Translation Of Nrf2 Research Articles

Ozone therapy mitigates parthanatos after ischemic stroke

BackgroundStroke is a leading cause of death worldwide, with oxidative stress and calcium overload playing significant roles in the pathophysiology of the disease. Ozone, renowned for its potent antioxidant properties, is commonly employed as an adjuvant therapy in clinical settings. Nevertheless, it remains unclear whether ozone therapy on parthanatos in cerebral ischemia–reperfusion injury (CIRI). This study aims to investigate the impact of ozone therapy on reducing parthanatos during CIRI and to elucidate the underlying mechanism.MethodsHydrogen peroxide (H2O2) was utilized to mimic the generation of reactive oxygen species (ROS) in SH-SY5Y cell reperfusion injury in vitro, and an in vivo ischemic stroke model was established. Ozone saline was introduced for co-culture or intravenously administered to mice. Apoptosis and oxidative stress were assessed using flow cytometry and immunofluorescence. Western blotting was utilized to examine the expression of parthanatos signature proteins. The mechanism by which ozone inhibits parthanatos was elucidated through inhibiting PPARg or Nrf2 activity. ResultsThe findings demonstrated that ozone mitigated H2O2-induced parthanatos by either upregulating nuclear factor erythroid 2-related factor 2 (Nrf2) or activating peroxisome proliferator-activated receptorg (PPARg). Furthermore, through the use of calcium chelators and ROS inhibitors, it was discovered that ROS directly induced parthanatos and facilitated intracellular calcium elevation. Notably, a malignant feedback loop between ROS and calcium was identified, further amplifying the induction of parthanatos. Ozone therapy exhibited its efficacy by increasing PPARg activity or enhancing the Nrf2 translation, thereby inhibiting ROS production induced by H2O2. Concurrently, our study demonstrated that ozone treatment markedly inhibited parthanatos in stroke-afflicted mice. Additionally, ozone therapy demonstrated significant neuroprotective effects on cortical neurons, effectively suppressing parthanatos.ConclusionsThese findings contribute valuable insights into the potential of ozone therapy as a therapeutic strategy for reducing parthanatos during CIRI, highlighting its impact on key molecular pathways associated with oxidative stress and calcium regulation.

NRF2 translation block by inhibition of cap-dependent initiation sensitizes lymphoma cells to ferroptosis and CAR-T immunotherapy.

Cancers coopt stress-response pathways to drive oncogenesis, dodge immune surveillance, and resist cytotoxic therapies. Several of these provide protection from ferroptosis, iron-mediated oxidative cell death. Here, we found dramatic sensitization to ferroptosis upon disruption of cap-dependent translation in diffuse large B-cell lymphoma (DLBCL). Specifically, rocaglate inhibitors of the eIF4A1 RNA helicase synergized with pharmacologic ferroptosis inducers, driven by a collapse of glutathione production that protects polyunsaturated fatty acids from ferroptotic oxidation. These effects occur despite initial up-regulation of specific protective factors. We find lost translation of NRF2, oncogenic master regulator of antioxidant gene-expression, is a key consequence of eIF4A1 inhibition. In vivo, combination of the clinical rocaglate zotatifin with a pharmacologically optimized ferroptosis inducer eradicated DLBCL patient derived xenografts. Moreover, we found zotatifin pre-exposure sensitized DLBCL to CD19-directed chimeric antigen receptor (CAR-19) T cells. Translational disruption therefore provides new opportunities to leverage therapeutic impacts of ferroptosis inducers including cytotoxic immunotherapies.

