Induced Nrf2 Activation Research Articles

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

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.

P2 - In vitro glycation and glycoxidation of phosphatidylethanolamines

Formation and accumulation of advanced glycation end products (AGEs) appear to correlate with many human diseases, such as atherosclerosis and inflammation. Whereas AGE-modified proteins relatively well studied, aminophospholipid AGE-adducts have been less intensively investigated. At elevated concentrations, glucose can react with amino groups of phosphatidylethanolamines (PE) to form Schiff bases or Amadori products, which can be further converted, under oxidative conditions, to various AGEs. Many of these products such as carboxymethylamine (CMA) and carboxyethylamine (CEA) can be formed by oxidative degradation of Amadori-products. The aim of this work was to investigate the different glycation and glycoxidation PE-adducts and to elucidate the most prominent mechanisms of AGE-PE formation.Four different aminophospholipids,[dipalmitoyl-(DPPE), palmitoyl-oleoyl-(POPE), palmitoyl-linoleoyl-(PLPE) or palmitoyl-arachidonoyl-phosphatidylethanolamine(PAPE); all 1mmol/L] were glycated in vitro (5mmol/L glucose) and oxidized by the Fenton reaction (80µmol/L FeSO4, 50mmol/L H2O2) or using electrochemical oxidation (Roxy EC System, Antec, Leiden, Netherlands). High resolution mass spectrometry (MS) in combination with MSn fragmentation allowed the identification of major products, such as CMA, CEA and oxo-glucuronic acid. The mechanism of CMA/CME formation by oxidative degradation of the Amadori product was confirmed by co-oxidation (Fenton reaction) of purified glycated (gPOPE) with nonglycated DPPE (Fenton reaction) and by electrochemical oxidation of purified gPOPE, whereas the reaction with glyoxal/methylglyoxal formed by glucose oxidation was excluded. In both cases CMA/CEA-POPE adducts were detected. When glycated with 1,2-13C glucose, DPPE-CMA/CME adducts contained two 13C atoms further confirming the proposed mechanism. Finally, the ability of glycated PE when co-incubated with cells (HeLa) to induce Nrf2 activation was also addressed.

AICAR induces Nrf2 activation by an AMPK-independent mechanism in hepatocarcinoma cells

Hepatocellular carcinoma is one of the most frequent tumor types worldwide and oxidative stress represents a major risk factor in pathogenesis of liver diseases leading to HCC. Nuclear factor erythroid 2-related factor (Nrf2) is a transcription factor activated by oxidative stress that governs the expression of many genes which constitute the antioxidant defenses of the cell. In addition, oxidative stress activates AMP-activated protein kinase (AMPK), which has emerged in recent years as a kinase that controls the redox-state of the cell. Since both AMPK and Nrf2 are involved in redox homeostasis, we investigated whether there was a crosstalk between the both signaling systems in hepatocarcinoma cells. Here, we demonstrated that AMPK activator AICAR, in contrary to the A769662 allosteric activator, induces Nrf2 activation and concomitantly modulates the basal redox state of the hepatocarcinoma cells.When the expression of Nrf2 is knocked down, AICAR failed to induce its effect on redox state. These data highlight a major role of Nrf2 signaling pathway in mediating the AICAR effect on basal oxidative state. Furthermore, we demonstrated that AICAR metabolization by the cell is required to induce Nrf2 activation while, the silencing of AMPK does not have any effect on Nrf2 activation. This suggests that AICAR-induced Nrf2 activation is independent of AMPK activity. In conclusion, we identified AICAR as a potent modulator of the redox state of human hepatocarcinoma cells, via the Nrf2 signaling pathway and in an AMPK-independent mechanism.

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

BackgroundNuclear 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.MethodA 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.ResultMicromolarity (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.ConclusionThese 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.

