Antioxidant Response Element Sequence Research Articles

P62/SQSTM1 upregulates NQO1 transcription via Nrf2/Keap1a signaling pathway to resist microcystins-induced oxidative stress in freshwater mussel Cristaria plicata

Microcystins (MCs) are the most frequent and widely distributed type of cyanotoxin in aquatic systems, and they cause an imbalance of the body's oxidative system. In a previous experiment, we demonstrated that the mollusk Cristaria plicata can protect against MC-induced oxidative damage through the nuclear factor erythroid 2-related factor 2(Nrf2)/Kelch-like epichlorohydrin-related protein-1 (Keap1) pathway. Here, we evaluated whether selective autophagy affects the Nrf2/Keap1a anti-oxidative stress pathway in C. plicata. Full-length cDNA sequences of p62/SQSTM1 from C. plicata (Cpp62) were divided into 2484 bp fragments. From N-terminal to C-terminal, the amino acid sequence of Cpp62 contained PB1 (Phox and Bem1p domain), ZNF (zinc finger domain) chain, LIR (LC3 interacting region) and UBA (ubiquitin-associated domain) domains, but not the KIR (Keap1 interacting region) domain. We confirmed that Cpp62 did not bind to CpKeap1a in vitro, and the relative level of Cpp62 was the highest in the hepatopancreas. Moreover, MCs significantly upregulated the mRNA and protein levels of Cpp62 in the hepatopancreas after CpKeap1a knockdown, whereas Nrf2 upregulated the transcription levels of Cpp62, suggesting that MCs increased Cpp62 expression via the Nrf2/Keap1a signaling pathway. Moreover, Cpp62 and CpNrf2 proteins have a strong affinity for the NQO1 promoter, but MCs inhibited the ability of CpNrf2 and Cpp62 to upregulate luciferase activity. The results show that Nrf2 and the p62 protein induced p62 expression by binding to ARE (antioxidant response element) sequences in the p62 promoter of C. plicata, thereby promoting p62 to resist MC-induced oxidative stress. Therefore, we speculate that MCs induce p62-dependent autophagy in C. plicata, resulting in the inhibition of Nrf2 transcription and Cpp62 promoter activity. These findings help to reveal the mechanism by which the p62-Nrf2/Keap1 pathway mitigates MC-induced oxidative damage in mussels.

Vitamin D Impacts Nrf‐2 Signaling and Antioxidation Pathways that Modulate Cellular Aging

Recent studies suggest that the biologically active form of vitamin D, 1,25‐dihydroxyvitamin D (1,25D), plays a role in potentially delaying the aging process, functions as an anti‐carcinogenic agent, and also prevents oxidative damage. 1,25D binding to the vitamin D receptor (VDR) promotes heterodimerization of VDR with the retinoid X receptor (RXR) to stimulate transcription of numerous vitamin D target genes. The VDR‐RXR complex controls transcriptional activity targeting various tissues such as the kidney and colon, regulates immune defenses, and controls cellular proliferation. The nuclear factor (erythroid‐derived 2)‐like 2 (Nrf‐ 2), is a transcriptional regulator that stimulates expression of genes encoding antioxidant enzymes via a DNA sequence referred to as an antioxidant‐responsive element (ARE). Nutraceuticals such as urolithin‐A have been proposed to play a role in this pathway. The current study was designed to investigate how vitamin D‐VDR signaling influences Nrf‐2 activity. Therefore, we utilized an ARE‐luciferase reporter plasmid to measure Nrf‐2 activity in human embryonic kidney cells (HEK‐293) in the presence of VDR. Our results indicate that VDR does not target genes with an ARE sequence directly. Instead, when cells were transfected with both Nrf‐2 and VDR, Nrf‐2 activity was significantly inhibited in a 1,25D‐ and VDR‐dependent manner. However, when cells were treated with urolithin‐A alone, or with vitamin D in conjunction with urolithin‐A, a nutraceutical that is hypothesized to cooperate with vitamin D, Nrf‐2 activity was upregulated. Additionally, real‐time quantitative polymerase chain reaction (qPCR) was employed to test the expression levels of GCLC and HMOX1 (two Nrf‐2 target genes), in the absence and presence of 1,25D, to assess if the mRNA expression results were consistent with the luciferase reporter assays. Thus, the current study reveals that VDR likely targets Nrf‐2 genes indirectly by influencing the activity of the Nrf‐2 transfactor. Collectively, our results demonstrate, for the first time, that VDR either binds directly to the Nrf‐2 protein to inhibit its activity, or 1,25D‐VDR represses Nrf‐2 gene expression resulting in a diminution of Nrf‐2 protein production. Modulation of Nrf‐2 activity by the vitamin D pathway appears to have a regulatory role in anti‐oxidation and cellular aging pathways.Support or Funding InformationNational Institutes of Health; grant numbers: DK033351 and CA140285.