N6-methyladenosine mediates Nrf2 protein expression involved in PM2.5-induced pulmonary fibrosis

It has been reported that particulate matter with an aerodynamic diameter of <2.5 µm (PM2.5) could induce epithelial–mesenchymal transition (EMT)- and extracellular matrix (ECM)-related pulmonary fibrosis (PF). The transcription factor Nrf2 alleviated PM2.5-induced PF by antagonizing oxidative stress. The N6-methyladenosine (m6A) modification plays a significant role in the stress response. However, the effect of m6A modification on the mechanisms of Nrf2-mediated defense against PM2.5-induced PF remained unknown. Here, we explored the role and the underlying molecular mechanisms of m6A methylation of Nrf2 mRNA in PM2.5-induced PF. We established filtered air (FA), unfiltered air (UA), and concentrated PM2.5 air (CA) group mice model and 0, 50, and 100 μg/mL PM2.5-treated 16HBE cell models. The extent of lung fibrosis in mice and fibrosis indicators were detected by histopathological analysis, immunohistochemical staining and western blotting. The molecular mechanism of m6A-modified Nrf2 was demonstrated by m6A-methylated RNA immunoprecipitation (MeRIP), RNA immunoprecipitation (RIP), qRT-PCR and T3 ligase-based PCR. Our data showed that PM2.5 exposure for 16 weeks could induce pulmonary fibrosis and activate Nrf2 signaling pathway. m6A methyltransferase METTL3 was upregulated after PM2.5 treatment in vivo and in vitro. Moreover, METTL3 mediated m6A modification of Nrf2 mRNA and promoted Nrf2 translation in mice and 16HBE cells after PM2.5 exposure. Mechanistically, three m6A-modified sites (1317, 1376 and 935; numbered relative to the first nucleotide of 3’UTR) of Nrf2 mRNA were identified in PM2.5-treatment 16HBE cells. Furthermore, the m6A binding proteins YTHDF1/IGF2BP1 promoted Nrf2 translation by binding to m6A residues of Nrf2 mRNA. Our results revealed the mechanism of m6A mediated Nrf2 signaling pathway against oxidative stress, which affected the development of PM2.5-induced PF.

Hyperoside Ameliorates Renal Tubular Oxidative Damage and Calcium Oxalate Deposition in Rats through AMPK/Nrf2 Signaling Axis.

Nephrolithiasis is a common disease that seriously affects the health and life quality of patients. Despite the reported effect of hyperoside (Hyp) against nephrolithiasis, the specific mechanism has not been clarified. Therefore, this study is aimed at investigating the effect and potential mechanism of Hyp on renal injury and calcium oxalate (CaOx) crystal deposition. Rat and cell models of renal calculi were constructed by ethylene glycol (EG) and CaOx induction, respectively. The renal histopathological damage, CaOx crystal deposition, and renal function damage of rats were assessed by HE staining, Pizzolato staining, and biochemical detection of blood and urine parameters. MTT and crystal-cell adhesion assays were utilized to determine the activity of HK-2 cells and crystal adhesion ability, biochemical detection and enzyme-linked immunosorbent assay (ELISA) to measure the levels of oxidative stress-related substances and inflammatory factors, and western blot to test the expression levels of proteins related to the AMPK/Nrf2 signaling pathway. Briefly speaking, Hyp could improve the renal histopathological injury and impaired renal function, reduce the deposition of CaOx crystals in the renal tissue of rats with renal calculi, and decrease the adhesion of crystals to CaOx-treated HK-2 cells. Besides, Hyp also significantly inhibited oxidative stress response. Furthermore, Hyp was associated with the downregulation of malondialdehyde, lactate dehydrogenase, and reactive oxygen species and upregulation of superoxide dismutase activity. Additionally, Hyp treatment also suppressed inflammatory response and had a correlation with declined levels of interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor. Further exploration of mechanism manifested that Hyp might play a protective role through promoting AMPK phosphorylation and nuclear translation of Nrf2 to activate the AMPK/Nrf2 signaling pathway. Hyp can improve renal pathological and functional damage, decrease CaOx crystal deposition, and inhibit oxidative stress and inflammatory response. Such effects may be achieved by activating the AMPK/Nrf2 signaling pathway.