CO-releasing molecules and increased heme oxygenase-1 induce protein S-glutathionylation to modulate NF-κB activity in endothelial cells

Protein glutathionylation is a protective mechanism that functions in response to mild oxidative stress. Carbon monoxide (CO) can increase the reactive oxygen species concentration from a low level via the inhibition of cytochrome c oxidase. We therefore hypothesized that CO would induce NF-κB-p65 glutathionylation and then show anti-inflammatory effects. In this study, we found that CO-releasing molecules suppress TNFα-induced monocyte adhesion to endothelial cells (ECs) and reduce ICAM-1 expression. Moreover, CO donors were further found to exert their inhibitory effects by blocking NF-κB-p65 nuclear translocation, but do so independent of IκBα degradation, in TNFα-treated ECs. In addition, p65 protein glutathionylation represents the response signal to CO donors and is reversed by the reducing agent dithiothreitol. Thiol modification of the cysteine residue in the p65 RHD region was required for the CO-modulated NF-κB activation. The suppression of p65 glutathionylation by a GSH synthesis inhibitor, BSO, and by catalase could also attenuate TNFα-induced p65 nuclear translocation and ICAM-1 expression. CO donors induce Nrf2 activation and Nrf2 siRNA suppresses CO-induced p65 glutathionylation and inhibition. Furthermore, we found that the CO donors induce heme oxygenase-1 (HO-1) expression, which increases p65 glutathionylation. In contrast, HO-1 siRNA attenuates CO donor- and hemin-induced p65 glutathionylation. Our results thus indicate that the glutathionylation of p65 is likely to be responsible for CO-mediated NF-κB inactivation and that the HO-1-dependent pathway may prolong the inhibitory effects of CO donors upon TNFα treatment of ECs.

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

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

Nrf2/ARE pathway activation, HO-1 and NQO1 induction by polychlorinated biphenyl quinone is associated with reactive oxygen species and PI3K/AKT signaling

Nrf2/ARE pathway plays an important role in adapt to oxidative stress caused by pro-oxidants and electrophiles through up-regulating phase II detoxifying enzymes. Our previous study has demonstrated that PCB quinone exposure causes severe cellular oxidative stress (Toxicology In Vitro 26 (2012) 841–848). There are no reports describing the ability of PCB quinone on Nrf2/ARE activation. In the present study, we found that exposure to PCB29-pQ resulted in a significant increase in Nrf2 and Keap1 expression in total protein, as well as the Nrf2 targeting genes, including NQO1 and HO-1. Next, immunocytochemistry analysis identified the accumulation of Nrf2 in nucleus subsequent to PCB29-pQ treatment. The increased Nrf2 and constant Keap1 expression in nucleus suggested the dissociation of Nrf2/Keap1 complex. Similarly, mRNA level of Nrf2 was elevated significantly with PCB29-pQ treatment, but not Keap1. Additionally, PCB29-pQ treatment led to significant up-regulation of the mRNA level of antioxidant enzymes, NQO1 and HO-1, in a concentration-dependent manner. Electrophoretic mobility shift assay and luciferase reporter assay further confirmed the formation of Nrf2–ARE complex. PCB29-pQ treatment has no effect on mitogen-activated protein kinase signaling, however, phospho-AKT was up-regulated and GSK-3β was down-regulated. Pretreatment with LY294002, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K), suppressed the phosphorylation of AKT and inhibited PCB29-pQ induced Nrf2/HO-1 activation, meanwhile, GSK-3β expression was increased accordingly. At last, reactive oxygen species (ROS) scavengers inhibited PCB29-pQ induced Nrf2 activation partly. These results suggested that Nrf2 activation by PCB29-pQ in HepG2 cells is associated with ROS and AKT pathway but not MAPK signaling, the activation of Nrf2/ARE may be an adaptive response to oxidative stress.

