We hypothesized that its function in cytoprotection may be regulated by NFE2-related factor 2 (Nrf2), the master transcriptional controller of cellular stress defenses. In this report, we show that hepcidin regulation is inextricably linked to the acute stress response through Nrf2 signaling. Nrf2 regulates hepcidin expression from a prototypical antioxidant response element in its promoter, and by synergizing with other basic leucine-zipper transcription factors. We also show that polyphenolic small molecules or phytoestrogens commonly found in fruits and vegetables including the red wine constituent resveratrol can induce hepcidin expression in vitro and post-prandially, with concomitant reductions in circulating iron levels and transferrin saturation by one such polyphenol quercetin. Furthermore, these molecules derepress hepcidin promoter activity when its transcription by Nrf2 is repressed by Keap1. Taken together, the data show that hepcidin is a prototypical antioxidant response or cytoprotective gene within the Nrf2 transcriptional circuitry. The ability of phytoestrogens to modulate hepcidin expression in vivo suggests a novel mechanism by which diet may impact iron homeostasis. PMID:26546695

  4. NRF2-mediated Notch pathway activation enhances hematopoietic reconstitution following myelosuppressive radiation

    PubMed Central

    Kim, Jung-Hyun; Thimmulappa, Rajesh K.; Kumar, Vineet; Cui, Wanchang; Kumar, Sarvesh; Kombairaju, Ponvijay; Zhang, Hao; Margolick, Joseph; Matsui, William; Macvittie, Thomas; Malhotra, Sanjay V.; Biswal, Shyam

    2014-01-01

    A nuclear disaster may result in exposure to potentially lethal doses of ionizing radiation (IR). Hematopoietic acute radiation syndrome (H-ARS) is characterized by severe myelosuppression, which increases the risk of infection, bleeding, and mortality. Here, we determined that activation of nuclear factor erythroid-2–related factor 2 (NRF2) signaling enhances hematopoietic stem progenitor cell (HSPC) function and mitigates IR-induced myelosuppression and mortality. Augmenting NRF2 signaling in mice, either by genetic deletion of the NRF2 inhibitor Keap1 or by pharmacological NRF2 activation with 2-trifluoromethyl-2′-methoxychalone (TMC), enhanced hematopoietic reconstitution following bone marrow transplantation (BMT). Strikingly, even 24 hours after lethal IR exposure, oral administration of TMC mitigated myelosuppression and mortality in mice. Furthermore, TMC administration to irradiated transgenic Notch reporter mice revealed activation of Notch signaling in HSPCs and enhanced HSPC expansion by increasing Jagged1 expression in BM stromal cells. Administration of a Notch inhibitor ablated the effects of TMC on hematopoietic reconstitution. Taken together, we identified a mechanism by which NRF2-mediated Notch signaling improves HSPC function and myelosuppression following IR exposure. Our data indicate that targeting this pathway may provide a countermeasure against the damaging effects of IR exposure. PMID:24463449

  5. Maneb causes pro-oxidant effects in the hippocampus of Nrf2 knockout mice.

    PubMed

    Kurzatkowski, Daniela M; Trombetta, Louis D

    2013-09-01

    The effects of maneb were investigated in C57BL/6 Nrf2 wildtype and knockout mice. Treated KO mice showed significant weight loss as compared to WT counterparts. ICPAAS analysis demonstrated a significant increase in manganese concentration in the tissues of treated KO mice as compared to WT. Biochemical analysis revealed significant decreases of antioxidants including glutathione, glutathione reductase and heme oxygenase-1. Levels of TBARS were significantly increased in hippocampal tissue in Nrf2 KO mice at the 30 and 60mg doses. qPCR demonstrated that the only gene mediated by the Nrf2 transcription pathway that was significantly modulated by at least 1.5 fold was glutathione peroxidase 4. GPX4 was significantly upregulated in Nrf2 WT mice treated with 30mg/kg maneb and significantly downregulated in Nrf2 KO mice treated with the same dose. Microscopy revealed neuronal pyknosis and eosinophilia of the cytoplasm in the hippocampi of both WT and KO animals treated with 60mg/kg maneb. PMID:23764462

  6. Nrf2 silencing to inhibit proteolytic defense induced by hyperthermia in HT22 cells.

    PubMed

    Bozaykut, Perinur; Ozer, Nesrin Kartal; Karademir, Betul

    2016-08-01

    Nrf2 pathway has been known to be protective against cancer progression however recent studies have revealed that the antioxidant activity of Nrf2 contributes to chemotherapy resistance. For many years, hyperthermia has been used as an additional therapy to increase the efficiency of chemotherapy and radiotherapy. Besides the positive effects of hyperthermia during treatment procedure, thermotolerance has been found to develop against heat treatment. Although the involved molecular mechanisms have not been fully clarified, heat shock proteins (HSP) and proteasome activity are known to be involved in the acquisition of thermotolerance. The aim of this study was to investigate the potential beneficial effects of combining hyperthermia with Nrf2 silencing to inhibit molecular mechanisms leading to induction of defense mechanisms in transcription level. Following heat treatment of HT22 cells, HSP70 and the proteasome levels and as well as proteasome activity were found to be elevated in the nucleus. Our results demonstrated that Nrf2 silencing reduced defense mechanisms against heat treatment both in antioxidant and proteolytic manner and Nrf2 may be a potential target for therapeutic approach in order to improve the beneficial effects of hyperthermia in cancer therapy. PMID:26966891

  7. Multiple regulations of Keap1/Nrf2 system by dietary phytochemicals.

    PubMed

    Qin, Si; Hou, De-Xing

    2016-08-01

    Keap1/Nrf2 system plays a critical role on cellular protection by regulating many antioxidant and detoxification enzyme genes through the antioxidant response element (ARE). Thus, it must work constantly to prevent the accumulation of reactive oxygen species (ROS) because excess ROS are associated with many diseases such as cancer, cardiovascular complications, inflammation, and neurodegeneration. Dietary phytochemicals widely distributing in fruits and vegetables have been considered to possess cancer chemopreventive potential through the induction of Keap1/Nrf2 system-mediated antioxidant and detoxification enzymes in a variety of manners. The data are extensive and are not well classified on the molecular mechanisms. In this review, we first briefly introduce the current knowledge on Keap1/Nrf2 system regulation including Keap1-dependent and Keap1-independent cascades, and epigenetic pathway. Then, we summarize the molecular targets of Keap1/Nrf2 system by dietary phytochemicals, and finally review the crosstalk between Keap1/Nrf2 system and other cellular signaling pathways to regulate diverse chronic diseases by dietary phytochemicals. These comprehensive data will help us to understand the potential effects of dietary phytochemicals on the prevention of chronic diseases and maintenance of human health. PMID:27523917

  8. Nrf2 as molecular target for polyphenols: A novel therapeutic strategy in diabetic retinopathy.

    PubMed

    Nabavi, Seyed Fazel; Barber, Alistair J; Spagnuolo, Carmela; Russo, Gian Luigi; Daglia, Maria; Nabavi, Seyed Mohammad; Sobarzo-Sánchez, Eduardo

    2016-10-01

    Diabetic retinopathy is a microvascular complication of diabetes that is considered one of the leading causes of blindness among adults. More than 4.4 million people suffer from this disorder throughout the world. Growing evidence suggests that oxidative stress plays a crucial role in the pathophysiology of diabetic retinopathy. Nuclear factor erythroid 2-related factor 2 (Nrf2), a redox sensitive transcription factor, plays an essential protective role in regulating the physiological response to oxidative and electrophilic stress via regulation of multiple genes encoding antioxidant proteins and phase II detoxifying enzymes. Many studies suggest that dozens of natural compounds, including polyphenols, can supress oxidative stress and inflammation through targeting Nrf2 and consequently activating the antioxidant response element-related cytoprotective genes. Therefore, Nrf2 may provide a new therapeutic target for treatment of diabetic retinopathy. In the present article, we will focus on the role of Nrf2 in diabetic retinopathy and the ability of polyphenols to target Nrf2 as a therapeutic strategy. PMID:26926494

  9. Sulforaphane and Other Nutrigenomic Nrf2 Activators: Can the Clinician's Expectation Be Matched by the Reality?

    PubMed Central

    Houghton, Christine A.; Fassett, Robert G.; Coombes, Jeff S.

    2016-01-01

    The recognition that food-derived nonnutrient molecules can modulate gene expression to influence intracellular molecular mechanisms has seen the emergence of the fields of nutrigenomics and nutrigenetics. The aim of this review is to describe the properties of nutrigenomic activators of transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2), comparing the potential for sulforaphane and other phytochemicals to demonstrate clinical efficacy as complementary medicines. Broccoli-derived sulforaphane emerges as a phytochemical with this capability, with oral doses capable of favourably modifying genes associated with chemoprevention. Compared with widely used phytochemical-based supplements like curcumin, silymarin, and resveratrol, sulforaphane more potently activates Nrf2 to induce the expression of a battery of cytoprotective genes. By virtue of its lipophilic nature and low molecular weight, sulforaphane displays significantly higher bioavailability than the polyphenol-based dietary supplements that also activate Nrf2. Nrf2 activation induces cytoprotective genes such as those playing key roles in cellular defense mechanisms including redox status and detoxification. Both its high bioavailability and significant Nrf2 inducer capacity contribute to the therapeutic potential of sulforaphane-yielding supplements. PMID:26881038

  10. Ascorbic acid reduces HMGB1 secretion in lipopolysaccharide-activated RAW 264.7 cells and improves survival rate in septic mice by activation of Nrf2/HO-1 signals.

    PubMed

    Kim, So Ra; Ha, Yu Mi; Kim, Young Min; Park, Eun Jung; Kim, Jung Whan; Park, Sang Won; Kim, Hye Jung; Chung, Hun Taeg; Chang, Ki Churl

    2015-06-15

    High mobility group box 1 (HMGB1) is now recognized as a late mediator of sepsis. We tested hypothesis that ascorbic acid (AscA) induces heme oxygenase (HO)-1 which inhibits HMGB1 release in lipopolysaccharide (LPS)-stimulated cells and increases survival of septic mice. AscA increased HO-1 protein expression in a concentration- and time-dependent manner via Nrf2 activation in RAW 264.7 cells. HO-1 induction by AscA was significantly reduced by Nrf2 siRNA-transfected cells. Mutation of cysteine to serine of keap-1 proteins (C151S, C273S, and C288S) lost the ability of HO-1 induction by AscA, due to failure of translocation of Nrf-2 to nucleus. The PI3 kinase inhibitor, LY294002, inhibited HO-1 induction by AscA. Oxyhemoglobin (HbO2), LY294002, and ZnPPIX (HO-1 enzyme inhibitor) reversed effect of AscA on HMGB1 release. Most importantly, administration of AscA (200mg/kg, i.p.) significantly increased survival in LPS-induced endotoxemic mice. In cecal ligation and puncture (CLP)-induced septic mice, AscA reduced hepatic injury and serum HMGB1 and plasminogen activator inhibitor (PAI)-1 in a ZnPPIX-sensitive manner. In addition, AscA failed to increase survival in Nrf2 knockout mice by LPS. Thus, we concluded that high dose of AscA may be useful in the treatment of sepsis, at least, by activation of Nrf2/HO-1 signals. PMID:25896849

  11. Mechanism of progestin resistance in endometrial precancer/cancer through Nrf2-AKR1C1 pathway.

    PubMed

    Wang, Yiying; Wang, Yue; Zhang, Zhenbo; Park, Ji-Young; Guo, Donghui; Liao, Hong; Yi, Xiaofang; Zheng, Yu; Zhang, Donna; Chambers, Setsuko K; Zheng, Wenxin

    2016-03-01

    Progestin resistance is a main obstacle for endometrial precancer/cancer conservative therapy. Therefore, biomarkers to predict progestin resistance and studies to gain a more detailed understanding of the mechanism are needed. The antioxidant Nrf2-AKR1C1 signal pathway exerts chemopreventive activity. However whether it plays a role in progestin resistance has not been explored. In this study, elevated levels of AKR1C1 and Nrf2 were found in progestin-resistant endometrial epithelia, but not in responsive endometrial glands. Exogenous overexpression of Nrf2/AKR1C1 resulted in progestin resistance. Inversely, silencing of Nrf2 or AKR1C1 rendered endometrial cancer cells more susceptible to progestin treatment. Moreover, medroxyprogesterone acetate withdrawal resulted in suppression of Nrf2/AKR1C1 expression accompanied by a reduction of cellular proliferative activity. In addition, brusatol and metformin overcame progestin resistance by down-regulating Nrf2/AKR1C1 expression. Our findings suggest that overexpression of Nrf2 and AKR1C1 in endometrial precancer/cancer may be part of the molecular mechanisms underlying progestin resistance. If validated in a larger cohort, overexpression of Nrf2 and AKR1C1 may prove to be useful biomarkers to predict progestin resistance. Targeting the Nrf2/AKR1C1 pathway may represent a new therapeutic strategy for treatment of endometrial hyperplasia/cancer. PMID:26824415

  12. Suppression in PHLPP2 induction by morin promotes Nrf2-regulated cellular defenses against oxidative injury to primary rat hepatocytes

    PubMed Central

    Rizvi, Fatima; Mathur, Alpana; Krishna, Shagun; Siddiqi, Mohammad Imran; Kakkar, Poonam

    2015-01-01

    Recent advances indicate a possible role of phytochemicals as modulatory factors in signaling pathways. We have previously demonstrated PHLPP2-mediated suppression of Nrf2 responses during oxidant attack. The present study was designed to explore Nrf2-potentiating mechanism of morin, a flavonol, via its possible role in intervening PHLPP2-regulated Akt/GSK3β/Fyn kinase axis. Efficacy of morin was evaluated against oxidative stress-mediated damage to primary hepatocytes by tert-butyl hydroperoxide (tBHP) and acetaminophen. The anti-cytotoxic effects of morin were found to be a consequence of fortification of Nrf2-regulated antioxidant defenses since morin failed to sustain activities of redox enzyme in Nrf2 silenced hepatocytes. Morin promoted Nrf2 stability and its nuclear retention by possibly modulating PHLPP2 activity which subdues cellular Nrf2 responses by activating Fyn kinase. Pull-down assay using morin-conjugated beads indicated the binding affinity of morin towards PHLPP2. Molecular docking also revealed the propensity of morin to occupy the active site of PHLPP2 enzyme. Thus, dietary phytochemical morin was observed to counteract oxidant-induced hepatocellular damage by promoting Nrf2-regulated transcriptional induction. The findings support the novel role of morin in potentiating Nrf2 responses by limiting PHLPP2 and hence Fyn kinase activation. Therefore, morin may be exploited in developing novel therapeutic strategy aimed at enhancing Nrf2 responses. PMID:26513344