Mechanisms underlying Nrf2 nuclear translocation by non-lethal levels of hydrogen peroxide: p38 MAPK-dependent neutral sphingomyelinase2 membrane trafficking and ceramide/PKCζ/CK2 signaling.

Hydrogen peroxide is an aerobic metabolite playing a central role in redox signaling and oxidative stress. H2O2 could activate redox sensitive transcription factors, such as Nrf2, AP-1 and NF-κB by different manners. In some cells, treatment with non-lethal levels of H2O2 induces rapid activation of Nrf2, which upregulates expression of a set of genes involved in glutathione (GSH) synthesis and defenses against oxidative damage. It depends on two steps, the rapid translational activation of Nrf2 and facilitation of Nrf2 nuclear translocation. We review the molecular mechanisms by which H2O2 induces nuclear translocation of Nrf2 in cultured cells by highlighting the role of neutral sphingomyelinase 2 (nSMase2), a GSH sensor. H2O2 enters cells through aquaporin channels in the plasma membrane and is rapidly reduced to H2O by GSH peroxidases to consume cellular GSH, resulting in nSMase2 activation to generate ceramide. H2O2 also activates p38 MAP kinase, which enhances transfer of nSMase2 from perinuclear regions to plasma membrane lipid rafts to accelerate ceramide generation. Low levels of ceramide activate PKCζ, which then activates casein kinase 2 (CK2). These protein kinases are able to phosphorylate Nrf2 to stabilize and activate it. Notably, Nrf2 also binds to caveolin-1 (Cav1), which protects Nrf2 from Keap1-mediated degradation and limits Nrf2 nuclear translocation. We propose that Cav1serves as a signaling hub for the control of H2O2-mediated phosphorylation of Nrf2 by kinases, which results in release of Nrf2 from Cav1 to facilitate nuclear translocation. In summary, H2O2 induces GSH depletion which is recovered by Nrf2 activation dependent on p38/nSMase2/ceramide signaling.

Extracellular Vesicles Regulate Sympatho-Excitation by Nrf2 in Heart Failure.

Chronic heart failure (CHF) is associated with redox imbalance. Downregulation of Nrf2 (nuclear factor [erythroid-derived 2]-like 2) plays important roles in disrupting myocardial redox homeostasis and mediating sympathetic nerve activity in the setting of CHF. However, it is unclear if circulating extracellular vesicles (EVs) elicit sympathetic excitation in CHF by disrupting central redox homeostasis. We tested the hypothesis that cardiac-derived EVs circulate to the presympathetic rostral ventrolateral medulla and contribute to oxidative stress and sympathetic excitation via EV-enriched microRNA-mediated Nrf2 downregulation. Data were collected on rats with CHF post-myocardial infarction (MI) and on human subjects with ischemic CHF. EVs were isolated from tissue and plasma, and we determined the miRNAs cargo that related to targeting Nrf2 translation. We tracked the distribution of cardiac-derived EVs using in vitro labeled circulating EVs and cardiac-specific membrane GFP+ transgenic mice. Finally, we tested the impact of exogenously loading of antagomirs to specific Nrf2-related miRNAs on CHF-EV-induced pathophysiological phenotypes in normal rats (eg, sympathetic and cardiac function). Nrf2 downregulation in CHF rats was associated with an upregulation of Nrf2-targeting miRNAs, which were abundant in cardiac-derived and circulating EVs from rats and humans. EVs isolated from the brain of CHF rats were also enriched with Nrf2-targeting miRNAs and cardiac-specific miRNAs. Cardiac-derived EVs were taken up by neurons in the rostral ventrolateral medulla. The administration of cardiac-derived and circulating EVs from CHF rats into the rostral ventrolateral medulla of normal rats evoked an increase in renal sympathetic nerve activity and plasma norepinephrine compared with Sham-operated rats, which were attenuated by exogenously preloading CHF-EVs with antagomirs to Nrf2-targeting miRNAs. Cardiac microRNA-enriched EVs from animals with CHF can mediate crosstalk between the heart and the brain in the regulation of sympathetic outflow by targeting the Nrf2/antioxidant signaling pathway. This new endocrine signaling pathway regulating sympathetic outflow in CHF may be exploited for novel therapeutics.