Transcriptional responses of neonatal mouse lung to hyperoxia by Nrf2 status

Hyperoxia exposure can inhibit alveolar growth in the neonatal lung through induction of p21/p53 pathways and is a risk factor for the development of bronchopulmonary dysplasia (BPD) in preterm infants. We previously found that activation of nuclear factor erythroid 2 p45-related factor (Nrf2) improved survival in neonatal mice exposed to hyperoxia likely due to increased expression of anti-oxidant response genes. It is not known however, whether hyperoxic induced Nrf2 activation attenuates the growth impairment caused by hyperoxia in neonatal lung. To determine if Nrf2 activation modulates cell cycle regulatory pathway genes associated with growth arrest we examined the gene expression in the lungs of Nrf2−/− and Nrf2+/+ neonatal mice at one and 3days of hyperoxia exposure. MethodsMicroarray analysis was performed in neonatal Nrf2+/+ and Nrf2−/− lungs exposed to one and 3days of hyperoxia. Sulforaphane, an inducer of Nrf2 was given to timed pregnant mice to determine if in utero exposure attenuated p21 and IL-6 gene expression in wildtype neonatal mice exposed to hyperoxia. ResultsCell cycle regulatory genes were induced in Nrf2−/− lung at 1day of hyperoxia. At 3days of hyperoxia, induction of cell cycle regulatory genes was similar in Nrf2+/+ and Nrf2−/− lungs, despite higher inflammatory gene expression in Nrf2−/− lung. Conclusionp21/p53 pathways gene expression was not attenuated by Nrf2 activation in neonatal lung. In utero SUL did not attenuate p21 expression in wildtype neonatal lung exposed to hyperoxia. These findings suggest that although Nrf2 activation induces expression of anti-oxidant genes, it does not attenuate alveolar growth arrest caused by exposure to hyperoxia.

Temozolomide and irradiation combined treatment-induced Nrf2 activation increases chemoradiation sensitivity in human glioblastoma cells

Resistance to chemoradiotherapy is a major obstacle to successful treatment of glioblastoma. Recently, the role of NF-E2-related factor 2 (Nrf2) in enhancing chemoradiation sensitivity has been reported in several types of cancers. Here, we investigated whether temozolomide (TMZ) and irradiation (IR) combined treatment induced Nrf2 activation in human glioblastoma cells. And we further performed a preliminary study about the effect of Nrf2 on chemoradiation sensitivity. Immunohistochemical staining for Nrf2 in paired clinical specimens showed that TMZ and IR combined treatment increased the expression and nuclear localization of Nrf2 in human glioblastoma tissues. Moreover, we found nuclear Nrf2 expression in the glioblastoma tissues obtained from the patients undergoing TMZ and IR combined treatment was associated with the time to tumor recurrence. In vitro, we further verified these findings. First, we detected increased nuclear localization of Nrf2 following treatment with TMZ+IR in human glioblastoma cell lines. Second, we demonstrated TMZ+IR increased the levels of Nrf2 protein in both nuclear and cytoplasmic fractions of U251 cells and induced Nrf2 target genes expression. Finally, downregulating Nrf2 expression increased TMZ+IR-induced cell death in the U251 cells. These findings suggest TMZ+IR combined treatment induces Nrf2 activation in human glioblastoma cells. The activation of Nrf2 may be associate with enhancing chemoradiation sensitivity in human glioblastoma cell. Blocking Nrf2 activation may be a promising method enhancing chemoradiation sensitivity of glioblastoma cells.

Curcumin ameliorates TNF-α-induced ICAM-1 expression and subsequent THP-1 adhesiveness via the induction of heme oxygenase-1 in the HaCaT cells