Genetic Activation of NRF2 By KEAP1 Inhibition Induces Fetal Hemoglobin Expression and Triggers Anti-Oxidant Stress Response in Erythroid Cells

Nuclear factor (erythroid derived-2)-like 2 (NRF2), a basic leucine zipper transcription factor, is sequestered in the cell cytosol by Keap1, a kelch domain protein. Under steady state, this interaction results in ubiquitination and proteasomal degradation of the NRF2 protein. Modification of critical cysteine residues leads to conformational changes in KEAP1, resulting in nuclear translocation of newly synthesized NRF2 and modulation of downstream gene expression. NRF2 activation by compounds that modify KEAP1 such as dimethyl fumarate has been shown to induce γ-globin in erythroid cell systems. Moreover, NRF2 alleviates oxidative stress associated with SCD by activating antioxidant enzymes including catalase, glutathione peroxidase and superoxide dismutase (SOD), that scavenge free radicals (Chirico and Pialoux 2012, Belcher, Chen et al. 2017, Krishnamoorthy, Pace et al. 2017). In the present work, we employed CRISPR Cas9 mediated knockout (KO) of KEAP1 in HUDEP2 cells and CD34+ derived erythroid cells to study its impact on downstream gene and protein expression relevant to sickle cell disease such as fetal hemoglobin (HbF) induction and anti-oxidant stress responses. Genetic KO of KEAP1 by six different gRNAs resulted in populations with indel levels from 30 to 95%, as determined by a mutation specific digital droplet PCR (ddPCR) and next-generation sequencing (NGS). LC/MS confirmed KEAP1 knockdown at the protein level. Consequently, NRF2 protein level in the nuclear extract was elevated in KEAP1 KO cells as demonstrated by nuclear NRF2 bound to antioxidant response element (ARE) in a DNA-binding assay. qPCR analysis revealed a robust and significant induction of the NRF2 dependent enzyme, NAD(P)H quinone dehydrogenase 1 (NQO1) and γ-globin. Gene expression levels of NQO1 and γ-globin inductions correlated significantly with the indel percentages; cell populations with ~80% indels showed about 20-fold induction in NQO1, and about 10-fold induction in γ-globin gene expression levels. Upon differentiation for 7 days in culture, KEAP1 KO cells showed up to 4-fold induction in γ-globin protein levels measured by flow cytometry and HPLC. Up-regulation of HbF and NQO1 protein were confirmed using LC/MS analysis. Additionally, KEAP1 KO clonal populations continued to show robust induction of NQO1 and HbF at gene and protein expression levels. To elucidate the mechanism of γ-globin induction, chromatin immunoprecipitation (ChIP) analysis in the KEAP1 KO cells demonstrated enrichment of NRF2 recruitment in the ARE sequences of the promoter regions of NQO1 and γ-globin genes. Next, KEAP1 KO was carried out in bone marrow derived CD34+ cells using a CRISPR Cas9 RNP system. In agreement with our observations in HUDEP2 cells, knockdown of KEAP1 in CD34+ cells resulted in induction of the expression of γ-globin, NQO1 and HO1. Additionally, treatment with CDDO-Me, an NRF2 activator compound, resulted in a 4-fold induction in γ-globin mRNA levels in primary SCD patient derived CD34+ cells (by ddPCR), a 2.5-fold induction in HbF (by flow cytometry), and a 2.5-fold up-regulation in both Aγ and Gγ globin levels (by ultra-performance liquid chromatography). When CDDO-Me treated cells were subjected to ex-vivo hypoxia challenge, we observed a dose dependent inhibition in sickling with a maximal decrease of 55% at 250 nM, as evaluated by IDEAS imaging flow cytometry. We further evaluated the antioxidant response with NRF2 activation in these cell systems. KEAP1 KO in HUDEP2 cells evaluated using RNA-sequencing showed enrichment of genes that participate in various protective mechanisms including NRF2 mediated oxidative stress response and glutathione redox reaction as analyzed by ingenuity pathway analysis (IPA). We subjected KEAP1 KO CD34+ cells to 4% hypoxia for 2 hours and observed a reduction in total reactive oxygen species levels as measured by flow cytometry compared to the control cells. These findings suggest that Nrf2 activation in erythroid cells offer a protective phenotype by improving the oxidative stress defense mechanisms, a well established pathophysiological manifestation of SCD. In conclusion, KEAP1 inhibition in erythroid cell lineage and subsequent NRF2 activation leads to HbF induction and improvement in the antioxidant stress enzymes. Overall these data demonstrate a multi-faceted potential benefit of NRF2 activation in sickle cell disease. Disclosures Gupta: Sanofi: Employment. Lessard:Sanofi: Employment. Moore:Sanofi: Employment. Duan:Sanofi: Employment. Hicks:Sanofi: Employment. Light:Sanofi: Employment. Krishnamoorthy:Sanofi: Employment.

Identification of a functional antioxidant response element at the HIF1A locus

Reactive oxygen species (ROS), which are a byproduct of oxidative metabolism, serve as signaling molecules in a number of physiological settings. However, if their levels are not tightly maintained, excess ROS lead to potentially cytotoxic oxidative stress. Accordingly, several transcriptional regulatory networks have evolved to include components that are highly ROS-responsive. Depending on the context, these regulatory networks can leverage ROS to respond to nutrient conditions, metabolism, or other physiological signals, or to respond to oxidative stress. However, ROS signaling is complex, so regulatory interactions between various ROS-responsive transcription factors are still being mapped out. Here we show that the transcription factor NRF2, a key regulator of the adaptive response to oxidative stress, directly regulates expression of HIF1A, which encodes HIF1α, a key transcriptional regulator of the adaptive response to hypoxia. We used an integrative genomics approach to identify HIF1A as a ROS-responsive transcript and we found an NRF2-bound antioxidant response element (ARE) approximately 30 kilobases upstream of HIF1A. This ARE sequence is deeply conserved, and we verified that it is directly bound and activated by NRF2. In addition, we found that HIF1A is upregulated in breast and bladder tumors with high NRF2 activity. Taken together, our results demonstrate that NRF2 targets a functional ARE at the HIF1A locus, and reveal a direct regulatory connection between two important oxygen responsive transcription factors.

Alterations in System xc- Expression in the Retina of Type 1 Diabetic Rats and the Role of Nrf2.