  13. The Relevance of Nrf2 Pathway and Autophagy in Pancreatic Cancer Cells upon Stimulation of Reactive Oxygen Species

    PubMed Central

    2016-01-01

    Nrf2 (NF-E2-related factor 2) pathway and autophagy both can respond to oxidative stress to promote cancer cells to survive in the tumor microenvironment. We, therefore, explored the relevance between Nrf2 pathway and autophagy in pancreatic cancer cells upon stimulation of reactive oxygen species (ROS). Pancreatic cancer cells were cultured under controlled ROS stressing condition or basal condition. Different inhibitors were used to prevent autophagy at particular stages. Nrf2 siRNA was used to inhibit Nrf2 pathway activation. Ad-mRFP-GFP-LC3 infection was used to monitor autophagic flux. The result shows that a small amount of exogenous hydrogen peroxide (H2O2) can significantly improve the level of intracellular ROS. Moreover, our findings indicate that ROS promotes the activation of both Nrf2 pathway and autophagy in pancreatic cancer cells. Moreover, our data demonstrate that suppression of autophagic activity at particular stages results in an increased promotion of Nrf2 pathway activation upon ROS stimulation. Furthermore, we found that silencing of Nrf2 promotes autophagy upon ROS stimulation. In addition, Nrf2 interference effectively promotes autophagic flux upon ROS stimulation. In summary, our findings suggest that Nrf2 pathway and autophagy have a negative interaction with each other upon ROS stimulation. PMID:26682003

  14. Mechanism of progestin resistance in endometrial precancer/cancer through Nrf2-AKR1C1 pathway

    PubMed Central

    Wang, Yiying; Wang, Yue; Zhang, Zhenbo; Park, Ji-Young; Guo, Donghui; Liao, Hong; Yi, Xiaofang; Zheng, Yu; Zhang, Donna; Chambers, Setsuko K.; Zheng, Wenxin

    2016-01-01

    Progestin resistance is a main obstacle for endometrial precancer/cancer conservative therapy. Therefore, biomarkers to predict progestin resistance and studies to gain a more detailed understanding of the mechanism are needed. The antioxidant Nrf2-AKR1C1 signal pathway exerts chemopreventive activity. However whether it plays a role in progestin resistance has not been explored. In this study, elevated levels of AKR1C1 and Nrf2 were found in progestin-resistant endometrial epithelia, but not in responsive endometrial glands. Exogenous overexpression of Nrf2/AKR1C1 resulted in progestin resistance. Inversely, silencing of Nrf2 or AKR1C1 rendered endometrial cancer cells more susceptible to progestin treatment. Moreover, medroxyprogesterone acetate withdrawal resulted in suppression of Nrf2/AKR1C1 expression accompanied by a reduction of cellular proliferative activity. In addition, brusatol and metformin overcame progestin resistance by down-regulating Nrf2/AKR1C1 expression. Our findings suggest that overexpression of Nrf2 and AKR1C1 in endometrial precancer/cancer may be part of the molecular mechanisms underlying progestin resistance. If validated in a larger cohort, overexpression of Nrf2 and AKR1C1 may prove to be useful biomarkers to predict progestin resistance. Targeting the Nrf2/AKR1C1 pathway may represent a new therapeutic strategy for treatment of endometrial hyperplasia/cancer. PMID:26824415

  15. Activation of NRF2 by nitrosative agents and H2O2 involves KEAP1 disulfide formation.

    PubMed

    Fourquet, Simon; Guerois, Raphaël; Biard, Denis; Toledano, Michel B

    2010-03-12

    The NRF2 transcription factor regulates a major environmental and oxidative stress response. NRF2 is itself negatively regulated by KEAP1, the adaptor of a Cul3-ubiquitin ligase complex that marks NRF2 for proteasomal degradation by ubiquitination. Electrophilic compounds activate NRF2 primarily by inhibiting KEAP1-dependent NRF2 degradation, through alkylation of specific cysteines. We have examined the impact on KEAP1 of reactive oxygen and nitrogen species, which are also NRF2 inducers. We found that in untreated cells, a fraction of KEAP1 carried a long range disulfide linking Cys(226) and Cys(613). Exposing cells to hydrogen peroxide, to the nitric oxide donor spermine NONOate, to hypochlorous acid, or to S-nitrosocysteine further increased this disulfide and promoted formation of a disulfide linking two KEAP1 molecules via Cys(151). None of these oxidants, except S-nitrocysteine, caused KEAP1 S-nitrosylation. A cysteine mutant preventing KEAP1 intermolecular disulfide formation also prevented NRF2 stabilization in response to oxidants, whereas those preventing intramolecular disulfide formation were functionally silent. Further, simultaneously inactivating the thioredoxin and glutathione pathways led both to major constitutive KEAP1 oxidation and NRF2 stabilization. We propose that KEAP1 intermolecular disulfide formation via Cys(151) underlies the activation of NRF2 by reactive oxygen and nitrogen species. PMID:20061377

  16. Ginsenoside Rb1 protects against 6-hydroxydopamine-induced oxidative stress by increasing heme oxygenase-1 expression through an estrogen receptor-related PI3K/Akt/Nrf2-dependent pathway in human dopaminergic cells

    SciTech Connect

    Hwang, Yong Pil; Jeong, Hye Gwang

    2010-01-01

    Phytoestrogens are polyphenolic non-steroidal plant compounds with estrogen-like biological activity. Ginseng, the root of Panax ginseng C.A. Meyer (Araliaceae), is a popular traditional herbal medicine. Ginsenoside Rb1 (Rb1), an active component commonly found in ginseng root, is a phytoestrogen that exerts estrogen-like activity. In this study, we demonstrate that the phytoestrogen Rb1 inhibits 6-hydroxydopamine (6-OHDA)-induced oxidative injury via an ER-dependent Gbeta1/PI3K/Akt and heme oxygenase-1 (HO-1) pathway. Pretreatment of SH-SY5Y cells with Rb1 significantly reduced 6-OHDA-induced caspase-3 activation and subsequent cell death. Rb1 also up-regulated HO-1 expression, which conferred cytoprotection against 6-OHDA-induced oxidative injury. Moreover, Rb1 induced both Nrf2 nuclear translocation, which is upstream of HO-1 expression and PI3K activation, a pathway that is involved in induced Nrf2 nuclear translocation, HO-1 expression and cytoprotection. Also, Rb1-mediated increases in PI3K activation and HO-1 induction were reversed by co-treatment with ICI 182,780 and pertussis toxin. Taken together, these results suggest that Rb1 augments the cellular antioxidant defenses through ER-dependent HO-1 induction via the Gbeta1/PI3K/Akt-Nrf2 signaling pathway, thereby protecting cells from oxidative stress. Thus our study indicates that Rb1 has a partial cytoprotective role in dopaminergic cell culture systems.

  17. Recent Updates on Acetaminophen Hepatotoxicity: The Role of Nrf2 in Hepatoprotection

    PubMed Central

    Gum, Sang Il

    2013-01-01

    Acetaminophen (APAP) known as paracetamol is the main ingredient in Tylenol, which has analgesic and anti-pyretic properties. Inappropriate use of APAP causes major morbidity and mortality secondary to hepatic failure. Overdose of APAP depletes the hepatic glutathione (GSH) rapidly, and the metabolic intermediate leads to hepatocellular death. This article reviews the mechanisms of hepatotoxicity and provides an overview of current research studies. Pharmacokinetics including metabolism (activation and detoxification), subsequent transport (efflux)-facilitating excretion, and some other aspects related to toxicity are discussed. Nuclear factor erythroid 2-related factor 2 (Nrf2)-regulated gene battery plays a critical role in the multiple steps associated with the mitigation of APAP toxicity. The role of Nrf2 as a protective target is described, and potential natural products inhibiting APAP toxicity are outlined. This review provides an update on the mechanism of APAP toxicity and highlights the beneficial role of Nrf2 and specific natural products in hepatoprotection. PMID:24386516

  18. Nrf2 in health and disease: current and future clinical implications.

    PubMed

    Al-Sawaf, Othman; Clarner, Tim; Fragoulis, Athanassios; Kan, Yuet Wai; Pufe, Thomas; Streetz, Konrad; Wruck, Christoph Jan

    2015-12-01

    The transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2) is a major regulator of oxidative stress defence in the human body. As Nrf2 regulates the expression of a large battery of cytoprotective genes, it plays a crucial role in the prevention of degenerative disease in multiple organs. Thus it has been the focus of research as a pharmacological target that could be used for prevention and treatment of chronic diseases such as multiple sclerosis, chronic kidney disease or cardiovascular diseases. The present review summarizes promising findings from basic research and shows which Nrf2-targeting therapies are currently being investigated in clinical trials and which agents have already entered clinical practice. PMID:26386022

  19. Essential role of Nrf2 in keratinocyte protection from UVA by quercetin

    SciTech Connect

    Kimura, Shintarou; Warabi, Eiji; Yanagawa, Toru; Ma, Dongmei; Itoh, Ken; Ishii, Yoshiyuki; Kawachi, Yasuhiro; Ishii, Tetsuro

    2009-09-11

    Much of the cell injury caused by ultraviolet A (UVA) irradiation is associated with oxidative stress. Quercetin is a major natural polyphenol that is known to protect cells from UVA-induced damage. Here, we investigated the molecular mechanism of this protection. Quercetin pretreatment strongly suppressed UVA-induced apoptosis in human keratinocyte HaCaT cells, markedly increased protein levels of the transcription factor Nrf2, induced the expression of antioxidative genes, and dramatically reduced the production of reactive oxygen species following UVA irradiation. Importantly, these beneficial effects were greatly attenuated by downregulating Nrf2 expression. Thus, quercetin protects cells from UVA damage mainly by elevating intracellular antioxidative activity via the enhanced accumulation of a transcription factor for antioxidant genes, Nrf2.

  20. Repurposing the NRF2 Activator Dimethyl Fumarate as Therapy Against Synucleinopathy in Parkinson's Disease

    PubMed Central

    García-Yagüe, Angel J.; Scannevin, Robert H.; Casarejos, María J.; Kügler, Sebastian; Rábano, Alberto

    2016-01-01

    Abstract Aims: This preclinical study was aimed at determining whether pharmacological targeting of transcription factor NRF2, a master controller of many homeostatic genes, might provide a disease-modifying therapy in the animal model of Parkinson's disease (PD) that best reproduces the main hallmark of this pathology, that is, α-synucleinopathy, and associated events, including nigral dopaminergic cell death, oxidative stress, and neuroinflammation. Results: Pharmacological activation of NRF2 was achieved at the basal ganglia by repurposing dimethyl fumarate (DMF), a drug already in use for the treatment of multiple sclerosis. Daily oral gavage of DMF protected nigral dopaminergic neurons against α-SYN toxicity and decreased astrocytosis and microgliosis after 1, 3, and 8 weeks from stereotaxic delivery to the ventral midbrain of recombinant adeno-associated viral vector expressing human α-synuclein. This protective effect was not observed in Nrf2-knockout mice. In vitro studies indicated that this neuroprotective effect was correlated with altered regulation of autophagy markers SQTSM1/p62 and LC3 in MN9D, BV2, and IMA 2.1 and with a shift in microglial dynamics toward a less pro-inflammatory and a more wound-healing phenotype. In postmortem samples of PD patients, the cytoprotective proteins associated with NRF2 expression, NQO1 and p62, were partly sequestered in Lewy bodies, suggesting impaired neuroprotective capacity of the NRF2 signature. Innovation: These experiments provide a compelling rationale for targeting NRF2 with DMF as a therapeutic strategy to reinforce endogenous brain defense mechanisms against PD-associated synucleinopathy. Conclusion: DMF is ready for clinical validation in PD. Antioxid. Redox Signal. 25, 61–77. PMID:27009601

  1. Electrophilic Nitro-fatty Acids Activate NRF2 by a KEAP1 Cysteine 151-independent Mechanism*

    PubMed Central

    Kansanen, Emilia; Bonacci, Gustavo; Schopfer, Francisco J.; Kuosmanen, Suvi M.; Tong, Kit I.; Leinonen, Hanna; Woodcock, Steven R.; Yamamoto, Masayuki; Carlberg, Carsten; Ylä-Herttuala, Seppo; Freeman, Bruce A.; Levonen, Anna-Liisa

    2011-01-01

    Nitro-fatty acids (NO2-FAs) are electrophilic signaling mediators formed in vivo via nitric oxide (NO)- and nitrite (NO2−)-dependent reactions. Nitro-fatty acids modulate signaling cascades via reversible covalent post-translational modification of nucleophilic amino acids in regulatory proteins and enzymes, thus altering downstream signaling events, such as Keap1-Nrf2-antioxidant response element (ARE)-regulated gene expression. In this study, we investigate the molecular mechanisms by which 9- and 10-nitro-octadec-9-enoic acid (OA-NO2) activate the transcription factor Nrf2, focusing on the post-translational modifications of cysteines in the Nrf2 inhibitor Keap1 by nitroalkylation and its downstream responses. Of the two regioisomers, 9-nitro-octadec-9-enoic acid was a more potent ARE inducer than 10-nitro-octadec-9-enoic acid. The most OA-NO2-reactive Cys residues in Keap1 were Cys38, Cys226, Cys257, Cys273, Cys288, and Cys489. Of these, Cys273 and Cys288 accounted for ∼50% of OA-NO2 reactions in a cellular milieu. Notably, Cys151 was among the least OA-NO2-reactive of the Keap1 Cys residues, with mutation of Cys151 having no effect on net OA-NO2 reaction with Keap1 or on ARE activation. Unlike many other Nrf2-activating electrophiles, OA-NO2 enhanced rather than diminished the binding between Keap1 and the Cul3 subunit of the E3 ligase for Nrf2. OA-NO2 can therefore be categorized as a Cys151-independent Nrf2 activator, which in turn can influence the pattern of gene expression and therapeutic actions of nitroalkenes. PMID:21357422

  2. Nrf2/p62 signaling in apoptosis resistance and its role in cadmium-induced carcinogenesis.

    PubMed

    Son, Young-Ok; Pratheeshkumar, Poyil; Roy, Ram Vinod; Hitron, John Andrew; Wang, Lei; Zhang, Zhuo; Shi, Xianglin