Adhesion molecules such as ICAM-1 are important in the infiltration of leukocytes into the site of inflammation. In this study, we investigated the inhibitory effects of curcumin on ICAM-1 expression and monocyte adhesiveness as well as its underlying action mechanism in the TNF-α-stimulated keratinocytes. Curcumin induced expression of heme oxygenase-1 (HO-1) in the human keratinocyte cell line HaCaT. In addition, curcumin induced Nrf2 activation in dose- and time-dependent manners in the HaCaT cells. Curcumin suppressed TNF-α- induced ICAM-1 expression and subsequent monocyte adhesion, which were reversed by the addition of tin protoporphyrin IX (SnPP), a specific inhibitor of HO-1, or HO-1 knockdown using siRNA. Furthermore, Nrf2 knockdown using siRNA reversed the inhibitory effect of curcumin on the TNF-α-induced ICAM-1 expression and adhesion of monocytes to keratinocytes. These results suggest that curcumin may exert its anti-inflammatory activity by suppressing the TNF-α-induced ICAM-1 expression and subsequent monocyte adhesion via expression of HO-1 in the keratinocytes. [BMB Reports 2013;46(8): 410-415]

The Nuclear Factor Erythroid 2–Related Factor 2 Activator Oltipraz Attenuates Chronic Hypoxia–Induced Cardiopulmonary Alterations in Mice

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key regulator that activates many antioxidant enzymes. Oxidative stress, which accumulates in diseased lungs associated with pulmonary hypertension (PH), is thought to be responsible for the progression of cardiopulmonary changes. To test whether Nrf2 activation would exert therapeutic efficacy against cardiopulmonary changes in a hypoxia-induced PH model, wild-type (WT) and Nrf2-deficient mice as well as Kelch-like ECH associating protein 1 (Keap1) (negative regulator of Nrf2) knockdown mutant mice were exposed to hypobaric hypoxia for 3 weeks. This chronic hypoxia exacerbated right ventricular systolic pressure, right ventricular hypertrophy (RVH), and pulmonary vascular remodeling in the WT mice. These pathological changes were associated with aberrant accumulation of Tenascin-C, a disease-indicative extracellular glycoprotein. Simultaneous administration of oltipraz, a potent Nrf2 activator, significantly attenuated RVH and pulmonary vascular remodeling and concomitantly ameliorated Tenascin-C accumulation in the hypoxic mice. Hypoxia-exposed Nrf2-deficient mice developed more pronounced RVH than WT mice, whereas hypoxia-exposed Keap1-knockdown mice showed less RVH and pulmonary vascular remodeling than WT mice, underscoring the beneficial potency of Nrf2 activity against PH. We also demonstrated that expression of the Nrf2-regulated antioxidant enzymes was decreased in a patient with chronic obstructive pulmonary disease associated with PH. The decreased antioxidant enzymes may underlie the pathogenesis of cardiopulmonary changes in the patient with chronic obstructive pulmonary disease and PH. The pharmacologically or genetically induced Nrf2 activity clearly decreased RVH and pulmonary vascular remodeling in the hypoxic PH model. The efficacy of oltipraz highlights a promising therapeutic potency of Nrf2 activators for the prevention of PH in patients with hypoxemic lung disease.

Involvement of ERK-Nrf-2 Signaling in Ionizing Radiation Induced Cell Death in Normal and Tumor Cells

Prolonged oxidative stress favors tumorigenic environment and inflammation. Oxidative stress may trigger redox adaptation mechanism(s) in tumor cells but not normal cells. This may increase levels of intracellular antioxidants and establish a new redox homeostasis. Nrf-2, a master regulator of battery of antioxidant genes is constitutively activated in many tumor cells. Here we show that, murine T cell lymphoma EL-4 cells show constitutive and inducible radioresistance via activation of Nrf-2/ERK pathway. EL-4 cells contained lower levels of ROS than their normal counterpart murine splenic lymphocytes. In response to radiation, the thiol redox circuits, GSH and thioredoxin were modified in EL-4 cells. Pharmacological inhibitors of ERK and Nrf-2 significantly enhanced radiosensitivity and reduced clonogenic potential of EL-4 cells. Unirradiated lymphoma cells showed nuclear accumulation of Nrf-2, upregulation of its dependent genes and protein levels. Interestingly, MEK inhibitor abrogated its nuclear translocation suggesting role of ERK in basal and radiation induced Nrf-2 activation in tumor cells. Double knockdown of ERK and Nrf-2 resulted in higher sensitivity to radiation induced cell death as compared to individual knockdown cells. Importantly, NF-kB which is reported to be constitutively active in many tumors was not present at basal levels in EL-4 cells and its inhibition did not influence radiosensitivity of EL-4 cells. Thus our results reveal that, tumor cells which are subjected to heightened oxidative stress employ master regulator cellular redox homeostasis Nrf-2 for prevention of radiation induced cell death. Our study reveals the molecular basis of tumor radioresistance and highlights role of Nrf-2 and ERK.