Nrf2 (nuclear factor erythroid 2-related factor 2), a transcription factor that controls expression of several proteins that are related to cellular antioxidant capacity, such as the subunit xCT of the system xc-, is dysregulated in diabetes. Recently, it was described that system xc- is decreased in the retina after 3weeks of diabetes. So, in the present work, the temporal relationship between xCT and Nrf2 in the retina of diabetic animals was investigated. Diabetes was induced in male Wistar rats (200g) by a single injection of streptozotocin, and retinas were collected after 1, 2, and 6months of diabetes induction. Expression of xCT, Nrf2 activity, and binding to antioxidant-responsive element (ARE) sequence were evaluated. Glutathione and reactive oxygen species (ROS) levels were also assessed. After 1month of diabetes, Nrf2 activity, xCT expression, and glutathione levels were reduced whereas ROS were increased. Although glutathione and ROS levels remain unchanged until later stages, Nrf2 activity and xCT expression returned to normal levels after 2months. However, they were decreased again at 6months of diabetes. Accordingly, Nrf2 binding to xCT ARE sequence followed the same pattern of Nrf2 activity and xCT expression. These data showed that retinal xCT expression is regulated by Nrf2 in diabetic condition. The results also demonstrated a temporal relationship between Nrf2 and system xc- which could be implicated in the initiation of oxidative stress in retina in diabetes.

Redox Homeostasis Plays Important Roles in the Maintenance of the Drosophila Testis Germline Stem Cells.

SummaryOxidative stress influences stem cell behavior by promoting the differentiation, proliferation, or apoptosis of stem cells. Thus, characterizing the effects of reactive oxygen species (ROS) on stem cell behavior provides insights into the significance of redox homeostasis in stem cell-associated diseases and efficient stem cell expansion for cellular therapies. We utilized the Drosophila testis as an in vivo model to examine the effects of ROS on germline stem cell (GSC) maintenance. High levels of ROS induced by alteration in Keap1/Nrf2 activity decreased GSC number by promoting precocious GSC differentiation. Notably, high ROS enhanced the transcription of the EGFR ligand spitz and the expression of phospho-Erk1/2, suggesting that high ROS-mediated GSC differentiation is through EGFR signaling. By contrast, testes with low ROS caused by Keap1 inhibition or antioxidant treatment showed an overgrowth of GSC-like cells. These findings suggest that redox homeostasis regulated by Keap1/Nrf2 signaling plays important roles in GSC maintenance.

Transcription factor NFE2L2/NRF2 is a regulator of macroautophagy genes

ABSTRACTAutophagy is a highly coordinated process that is controlled at several levels including transcriptional regulation. Here, we identify the transcription factor NFE2L2/NRF2 (nuclear factor, erythroid 2 like 2) as a regulator of autophagy gene expression and its relevance in a mouse model of Alzheimer disease (AD) that reproduces impaired APP (amyloid β precursor protein) and human (Hs)MAPT/TAU processing, clearance and aggregation. We screened the chromatin immunoprecipitation database ENCODE for 2 proteins, MAFK and BACH1, that bind the NFE2L2-regulated enhancer antioxidant response element (ARE). Using a script generated from the JASPAR's consensus ARE sequence, we identified 27 putative AREs in 16 autophagy-related genes. Twelve of these sequences were validated as NFE2L2 regulated AREs in 9 autophagy genes by additional ChIP assays and quantitative RT-PCR on human and mouse cells after NFE2L2 activation with sulforaphane. Mouse embryo fibroblasts of nfe2l2-knockout mice exhibited reduced expression of autophagy genes, which was rescued by an NFE2L2 expressing lentivirus, and impaired autophagy flux when exposed to hydrogen peroxide. NFE2L2-deficient mice co-expressing HsAPPV717I and HsMAPTP301L, exhibited more intracellular aggregates of these proteins and reduced neuronal levels of SQSTM1/p62, CALCOCO2/NDP52, ULK1, ATG5 and GABARAPL1. Also, colocalization of HsAPPV717I and HsMAPTP301L with the NFE2L2-regulated autophagy marker SQSTM1/p62 was reduced in the absence of NFE2L2. In AD patients, neurons expressing high levels of APP or MAPT also expressed SQSTM1/p62 and nuclear NFE2L2, suggesting their attempt to degrade intraneuronal aggregates through autophagy. This study shows that NFE2L2 modulates autophagy gene expression and suggests a new strategy to combat proteinopathies.