    2014-10-10

    The cadmium-transformed human lung bronchial epithelial BEAS-2B cells exhibit a property of apoptosis resistance as compared with normal non-transformed BEAS-2B cells. The level of basal reactive oxygen species (ROS) is extremely low in transformed cells in correlation with elevated expressions of both antioxidant enzymes (catalase, SOD1, and SOD2) and antiapoptotic proteins (Bcl-2/Bcl-xL). Moreover, Nrf2 and p62 are highly expressed in these transformed cells. The knockdown of Nrf2 or p62 by siRNA enhances ROS levels and cadmium-induced apoptosis. The binding activities of Nrf2 on the antioxidant response element promoter regions of p62/Bcl-2/Bcl-xL were dramatically increased in the cadmium-exposed transformed cells. Cadmium exposure increased the formation of LC3-II and the frequency of GFP-LC3 punctal cells in non-transformed BEAS-2B cells, whereas these increases are not shown in transformed cells, an indication of autophagy deficiency of transformed cells. Furthermore, the expression levels of Nrf2 and p62 are dramatically increased during chronic long term exposure to cadmium in the BEAS-2B cells as well as antiapoptotic proteins and antioxidant enzymes. These proteins are overexpressed in the tumor tissues derived from xenograft mouse models. Moreover, the colony growth is significantly attenuated in the transformed cells by siRNA transfection specific for Nrf2 or p62. Taken together, this study demonstrates that cadmium-transformed cells have acquired autophagy deficiency, leading to constitutive p62 and Nrf2 overexpression. These overexpressions up-regulate the antioxidant proteins catalase and SOD and the antiapoptotic proteins Bcl-2 and Bcl-xL. The final consequences are decrease in ROS generation, apoptotic resistance, and increased cell survival, proliferation, and tumorigenesis. PMID:25157103

  3. Repeated Nrf2 stimulation using sulforaphane protects fibroblasts from ionizing radiation

    SciTech Connect

    Mathew, Sherin T.; Bergström, Petra; Hammarsten, Ola

    2014-05-01

    Most of the cytotoxicity induced by ionizing radiation is mediated by radical-induced DNA double-strand breaks. Cellular protection from free radicals can be stimulated several fold by sulforaphane-mediated activation of the transcription factor Nrf2 that regulates more than 50 genes involved in the detoxification of reactive substances and radicals. Here, we report that repeated sulforaphane treatment increases radioresistance in primary human skin fibroblasts. Cells were either treated with sulforaphane for four hours once or with four-hour treatments repeatedly for three consecutive days prior to radiation exposure. Fibroblasts exposed to repeated-sulforaphane treatment showed a more pronounced dose-dependent induction of Nrf2-regulated mRNA and reduced amount of radiation-induced free radicals compared with cells treated once with sulforaphane. In addition, radiation- induced DNA double-strand breaks measured by gamma-H2AX foci were attenuated following repeated sulforaphane treatment. As a result, cellular protection from ionizing radiation measured by the 5-ethynyl-2′-deoxyuridine (EdU) assay was increased, specifically in cells exposed to repeated sulforaphane treatment. Sulforaphane treatment was unable to protect Nrf2 knockout mouse embryonic fibroblasts, indicating that the sulforaphane-induced radioprotection was Nrf2-dependent. Moreover, radioprotection by repeated sulforaphane treatment was dose-dependent with an optimal effect at 10 uM, whereas both lower and higher concentrations resulted in lower levels of radioprotection. Our data indicate that the Nrf2 system can be trained to provide further protection from radical damage. - Highlights: • Repeated treatment with sulforaphane protects fibroblasts from ionizing radiation • Repeated sulforaphane treatment attenuates radiation induced ROS and DNA damage • Sulforaphane mediated protection is Nrf2 dependent.

  4. Dimethylfumarate attenuates restenosis after acute vascular injury by cell-specific and Nrf2-dependent mechanisms

    PubMed Central

    Oh, Chang Joo; Park, Sungmi; Kim, Joon-Young; Kim, Han-Jong; Jeoung, Nam Ho; Choi, Young-Keun; Go, Younghoon; Park, Keun-Gyu; Lee, In-Kyu

    2014-01-01

    Excessive proliferation of vascular smooth muscle cells (VSMCs) and incomplete re-endothelialization is a major clinical problem limiting the long-term efficacy of percutaneous coronary angioplasty. We tested if dimethylfumarate (DMF), an anti-psoriasis drug, could inhibit abnormal vascular remodeling via NF−E2-related factor 2 (Nrf2)-NAD(P)H quinone oxidoreductase 1 (NQO1) activity. DMF significantly attenuated neointimal hyperplasia induced by balloon injury in rat carotid arteries via suppression of the G1 to S phase transition resulting from induction of p21 protein in VSMCs. Initially, DMF increased p21 protein stability through an enhancement in Nrf2 activity without an increase in p21 mRNA. Later on, DMF stimulated p21 mRNA expression through a process dependent on p53 activity. However, heme oxygenase-1 (HO-1) or NQO1 activity, well-known target genes induced by Nrf2, were dispensable for the DMF induction of p21 protein and the effect on the VSMC proliferation. Likewise, DMF protected endothelial cells from TNF-α-induced apoptosis and the dysfunction characterized by decreased eNOS expression. With knock-down of Nrf2 or NQO1, DMF failed to prevent TNF-α-induced cell apoptosis and decreased eNOS expression. Also, CD31 expression, an endothelial specific marker, was restored in vivo by DMF. In conclusion, DMF prevented abnormal proliferation in VSMCs by G1 cell cycle arrest via p21 upregulation driven by Nrf2 and p53 activity, and had a beneficial effect on TNF-α-induced apoptosis and dysfunction in endothelial cells through Nrf2–NQO1 activity suggesting that DMF might be a therapeutic drug for patients with vascular disease. PMID:25009787

  5. De-Differentiation Confers Multidrug Resistance Via Noncanonical PERK-Nrf2 Signaling

    PubMed Central

    Del Vecchio, Catherine A.; Feng, Yuxiong; Sokol, Ethan S.; Tillman, Erik J.; Sanduja, Sandhya; Reinhardt, Ferenc; Gupta, Piyush B.

    2014-01-01

    Malignant carcinomas that recur following therapy are typically de-differentiated and multidrug resistant (MDR). De-differentiated cancer cells acquire MDR by up-regulating reactive oxygen species (ROS)–scavenging enzymes and drug efflux pumps, but how these genes are up-regulated in response to de-differentiation is not known. Here, we examine this question by using global transcriptional profiling to identify ROS-induced genes that are already up-regulated in de-differentiated cells, even in the absence of oxidative damage. Using this approach, we found that the Nrf2 transcription factor, which is the master regulator of cellular responses to oxidative stress, is preactivated in de-differentiated cells. In de-differentiated cells, Nrf2 is not activated by oxidation but rather through a noncanonical mechanism involving its phosphorylation by the ER membrane kinase PERK. In contrast, differentiated cells require oxidative damage to activate Nrf2. Constitutive PERK-Nrf2 signaling protects de-differentiated cells from chemotherapy by reducing ROS levels and increasing drug efflux. These findings are validated in therapy-resistant basal breast cancer cell lines and animal models, where inhibition of the PERK-Nrf2 signaling axis reversed the MDR of de-differentiated cancer cells. Additionally, analysis of patient tumor datasets showed that a PERK pathway signature correlates strongly with chemotherapy resistance, tumor grade, and overall survival. Collectively, these results indicate that de-differentiated cells up-regulate MDR genes via PERK-Nrf2 signaling and suggest that targeting this pathway could sensitize drug-resistant cells to chemotherapy. PMID:25203443

  6. The role of transcription factor Nrf2 in skin cells metabolism.

    PubMed

    Gęgotek, Agnieszka; Skrzydlewska, Elżbieta

    2015-07-01

    Skin, which is a protective layer of the body, is in constant contact with physical and chemical environmental factors. Exposure of the skin to highly adverse conditions often leads to oxidative stress. Moreover, it has been observed that skin cells are also exposed to reactive oxygen species generated during cell metabolism particularly in relation to the synthesis of melanin or the metabolism in immune system cells. However, skin cells have special features that protect them against oxidative modifications including transcription factor Nrf2, which is responsible for the transcription of the antioxidant protein genes such as antioxidant enzymes, small molecular antioxidant proteins or interleukins, and multidrug response protein. In the present study, the mechanisms of Nrf2 activation have been compared in the cells forming the various layers of the skin: keratinocytes, melanocytes, and fibroblasts. The primary mechanism of control of Nrf2 activity is its binding by cytoplasmic inhibitor Keap1, while cells have also other controlling mechanisms, such as phosphorylation of Nrf2 and modifications of its activators (e.g., Maf, IKKβ) or inhibitors (e.g., Bach1, caveolae, TGF-β). Moreover, there are a number of drugs (e.g., ketoconazole) used in the pharmacotherapy of skin diseases based on the activation of Nrf2, but they may also induce oxidative stress. Therefore, it is important to look for compounds that cause a selective activation of Nrf2 particularly natural substances such as curcumin, sulforaphane, or extracts from the broccoli leaves without side effects. These findings could be helpful in the searching for new drugs for people with vitiligo or even melanoma. PMID:25708189

  7. Sofalcone, a gastric mucosa protective agent, increases vascular endothelial growth factor via the Nrf2-heme-oxygenase-1 dependent pathway in gastric epithelial cells

    SciTech Connect

    Shibuya, Akiko; Onda, Kenji; Kawahara, Hirofumi; Uchiyama, Yuka; Nakayama, Hiroko; Omi, Takamasa; Nagaoka, Masayoshi; Matsui, Hirofumi; Hirano, Toshihiko

    2010-07-30

    Research highlights: {yields} Sofalcone increases HO-1 in gastric epithelial cells. {yields} The induction of HO-1 by sofalcone treatment follows the activation of Nrf2. {yields} The production of VEGF by sofalcone treatment is mediated by HO-1 induction. -- Abstract: Sofalcone, 2'-carboxymethoxy-4,4-bis(3-methyl-2-butenyloxy)chalcone, is an anti-ulcer agent that is classified as a gastric mucosa protective agent. Recent studies indicate heat shock proteins such as HSP32, also known as heme-oxygenase-1(HO-1), play important roles in protecting gastrointestinal tissues from several stresses. We have previously reported that sofalcone increases the expression of HO-1 in adipocytes and pre-adipocytes, although the effect of sofalcone on HO-1 induction in gastrointestinal tissues is not clear. In the current study, we investigated the effects of sofalcone on the expression of HO-1 and its functional role in rat gastric epithelial (RGM-1) cells. We found that sofalcone increased HO-1 expression in RGM-1 cells in both time- and concentration-dependent manners. The HO-1 induction was associated with the nuclear translocation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in RGM-1 cells. We also observed that sofalcone increased vascular endothelial growth factor (VEGF) production in the culture medium. Treatment of RGM-1 cells with an HO-1 inhibitor (tin-protoporphyrin), or HO-1 siRNA inhibited sofalcone-induced VEGF production, suggesting that the effect of sofalcone on VEGF expression is mediated by the HO-1 pathway. These results suggest that the gastroprotective effects of sofalcone are partly exerted via Nrf2-HO-1 activation followed by VEGF production.

  8. Tenuigenin exhibits anti-inflammatory activity via inhibiting MAPK and NF-κB and inducing Nrf2/HO-1 signaling in macrophages.

    PubMed

    Lv, Hongming; Ren, Wenzhi; Zheng, Yuwei; Wang, Lidong; Lu, Gejin; Yi, Pengfei; Ci, Xinxin

    2016-01-01

    Tenuigenin (TNG), isolated from the root of the Chinese herb Polygala tenuifolia, possesses various biological and pharmacological activities, including anti-oxidation and anti-inflammation activities. In this study, we aimed to further investigate whether its anti-inflammatory activity is associated with the inhibition of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression in lipopolysaccharide (LPS)-induced RAW 264.7 cells. Our results showed that TNG treatment dramatically reduced prostaglandin E2 (PGE2) and NO production, decreased iNOS and COX-2 gene expression, inhibited JNK1/2, ERK1/2, p38 and NF-κB (p65) phosphorylation, and blocked IκBα phosphorylation and degradation. Further studies revealed that TNG dramatically up-regulated heme oxygenase (HO)-1 and nuclear factor erythroid 2-related factor 2 (Nrf2) expression, which was related to the induction of Nrf2 nuclear translocation and decreased Keap1 protein expression. Additionally, treatment with JNK1/2, ERK1/2 or p38 inhibitors had no effect on the TNG-induced HO-1 protein expression. Furthermore, the LPS-induced iNOS and COX-2 expression levels were inhibited by TNG, which was partially reversed by the HO-1-siRNA and HO-1 inhibitors. Together, these results showed that TNG's anti-inflammatory activity is related to the inhibition of iNOS and COX-2 expression via down-regulation of the MAPK and NF-κB, and up-regulation of the Nrf2/HO-1 signaling pathways. PMID:26499342

  9. Mangiferin exerts hepatoprotective activity against D-galactosamine induced acute toxicity and oxidative/nitrosative stress via Nrf2–NFκB pathways

    SciTech Connect

    Das, Joydeep; Ghosh, Jyotirmoy; Roy, Anandita; Sil, Parames C.

    2012-04-01

    Mangiferin, a xanthone glucoside, is well known to exhibit antioxidant, antiviral, antitumor, anti-inflammatory and gene-regulatory effects. In the present study, we isolated mangiferin from the bark of Mangifera indica and assessed its beneficial role in galactosamine (GAL) induced hepatic pathophysiology. GAL (400 mg/kg body weight) exposed hepatotoxic rats showed elevation in the activities of serum ALP, ALT, levels of triglycerides, total cholesterol, lipid-peroxidation and reduction in the levels of serum total proteins, albumin and cellular GSH. Besides, GAL exposure (5 mM) in hepatocytes induced apoptosis and necrosis, increased ROS and NO production. Signal transduction studies showed that GAL exposure significantly increased the nuclear translocation of NFκB and elevated iNOS protein expression. The same exposure also elevated TNF-α, IFN-γ, IL-1β, IL-6, IL-12, IL-18 and decreased IL-10 mRNA expressions. Furthermore, GAL also decreased the protein expression of Nrf2, NADPH:quinine oxidoreductase-1, heme oxygenase-1 and GSTα. However, mangiferin administration in GAL intoxicated rats or coincubation of hepatocytes with mangiferin significantly altered all these GAL-induced adverse effects. In conclusion, the hepatoprotective role of mangiferin was due to induction of antioxidant defense via the Nrf2 pathway and reduction of inflammation via NFκB inhibition. Highlights: ►Galactosamine induces hepatocytes death via oxidative and nitrosative stress. ►Mangiferin exerts hepatoprotective effect/antioxidant defense via Nrf2 pathway. ►Mangiferin exerts anti-inflammatory responses by inhibiting NF-κB. ►Mangiferin suppresses galactosamine-induced repression of IL-10 mRNA.