ERK and PI3K signaling cascades induce Nrf2 activation and regulate cell viability partly through Nrf2 in human glioblastoma cells

The ERK and PI3K signaling cascades are aberrantly activated in human glioblastoma cells, resulting in the dysregulation of numerous downstream transcription factors. The dark side of the transcription factor, NF-E2-related factor2 (Nrf2) in human cancer has been revealed. It has been accepted that high levels of Nrf2 promote tumor progression. In the present study, we investigated the effect of the ERK and PI3K signaling cascades on Nrf2 in human glioblastoma cells. Immunohistochemical staining for Nrf2 in clinical specimens showed that the expression and nuclear localization of Nrf2 were increased in human glioblastoma tissues when compared to peritumoral normal tissues. In addition, we detected decreased nuclear localization of Nrf2 following combined treatment with ERK and PI3K inhibitors in three human glioblastoma cell lines and selected the cell line (U251) most sensitive to the inhibitors for further study. Our data demonstrated that inhibition of ERK and PI3K not only suppressed the nuclear accumulation of Nrf2 protein but also decreased the expression of the Nrf2 protein. In addition, combined inhibition of ERK and PI3K also decreased the mRNA levels of Nrf2 target genes. Finally, we found that Nrf2 overexpression partly reversed the ERK and PI3K inhibitor-induced inhibition of cell viability. Therefore, the ERK and PI3K signaling cascades regulate the expression and activation of Nrf2 and control cell viability partly through Nrf2 in U251 human glioblastoma cells. Thus, targeting the ERK and PI3K signaling cascades for Nrf2 activation may provide new methods for the treatment of glioblastoma.

Ethanol Extract of Peanut Sprout Induces Nrf2 Activation and Expression of Antioxidant and Detoxifying Enzymes in Human Dermal Fibroblasts: Implication for its Protection Against UVB‐irradiated Oxidative Stress

A peanut sprout is known to contain a significant level of resveratrol, which was reported to have beneficial effects in our body due to its antioxidant activities. The purpose of this study was to evaluate the cytoprotective activity of ethanol extract of peanut sprout (EPS) from ultraviolet B (UVB)-induced oxidative stress in human dermal fibroblasts (HDF). EPS was revealed to contain 54.2μgg(-1) of trans-resveratrol. The DCF-DA-positive reactive oxygen species level was increased by 50mJcm(-2) of UVB irradiation (2150±450% of nonirradiated control), which was markedly suppressed by EPS treatment (180±42% of control). Annexin V-positive apoptotic cell death induced by UVB irradiation (16.4±4.5%) was also significantly inhibited by EPS treatment (6.7±2.5%). EPS induced up-regulation and nuclear translocation of Nrf2, a transcription factor for antioxidant and detoxifying enzymes, in HDF as a dose-dependent manner. UVB irradiation up-regulated Nrf2-dependent enzymes of heme oxygenase-1, NAD(P)H:quinine oxidoreductase-1 and glutathione-S-transferase pi, and they were further stimulated by EPS treatment. Taken together, EPS is an efficient cytoprotective agent against UVB-induced oxidative stress by activation of Nrf2 and upregulation of Nrf2-relating antioxidant and detoxifying enzymes in HDF.