Dimethyl Fumarate Induces Fetal Hemoglobin in Sickle Cell Disease

Sickle cell disease (SCD) is caused by a point mutation in the β-chain of hemoglobin, which triggers a complex pathophysiology resulting in recurrent, painful vaso-occlusive crises (VOC) and chronic hemolytic anemia. Fetal hemoglobin (HbF) is the major species of hemoglobin during fetal and neonatal development, the expression of which is replaced by the adult beta globin perinatally. Reactivation of HbF in adult SCD patients is considered beneficial in ameliorating the symptoms of the disease. This is exemplified by a condition known as hereditary persistence of fetal hemoglobin (HPFH) in heterozygous sickle disease patients where symptoms of SCD are absent and the typical HbF level is over 30% (Steinberg, M.H. et al, 2014). Strategies to reactivate HbF have been used successfully in mouse models of SCD and led to amelioration of the disease phenotype, which is also partly the mechanism of action of hydroxyurea, the only FDA approved drug for SCD. Up-regulation of HbF can be achieved by several different approaches including pharmacologic or genetic manipulation of transcription activators or repressors of HbF. Nuclear factor (erythroid derived-2)-like 2 (Nrf2) is a basic leucine zipper transcription factor that has been shown to activate γ-globin transcription and increase HbF levels in cultured CD34+ erythroid cells. Nrf2 is well established for its role in cytoprotective and anti-oxidant actions by transcriptionally activating target genes that confer protection from oxidative damage triggered from injury and inflammation. Nrf2 is normally sequestered in the cytoplasm by Keap1, a Kelch-domain protein. Release of Nrf2 from Keap1 allows nuclear translocation of Nrf2 and activation of target genes via its binding to an anti-oxidant response element (ARE) in the promoter region of Nrf2 target genes. Previous studies (Macari and Lowrey, 2011) have demonstrated that the γ-globin promoter contains an ARE sequence supporting Nrf2 as an inducer of HbF. We investigated the role of dimethyl fumarate (DMF), a small molecule Nrf2 agonist, in activating γ-globin transcription and enhancing levels of HbF in tissue culture and murine SCD models. Delayed-release DMF, approved by the FDA as Tecfidera, is an oral therapeutic for the treatment of relapsing multiple sclerosis (MS). After oral administration of DMF, human exposure occurs to both DMF and the bioactive primary metabolite, monomethyl fumarate (MMF). We assessed the ability of increasing concentrations of DMF to induce γ-globin mRNA in human erythroleukemia cells, CD34+ cells isolated from bone marrow of non-SCD volunteer donors, and peripheral blood mononuclear cells from SCD volunteer donors. γ-globin mRNA increased by 3-4 fold compared to control as quantitated using real-time PCR. DMF incubation also resulted in 2-fold upregulation in HbF protein levels as demonstrated by UPLC and western blotting analysis and also a 2-fold induction in percentage of RBC containing HbF (F-cells) by flow cytometry. In addition, co-incubation of DMF with hydroxyurea produced an additive effect on γ-globin mRNA (8 - 10 fold in non-SCD; 2-4 fold in SCD) and F-cell induction in CD34+ stem cell derived erythroid progenitors (3-4 fold in both SCD and non-SCD). DMF treatment of KU812 erythroleukemia cells induced Nrf2 activation resulting in Nrf2 nuclear translocation and occupancy of the ARE in the γ-globin promoter by ChIP assay. Moreover, siRNA based knockdown of Keap1 resulted in up-regulation of HbF by flow cytometry, demonstrating that activation of the Nrf2 pathway in erythroid cells can result in the up-regulation of HbF. The effect of DMF on HbF induction was assessed in the Townes SCD mouse model. Townes mice were administered DMF at 100 mg/kg IP for 5 day per week for one month duration and compared to treatment with vehicle control and to treatment with hydroxyurea. This dose and route of administration of DMF resulted in clinically relevant plasma levels of MMF in Townes mice. After a month of dosing, there was a 3- to 4-fold increase in circulating F-cells in the DMF treated mice which was comparable to the hydroxyurea treated mice. Based on these findings, DMF may have potential as an HbF inducer in the therapy of SCD, in addition to its vascular protective and heme detoxifying properties (Belcher et al, ASH 2014). Further clinical studies are needed to evaluate the safety and efficacy of DMF in SCD. DisclosuresKrishnamoorthy:Biogen: Employment, Equity Ownership. Gupta:Biogen: Employment, Equity Ownership. Sturtevant:Biogen: Employment. Li:Georgia Regents University: Employment. Makala:Georgia Regents University: Employment. Hobbs:Biogen: Employment, Equity Ownership. Light:Biogen: Employment, Equity Ownership.

Tanshinone IIA protects dopaminergic neurons against 6-hydroxydopamine-induced neurotoxicity through miR-153/NF-E2-related factor 2/antioxidant response element signaling pathway

Parkinson’s disease (PD) is the second most common progressive neurodegenerative disorder with increased oxidative stress, the underlying vital process contributing to cell death. Tanshinone IIA (Tan IIA), a major bioactive diterpene quinone of Salva miltiorrhiza, had been proved effective in the MPTP model through its anti-inflammatory activity. Here in this research, we found that Tan IIA prevented the loss of nigrostriatal dopaminergic neurons by activating the NF-E2-related factor 2 (Nrf2)–antioxidant response element (ARE) pathway. The cytotoxicity of 6-hydroxydopamine (6-OHDA) was attenuated by the treatment of Tan IIA in SH-SY5Y cells, which significantly reduced 6-OHDA-induced lactic dehydrogenase release and reactive oxygen species production. Further study indicated that Tan IIA contributed to the nuclear accumulation of Nrf2, which bound to the ARE sequence, and activated ARE-regulated genes, including heme oxygenase-1, glutamate cysteine ligase catalytic subunit (GCLC) and glutamate cysteine ligase modifier subunit (GCLM). Tan IIA also protected against damage to mitochondrial membrane potential, reduced the translocation of cytochrome c from the mitochondria to the cytoplasm and the activation of Caspase-9 and Caspase-3. Moreover, we demonstrated the above effects were performed in Nrf2-dependent manner. Further studies revealed that Tan IIA reduced the enhancement of miR-153 by 6-OHDA, which targeted the 3′-UTR of Nrf2, and suppressed its expression and activation. Additionally, neurodegeneration caused by in vivo stereotaxic injection of 6-OHDA could also be ameliorated by the administration of Tan IIA. Taken together, our results strongly suggest that Tan IIA may be beneficial for the treatment of PD, and also confirm that targeting the Nrf2/ARE pathway is a promising strategy for therapeutic intervention in PD.

Identification of a functional antioxidant response element within the eighth intron of the human ABCC3 gene.