  10. Anti-inflammatory potential of allyl-isothiocyanate--role of Nrf2, NF-(κ) B and microRNA-155.

    PubMed

    Wagner, Anika Eva; Boesch-Saadatmandi, Christine; Dose, Janina; Schultheiss, Gerhard; Rimbach, Gerald

    2012-04-01

    In this study, the underlying mechanisms of the potential anti-inflammatory properties of allyl-isothiocyanate (AITC) were analysed in vitro and in vivo. Murine RAW264.7 macrophages stimulated with lipopolysaccharide (LPS) were supplemented with increasing concentrations of AITC. In addition, C57BL/6 mice (n= 10 per group) were fed a pro-inflammatory high-fat diet and AITC was administered orally via gavage for 7 days. Biomarkers of inflammation were determined both in cultured cells and in mice. AITC significantly decreased tumour necrosis factor α mRNA levels and its secretion in LPS stimulated RAW264.7 macrophages. Furthermore, gene expression of other pro-inflammatory markers including interleukin-1β and inducible nitric oxide synthase were down-regulated following AITC treatment. AITC decreased nuclear p65 protein levels, a subunit of the transcription factor NF-κB. Importantly, our data indicate that AITC significantly attenuated microRNA-155 levels in LPS-stimulated RAW264.7 macrophages in a dose-dependent manner. The anti-inflammatory effects of AITC were accompanied by an increase in Nrf2 nuclear translocation and consequently by an increase of mRNA and protein levels of the Nrf2 target gene heme-oxygenase 1. AITC was slightly less potent than sulforaphane (used as a positive control) in down-regulating inflammation in LPS-stimulated macrophages. A significant increase in nuclear Nrf2 and heme-oxygenase 1 gene expression and only a moderate down-regulation of interleukin-1β and microRNA-155 levels due to AITC was found in mouse liver. Present data suggest that AITC exhibits potent anti-inflammatory activity in cultured macrophages in vitro but has only little anti-inflammatory activity in mice in vivo. PMID:21692985

  11. Plumbagin suppresses epithelial to mesenchymal transition and stemness via inhibiting Nrf2-mediated signaling pathway in human tongue squamous cell carcinoma cells

    PubMed Central

    Pan, Shu-Ting; Qin, Yiru; Zhou, Zhi-Wei; He, Zhi-Xu; Zhang, Xueji; Yang, Tianxin; Yang, Yin-Xue; Wang, Dong; Zhou, Shu-Feng; Qiu, Jia-Xuan

    2015-01-01

    Tongue squamous cell carcinoma (TSCC) is the most common malignancy in oral and maxillofacial tumors with highly metastatic characteristics. Plumbagin (5-hydroxy-2-methyl-1, 4-naphthoquinone; PLB), a natural naphthoquinone derived from the roots of Plumbaginaceae plants, exhibits various bioactivities, including anticancer effects. However, the potential molecular targets and underlying mechanisms of PLB in the treatment of TSCC remain elusive. This study employed stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomic approach to investigate the molecular interactome of PLB in human TSCC cell line SCC25 and elucidate the molecular mechanisms. The proteomic data indicated that PLB inhibited cell proliferation, activated death receptor-mediated apoptotic pathway, remodeled epithelial adherens junctions pathway, and manipulated nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated oxidative stress response signaling pathway in SCC25 cells with the involvement of a number of key functional proteins. Furthermore, we verified these protein targets using Western blotting assay. The verification results showed that PLB markedly induced cell cycle arrest at G2/M phase and extrinsic apoptosis, and inhibited epithelial to mesenchymal transition (EMT) and stemness in SCC25 cells. Of note, N-acetyl-l-cysteine (NAC) and l-glutathione (GSH) abolished the effects of PLB on cell cycle arrest, apoptosis induction, EMT inhibition, and stemness attenuation in SCC25 cells. Importantly, PLB suppressed the translocation of Nrf2 from cytosol to nucleus, resulting in an inhibition in the expression of downstream targets. Taken together, these results suggest that PLB may act as a promising anticancer compound via inhibiting Nrf2-mediated oxidative stress signaling pathway in SCC25 cells. This study provides a clue to fully identify the molecular targets and decipher the underlying mechanisms of PLB in the treatment of TSCC. PMID:26491260

  12. Mycophenolate mofetil and curcumin provide comparable therapeutic benefit in experimental chronic kidney disease: role of Nrf2-Keap1 and renal dopamine pathways.

    PubMed

    Tapia, Edilia; García-Arroyo, Fernando; Silverio, Octaviano; Rodríguez-Alcocer, Alma N; Jiménez-Flores, Ana B; Cristobal, Magdalena; Arellano, Abraham S; Soto, Virgilia; Osorio-Alonso, Horacio; Molina-Jijón, Eduardo; Pedraza-Chaverri, José; Sanchez-Lozada, Laura G

    2016-07-01

    Increased oxidative stress and inflammation have an important role in the pathophysiology of chronic kidney disease (CKD). On the other hand, more affordable therapeutic alternatives for treating this disease are urgently needed. Therefore, we compared the therapeutic efficacy of curcumin and mycophenolate mofetil (MMF) in 5/6 nephrectomy (5/6 Nx) model of CKD. Also, we evaluated whether both compounds provide benefit through the preservation of similar antioxidant mechanisms. Four groups of male Wistar were studied over a period of 4 wk. Control sham group (n= 12), 5/6 Nx (n = 12), 5/6 Nx + MMF (30 mg/k BW/day, n = 11) and 5/6 Nx + Curcumin (120 mg/k BW/day, n = 12). Renal function and markers of oxidative stress and inflammation were evaluated. Also Nrf2-Keap1 and renal dopamine, antioxidant pathways were assessed. 5/6 Nx induced an altered renal autoregulation response, proteinuria, and hypertension; these effects were in association with increased oxidative stress, endothelial dysfunction and renal inflammation. The mechanisms associated with these alterations included a reduced nuclear translocation of Nrf2 and hyperphosphorylation of dopamine D1 receptor with a concurrent overactivation of renal NADPH oxidase. Treatments with MMF and curcumin provided equivalent therapeutic efficacy as both prevented functional renal alterations as well as preserved antioxidant capacity and avoided renal inflammatory infiltration. Moreover, both treatments preserved Nrf2-Keap1 and renal dopamine antioxidant pathways. In summary, therapeutic strategies aimed to preserve renal antioxidant pathways can help to retard the progression of CKD. PMID:27050624

  13. Network Inference Algorithms Elucidate Nrf2 Regulation of Mouse Lung Oxidative Stress

    PubMed Central

    Singhal, Mudita; Malhotra, Deepti; Biswal, Shyam

    2008-01-01

    A variety of cardiovascular, neurological, and neoplastic conditions have been associated with oxidative stress, i.e., conditions under which levels of reactive oxygen species (ROS) are elevated over significant periods. Nuclear factor erythroid 2-related factor (Nrf2) regulates the transcription of several gene products involved in the protective response to oxidative stress. The transcriptional regulatory and signaling relationships linking gene products involved in the response to oxidative stress are, currently, only partially resolved. Microarray data constitute RNA abundance measures representing gene expression patterns. In some cases, these patterns can identify the molecular interactions of gene products. They can be, in effect, proxies for protein–protein and protein–DNA interactions. Traditional techniques used for clustering coregulated genes on high-throughput gene arrays are rarely capable of distinguishing between direct transcriptional regulatory interactions and indirect ones. In this study, newly developed information-theoretic algorithms that employ the concept of mutual information were used: the Algorithm for the Reconstruction of Accurate Cellular Networks (ARACNE), and Context Likelihood of Relatedness (CLR). These algorithms captured dependencies in the gene expression profiles of the mouse lung, allowing the regulatory effect of Nrf2 in response to oxidative stress to be determined more precisely. In addition, a characterization of promoter sequences of Nrf2 regulatory targets was conducted using a Support Vector Machine classification algorithm to corroborate ARACNE and CLR predictions. Inferred networks were analyzed, compared, and integrated using the Collective Analysis of Biological Interaction Networks (CABIN) plug-in of Cytoscape. Using the two network inference algorithms and one machine learning algorithm, a number of both previously known and novel targets of Nrf2 transcriptional activation were identified. Genes predicted as

  14. PCB 126 toxicity is modulated by cross-talk between caveolae and Nrf2 signaling

    SciTech Connect

    Petriello, Michael C.; Han, Sung Gu; Newsome, Bradley J.; Hennig, Bernhard

    2014-06-01

    Environmental toxicants such as polychlorinated biphenyls (PCBs) have been implicated in the promotion of multiple inflammatory disorders including cardiovascular disease, but information regarding mechanisms of toxicity and cross-talk between relevant cell signaling pathways is lacking. To examine the hypothesis that cross-talk between membrane domains called caveolae and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathways alters PCB-induced inflammation, caveolin-1 was silenced in vascular endothelial cells, resulting in a decreased PCB-induced inflammatory response. Cav-1 silencing (siRNA treatment) also increased levels of Nrf2-ARE transcriptional binding, resulting in higher mRNA levels of the antioxidant genes glutathione s-transferase and NADPH dehydrogenase quinone-1 in both vehicle and PCB-treated systems. Along with this upregulated antioxidant response, Cav-1 siRNA treated cells exhibited decreased mRNA levels of the Nrf2 inhibitory protein Keap1 in both vehicle and PCB-treated samples. Silencing Cav-1 also decreased protein levels of Nrf2 inhibitory proteins Keap1 and Fyn kinase, especially in PCB-treated cells. Further, endothelial cells from wildtype and Cav-1 −/− mice were isolated and treated with PCB to better elucidate the role of functional caveolae in PCB-induced endothelial inflammation. Cav-1 −/− endothelial cells were protected from PCB-induced cellular dysfunction as evidenced by decreased vascular cell adhesion molecule (VCAM-1) protein induction. Compared to wildtype cells, Cav-1 −/− endothelial cells also allowed for a more effective antioxidant response, as observed by higher levels of the antioxidant genes. These data demonstrate novel cross-talk mechanisms between Cav-1 and Nrf2 and implicate the reduction of Cav-1 as a protective mechanism for PCB-induced cellular dysfunction and inflammation. - Highlights: • Reduction of caveolin-1 protein protects against polychlorinated biphenyl toxicity. • Decreasing

  15. Oxaliplatin activates the Keap1/Nrf2 antioxidant system conferring protection against the cytotoxicity of anticancer drugs.

    PubMed

    Wang, Xiu Jun; Li, Yinyan; Luo, Lin; Wang, Hongyan; Chi, Zhexu; Xin, Ai; Li, Xin; Wu, Jiaguo; Tang, Xiuwen

    2014-05-01

    Oxaliplatin is an important drug in the treatment of advanced metastatic colorectal cancer. NF-E2 p45-related factor 2 (Nrf2) is a key transcription factor that controls genes encoding cytoprotective and detoxifying enzymes through antioxidant-response elements (AREs) in their regulatory regions. Here, we report that oxaliplatin is an activator of the Nrf2 signaling pathway, with upregulation of ARE-driven genes and glutathione elevation. An injection of oxaliplatin into mice enhanced the expression of glutathione transferases and antioxidant enzymes in the small and large intestines of wild-type (WT) mice but not Nrf2(-/-) mice, indicating that oxaliplatin activates Nrf2 in vivo. Oxaliplatin failed to increase Nrf2 accumulation in non-small-cell lung cancer A549 cells, which harbor a dysfunctional somatic mutation of KEAP1. However, forced expression of WT mKeap1 restored the ability of oxaliplatin to activate the transcription factor. Cys(151) in Keap1 was required for the response stimulated by oxaliplatin. In addition, dichloro(1,2-diaminocyclohexane) platinum, a metabolite of oxaliplatin, was found to have the same effect in activating the ARE-gene battery as its parent drug, whereas another metabolite, oxalate, was ineffective. Moreover, two other platinum derivatives, cisplatin and carboplatin, had no effect on the Keap1/Nrf2 system. Furthermore, activation of Nrf2 by oxaliplatin reduced the sensitivity of colon cancer cells to therapeutic drugs. Conversely, knockdown of Nrf2 by Nrf2 siRNA reduced oxaliplatin-induced chemoresistance. Our study showed that oxaliplatin exerts protection against the cytotoxicity of anticancer drugs via Nrf2, indicating an important role of Nrf2 in oxaliplatin-based chemotherapy. PMID:24556415

  16. Enhanced expression of Nrf2 in mice attenuates the fatty liver produced by a methionine- and choline-deficient diet☆

    PubMed Central

    Zhang, Yu-Kun Jennifer; Yeager, Ronnie L.; Tanaka, Yuji; Klaassen, Curtis D.

    2011-01-01

    Oxidative stress has been proposed as an important promoter of the progression of fatty liver diseases. The current study investigates the potential functions of the Nrf2–Keap1 signaling pathway, an important hepatic oxidative stress sensor, in a rodent fatty liver model. Mice with no (Nrf2-null), normal (wild type, WT), and enhanced (Keap1 knockdown, K1-kd) expression of Nrf2 were fed a methionine- and choline-deficient (MCD) diet or a control diet for 5 days. Compared to WT mice, the MCD diet-caused hepatosteatosis was more severe in the Nrf2-null mice and less in the K1-kd mice. The Nrf2-null mice had lower hepatic glutathione and exhibited more lipid peroxidation, whereas the K1-kd mice had the highest amount of glutathione in the liver and developed the least lipid peroxidation among the three genotypes fed the MCD diet. The Nrf2 signaling pathway was activated by the MCD diet, and the Nrf2-targeted cytoprotective genes Nqo1 and Gstα1/2 were induced in WT and even more in K1-kd mice. In addition, Nrf2-null mice on both control and MCD diets exhibited altered expression profiles of fatty acid metabolism genes, indicating Nrf2 may influence lipid metabolism in liver. For example, mRNA levels of long chain fatty acid translocase CD36 and the endocrine hormone Fgf21 were higher in livers of Nrf2-null mice and lower in the K1-kd mice than WT mice fed the MCD diet. Taken together, these observations indicate that Nrf2 could decelerate the onset of fatty livers caused by the MCD diet by increasing hepatic antioxidant and detoxification capabilities. PMID:20350562

  17. Enhanced expression of Nrf2 in mice attenuates the fatty liver produced by a methionine- and choline-deficient diet

    SciTech Connect

    Zhang, Yu-Kun Jennifer; Yeager, Ronnie L.; Tanaka, Yuji; Klaassen, Curtis D.

    2010-06-15

    Oxidative stress has been proposed as an important promoter of the progression of fatty liver diseases. The current study investigates the potential functions of the Nrf2-Keap1 signaling pathway, an important hepatic oxidative stress sensor, in a rodent fatty liver model. Mice with no (Nrf2-null), normal (wild type, WT), and enhanced (Keap1 knockdown, K1-kd) expression of Nrf2 were fed a methionine- and choline-deficient (MCD) diet or a control diet for 5 days. Compared to WT mice, the MCD diet-caused hepatosteatosis was more severe in the Nrf2-null mice and less in the K1-kd mice. The Nrf2-null mice had lower hepatic glutathione and exhibited more lipid peroxidation, whereas the K1-kd mice had the highest amount of glutathione in the liver and developed the least lipid peroxidation among the three genotypes fed the MCD diet. The Nrf2 signaling pathway was activated by the MCD diet, and the Nrf2-targeted cytoprotective genes Nqo1 and Gst{alpha}1/2 were induced in WT and even more in K1-kd mice. In addition, Nrf2-null mice on both control and MCD diets exhibited altered expression profiles of fatty acid metabolism genes, indicating Nrf2 may influence lipid metabolism in liver. For example, mRNA levels of long chain fatty acid translocase CD36 and the endocrine hormone Fgf21 were higher in livers of Nrf2-null mice and lower in the K1-kd mice than WT mice fed the MCD diet. Taken together, these observations indicate that Nrf2 could decelerate the onset of fatty livers caused by the MCD diet by increasing hepatic antioxidant and detoxification capabilities.