Nrf2-Keap1 Antioxidant Defense and Cell Survival Signaling Are Upregulated by 17β-Estradiol in Homocysteine-Treated Dopaminergic SH-SY5Y Cells

Background and Aims: A recent neuroimaging study discovered the neurotoxicity effects of homocysteine (Hcy), which is only seen in elderly women. Estrogens exert a variety of actions on brain function that influence cognitive function, mood, and neuroprotection. The Kelch-like ECH-associated protein 1 (Keap1)/nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidant defense pathway has been well-known to afford neuroprotection. Here, we first demonstrate the roles of Nrf2-Keap1 in 17β-estradiol (E₂) cytoprotection and Hcy toxicity and the protective mechanisms of E₂ on Hcy cytotoxicity in human dopaminergic SH-SY5Y cells. Methods: Cell viability was determined by trypan blue method. Protein expression was determined by Western blot analysis. Superoxide dismutase (SOD) activity was determined by ELISA. Reactive oxygen species (ROS) production was determined by flow cytometry. Results: In Hcy-treated SH-SY5Y cells, E₂ increased cell viability, attenuated ROS production, activated Akt signaling and inhibited glycogen synthase kinase-3β (GSK-3β), a kinase known to participate in neurodegeneration. Moreover, E₂ treatment led to Nrf2 dissociation from Keap1, the main negative regulator of Nrf2 activity in the cytoplasm, and increased the protein level of Nrf2 in the nucleus, with a significant increase in HO-1 expression and SOD activity in Hcy-treated cells. E₂-induced Nrf2 activation was attenuated by the PI3K inhibitor LY294002 and the estrogen receptor antagonist ICI 182,780. Further, E₂ decreased Hcy-induced apoptotic death by upregulating the antiapoptotic protein Bcl-2, decreasing cytochrome c release from mitochondria, and attenuating apoptotic cascade activation (Bax, caspase-9, and caspase-3). Conclusion: E₂ activates cell survival signaling and Nrf2-Keap1 antioxidant defense pathway and attenuates Hcy cytotoxicity.

Laminar Flow Activation of ERK5 Protein in Vascular Endothelium Leads to Atheroprotective Effect via NF-E2-related Factor 2 (Nrf2) Activation

Laminar flow protects from atherosclerosis in endothelium. Laminar flow induces Nrf2 activation dependent on ERK5 activation, leading to up-regulation of downstream genes of Nrf2. ERK5 requires Nrf2 activation to exert cytoprotective effect on HUVEC. ERK5 inhibitor BIX02189 regulates Nrf2 activation in vivo. Identifying ERK5 as a molecular target for regulating flow-mediating Nrf2-dependent gene expression may have significant therapeutic potential for treating atherosclerosis. Atherosclerosis is often observed in areas where disturbed flow is formed, whereas atheroprotective region is found in areas where steady laminar flow is developed. It has been reported that some genes activated by blood flow play important roles in vascular function and pathogenesis of atherosclerosis. Extracellular signal-regulated kinase 5 (ERK5) has been reported to regulate endothelial integrity and protect from vascular dysfunction and disease under laminar flow. Krüppel-like factor 2 (KLF2) and NF-E2-related factor 2 (Nrf2) are major transcriptional factors that contribute to anti-atherogenic responses under laminar flow. Implication of ERK5 in laminar flow-mediated regulation of KLF2-dependent gene has been established, whereas the role of ERK5 in laminar flow-mediated activation of Nrf2 pathway has not been addressed yet. In this study, we found that the blockage of ERK5 either by genetic depletion with siRNA or by biochemical inactivation with a specific chemical compound inhibited laminar flow-induced up-regulation of Nrf2-dependent gene expressions, whereas activation of ERK5 increased transcriptional activity and nuclear translocation of Nrf2, which suggests that ERK5 mediates laminar flow-induced up-regulation of Nrf2-dependent gene expression. Further functional studies showed that ERK5 provides protection against oxidative stress-induced cytotoxicity dependent on Nrf2. Molecular interaction between ERK5 and Nrf2 was further induced by laminar flow. Finally, flow-dependent nuclear localization of Nrf2 was inhibited by BIX02189, a specific inhibitor of MEK5, in aorta of mice in vivo. Collectively, these data demonstrate that laminar flow-induced activation of ERK5-Nrf2 signal pathway plays a critical role for anti-inflammatory and anti-apoptotic mechanism in endothelial cells.