The ATP-binding cassette (ABC) family of transporters, including ABCC3, is a large family of efflux pumps that plays a pivotal role in the elimination of xenobiotics from the body. ABCC3 has been reported to be induced during hepatic stress conditions and through the progression of some forms of cancer. Several lines of evidence have implicated the transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in this induction. However, although rodent models have been investigated, a functional antioxidant response element (ARE) in the human ABCC3 gene has not been identified. The purpose of this study was to identify and characterize the ARE(s) responsible for mediating the Nrf2-dependent induction of the human ABCC3 gene. A high-throughput chromatin immunoprecipitation-sequencing analysis performed in A549 cells revealed a specific interaction between Nrf2 and the eighth intron of the human ABCC3 gene rather than the more prototypical flanking region of the gene. Subsequent in silico analysis of the intron identified two putative ARE elements that contained the core consensus ARE sequence commonly found in several Nrf2-responsive genes. Functional characterization of these two AREs using luciferase-reporter constructs with ARE mutant constructs revealed that one of these putative AREs is functionally active. Finally, DNA pull-down assays confirmed specific binding of these intronic AREs by Nrf2 in vitro. Our findings identify a functional Nrf2 response element within the eighth intron of the ABCC3 gene, which may provide mechanistic insight into the induction of ABCC3 during antioxidant response stimuli.

Abstract 506: H2O2 upregulates xCT in human breast cancer cells via NRF-2

Abstract Cancer cells adapt to high levels of oxidative stress in order to survive and proliferate, making the transcription factors involved in antioxidant defense regulation targets of interest. The transcription factor NF E2 Related Factor 2 (NRF-2) regulates cellular defense genes including those encoding intracellular redox-balancing proteins such as enzymes involved in glutathione metabolism. Glutathione in particular is an important intracellular antioxidant molecule. NRF-2 binds to the Antioxidant Response Element (ARE) in the promoter of its target genes. Under basal conditions, KEAP1 acts as an inhibitor that targets NRF-2 for ubiquitination. During oxidative stress, NRF-2 dissociates from KEAP1 and enters the nucleus to bind to the ARE sequence. It is hypothesized that the elevated Reactive Oxygen Species may be depleting the glutathione levels within the cancer cell. System xc- is a cysteine/glutamate antiporter that exports glutamate while importing cysteine to synthesize glutathione. In response to oxidative stress the cells increase system xc- activity in order to provide cysteine for glutathione synthesis. There is evidence that expression of xCT the specific subunit of System X is regulated by NRF-2. However this has not yet been demonstrated in human cancer cells, which is the focus of this project. Basal expression of NRF-2, Keap1 and xCT was characterized in two breast cancer cell lines (MDA MB 231 and MCF-7) compared to normal human breast epithelial 184B5 cells. Compared to 184B5 cells, Keap-1, NRF-2 and xCT were upregulated in MDA MB 231 cells, but down regulated in MCF-7 cells. MCF-7 cells were treated with H202 resulting in NRF-2 protein accumulation in the nucleus and decreased levels in the cytosol. Under the same treatment conditions, Keap-1 protein decreased in the nucleus and increased in the cytosol. With H202 treatment, xCT mRNA levels increased in MCF-7 cells. We have cloned approximately 2.6 kilobase (-2329 to +278 bp) of the human xCT promoter region from genomic DNA isolated from MDA MB 231 breast cancer cells. We found that the xCT promoter was responsive to treatment with H2O2, which increased transcriptional activity. This is consistent with results described for the mouse promoter, which is known to contain an antioxidant response element that can be bound by NRF-2 during oxidative stress. Co transfection of MDA MB 231 with the xCT promoter and an NRF-2 expression vector also increased promoter activity. This data suggests that under oxidative stress, NRF-2 is localized to the nucleus and transcriptionally upregulates xCT. This is the first study in which the regulation of xCT has been linked to oxidative stress via NRF-2 in human breast cancer cells. This work was supported by the Canadian Institutes for Health Research. Citation Format: Eric Habib, Katja Linher-Melville, Gurmit Singh. H2O2 upregulates xCT in human breast cancer cells via NRF-2. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 506. doi:10.1158/1538-7445.AM2014-506

Study on anti-oxidative effect of extracts from Cichorium endivia on HepG2 cells and its mechanism

To investigate the protective effect of extracts from Cichorium endivia (CEE) in H2O2-induced HepG2 cell oxidative stress injury, and explore the antioxidant mechanism of CEE in HepG2 cells. The viability of H2O2-induced HepG2 cells and the intracellular ROS level were measured by MTT assay and DCFH-DA fluorescence staining assay. The antioxidant-response element (ARE)-Luciferase activity was tested in HepG2 cells stably transected by ARE reporter gene. The fluorescence quantitative RT-PCR was adopted to determine the mRNA expressions of genes containing ARE sequence in HepG2 cells. The cell viability reduced, while the ROS level increased after HepG2 cells were treated by H2O2. Different concentrations of CEE could be added to significantly improve the above results. After HepG2 cells transected by ARE reporter gene were treated with different concentrations of CEE, the intracellular ARE activity could increase in a concentration-dependent manner. In addition, the mRNA expressions of regulatory genesGCLC, GCLM and HMOX-1 containing ARE sequence in HepG2 cells were up-regulated in a concentration-dependent manner by CEE. CEE inhibited the H2O2-injured HepG2 cells by reducing the ROS level. CEE's antioxidant mechanism for HepG2 cells may be closely related to the antioxidant defense system associated with its effect of activating Nrf2-ARE pathway in HepG2 cells.