  18. Hydrangenol inhibits lipopolysaccharide-induced nitric oxide production in BV2 microglial cells by suppressing the NF-κB pathway and activating the Nrf2-mediated HO-1 pathway.

    PubMed

    Kim, Hee-Ju; Kang, Chang-Hee; Jayasooriya, Rajapaksha Gedara Prasad Tharanga; Dilshara, Matharage Gayani; Lee, Seungheon; Choi, Yung Hyun; Seo, Yong Taek; Kim, Gi-Young

    2016-06-01

    We previously demonstrated the anti-inflammatory effect of water extract of Hydrangea macrophylla in lipopolysaccharide (LPS)-stimulated macrophage cells. Here, we investigated whether hydrangenol, a bioactive component of H. macrophylla, attenuates the expression of nitric oxide (NO) and its associated gene, inducible NO synthase (iNOS), in LPS-stimulated BV2 microglial cells. Our data showed that low dosages of hydrangenol inhibited LPS-stimulated NO release and iNOS expression without any accompanying cytotoxicity. Hydrangenol also suppressed LPS-induced nuclear translocation of nuclear factor-κB (NF-κB) subunits, consequently inhibiting DNA-binding activity of NF-κB. Additionally, the NF-κB inhibitors, pyrrolidine dithiocarbamate (PDTC) and PS-1145, significantly attenuated LPS-induced iNOS expression, indicating that hydrangenol-induced NF-κB inhibition might be a key regulator of iNOS expression. Furthermore, our data showed that hydrangenol suppresses NO production by inducing heme oxygenase-1 (HO-1). The presence of cobalt protoporphyrin, a specific HO-1 inducer, potently suppressed LPS-induced NO production. Hydrangenol also promoted nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and subsequently increased its binding activity at the specific antioxidant response element sites. Additionally, transient knockdown of Nrf2 significantly downregulated hydrangenol-induced HO-1 expression, indicating that hydrangenol-induced Nrf2 is an upstream regulator of HO-1. Taken together, these data suggest that hydrangenol attenuates NO production and iNOS expression in LPS-stimulated BV2 microglial cells by inhibiting NF-κB activation and by stimulating the Nrf2-mediated HO-1 signaling pathway. Therefore, hydrangenol is a promising therapeutic agent for treatment of LPS-mediated inflammatory diseases. PMID:27032067

  19. Genetic Ablation of Nrf2/Antioxidant Response Pathway in Alexander Disease Mice Reduces Hippocampal Gliosis but Does Not Impact Survival

    PubMed Central

    Hagemann, Tracy L.; Jobe, Emily M.; Messing, Albee

    2012-01-01

    In Alexander disease (AxD) the presence of mutant glial fibrillary acidic protein (GFAP), the major intermediate filament of astrocytes, triggers protein aggregation, with marked induction of a stress response mediated by the transcription factor, Nrf2. To clarify the role of Nrf2 in AxD, we have crossed Gfap mutant and transgenic mouse models into an Nrf2 null background. Deletion of Nrf2 eliminates the phase II stress response normally present in mouse models of AxD, but causes no change in body weight or lifespan, even in a severe lethal model. AxD astrocytes without Nrf2 retain features of reactivity, such as expression of the endothelin-B receptor, but have lower Gfap levels, a decrease in p62 protein and reduced iron accumulation, particularly in hippocampus. Microglial activation, indicated by Iba1 expression, is also diminished. Although the Nrf2 response is generally considered beneficial, these results show that in the context of AxD, loss of the antioxidant pathway has no obvious negative effects, while actually decreasing Gfap accumulation and pathology. Given the attention Nrf2 is receiving as a potential therapeutic target in AxD and other neurodegenerative diseases, it will be interesting to see whether induction of Nrf2, beyond the endogenous response, is beneficial or not in these same models. PMID:22693571

  20. The circadian clock regulates rhythmic activation of the NRF2/glutathione-mediated antioxidant defense pathway to modulate pulmonary fibrosis.

    PubMed

    Pekovic-Vaughan, Vanja; Gibbs, Julie; Yoshitane, Hikari; Yang, Nan; Pathiranage, Dharshika; Guo, Baoqiang; Sagami, Aya; Taguchi, Keiko; Bechtold, David; Loudon, Andrew; Yamamoto, Masayuki; Chan, Jefferson; van der Horst, Gijsbertus T J; Fukada, Yoshitaka; Meng, Qing-Jun

    2014-03-15

    The disruption of the NRF2 (nuclear factor erythroid-derived 2-like 2)/glutathione-mediated antioxidant defense pathway is a critical step in the pathogenesis of several chronic pulmonary diseases and cancer. While the mechanism of NRF2 activation upon oxidative stress has been widely investigated, little is known about the endogenous signals that regulate the NRF2 pathway in lung physiology and pathology. Here we show that an E-box-mediated circadian rhythm of NRF2 protein is essential in regulating the rhythmic expression of antioxidant genes involved in glutathione redox homeostasis in the mouse lung. Using an in vivo bleomycin-induced lung fibrosis model, we reveal a clock "gated" pulmonary response to oxidative injury, with a more severe fibrotic effect when bleomycin was applied at a circadian nadir in NRF2 levels. Timed administration of sulforaphane, an NRF2 activator, significantly blocked this phenotype. Moreover, in the lungs of the arrhythmic Clock(Δ19) mice, the levels of NRF2 and the reduced glutathione are constitutively low, associated with increased protein oxidative damage and a spontaneous fibrotic-like pulmonary phenotype. Our findings reveal a pivotal role for the circadian control of the NRF2/glutathione pathway in combating oxidative/fibrotic lung damage, which might prompt new chronotherapeutic strategies for the treatment of human lung diseases, including idiopathic pulmonary fibrosis. PMID:24637114

  1. Activated AMPK boosts the Nrf2/HO-1 signaling axis—A role for the unfolded protein response

    PubMed Central

    Zimmermann, Kristin; Baldinger, Johannes; Mayerhofer, Barbara; Atanasov, Atanas G.; Dirsch, Verena M.; Heiss, Elke H.

    2015-01-01

    In light of the emerging interplay between redox and metabolic signaling pathways we investigated the potential cross talk between nuclear factor E2-related factor 2 (Nrf2) and AMP-activated kinase (AMPK), central regulators of the cellular redox and energy balance, respectively. Making use of xanthohumol (XN) as an activator of both the AMPK and the Nrf2 signaling pathway we show that AMPK exerts a positive influence on Nrf2/heme oxygenase (HO)-1 signaling in mouse embryonic fibroblasts. Genetic ablation and pharmacological inhibition of AMPK blunts Nrf2-dependent HO-1 expression by XN already at the mRNA level. XN leads to AMPK activation via interference with mitochondrial function and activation of liver kinase B1 as upstream AMPK kinase. The subsequent AMPK-mediated enhancement of the Nrf2/HO-1 response does not depend on inhibition of the mammalian target of rapamycin, inhibition of glycogen synthase kinase 3β, or altered abundance of Nrf2 (total and nuclear). However, reduced endoplasmic reticulum stress was identified and elaborated as a step in the AMPK-augmented Nrf2/HO-1 response. Overall, we shed more light on the hitherto incompletely understood cross talk between the LKB1/AMPK and the Nrf2/HO-1 axis revealing for the first time involvement of the unfolded protein response as an additional player and suggesting tight cooperation between signaling pathways controlling cellular redox, energy, or protein homeostasis. PMID:25843659

  2. Gestational diabetes mellitus impairs Nrf2-mediated adaptive antioxidant defenses and redox signaling in fetal endothelial cells in utero.

    PubMed

    Cheng, Xinghua; Chapple, Sarah J; Patel, Bijal; Puszyk, William; Sugden, David; Yin, Xiaoke; Mayr, Manuel; Siow, Richard C M; Mann, Giovanni E

    2013-12-01

    In utero exposure to gestational diabetes mellitus (GDM) is associated with an increased risk of type 2 diabetes and cardiovascular disease in later life, yet the underlying mechanisms remain to be elucidated. We examined the effects of GDM on the proteome, redox status, and nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated antioxidant gene expression in human fetal endothelial cells. Proteomic analysis revealed that proteins involved in redox homeostasis were significantly altered in GDM and associated with increased mitochondrial superoxide generation, protein oxidation, DNA damage, and diminished glutathione (GSH) synthesis. In GDM cells, the lipid peroxidation product 4-hydroxynonenal (HNE) failed to induce nuclear Nrf2 accumulation and mRNA and/or protein expression of Nrf2 and its target genes NAD(P)H:quinone oxidoreductase 1 (NQO1), Bach1, cystine/glutamate transporter, and glutamate cysteine ligase. Although methylation of CpG islands in Nrf2 or NQO1 promoters was unaltered by GDM, decreased DJ-1 and increased phosphorylated glycogen synthase kinase 3β levels may account for impaired Nrf2 signaling. HNE-induced increases in GSH and NQO1 levels were abrogated by Nrf2 small interfering RNA in normal cells, and overexpression of Nrf2 in GDM cells partially restored NQO1 induction. Dysregulation of Nrf2 in fetal endothelium may contribute to the increased risk of type 2 diabetes and cardiovascular disease in offspring. PMID:23974919

  3. The circadian clock regulates rhythmic activation of the NRF2/glutathione-mediated antioxidant defense pathway to modulate pulmonary fibrosis

    PubMed Central

    Pekovic-Vaughan, Vanja; Gibbs, Julie; Yoshitane, Hikari; Yang, Nan; Pathiranage, Dharshika; Guo, Baoqiang; Sagami, Aya; Taguchi, Keiko; Bechtold, David; Loudon, Andrew; Yamamoto, Masayuki; Chan, Jefferson; van der Horst, Gijsbertus T.J.; Fukada, Yoshitaka; Meng, Qing-Jun

    2014-01-01

    The disruption of the NRF2 (nuclear factor erythroid-derived 2-like 2)/glutathione-mediated antioxidant defense pathway is a critical step in the pathogenesis of several chronic pulmonary diseases and cancer. While the mechanism of NRF2 activation upon oxidative stress has been widely investigated, little is known about the endogenous signals that regulate the NRF2 pathway in lung physiology and pathology. Here we show that an E-box-mediated circadian rhythm of NRF2 protein is essential in regulating the rhythmic expression of antioxidant genes involved in glutathione redox homeostasis in the mouse lung. Using an in vivo bleomycin-induced lung fibrosis model, we reveal a clock “gated” pulmonary response to oxidative injury, with a more severe fibrotic effect when bleomycin was applied at a circadian nadir in NRF2 levels. Timed administration of sulforaphane, an NRF2 activator, significantly blocked this phenotype. Moreover, in the lungs of the arrhythmic ClockΔ19 mice, the levels of NRF2 and the reduced glutathione are constitutively low, associated with increased protein oxidative damage and a spontaneous fibrotic-like pulmonary phenotype. Our findings reveal a pivotal role for the circadian control of the NRF2/glutathione pathway in combating oxidative/fibrotic lung damage, which might prompt new chronotherapeutic strategies for the treatment of human lung diseases, including idiopathic pulmonary fibrosis. PMID:24637114

  4. Gestational Diabetes Mellitus Impairs Nrf2-Mediated Adaptive Antioxidant Defenses and Redox Signaling in Fetal Endothelial Cells In Utero

    PubMed Central

    Cheng, Xinghua; Chapple, Sarah J.; Patel, Bijal; Puszyk, William; Sugden, David; Yin, Xiaoke; Mayr, Manuel; Siow, Richard C.M.; Mann, Giovanni E.

    2013-01-01

    In utero exposure to gestational diabetes mellitus (GDM) is associated with an increased risk of type 2 diabetes and cardiovascular disease in later life, yet the underlying mechanisms remain to be elucidated. We examined the effects of GDM on the proteome, redox status, and nuclear factor erythroid 2–related factor 2 (Nrf2)-mediated antioxidant gene expression in human fetal endothelial cells. Proteomic analysis revealed that proteins involved in redox homeostasis were significantly altered in GDM and associated with increased mitochondrial superoxide generation, protein oxidation, DNA damage, and diminished glutathione (GSH) synthesis. In GDM cells, the lipid peroxidation product 4-hydroxynonenal (HNE) failed to induce nuclear Nrf2 accumulation and mRNA and/or protein expression of Nrf2 and its target genes NAD(P)H:quinone oxidoreductase 1 (NQO1), Bach1, cystine/glutamate transporter, and glutamate cysteine ligase. Although methylation of CpG islands in Nrf2 or NQO1 promoters was unaltered by GDM, decreased DJ-1 and increased phosphorylated glycogen synthase kinase 3β levels may account for impaired Nrf2 signaling. HNE-induced increases in GSH and NQO1 levels were abrogated by Nrf2 small interfering RNA in normal cells, and overexpression of Nrf2 in GDM cells partially restored NQO1 induction. Dysregulation of Nrf2 in fetal endothelium may contribute to the increased risk of type 2 diabetes and cardiovascular disease in offspring. PMID:23974919

  5. Nrf2 plays a pivotal role in protection against burn trauma-induced intestinal injury and death

    PubMed Central

    Ma, Liang; Ni, Yiming; Zhao, Haige

    2016-01-01

    Nuclear factor (erythroid-derived 2)–like 2 (NRF2) is a basic leucine zipper transcription factor that principally defends against oxidative stress and also plays a unique role in severe sepsis. However, its contribution to intestinal injury and death after burn trauma is unclear.In this study, wild-type (Nrf2+/+) and Nrf2-deficient (Nrf2−/−) mice were subjected to 15% or 30% total body surface area burn or sham injury. Survival, systemic inflammation, and gut injury were determined. Nrf2−/− mice were more susceptible to burn-induced intestinal injury, as characterized by increases in damage to the gut structure and in intestinal permeability. This exacerbation was associated with an increase in the intestinal mRNA expression of inflammatory cytokines (interleukin [IL]–6, IL-1B, monocyte chemotactic protein 1, intercellular adhesion molecule, and vascular cell adhesion molecule) and a decrease in the intestinal mRNA expression of Nrf2-regulated genes (NAD(P)H dehydrogenasequinine-1 and glutamate-cysteine ligase modifier subunit). Nrf2-deficient mice also showed a lower survival rate and higher levels of systemic cytokines (IL-6 and IL-1B) and high-mobility group protein B1 than wild-type mice. This study demonstrates for the first time that mice that lack Nrf2 are more susceptible to burn-induced intestinal injury and have more systemic inflammation and a lower survival rate. PMID:27009867