Effect of auranofin on oxidative and endoplasmic reticulum stress as well as anti-CLL activity with proteasome inhibitor.

e13568 Background: The gold-containing compound auranofin (AF) (Ridaura), is a treatment of rheumatoid arthritis. Recently, AF was shown to inhibit thioredoxin reductase (TRR), increase reactive oxygen species (ROS) and induce apoptosis in cancer cells. Methods: We determined the ability of AF (250 to 1000 nM) to induce oxidative, proteotoxic and lethal endoplasmic reticulum (ER) stress in CD19+ primary CLL cells, including those with deletion of chromosome 17p. Results: Treatment with AF induced 20 to 40% increase in ROS levels, decreased TRR activity (mean of 45%), but not its protein expression, in CLL cells. AF-mediated oxidative stress induced NRF2 activity with increase in hemeoxygenase-1 (HO-1) and glutamate cysteine ligase modifier (GCLM) levels. AF induced ER stress, associated with increase in GRP78 and the pro-apoptotic transcription factor CHOP protein levels. AF also induced the pro-apoptotic BH3-only domain protein BIM. Exposure to AF increased the intracellular levels of misfolded polyubiquitylated proteins. This disrupted the binding of heat shock protein (hsp) 90 with histone deacetylase 6 (HDAC6), resulting in hyperacetylation and inhibition of the chaperone function of hsp90. This led to proteasomal degradation of CLL-relevant hsp90 client proteins, including ZAP70, c-Raf and AKT. Nuclear STAT3 levels also declined. Exposure to AF induced significantly more apoptosis (range 40 to 60%) in primary CLL cells, as compared to CD19+ normal B cells and CD34+ human cord blood and bone marrow progenitor cells (< 15% apoptosis) (p < 0.01). AF treatment also induced apoptosis in CD19+ cells from patients with poor prognosis CLL with deletion of 17p or with deletion of 13q. Co-treatment with AF and the proteasome inhibitor carfilzomib or the GCLM antagonist buthionine sulfoximine (BSO) synergistically induced apoptosis in primary CLL cells. A FDA IND-supported phase II clinical trial of AF (6 to 9 mg PO/day) with correlative biomarker analysis (as above) is underway and three patients are enrolled. Conclusions: These findings support the full evaluation of clinical activity and predictive biomarkers of response to the re-purposed AF in patients with CLL.

Functional dissection of Nrf2-dependent phase II genes in vascular inflammation and endotoxic injury using Keap1 siRNA

Keap1 is a cytoplasmic repressor of the transcription factor Nrf2, and its degradation induces Nrf2 activation, leading to upregulation of antioxidant phase II genes. We investigated the roles of phase II genes in vascular inflammation and septic injury using Keap1 siRNA and elucidated its underlying mechanism. Selective knockdown of Keap1 with siRNA promoted Nrf2-dependent expression of phase II genes in endothelial cells, such as heme oxygenase-1 (HO-1), glutamate–cysteine ligase (GCL), and peroxiredoxin-1 (Prx1), resulting in the elevation of cellular glutathione levels and suppression of tumor necrosis factor (TNF)-α-induced intracellular H2O2 accumulation. Keap1 knockdown inhibited TNF-α-induced expression of intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) by suppressing NF-κB activation via inhibition of its upstream modulators, Akt, NIK, and IKK, resulting in the elevation of monocyte adhesion to endothelial cells. Importantly, these events were reversed by HO-1 and GCL inhibitors and Prx1-specific siRNA. Keap1 knockdown also inhibited endotoxin-induced expression of inducible nitric oxide synthase (iNOS) and TNF-α by upregulating HO-1, GCL, and Prx1 expression in macrophages. Moreover, in vivo Keap1 knockdown increased the expression of phase II genes and suppressed the expression of ICAM-1, VCAM-1, iNOS, and TNF-α in an endotoxemic mouse model, resulting in significant protection against liver and lung injuries and lethality. Our results indicate that Keap1 knockdown prevents NF-κB-mediated vascular inflammation and endotoxic shock by suppressing NF-κB-mediated inflammatory gene expression via upregulation of Nrf2-mediated antioxidant genes. Thus, siRNA targeting Keap1 may provide a new therapeutic approach for inflammation-associated vascular diseases and sepsis.