The adjuvant component α-tocopherol triggers via modulation of Nrf2 the expression and turnover of hypocretin in vitro and its implication to the development of narcolepsy

BackgroundAfter the H1N1 swine flu vaccination campaign an increased number of narcolepsy cases in children and adolescents was observed in Scandinavian and later in further European countries that correlated with the vaccination by an AS03-adjuvanted influenza vaccine (Pandemrix). Narcolepsy is a chronic sleep disorder characterized by the loss of hypocretin in the cerebrospinal fluid due to selective destruction of hypocretin-producing neurons in the perifornical hypothalamus. In >99% of the cases narcolepsy is associated with the HLA-subtype DQB1*602 giving the link to an autoimmune process. In contrast to other squalene-based adjuvants, for which no association with narcolepsy was reported so far, ASO3 contains in addition α-tocopherol. It could be observed recently that α-tocopherol activates the transcription factor Nrf2. Nrf2 triggers the expression of cytoprotective genes, i.e. the catalytic active subunits of the constitutive proteasome, by binding to the antioxidant response element (ARE). It was hypothesized that α-tocopherol via activation of Nrf2 affects expression and turnover of hypocretin, leading to an increased amount of hypocretinα-specific fragments that bind to DQB1*602. Resultsα-Tocopherol activates Nrf2 in neuronal cells in vitro. Promoter analysis revealed an ARE sequence in the hypocretin promoter. Indeed, α-tocopherol increases by activation of Nrf2 the expression of hypocretin. In parallel, α-tocopherol -dependent induction of Nrf2 augments expression of catalytic subunits of the proteasome leading to increased degradation of hypocretin. Moreover, elevated activation of Nrf2 is associated with a decreased activity of NF-κB that results in an increased sensitivity to apoptotic stimuli. ConclusionIn case of a genetic predisposition (DQB1*602) α-tocopherol could confer to development of narcolepsy by activation of Nrf2 that finally leads to an elevated formation of longer hypocretin-derived fragments that can be presented by HLA-subtype DQB1*602. These cells are recognized by the immune system and due to their increased sensitivity to apoptotic stimuli they can be destroyed, finally leading to a lack of hypocretin.

Membrane-Bound CYB5R3 Is a Common Effector of Nutritional and Oxidative Stress Response Through FOXO3a and Nrf2

Membrane-bound CYB5R3 deficiency in humans causes recessive hereditary methaemoglobinaemia (RHM), an incurable disease that is characterized by severe neurological disorders. CYB5R3 encodes for NADH-dependent redox enzyme that contributes to metabolic homeostasis and stress protection; however, how it is involved in the neurological pathology of RHM remains unknown. Here, the role and transcriptional regulation of CYB5R3 was studied under nutritional and oxidative stress. CYB5R3-deficient cells exhibited a decrease of the NAD(+)/NADH ratio, mitochondrial respiration rate, ATP production, and mitochondrial electron transport chain activities, which were associated with higher sensitivity to oxidative stress, and an increase in senescence-associated β-galactosidase activity. Overexpression of either forkhead box class O 3a (FOXO3a) or nuclear factor (erythroid-derived 2)-like2 (Nrf2) was associated with increased CYB5R3 levels, and genetic ablation of Nrf2 resulted in lower CYB5R3 expression. The presence of two antioxidant response element sequences in the CYB5R3 promoter led to chromatin immunoprecipitation studies, which showed that cellular stressors enhanced the binding of Nrf2 and FOXO3a to the CYB5R3 promoter. Our findings demonstrate that CYB5R3 contributes to regulate redox homeostasis, aerobic metabolism, and cellular senescence, suggesting that CYB5R3 might be a key effector of oxidative and nutritional stress pathways. The expression of CYB5R3 is regulated by the cooperation of Nrf2 and FOXO3a. CYB5R3 is an essential gene that appears as a final effector for both nutritional and oxidative stress responses through FOXO3a and Nrf2, respectively, and their interaction promotes CYB5R3 expression. These results unveil a potential mechanism of action by which CYB5R3 deficiency contributes to the pathophysiological underpinnings of neurological disorders in RHM patients.

Betanin, a beetroot component, induces nuclear factor erythroid-2-related factor 2-mediated expression of detoxifying/antioxidant enzymes in human liver cell lines

Our recent study has shown that beetroot juice protects against N-nitrosodimethylamine (NDEA)-induced liver injury and increases the activity of phase II enzymes, suggesting the activation of the nuclear factor erythroid-2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway. The aim of the present study was to further explore the mechanism of the activity of beetroot by evaluating the cytoprotective effects of its major component. The influence of betanin (BET) on the activation of Nrf2 and the expression of GSTA, GSTP, GSTM, GSTT, NQO1 and HO-1 was assessed in two hepatic cell lines: non-tumour THLE-2 and hepatoma-derived HepG2 cell lines. The level of the tumour suppressor p53 in both cell lines and the methylation of GSTP in HepG2 cells were also evaluated. Treatment of both cell lines with 2, 10 and 20μm of BET resulted in the translocation of Nrf2 from the cytosol to the nucleus. The mRNA and nuclear protein levels of Nrf2 and the binding of Nrf2 to ARE sequences were increased only in the THLE-2 cells and were accompanied by the phosphorylation of serine/threonine kinase (AKT), c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK). BET also significantly increased the mRNA and protein levels of GSTP, GSTT, GSTM and NQO1 in these cells. Conversely, besides the translocation of Nrf2 from the cytosol to the nucleus, BET did not modulate any of the other parameters measured in the HepG2 cells. BET did not change the methylation of GSTP1 in these cells either. These results indicate that BET through the activation of Nrf2 and subsequent induction of the expression of genes controlled by this factor may exert its hepatoprotective and anticarcinogenic effects. Moreover, the activation of mitogen-activated protein kinases may be responsible for the activation of Nrf2 in the THLE-2 cells.