The TIAR-mediated Nrf2 response to oxidative stress is mediated through the Nrf2 noncoding 3'untranslated region in Spodoptera litura

Nrf2 is a key regulator in the maintenance of cellular redox balance by regulating the expression of genes related to antioxidative responses and detoxification. Nrf2 protein levels are increased in response to oxidative stress. However, the regulation of the Nrf2 3′UTR on Nrf2 translation is unclear. Here, we report that the translational activity of the 3′UTR is required for Spodoptera litura Nrf2 protein expression. Experiments showed that the 3′UTR translation activity of S. litura Nrf2 was much higher than that of the 5′UTR. RNA interference (RNAi) of the expression of T cell internal antigen-related protein (TIAR), an RNA-binding protein that interacts with the 3′UTR of S. litura Nrf2, resulted in Nrf2 mRNA movement out of translationally active polysomes and a decrease in cellular Nrf2 protein levels. TIAR interacted with poly(A)-binding protein (PABP) and translation initiation factors eIF2-2 and eIF2-3 to enhance Nrf2 translation, indicating that the 3′UTR regulates Nrf2 translation. Diethyl maleate (DEM) treatment increased reactive oxygen species (ROS) in cells and enhanced Nrf2 levels, which had been reduced by cycloheximide (CHX), an inhibitor of de novo protein synthesis; Tiar RNAi increased ROS levels in DEM-treated cells, suggesting TIAR-mediated 3′UTR involvement in Nrf2 translation in response to DEM treatment. Thus, we reveal a posttranscriptional regulation mechanism of Nrf2, in which TIAR binds with the Nrf2 mRNA 3′UTR to enhance Nrf2 translation, facilitating the increase in Nrf2 protein levels in response to oxidative stress.

HACE1-mediated NRF2 activation causes enhanced malignant phenotypes and decreased radiosensitivity of glioma cells

HACE1, an E3 ubiquitin-protein ligase, is frequently inactivated and has been evidenced as a putative tumor suppressor in different types of cancer. However, its role in glioma remains elusive. Here, we observed increased expression of HACE1 in gliomas related to control subjects, and found a strong correlation of high HACE1 expression with poor prognosis in patients with WHO grade III and IV as well as low-grade glioma (LGG) patients receiving radiotherapy. HACE1 knockdown obviously suppressed malignant behaviors of glioma cells, while ectopic expression of HACE1 enhanced cell growth in vitro and in vivo. Further studies revealed that HACE1 enhanced protein stability of nuclear factor erythroid 2-related factor 2 (NRF2) by competitively binding to NRF2 with another E3 ligase KEAP1. Besides, HACE1 also promoted internal ribosome entry site (IRES)-mediated mRNA translation of NRF2. These effects did not depend on its E3 ligase activity. Finally, we demonstrated that HACE1 dramatically reduced cellular ROS levels by activating NRF2, thereby decreasing the response of glioma cells to radiation. Altogether, our data demonstrate that HACE1 causes enhanced malignant phenotypes and decreased radiosensitivity of glioma cells by activating NRF2, and indicate that it may act as the role of prognostic factor and potential therapeutic target in glioma.

USP10 inhibits the dopamine-induced reactive oxygen species–dependent apoptosis of neuronal cells by stimulating the antioxidant Nrf2 activity