  6. Nrf2 plays a pivotal role in protection against burn trauma-induced intestinal injury and death.

    PubMed

    Chen, Zhao; Zhang, Yiran; Ma, Liang; Ni, Yiming; Zhao, Haige

    2016-04-12

    Nuclear factor (erythroid-derived 2)-like 2 (NRF2) is a basic leucine zipper transcription factor that principally defends against oxidative stress and also plays a unique role in severe sepsis. However, its contribution to intestinal injury and death after burn trauma is unclear.In this study, wild-type (Nrf2+/+) and Nrf2-deficient (Nrf2-/-) mice were subjected to 15% or 30% total body surface area burn or sham injury. Survival, systemic inflammation, and gut injury were determined.Nrf2-/- mice were more susceptible to burn-induced intestinal injury, as characterized by increases in damage to the gut structure and in intestinal permeability. This exacerbation was associated with an increase in the intestinal mRNA expression of inflammatory cytokines (interleukin [IL]-6, IL-1B, monocyte chemotactic protein 1, intercellular adhesion molecule, and vascular cell adhesion molecule) and a decrease in the intestinal mRNA expression of Nrf2-regulated genes (NAD(P)H dehydrogenasequinine-1 and glutamate-cysteine ligase modifier subunit). Nrf2-deficient mice also showed a lower survival rate and higher levels of systemic cytokines (IL-6 and IL-1B) and high-mobility group protein B1 than wild-type mice. This study demonstrates for the first time that mice that lack Nrf2 are more susceptible to burn-induced intestinal injury and have more systemic inflammation and a lower survival rate. PMID:27009867

  7. Hepatic and Nephric NRF2 Pathway Up-Regulation, an Early Antioxidant Response, in Acute Arsenic-Exposed Mice

    PubMed Central

    Li, Jinlong; Duan, Xiaoxu; Dong, Dandan; Zhang, Yang; Li, Wei; Zhao, Lu; Nie, Huifang; Sun, Guifan; Li, Bing

    2015-01-01

    Inorganic arsenic (iAs), a proven human carcinogen, damages biological systems through multiple mechanisms, one of them being reactive oxygen species (ROS) production. NRF2 is a redox-sensitive transcription factor that positively regulates the genes of encoding antioxidant and detoxification enzymes to neutralize ROS. Although NRF2 pathway activation by iAs has been reported in various cell types, however, the experimental data in vivo are very limited and not fully elucidated in humans. The present investigation aimed to explore the hepatic and nephric NRF2 pathway upregulation in acute arsenic-exposed mice in vivo. Our results showed 10 mg/kg NaAsO2 elevated the NRF2 protein and increased the transcription of Nrf2 mRNA, as well as up-regulated NRF2 downstream targets HO-1, GST and GCLC time- and dose-dependently both in the liver and kidney. Acute NaAsO2 exposure also resulted in obvious imbalance of oxidative redox status represented by the increase of GSH and MDA, and the decrease of T-AOC. The present investigation reveals that hepatic and nephric NRF2 pathway expression is an early antioxidant defensive response upon iAs exposure. A better knowledge about the NRF2 pathway involvment in the cellular response against arsenic could help improve the strategies for reducing the cellular toxicity related to this metalloid. PMID:26473898

  8. Structural and Functional Characterization of Nrf2 Degradation by the Glycogen Synthase Kinase 3/β-TrCP Axis

    PubMed Central

    Rada, Patricia; Rojo, Ana I.; Evrard-Todeschi, Nathalie; Innamorato, Nadia G.; Cotte, Axelle; Jaworski, Tomasz; Tobón-Velasco, Julio C.; Devijver, Herman; García-Mayoral, María Flor; Van Leuven, Fred; Hayes, John D.

    2012-01-01

    The transcription factor NF-E2-related factor 2 (Nrf2) is a master regulator of a genetic program, termed the phase 2 response, that controls redox homeostasis and participates in multiple aspects of physiology and pathology. Nrf2 protein stability is regulated by two E3 ubiquitin ligase adaptors, Keap1 and β-TrCP, the latter of which was only recently reported. Here, two-dimensional (2D) gel electrophoresis and site-directed mutagenesis allowed us to identify two serines of Nrf2 that are phosphorylated by glycogen synthase kinase 3β (GSK-3β) in the sequence DSGISL. Nuclear magnetic resonance studies defined key residues of this phosphosequence involved in docking to the WD40 propeller of β-TrCP, through electrostatic and hydrophobic interactions. We also identified three arginine residues of β-TrCP that participate in Nrf2 docking. Intraperitoneal injection of the GSK-3 inhibitor SB216763 led to increased Nrf2 and heme oxygenase-1 levels in liver and hippocampus. Moreover, mice with hippocampal absence of GSK-3β exhibited increased levels of Nrf2 and phase 2 gene products, reduced glutathione, and decreased levels of carbonylated proteins and malondialdehyde. This study establishes the structural parameters of the interaction of Nrf2 with the GSK-3/β-TrCP axis and its functional relevance in the regulation of Nrf2 by the signaling pathways that impinge on GSK-3. PMID:22751928

  9. Inhibitory effects of geraniin on LPS-induced inflammation via regulating NF-κB and Nrf2 pathways in RAW 264.7 cells.

    PubMed

    Wang, Peng; Qiao, Qi; Li, Ji; Wang, Wei; Yao, Li-Ping; Fu, Yu-Jie

    2016-06-25

    Geraniin, a major polyphenolic compound of Geranium sibiricum L, has long been used as an important Chinese herbal medicine for the treatment of a variety of inflammatory pathologies. However, the underlying anti-inflammatory molecular mechanisms of this compound are not clear. The aim of the present study was to investigate the anti-inflammatory activities of geraniin and elucidate the underlying mechanisms. The anti-inflammatory effects of geraniin were studied by using lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Geraniin suppressed the inducible nitric oxide synthase (iNOS) expression, and inhibited reactive oxygen species (ROS) production. Subsequent studies demonstrated that geraniin effectively reduced production of NO and pro-inflammatory cytokines. These effects were mediated by impaired translocation of nuclear factor (NF)-κB and inhibition of the phosphorylation of Akt in LPS-stimulated RAW 264.7 cells. Furthermore, geraniin induced heme oxygenase-1 (HO-1) expression via activation of transcription factor Nrf2. This study gives scientific evidence that geraniin inhibits the LPS-induced expression of inflammatory mediators via suppression of Akt-mediated NF-κB pathway as well as up-regulation of Nrf2/HO-1 pathway, indicating that geraniin has a potential application in inflammatory conditions. PMID:27181634

  10. Paeonol and danshensu combination attenuates apoptosis in myocardial infarcted rats by inhibiting oxidative stress: Roles of Nrf2/HO-1 and PI3K/Akt pathway

    PubMed Central

    Li, Hua; Song, Fan; Duan, Lin-Rui; Sheng, Juan-Juan; Xie, Yan-Hua; Yang, Qian; Chen, Ying; Dong, Qian-Qian; Zhang, Bang-Le; Wang, Si-Wang

    2016-01-01

    Paeonol and danshensu is the representative active ingredient of traditional Chinese medicinal herbs Cortex Moutan and Radix Salviae Milthiorrhizae, respectively. Paeonol and danshensu combination (PDSS) has putative cardioprotective effects in treating ischemic heart disease (IHD). However, the evidence for the protective effect is scarce and the pharmacological mechanisms of the combination remain unclear. The present study was designed to investigate the protective effect of PDSS on isoproterenol (ISO)-induced myocardial infarction in rats and to elucidate the potential mechanism. Assays of creatine kinase-MB, cardiac troponin I and T and histopathological analysis revealed PDSS significantly prevented myocardial injury induced by ISO. The ISO-induced profound elevation of oxidative stress was also suppressed by PDSS. TUNEL and caspase-3 activity assay showed that PDSS significantly inhibited apoptosis in myocardia. In exploring the underlying mechanisms of PDSS, we found PDSS enhanced the nuclear translocation of Nrf2 in myocardial injured rats. Furthermore, PDSS increased phosphorylated PI3K and Akt, which may in turn activate antioxidative and antiapoptotic signaling events in rat. These present findings demonstrated that PDSS exerts significant cardioprotective effects against ISO-induced myocardial infarction in rats. The protective effect is, at least partly, via activation of Nrf2/HO-1 signaling and involvement of the PI3K/Akt cell survival signaling pathway. PMID:27021411

  11. Dioscin alleviates BDL- and DMN-induced hepatic fibrosis via Sirt1/Nrf2-mediated inhibition of p38 MAPK pathway.

    PubMed

    Gu, Lina; Tao, Xufeng; Xu, Youwei; Han, Xu; Qi, Yan; Xu, Lina; Yin, Lianhong; Peng, Jinyong

    2016-02-01

    Oxidative stress is involved in hepatic stellate cells (HSCs) activation and extracellular matrix overproduction. We previously reported the promising effects of dioscin against CCl4-induced liver fibrosis, but its effects and mechanisms on BDL- and DMN-induced liver fibrosis remain unknown. The results in the present study indicated that dioscin significantly inhibited HSCs activation and attenuated hepatic fibrosis in rats. Furthermore, dioscin markedly up-regulated the levels of sirtuin 1 (Sirt1), HO-1, GST, GCLC and GCLM via increasing the nuclear translocation of nuclear erythroid factor 2-related factor 2 (Nrf2), which in turn inhibited mitogen-activated protein kinase 14 (p38 MAPK) phosphorylation and reduced the levels of COL1A1, COL3A1, α-SMA and fibronectin. These results were further validated by knockdown of Sirt1 and Nrf2 using siRNAs silencing, and abrogation of p38 MAPK using SB-203580 (a p38 MAPK inhibitor) in HSC-T6 and LX-2 cells. Collectively, our findings confirmed the potent effects of dioscin against liver fibrosis and also provided novel insights into the mechanisms of this compound as a candidate for the prevention of liver fibrosis in the future. PMID:26747300

  12. 3,4-Dihydroxyphenylacetic acid, a microbiota-derived metabolite of quercetin, attenuates acetaminophen (APAP)-induced liver injury through activation of Nrf-2.

    PubMed

    Xue, Huiting; Xie, Wenyan; Jiang, Zhihui; Wang, Meng; Wang, Jian; Zhao, Hongqiong; Zhang, Xiaoying

    2016-10-01

    1. Acetaminophen (APAP) overdose leads to severe hepatotoxicity. 3,4-dihydroxyphenylacetic acid (DOPAC) is a scarcely studied microbiota-derived metabolite of quercetin. The aim of this study was to determine the protective effect of DOPAC against APAP-induced liver injury. 2. Mice were treated intragastrically with DOPAC (10, 20 or 50 mg/kg) for 3 days before APAP (300 mg/kg) injection. APAP alone caused increase in serum aminotransferase levels and changes in hepatic histopathology. APAP also promoted oxidative stress by increasing lipid peroxidation and decreasing anti-oxidant enzyme activities. These events led to hepatocellular necrosis and reduced liver function. DOPAC increased nuclear factor erythroid 2-related factor 2 (Nrf-2) translocation to the nucleus and enhanced the expression of phase II enzymes and anti-oxidant enzymes, and thereby reduced APAP hepatotoxicity and enhanced anti-oxidant ability. 3. Our data provide evidence that DOPAC protected the liver against APAP-induced injury, which is involved in Nrf-2 activation, implying that DOPAC can be considered as a potential natural hepatoprotective agent. PMID:26931552

  13. Dieckol enhances the expression of antioxidant and detoxifying enzymes by the activation of Nrf2-MAPK signalling pathway in HepG2 cells.

    PubMed

    Lee, Min-Sup; Lee, Bonggi; Park, Kyoung-Eun; Utsuki, Tadanobu; Shin, Taisun; Oh, Chul Woong; Kim, Hyeung-Rak

    2015-05-01

    Dieckol was previously reported to exhibit antioxidant and anticancer activities in vitro studies. In this study, we characterised the mechanism underlying the dieckol-mediated expression of antioxidant and detoxifying enzymes. Dieckol suppressed the production of intracellular reactive oxygen species in the presence or absence of H2O2 and increased glutathione level in HepG2 cells. Dieckol enhanced the activities of antioxidant enzymes, and the expression of detoxifying enzymes including heme oxygenase-1 (HO-1), NAD(P)H:quinine oxidoreductase 1 (NQO1), and glutathione S-transferase (GST) in HepG2 cells. Enhanced expression of antioxidant and detoxifying enzymes by dieckol was presumed to be the activation of the nuclear factor erythroid-derived 2-like 2 (Nrf2) demonstrated by its nuclear translocation and transcriptional activity via activation of mitogen-activated protein kinases in HepG2 cells. Furthermore, we demonstrated dieckol induced the expression of HO-1 in mouse liver. These results demonstrate that the dieckol-mediated cytoprotection in HepG2 cells is mediated through a ROS-independent up-regulation of antioxidant and detoxifying enzymes via Nrf2 activation as well as its intrinsic antioxidant activity, suggesting that dieckol may be used as a natural cytoprotective agent. PMID:25529716

  14. Protopanaxatriol Ginsenoside Rh1 Upregulates Phase II Antioxidant Enzyme Gene Expression in Rat Primary Astrocytes: Involvement of MAP Kinases and Nrf2/ARE Signaling

    PubMed Central

    Jung, Ji-Sun; Lee, Sang-Yoon; Kim, Dong-Hyun; Kim, Hee-Sun

    2016-01-01

    Oxidative stress activates several intracellular signaling cascades that may have deleterious effects on neuronal cell survival. Thus, controlling oxidative stress has been suggested as an important strategy for prevention and/or treatment of neurodegenerative diseases. In this study, we found that ginsenoside Rh1 inhibited hydrogen peroxide-induced reactive oxygen species generation and subsequent cell death in rat primary astrocytes. Rh1 increased the expression of phase II antioxidant enzymes, such as heme oxygenase-1 (HO-1), NAD(P)H:quinone oxidoreductase 1, superoxide dismutase-2, and catalase, that are under the control of Nrf2/ARE signaling pathways. Further mechanistic studies showed that Rh1 increased the nuclear translocation and DNA binding of Nrf2 and c-Jun to the antioxidant response element (ARE), and increased the ARE-mediated transcription activities in rat primary astrocytes. Analysis of signaling pathways revealed that MAP kinases are important in HO-1 expression, and act by modulating ARE-mediated transcriptional activity. Therefore, the upregulation of antioxidant enzymes by Rh1 may provide preventive therapeutic potential for various neurodegenerative diseases that are associated with oxidative stress. PMID:26759699

  15. Synergistic cardioprotective effects of Danshensu and hydroxysafflor yellow A against myocardial ischemia-reperfusion injury are mediated through the Akt/Nrf2/HO-1 pathway

    PubMed Central

    HU, TIANXIN; WEI, GUO; XI, MIAOMIAO; YAN, JIAJIA; WU, XIAOXIAO; WANG, YANHUA; ZHU, YANRONG; WANG, CHAO; WEN, AIDONG

    2016-01-01

    In clinical practice, the traditional Chinese medicinal herbs, Radix Salvia Miltiorrhiza and Carthamus tinctorius L., are usually prescribed in combination due to their significant cardioprotective effects. However, the mechanisms responsible for these combined effects remain unknown. Thus, in this study, we investigated the mechanisms responsible for the combined effects of Danshensu (DSS) and hydroxysafflor yellow A (HSYA) by establishing a rat model of myocardial ischemia/reperfusion (MI/R), as well as a model of hypoxia/reoxygenation (H/R) using H9c2 cells. The combination index (CI) was calculated using the median-effect method. DSS and HSYA in combination led to a CI value of <1 as regards infarct size in vivo and cell viability in vitro. The rats with MI/R injury that were treated with DSS and/or HSYA were found to have significantly lower levels of creatine kinase-MB (CK-MB) and cardiac troponin I (cTnI) and malondialdehyde (MDA), and a lower expressoin of 8-hydroxydeoxyguanosine (8-OHdG), and markedly enhanced superoxide dismutase (SOD) activity. Our in vitro experiments revealed that the cells treated with DSS and/or HSYA had a reduced lactate dehydrogenase (LDH) activity and a decreased percentage of cell apoptosis (increased Bcl-2/Bax ratio, decreased expression of cleaved caspase-3). DSS and HSYA increased the expression of heme oxygenase-1 (HO-1), the phosphorylation of Akt and the trans-location of nuclear factor erythroid 2-related factor 2 (Nrf2). Furthermore, the Akt inhibitor, LY294002, partially hampered the expression of Nrf2 and HO-1. The HO-1 inhibitor, zinc protoporphyrin IX (ZnPP-IX), did not decrease the expression of p-Akt and Nrf2, although it abolished the anti-apoptotic and antioxidant effects of DSS and HSYA. The findings of our study thus demonstrate that DSS and HSYA confer synergistic cardioprotective effects through the Akt/Nrf2/HO-1 signaling pathway, to certain extent, by enhancing the antioxidant defense system and exerting

  16. Synergistic cardioprotective effects of Danshensu and hydroxysafflor yellow A against myocardial ischemia-reperfusion injury are mediated through the Akt/Nrf2/HO-1 pathway.