Nrf2-ARE pathway regulates induction of Sestrin-2 expression

The Sestrin2 (Sesn2) gene encodes a conserved antioxidant protein that is induced on oxidative stress and protects cells against reactive oxygen species. NF-E2-related factor-2 (Nrf2) is an essential transcription factor that regulates expression of a variety of antioxidant genes via binding to the antioxidant-response element (ARE), but the role of Nrf2 in Sesn2 gene expression has not been elucidated yet. The present study investigated whether the Nrf2-ARE pathway regulates Sesn2 gene expression and the identification of the molecular mechanism. The Nrf2 activators, tert-butylhydroquinone (t-BHQ) and sulforaphane (SFN), up-regulated Sesn2 expression in a dose- and time-dependent manner in hepatocytes. Also, t-BHQ increased Sesn2 mRNA and luciferase gene activity, whereas the levels of Sesn1 and Sesn3 mRNA were not affected by t-BHQ treatment. The specific role of Nrf2 in Sesn2 induction was verified by using Nrf2 overexpression plasmid and Nrf2 knockout or knockdown cells. In silico analysis of the 5′ upstream region of Sesn2 gene identified a putative ARE sequence. Deletion of the putative ARE demonstrated that the ARE from −550 to −539bp in the human Sesn2 promoter was critical for the Nrf2-mediated response. Moreover, SFN injection increased Sesn2 mRNA and protein levels in the livers of mice. Knockdown experiments with Sesn2 siRNA showed that Sesn2 is required for the Nrf2-mediated cytoprotective activity against hydrogen peroxide. Our results suggest that the Nrf2-ARE pathway is critical for Sesn2 gene expression and might protect against oxidative stress.

HepG2 Cell에서 녹차씨박 에탄올 추출물의 암세포 증식 억제효과

본 연구는 간암세포주인 HepG2 cell line에서 녹차씨박 추출물을 에탄올, 석유 에테르, 에틸 아세테이트, 부탄올의 순으로 분획 및 열수 추출물을 조제하여 암세포 증식 억제능을 확인한 후 에탄올 추출물을 선정하여 항종양 및 항염증효과를 생화학적, 분자적 방법으로 분석하였다. 녹차씨박 에탄올추출물(DGTSE)의 polyphenol 함량을 HPLC로 분석한 결과 EGC(1039.1±15.26 ㎍/g)＞tannic acid(683.5±17.61 ㎍/g)＞EC(62.4±5.00 ㎍/g)＞ECG(24.4±7.81 ㎍/g)＞EGCG (20.9±0.96 ㎍/g)＞gallic acid(2.4±0.68 ㎍/g)의 순이었으나 caffeic acid는 검출되지 않았다. DGTSE의 농도가 10 ㎍/㎖ 이상에서는 84.13%의 세포 증식 억제능(IC??: 6.58 ㎍/㎖)을 보여 간암세포의 증식을 효과적으로 억제하였고 제1상효소계인 CYP1A1와 CYP1A2의 발현을 농도 의존적으로 감소시키는 것으로 나타났다. QR 활성은 DGTSE을 20 ㎍/㎖ 농도로 처리 시 대조군에 비해 2.6배 증가하였으며 reporter gene activity로 측정한 ARE 활성은 1.94배로 각각 증가하였다. DGTSE의 처리는 염증생성 인자인 PGE2의 생성과 TNF-α의 단백질 발현은 유의적으로 저해하였으며 cytokine 반응, 염증, 세포성장조절과 같은 다양한 단계에 참여하는 전사인자인 NFκB의 핵으로의 translocation을 억제하였다. 이상의 결과에서 DGTSE은 간암세포인 HepG2 세포계에서 암세포 증식 억제능을 가지며 해독효소인 QR, ARE의 활성을 증진시키고 NFκB의 translocation을 방해하고 TNF-α의 단백질 발현과 PGE2의 생성을 억제하여 암세포의 증식을 억제하였다. 따라서 녹차씨박 추출물에 함유된 암세포 증식효과를 나타내는 생리활성 물질들에 대한 연구가 앞으로 진행되어야 할 것으로 사료된다.

Abstract 1528: Activation of NRF2/ABCC1 axis confers resistance to topoisomerase II poisons in human oral malignancies

Abstract Etoposide (VP-16), a DNA topoisomerase II poison, is an important clinical used chemotherapeutic agent for human oral malignancies. An etoposide-resistant cell line, KB-7D, has been generated from human oral epidermoid carcinoma KB cells to investigate the mechanism of action of drug resistance in oral malignancies. Previous studies revealed that KB-7D cells were approximately 50-fold more resistant to etoposide as compared to parental KB cells. It also exhibited cross-resistant to chemotherapeutic agents such as doxorubicin. This multi-drug resistance may be caused by the over-expression of ABCC1, leading to the decrease in drug accumulation in KB-7D cells. Our current work continues the effort to investigate the mechanism of regulation of ABCC1 expression in the etoposide-derived drug resistant cells and subsequently find the molecular target that can be used to restore therapeutic efficacy in chemo-refratory cancers. Down-regulation of ABCC1 by RNA interference and a selective inhibitor, MK-571, significantly enhanced the chemosensitivity to etoposide and doxorubicin in KB and KB-7D cells. To further determine the possible transcriptional factors that regulate the ABCC1 transactivation, the promoter region of ABCC1 was examined. A NRF2 binding sequence, antioxidant responsive element (ARE), has been found locate in the ABCC1 promoter at -470 to -458 upstream from the transcription start site. Real-time RT-PCR and Western blot analyses showed that expression of NRF2 mRNA and protein in KB-7D cells was 1.4 to 1.8 folds higher than those expressed in the parental cells. In addition, Chromatin Immunoprecipitation (ChIP) analysis revealed that NRF2 directly targeted to the ARE sequence in the ABCC1 promoter region. Interestingly, down-regulation of NRF2 decreased the expression of ABCC1 and also increased the chemosensitivity of KB-7D cells against selected anti-cancer drugs. In addition, cells incubated with an NRF2 activator, tBHQ, induced nucleus accumulation of NRF2 and over-expression of ABCC1, resulting in the enhancement of drug-resistance against etoposide or doxorubcin in KB and KB-7D cells. In summary, constitutive activation of NRF2-dependent ABCC1 contributed to the causation of chemoresistance in etoposide-derived drug resistant cells. Blockage of ABCC1 expression by manipulation of the NRF2 signaling pathway enhanced the chemotherapeutic efficacy in cells. Therefore, targeting NRF2 may be able to reverse chemoresistance in chemo-refractory oral malignancies. In addition, development of NRF2 inhibitors may be a new strategy to overcome chemoresistance in human cancers. (The study was supported by grants of Department of Health DOH99-TD-C-111-004, Taiwan.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1528. doi:10.1158/1538-7445.AM2011-1528