Off Target therapeutic effects of commonly used drugs to treat acute mountain sickness

IntroductionAcute mountain sickness is a syndrome that effects ~60% of individuals ascending to high altitude, yet the etiology is still unclear. It has been proposed that reactive oxygen species (ROS) formed from acute high altitude exposure contribute to brain vascular leak and development of AMS. NF‐E2‐related factor 2 (Nrf2) is a transcription factor that regulates expression of greater than 90% of antioxidant genes.HypothesisWe postulated that some drugs currently used to treat AMS symptoms could induce Nrf2 activation and that by increasing anti‐oxidant status would decrease vascular damage from oxidative stress in hypoxic conditions.MethodsA MCF7 cell line stably transfected with a luciferase gene driven by an antioxidant response element, the known Nrf2 promoter, was used to screen. Drugs that showed favorable Nrf2 activation were further tested in brain microvascular endothelial cells to determine if they attenuated hypoxia induced ROS production and permeability. Finally, compounds were tested for their ability to induce Nrf2 and decrease brain leak (measured by Evans blue dye) in male Spraque Dawly rats exposed to high altitude (48 h, 19,000 ft).ResultsOf 9 drugs tested, Nifedipine and Methazolamide showed strong NRF2 activation. Cells in hypoxic conditions increased hydrogen peroxide production by two fold from normoxic. Pre‐treatment with Methazolamide, Nifedipine, and Protandim decreased ROS production back to normoxic concentrations. This response was blocked by siRNA against Nrf2. Resistance was decreased after 24 hr hypoxic exposure, which was restored to normoxic values with pre‐treatments of Protandim, Methazolamide, and Nifedipine. Only drugs that showed Nrf2 activation in‐vitro, decreased brain vascular leak, in‐vivo.ConclusionNrf2 activators are effective at reducing brain vascular leak from acute high altitude exposures.

Hepatitis C Virus Proteins Activate NRF2/ARE Pathway by Distinct ROS-Dependent and Independent Mechanisms in HUH7 Cells

Hepatitis C virus (HCV) is a highly pathogenic human virus associated with liver fibrosis, steatosis, and cancer. In infected cells HCV induces oxidative stress. Here, we show that HCV proteins core, E1, E2, NS4B, and NS5A activate antioxidant defense Nrf2/ARE pathway via several independent mechanisms. This was demonstrated by the analysis of transient co-expression in Huh7 cells of HCV proteins and luciferase reporters. Expression, controlled by the promoters of stress-response genes or their minimal Nrf2-responsive elements, was studied using luminescence assay, RT-qPCR and/or Western-blot analysis. All five proteins induced Nrf2 activation by protein kinase C in response to accumulation of reactive oxygen species (ROS). In addition, expression of core, E1, E2, NS4B, and NS5A proteins resulted in the activation of Nrf2 in a ROS-independent manner. The effect of core and NS5A was mediated through casein kinase 2 and phosphoinositide-3 kinase, whereas those of NS4B, E1, and E2, were not mediated by either PKC, CK2, PI3K, p38, or ERK. Altogether, on the earliest stage of expression HCV proteins induced a strong up-regulation of the antioxidant defense system. These events may underlie the harmful effects of HCV-induced oxidative stress during acute stage of hepatitis C.