    PubMed

    Hu, Tianxin; Wei, Guo; Xi, Miaomiao; Yan, Jiajia; Wu, Xiaoxiao; Wang, Yanhua; Zhu, Yanrong; Wang, Chao; Wen, Aidong

    2016-07-01

    In clinical practice, the traditional Chinese medicinal herbs, Radix Salvia Miltiorrhiza and Carthamus tinctorius L., are usually prescribed in combination due to their significant cardioprotective effects. However, the mechanisms responsible for these combined effects remain unknown. Thus, in this study, we investigated the mechanisms responsible for the combined effects of Danshensu (DSS) and hydroxysafflor yellow A (HSYA) by establishing a rat model of myocardial ischemia/reperfusion (MI/R), as well as a model of hypoxia/reoxygenation (H/R) using H9c2 cells. The combination index (CI) was calculated using the median-effect method. DSS and HSYA in combination led to a CI value of <1 as regards infarct size in vivo and cell viability in vitro. The rats with MI/R injury that were treated with DSS and/or HSYA were found to have significantly lower levels of creatine kinase-MB (CK-MB) and cardiac troponin I (cTnI) and malondialdehyde (MDA), and a lower expressoin of 8-hydroxydeoxyguanosine (8-OHdG), and markedly enhanced superoxide dismutase (SOD) activity. Our in vitro experiments revealed that the cells treated with DSS and/or HSYA had a reduced lactate dehydrogenase (LDH) activity and a decreased percentage of cell apoptosis (increased Bcl-2/Bax ratio, decreased expression of cleaved caspase-3). DSS and HSYA increased the expression of heme oxygenase-1 (HO-1), the phosphorylation of Akt and the translocation of nuclear factor erythroid 2-related factor 2 (Nrf2). Furthermore, the Akt inhibitor, LY294002, partially hampered the expression of Nrf2 and HO-1. The HO-1 inhibitor, zinc protoporphyrin IX (ZnPP‑IX), did not decrease the expression of p-Akt and Nrf2, although it abolished the anti-apoptotic and antioxidant effects of DSS and HSYA. The findings of our study thus demonstrate that DSS and HSYA confer synergistic cardioprotective effects through the Akt/Nrf2/HO-1 signaling pathway, to certain extent, by enhancing the antioxidant

  17. Disulfiram targeting lymphoid malignant cell lines via ROS-JNK activation as well as Nrf2 and NF-kB pathway inhibition

    PubMed Central

    2014-01-01

    Background Disulfiram (DS), an anti-alcoholism drug, demonstrates strong antitumor activity in a copper (Cu)-dependent manner. This study investigates the cytotoxicity of DS/Cu complex in lymphoid malignant cell lines in vitro and in vivo. Method Raji cells were subjected to different treatments and thereafter MTT assay, flow cytometry were used to determine IC50 and apoptotic status. We also tested the cytotoxicity of DS/Cu in acute lymphoblastic leukemia cell line Molt4 in vitro. In vivo experiments were also performed to demonstrate the anticancer efficacy of DS/Cu in Raji cells xenografted nude mice. Results In combination with a low concentration (1 μM) of Cu2+, DS induced cytotoxicity in Raji cells with an IC50 of 0.085 ± 0.015 μM and in Molt4 cells with an IC50 of 0.435 ± 0.109 μM. The results of our animal experiments also showed that the mean tumor volume in DS/Cu-treated mice was significantly smaller than that in DS or control group, indicating that DS/Cu inhibits the proliferation of Raji cells in vivo. DS/Cu also induced apoptosis in 2 lymphoid malignant cell lines. After exposure to DS (3.3 μM)/Cu (1 μM) for 24 hours, apoptosis was detected in 81.03 ± 7.91% of Raji cells. DS/Cu induced significant apoptosis in a concentration-dependent manner with the highest apoptotic proportion (DS/Cu: 89.867 ± 4.69%) at a concentration of 2 μM in Molt4 cells. After 24 h exposure, DS/Cu inhibits Nrf2 expression. Flow cytometric analysis shows that DS/Cu induced ROS generation. DS/Cu induced phosphorylation of JNK and inhibits p65 expression as well as Nrf2 expression both in vitro and in vivo. N-acetyl-L-cysteine (NAC), an antioxidant, can partially attenuate DS/Cu complex-induced apoptosis and block JNK activation in vitro. In addition, NAC is able to restore Nrf2 nuclear translocation and p65 expression. Conclusion Our study manifests that DS/Cu complex targets lymphoid malignant cells in vitro and in vivo. Generation of ROS might

  18. Nrf2 activators modulate oxidative stress responses and bioenergetic profiles of human retinal epithelial cells cultured in normal or high glucose conditions.

    PubMed

    Foresti, Roberta; Bucolo, Claudio; Platania, Chiara Maria Bianca; Drago, Filippo; Dubois-Randé, Jean-Luc; Motterlini, Roberto

    2015-09-01

    Retinal pigment epithelial cells exert an important supporting role in the eye and develop adaptive responses to oxidative stress or high glucose levels, as observed during diabetes. Endogenous antioxidant defences are mainly regulated by Nrf2, a transcription factor that is activated by naturally-derived and electrophilic compounds. Here we investigated the effect of the Nrf2 activators dimethylfumarate (DMF) and carnosol on antioxidant pathways, oxygen consumption rate and wound healing in human retinal pigment epithelial cells (ARPE-19) cultured in medium containing normal (NG, 5mM) or high (HG, 25 mM) glucose levels. We also assessed wound healing using an in vivo corneal epithelial injury model. We found that Nrf2 nuclear translocation and heme oxygenase activity increased in ARPE cells treated with 10 μM DMF or carnosol irrespective of glucose culture conditions. However, HG rendered retinal cells more sensitive to regulators of glutathione synthesis or inhibition and caused a decrease of both cellular and mitochondrial reactive oxygen species. Culture in HG also reduced ATP production and mitochondrial function as measured with the Seahorse XF analyzer and electron microscopy analysis revealed morphologically damaged mitochondria. Acute treatment with DMF or carnosol did not restore mitochondrial function in HG cells; conversely, the compounds reduced cellular maximal respiratory and reserve capacity, which were completely prevented by N-acetylcysteine thus suggesting the involvement of thiols in this effect. Interestingly, the scratch assay showed that wound closure was faster in cells cultured in HG than NG and was accelerated by carnosol. This effect was reversed by an inhibitor of heme oxygenase activity. Moreover, topical application of carnosol to the cornea of diabetic rats significantly accelerated wound healing. In summary, these data indicate that culture of retinal epithelial cells in HG does not affect the activation of the Nrf2/heme oxygenase

  19. Hyaluronic acid regulates a key redox control factor Nrf2 via phosphorylation of Akt in bovine articular chondrocytes

    PubMed Central

    Onodera, Yuta; Teramura, Takeshi; Takehara, Toshiyuki; Fukuda, Kanji

    2015-01-01

    One important pharmacological function of hyaluronic acid (HA) in chondrocytes is reduction of cellular superoxide generation and accumulation. Here we demonstrated a relationship between HA supplementation and accumulation of Nuclear factor-erythroid-2-related factor 2 (Nrf2), which is a master transcription factor in cellular redox reactions, in cultured chondrocytes derived from bovine joint cartilage. In HA-treated chondrocytes, expression of Nrf2 and its downstream genes was upregulated. In HA-treated chondrocytes, Akt was phosphorylated, and inhibition of Akt activity or suppression of HA receptors CD44 and/or RHAMM with siRNAs prevented HA-mediated Nrf2 accumulation. Furthermore, Nrf2 siRNA inhibited the HA effect on antioxidant enzymes. These results show that HA might contribute to ROS reduction through Nrf2 regulation by activating Akt. Our study suggests a new mechanism for extracellular matrix (ECM)-mediated redox systems in chondrocytes. PMID:26106522

  20. Nutritional strategies to modulate inflammation and oxidative stress pathways via activation of the master antioxidant switch Nrf2.

    PubMed

    Cardozo, Ludmila F M F; Pedruzzi, Liliana M; Stenvinkel, Peter; Stockler-Pinto, Milena B; Daleprane, Julio B; Leite, Maurilo; Mafra, Denise

    2013-08-01

    The nuclear factor E2-related factor 2 (Nrf2) plays an important role in cellular protection against cancer, renal, pulmonary, cardiovascular and neurodegenerative diseases where oxidative stress and inflammation are common conditions. The Nrf2 regulates the expression of detoxifying enzymes by recognizing the human Antioxidant Response Element (ARE) binding site and it can regulate antioxidant and anti-inflammatory cellular responses, playing an important protective role on the development of the diseases. Studies designed to investigate how effective Nrf2 activators or modulators are need to be initiated. Several recent studies have shown that nutritional compounds can modulate the activation of Nrf2-Keap1 system. This review aims to discuss some of the key nutritional compounds that promote the activation of Nrf2, which may have impact on the human health. PMID:23643732

  1. NRF2 Oxidative Stress Induced by Heavy Metals is Cell Type Dependent

    EPA Science Inventory

    Exposure to metallic environmental toxicants has been demonstrated to induce a variety of oxidative stress responses in mammalian cells. The transcription factor Nrf2 is activated in response to oxidative stress and coordinates the expression of antioxidant gene products. In this...

  2. The Nrf2-antioxidant response element pathway: a target for regulating energy metabolism

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The nuclear factor E2-related factor 2 (Nrf2) is a transcription factor that responds to oxidative stress by binding to the antioxidant response element (ARE) in the promoter of genes coding for antioxidant enzymes like NAD(P)H:quinone oxidoreductase 1 (NQO1) and proteins for glutathione synthesis. ...

  3. Nrf2 reduces levels of phosphorylated tau protein by inducing autophagy adaptor protein NDP52

    NASA Astrophysics Data System (ADS)

    Jo, Chulman; Gundemir, Soner; Pritchard, Susanne; Jin, Youngnam N.; Rahman, Irfan; Johnson, Gail V. W.

    2014-03-01

    Nuclear factor erythroid 2-related factor 2 (Nrf2) is a pivotal transcription factor in the defence against oxidative stress. Here we provide evidence that activation of the Nrf2 pathway reduces the levels of phosphorylated tau by induction of an autophagy adaptor protein NDP52 (also known as CALCOCO2) in neurons. The expression of NDP52, which we show has three antioxidant response elements (AREs) in its promoter region, is strongly induced by Nrf2, and its overexpression facilitates clearance of phosphorylated tau in the presence of an autophagy stimulator. In Nrf2-knockout mice, phosphorylated and sarkosyl-insoluble tau accumulates in the brains concurrent with decreased levels of NDP52. Moreover, NDP52 associates with phosphorylated tau from brain cortical samples of Alzheimer disease cases, and the amount of phosphorylated tau in sarkosyl-insoluble fractions is inversely proportional to that of NDP52. These results suggest that NDP52 plays a key role in autophagy-mediated degradation of phosphorylated tau in vivo.

  4. Value of monitoring Nrf2 activity for the detection of chemical and oxidative stress

    PubMed Central

    Mutter, Fiona E.; Park, B. Kevin; Copple, Ian M.