Nrf2 Induces Interleukin-6 (IL-6) Expression via an Antioxidant Response Element within the IL-6 Promoter

IL-6 gene expression is controlled by a promoter region containing multiple regulatory elements such as NF-κB, NF-IL6, CRE, GRE, and TRE. In this study, we demonstrated that TRE, found within the IL-6 promoter, is embedded in a functional antioxidant response element (ARE) matching an entire ARE consensus sequence. Further, point mutations of the ARE consensus sequence in the IL-6 promoter construct selectively eliminate ARE but not TRE activity. Nrf2 is a redox-sensitive transcription factor which provides cytoprotection against electrophilic and oxidative stress and is the most potent activator of ARE-dependent transcription. Using Nrf2 knock-out mice we demonstrate that Nrf2 is a potent activator of IL-6 gene transcription in vivo. Moreover, we show evidence that Nrf2 is the transcription factor that activates IL6 expression in a cholestatic hepatitis mouse model. Our findings suggest a possible role of IL-6 in oxidative stress defense and also give indication about an important function for Nrf2 in the regulation of hematopoietic and inflammatory processes.

Exploiting the NRF2/Antioxidant Response Element Signaling Pathway to Induce γ-Globin Gene Expression and HbF Production

AbstractAbstract 2074Induction of fetal hemoglobin (HbF) has proven therapeutic potential to treat sickle cell disease and β-thalassemia. However, agents known to be effective in humans, including hydroxyurea, DNMT inhibitors and butyrate derivatives are not ideal due to suppression of hematopoiesis and the possibility of long-term side effects. Two natural compounds, angelicin (Lampronti, et al, Eur J Hematol 2003) and resveratrol (Rodrigue, et al, Hematology 2001) have been found to induce γ-globin gene expression in K562 cells. These agents may be important lead compounds as they are generally non-cytotoxic and are being evaluated in ongoing human trials as cancer chemopreventative agents where these and several other agents are thought to work by activating antioxidant response pathway genes. The products of these genes are enzymes involved in antioxidant and detoxification activities and include NADPH-quinone oxireductase 1 (NQO1), glutamate-cysteine ligase (GCL) and glutathione S-transferase (GST). The activation of these genes is mediated by the transcription factor, NF-E2 related factor 2 (NRF2), which binds to a specific antioxidant response element (ARE) sequence (TGACnnnGCA) in target gene promoters. The proximal γ-globin promoter contains an ARE sequence between the two CAAT boxes, suggesting that it too may be activated by NRF2. This led us to hypothesize that drugs that activate the ARE/NRF2 pathway may provide a less toxic approach to HbF induction. To test this hypothesis, we treated K562 cells with various NRF2 pathway activators. We initially tested six compounds that are known to induce antioxidant response genes at doses that did not inhibit proliferation and found the most pronounced γ-globin induction with tert-butylhydroquinone (tBHQ) (2.8 fold). We next tested tBHQ in two different primary cell culture models: erythroid precursors isolated from normal human bone marrow and in vitro erythroid differentiation of primary human CD34+ cells. In both of these models, tBHQ treatment increased γ-globin steady state mRNA levels and induced expression of NRF2 target genes. Treatment of differentiating erythroid cells caused a dose dependent increase in γ/(γ+β) mRNA and % HbF. HPLC analysis revealed the highest non-toxic concentration of tBHQ, 5μM, produced 10% HbF while the positive control, 0.5μM of 5-Azacytidine, resulted in 12% HbF compared to the untreated control at 3%. Since similar mRNA induction was seen in K562 cells, we used these cells to characterize the mechanism of tBHQ induced γ-globin expression. First, we used siRNA to decrease NRF2 mRNA levels. This resulted in a greater than 75% knockdown of NRF2 mRNA and protein and reduced tBHQ induction of γ-globin and NQO1 gene expression by 90% and 75%, respectively, compared to samples transfected with scrambled siRNA (p < 0.01). Subsequent experiments showed that tBHQ treatment resulted in NRF2 translocation to the nucleus and binding to the NQO1 and γ-globin promoters but not at negative control sites. In addition, inducing NRF2 translocation by transiently suppressing levels of its inhibitor, KEAP1, did not result in full induction of γ-globin mRNA expression, suggesting that NRF2 translocation alone is not sufficient for γ-globin induction. However, when combined with tBHQ, suppression of KEAP1 did enhance γ-globin induction. Taken together, these results suggest that NRF2/ARE pathway activation induces γ-globin mRNA expression and HbF production in primary human erythroid cells and that this is a promising strategy for further pre-clinical development. Disclosures:No relevant conflicts of interest to declare.