    2015-01-01

    Beyond specific limits of exposure, chemical entities can provoke deleterious effects in mammalian cells via direct interaction with critical macromolecules or by stimulating the accumulation of reactive oxygen species (ROS). In particular, these chemical and oxidative stresses can underpin adverse reactions to therapeutic drugs, which pose an unnecessary burden in the clinic and pharmaceutical industry. Novel pre-clinical testing strategies are required to identify, at an earlier stage in the development pathway, chemicals and drugs that are likely to provoke toxicity in humans. Mammalian cells can adapt to chemical and oxidative stress via the action of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), which up-regulates the expression of numerous cell defence genes and has been shown to protect against a variety of chemical toxicities. Here, we provide a brief overview of the Nrf2 pathway and summarize novel experimental models that can be used to monitor changes in Nrf2 pathway activity and thus understand the functional consequences of such perturbations in the context of chemical and drug toxicity. We also provide an outlook on the potential value of monitoring Nrf2 activity for improving the pre-clinical identification of chemicals and drugs with toxic liability in humans. PMID:26551708

  5. Involvement of NRF2 Signaling in Doxorubicin Resistance of Cancer Stem Cell-Enriched Colonospheres

    PubMed Central

    Ryoo, In-geun; Kim, Geon; Choi, Bo-hyun; Lee, Sang-hwan; Kwak, Mi-Kyoung

    2016-01-01

    Cancer stem cells (CSCs) are a subset of tumor cells, which are characterized by resistance against chemotherapy and environmental stress, and are known to cause tumor relapse after therapy. A number of molecular mechanisms underlie the chemoresistance of CSCs, including high expression levels of drug efflux transporters. We investigated the role of the antioxidant transcription factor NF-E2-related factor 2 (NRF2) in chemoresistance development, using a CSC-enriched colonosphere system. HCT116 colonospheres were more resistant to doxorubicin-induced cell death and expressed higher levels of drug efflux transporters such as P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP) compared to HCT116 monolayers. Notably, levels of NRF2 and expression of its target genes were substantially elevated in colonospheres, and these increases were linked to doxorubicin resistance. When NRF2 expression was silenced in colonospheres, Pgp and BCRP expression was downregulated, and doxorubicin resistance was diminished. Collectively, these results indicate that NRF2 activation contributes to chemoresistance acquisition in CSC-enriched colonospheres through the upregulation of drug efflux transporters. PMID:27582554

  6. Involvement of NRF2 Signaling in Doxorubicin Resistance of Cancer Stem Cell-Enriched Colonospheres.

    PubMed

    Ryoo, In-Geun; Kim, Geon; Choi, Bo-Hyun; Lee, Sang-Hwan; Kwak, Mi-Kyoung

    2016-09-01

    Cancer stem cells (CSCs) are a subset of tumor cells, which are characterized by resistance against chemotherapy and environmental stress, and are known to cause tumor relapse after therapy. A number of molecular mechanisms underlie the chemoresistance of CSCs, including high expression levels of drug efflux transporters. We investigated the role of the antioxidant transcription factor NF-E2-related factor 2 (NRF2) in chemoresistance development, using a CSC-enriched colonosphere system. HCT116 colonospheres were more resistant to doxorubicin-induced cell death and expressed higher levels of drug efflux transporters such as P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP) compared to HCT116 monolayers. Notably, levels of NRF2 and expression of its target genes were substantially elevated in colonospheres, and these increases were linked to doxorubicin resistance. When NRF2 expression was silenced in colonospheres, Pgp and BCRP expression was downregulated, and doxorubicin resistance was diminished. Collectively, these results indicate that NRF2 activation contributes to chemoresistance acquisition in CSC-enriched colonospheres through the upregulation of drug efflux transporters. PMID:27582554

  7. Nrf2 Expression Modifies Influenza A Entry and Replication inNasal Epithelial Cells

    EPA Science Inventory

    Influenza infection is a major cause of morbidity and mortality worldwide, especially during pandemics outbreaks. Emerging data indicate that phase II antioxidant enzyme pathways could playa role in virus-associated inflammation and immune clearance. While Nrf2-dependent gene exp...

  8. Nrf2/ARE Pathway Involved in Oxidative Stress Induced by Paraquat in Human Neural Progenitor Cells.

    PubMed

    Dou, Tingting; Yan, Mengling; Wang, Xinjin; Lu, Wen; Zhao, Lina; Lou, Dan; Wu, Chunhua; Chang, Xiuli; Zhou, Zhijun

    2016-01-01

    Compelling evidences have shown that diverse environmental insults arising during early life can either directly lead to a reduction in the number of dopaminergic neurons or cause an increased susceptibility to neurons degeneration with subsequent environmental insults or with aging alone. Oxidative stress is considered the main effect of neurotoxins exposure. In this study, we investigated the oxidative stress effect of Paraquat (PQ) on immortalized human embryonic neural progenitor cells by treating them with various concentrations of PQ. We show that PQ can decrease the activity of SOD and CAT but increase MDA and LDH level. Furthermore, the activities of Cyc and caspase-9 were found increased significantly at 10 μM of PQ treatment. The cytoplasmic Nrf2 protein expressions were upregulated at 10 μM but fell back at 100 μM. The nuclear Nrf2 protein expressions were upregulated as well as the downstream mRNA expressions of HO-1 and NQO1 in a dose-dependent manner. In addition, the proteins expression of PKC and CKII was also increased significantly even at 1 μM. The results suggested that Nrf2/ARE pathway is involved in mild to moderate PQ-induced oxidative stress which is evident from dampened Nrf2 activity and low expression of antioxidant genes in PQ induced oxidative damage. PMID:26649146

  9. Lack of Nrf2 reduces voluntary exercise in mice: influences of sex and diet

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Exercise is generally accepted to increase the generation of reactive oxygen species (ROS). However, the regulatory mechanisms that are involved in the adaptations occurring during exercise are not well understood. The Nrf2/antioxidant response element pathway adapts cells to elevated ROS. We tested...

  10. Protective effects of 6-hydroxy-1-methylindole-3-acetonitrile on cisplatin-induced oxidative nephrotoxicity via Nrf2 inactivation.

    PubMed

    Moon, Ji Hee; Shin, Ji-Sun; Kim, Jong-Bin; Baek, Nam-In; Cho, Young-Wuk; Lee, Yong Sup; Kay, Hee Yeon; Kim, Soo-dong; Lee, Kyung-Tae

    2013-12-01

    We previously demonstrated the ethanol extract of the roots of Brassica rapa protects against cisplatin-induced nephrotoxicity by attenuating oxidative stress. Here, we investigated the nephroprotective effects of 6-hydroxy-1-methylindole-3-acetonitrile (6-HMA), which was isolated from the roots of B. rapa, on cisplatin-induced toxicity in renal epithelial LLC-PK1 cells and in rats with acute renal injury. Pretreatment of LLC-PK1 cells with 6-HMA ameliorated cisplatin-induced cytotoxicity caused by oxidative stress, as was demonstrated by reductions in the levels of reactive oxygen species (ROS) and malondialdehyde (MDA) and increased levels of glutathione (GSH). In addition, 6-HMA inhibited cisplatin-induced heme oxygenase-1 (HO-1) expression, possibly due to the suppression of the nuclear translocation and binding activity of NF-E2-related factor 2 (Nrf2). Furthermore, 6-HMA administered rats showed lower levels of blood urea nitrogen (BUN), creatinine, and urinary lactate dehydrogenase (LDH) than cisplatin alone-treated rats in cisplatin-induced renal injury model. Moreover, 6-HMA inhibited the cisplatin-induced formation of MDA and GSH depletion and increased the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione reductase (GR). Taken together, these findings indicate 6-HMA is a major active constituent from the roots of B. rapa to have a protective effect against cisplatin-induced nephrotoxicity by attenuating oxidative stress. PMID:23989062

  11. Genetic and Pharmacologic Targeting of Glycogen Synthase Kinase 3β Reinforces the Nrf2 Antioxidant Defense against Podocytopathy.

    PubMed

    Zhou, Sijie; Wang, Pei; Qiao, Yingjin; Ge, Yan; Wang, Yingzi; Quan, Songxia; Yao, Ricky; Zhuang, Shougang; Wang, Li Juan; Du, Yong; Liu, Zhangsuo; Gong, Rujun

    2016-08-01

    Evidence suggests that the glycogen synthase kinase 3 (GSK3)-dictated nuclear exclusion and degradation of Nrf2 is pivotal in switching off the self-protective antioxidant stress response after injury. Here, we examined the mechanisms underlying this regulation in glomerular disease. In primary podocytes, doxorubicin elicited cell death and actin cytoskeleton disorganization, concomitant with overactivation of GSK3β (the predominant GSK3 isoform expressed in glomerular podocytes) and minimal Nrf2 activation. SB216763, a highly selective small molecule inhibitor of GSK3, exerted a protective effect that depended on the potentiated Nrf2 antioxidant response, marked by increased Nrf2 expression and nuclear accumulation and augmented production of the Nrf2 target heme oxygenase-1. Ectopic expression of the kinase-dead mutant of GSK3β in cultured podocytes reinforced the doxorubicin-induced Nrf2 activation and prevented podocyte injury. Conversely, a constitutively active GSK3β mutant blunted the doxorubicin-induced Nrf2 response and exacerbated podocyte injury, which could be abolished by treatment with SB216763. In murine models of doxorubicin nephropathy or nephrotoxic serum nephritis, genetic targeting of GSK3β by doxycycline-inducible podocyte-specific knockout or pharmacologic targeting by SB216763 significantly attenuated albuminuria and ameliorated histologic signs of podocyte injury, including podocytopenia, loss of podocyte markers, podocyte de novo expression of desmin, and ultrastructural lesions of podocytopathy (such as foot process effacement). This beneficial outcome was likely attributable to an enhanced Nrf2 antioxidant response in glomerular podocytes because the selective Nrf2 antagonist trigonelline abolished the proteinuria-reducing and podocyte-protective effect. Collectively, our results suggest the GSK3β-regulated Nrf2 antioxidant response as a novel therapeutic target for protecting podocytes and treating proteinuric glomerulopathies. PMID

  12. Cancer related mutations in NRF2 impair its recognition by Keap1-Cul3 E3 ligase and promote malignancy

    PubMed Central

    Shibata, Tatsuhiro; Ohta, Tsutomu; Tong, Kit I.; Kokubu, Akiko; Odogawa, Reiko; Tsuta, Koji; Asamura, Hisao; Yamamoto, Masayuki; Hirohashi, Setsuo

    2008-01-01

    The nuclear factor E2-related factor 2 (Nrf2) is a master transcriptional activator of genes encoding numerous cytoprotective enzymes that are induced in response to environmental and endogenously derived oxidative/electrophilic agents. Under normal, nonstressed circumstances, low cellular concentrations of Nrf2 are maintained by proteasomal degradation through a Keap1-Cul3-Roc1-dependent mechanism. A model for Nrf2 activation has been proposed in which two amino-terminal motifs, DLG and ETGE, promote efficient ubiquitination and rapid turnover; known as the two-site substrate recognition/hinge and latch model. Here, we show that in human cancer, somatic mutations occur in the coding region of NRF2, especially among patients with a history of smoking or suffering from squamous cell carcinoma; in the latter case, this leads to poor prognosis. These mutations specifically alter amino acids in the DLG or ETGE motifs, resulting in aberrant cellular accumulation of Nrf2. Mutant Nrf2 cells display constitutive induction of cytoprotective enzymes and drug efflux pumps, which are insensitive to Keap1-mediated regulation. Suppression of Nrf2 protein levels by siRNA knockdown sensitized cancer cells to oxidative stress and chemotherapeutic reagents. Our results strongly support the contention that constitutive Nrf2 activation affords cancer cells with undue protection from their inherently stressed microenvironment and anti-cancer treatments. Hence, inactivation of the Nrf2 pathway may represent a therapeutic strategy to reinforce current treatments for malignancy. Congruously, the present study also provides in vivo validation of the two-site substrate recognition model for Nrf2 activation by the Keap1-Cul3-based E3 ligase. PMID:18757741

  13. Effects of different exercise durations on Keap1-Nrf2-ARE pathway activation in mouse skeletal muscle.

    PubMed

    Li, T; He, S; Liu, S; Kong, Z; Wang, J; Zhang, Y

    2015-10-01

    The purpose of this study was to investigate the effects of acute exercise stress on the nuclear factor-erythroid2 p45-related factor 2 (Nrf2)/antioxidant response element (ARE) transactivation, Kelch-like ECH-associated protein 1 (Keap1) cytosolic protein and Nrf2 nucleoprotein expressions, Nrf2 target genes mRNA expressions, and glutathione redox (GSH/GSSG) ratio level; with a particular focus on the changes in Keap1-Nrf2-ARE pathway activation following different durations of exercise. Wild-type mice (C57BL/6J, two months old) were separated into one-hour and six-hour treadmill running groups, as well as a non-exercise control group (n = 10 in each group). Measurements of Nrf2/ARE transactivation, Nrf2 nucleoprotein expressions, Keap1 cytosolic protein expression, Nrf2 target genes' mRNA expressions (superoxide dismutase-1 [SOD1], superoxide dismutase-2 [SOD2], γ-glutamyl cysteine ligase-modulatory [GCLm], γ-glutamyl cysteine ligase-catalytic [GCLc], glutathione reductase [GR], glutathione peroxidase-1 [Gpx1], catalase [CAT], and hemoxygenase-1 [Ho-1]), and GSH/GSSG ratio were carried out immediately after exercise. The results showed significant increases in Keap1-Nrf2-ARE pathway activation and the mRNA expressions of six measured enzymes in skeletal muscle after six hours of exercise; while in the one-hour exercise group, there was no change in Keap1-Nrf2-ARE pathway activation and only two enzymes' mRNA expressions were increased. It is suggested that the changes in Keap1-Nrf2-ARE pathway activation and its target genes' mRNA expressions were dependent on the exercise duration, with longer duration associated with higher responses. PMID:26118597

  14. ROLE OF NRF2 IN THE OXIDATIVE STRESS-DEPENDENT HYPERTENSION ASSOCIATED WITH THE DEPLETION OF DJ-1

    PubMed Central

    Cuevas, Santiago; Yang, Yu; Konkalmatt, Prasad; Asico, Laureano; Feranil, Jun; Jones, John; Villar, Van Anthony; Armando, Ines; Jose, Pedro A.

    2015-01-01

    Renal dopamine 2 receptor dysfunction is associated with oxidative stress and high blood pressure. We have reported that DJ-1, an oxidative stress response protein, is positively regulated by dopamine 2 receptor in the kidney. The transcription factor Nrf2 regulates the expression of several antioxidant genes. We tested the hypothesis that Nrf2 is involved in the renal DJ-1-mediated inhibition of reactive oxygen species production. We have reported that silencing dopamine 2 receptor in mouse renal proximal tubule cells decreases the expression of DJ-1. We now report that silencing DJ-1 or dopamine 2 receptor in mouse proximal tubule cells and mouse kidneys, decreases Nrf2 expression and activity and increases reactive oxygen species production; blood pressure is also increased in mice in which renal DJ-1 or dopamine 2 receptor is silenced. DJ-1−/− mice have decreased renal Nrf2 expression and activity, and increased nitro-tyrosine levels an dopamine 2 receptor d blood pressure. Silencing Nrf2 in mouse proximal tubule cells does not alter the expression of DJ-1 or dopamine 2 receptor, indicating that Nrf2 is downstream of dopamine 2 receptor and DJ-1. A Nrf2 inducer, bardoxolone, normalizes the systolic blood pressure and renal malondialdehyde levels in DJ-1−/− mice without affecting them in their wild-type littermates. Because Nrf2 ubiquitination is increased in DJ-1−/− mice, we conclude that the protective effect of DJ-1 on renal oxidative stress is mediated, in part, by preventing Nrf2 degradation. Moreover, renal dopamine 2 receptor and DJ-1 are necessary for normal Nrf2 activity to keep a normal redox balance and blood pressure. PMID:25895590

  15. Fenofibrate activates Nrf2 through p62-dependent Keap1 degradation

    SciTech Connect

    Park, Jeong Su; Kang, Dong Hoon; Lee, Da Hyun; Bae, Soo Han

    2015-09-25

    Peroxisome proliferator-activated receptor α (PPARα) activates the β-oxidation of fatty acids in the liver. Fenofibrate is a potent agonist of PPARα and is used in the treatment of hyperlipidemia. Fenofibrate treatment often induces the production of intracellular reactive oxygen species (ROS), leading to cell death. The nuclear factor erythroid 2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1