Nrf2 is an antioxidant transcriptional activator in many types of cells, and its dysfunction plays key roles in a variety of human disorders, including Parkinson's disease (PD). PD is characterized by the selective loss of dopaminergic neurons in PD-affected brain regions. Dopamine treatment of neuronal cells stimulates the production of reactive oxygen species (ROS) and increases ROS-dependent neuronal apoptosis. In this study, we found that the ubiquitin-specific protease 10 (USP10) protein reduces dopamine-induced ROS production of neuronal cells and ROS-dependent apoptosis by stimulating the antioxidant activity of Nrf2. USP10 interacted with the Nrf2 activator p62, increased the phosphorylation of p62, increased the interaction of p62 with the Nrf2 inhibitor Keap1, and stimulated Nrf2 antioxidant transcriptional activity. In addition, USP10 augmented dopamine-induced Nrf2 translation. Taken together, these results indicate that USP10 is a key regulator of Nrf2 antioxidant activity in neuronal cells and suggest that USP10 activators are promising therapeutic agents for oxidative stress–related diseases, including PD.

Isoorientin exerts a protective effect against 6-OHDA-induced neurotoxicity by activating the AMPK/AKT/Nrf2 signalling pathway.

Parkinson's disease (PD) is a progressive neurodegenerative disorder that is closely associated with oxidative stress. Nuclear factor erythroid 2 related factor 2 (Nrf2) is a key transcription factor that regulates oxidative stress. Isoorientin (IOT), as a dietary C-glucosyl flavone derived from rooibos tea, cereals and legumes, is thought to possess multiple pharmacological activities; however, the protective effect of IOT against 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in SH-SY5Y cells is still poorly understood. The present study focused on investigating whether IOT could ameliorate neurotoxicity and the underlying mechanisms. Our findings indicated that IOT significantly inhibited neurotoxicity reduced apoptotic cell numbers, reactive oxygen species (ROS) overproduction and mitochondrial membrane potential, and modulated the expression of apoptosis-related proteins, including Bcl-2, Bax and caspase-3, which were induced by 6-OHDA. Moreover, IOT also enhanced the expression of the GCLC, GCLM, HO-1, NQO1 and Trx-1 proteins, which mostly depends on the nuclear translation of Nrf2 and reduced expression of the Keap1 protein. IOT significantly increased the phosphorylation of AMPK, ERK, GSK3β, JNK, PI3K and AKT. In contrast, pretreatment with the inhibitors of AMPK and PI3K/AKT only suppressed the nuclear translocation of Nrf2. In addition, the expression of these proteins was effectively decreased by 6-OHDA, and this effect was reversed by IOT treatment. Importantly, the effect of IOT on improving 6-OHDA induced neurotoxicity was remarkably abrogated by the application of Nrf2 siRNA and, AMPK and PI3K/AKT inhibitors. In summary, IOT might play a protective role against 6-OHDA-induced neurotoxicity by inducing the expression of various antioxidant enzymes via the activation of the AMPK/AKT-Nrf2 signalling pathway.

Curcumin analogues attenuate Aβ25-35-induced oxidative stress in PC12 cells via Keap1/Nrf2/HO-1 signaling pathways

Beta-amyloid (Aβ) has pivotal functions in the pathogenesis of Alzheimer's Disease (AD). In the present study, we adopted an vitro model that involved Aβ25-35-induced oxidative damage in PC12 cells. Aβ25-35 (10 μΜ) treatment for 24 h induced significant cell death and oxidative stress in PC12 cells, as evidenced by cell viability reduction, LDH release, ROS accumulation and increased production MDA. (1E,4E)-1, 5-bis(4-hydroxy-3-methoxyphenyl) penta-1, 4-dien-3-one (CB) and (1E, 4E)-1-(3, 4-dimethoxyphenyl)-5-(4-hydroxy-3, 5-dime-thoxyphenyl) Penta-1, 4-dien-3-one (FE), two Curcumin (Cur) analogues displayed neuroprotective effects against Aβ25-35-induced oxidative damage and cellular apoptosis in PC12 cells. Here, we investigated three different treatment ways of CB and FE. It was interesting that post-treatment of CB and FE (restoring way) showed similar effect to the preventive way, while attenuating way did not show any protective effect. We found that low dose CB and FE increased transcriptional factor NF-E2-related factor 2 (Nrf2)/hemo oxygenase 1 (HO-1) protein expression and decreased Kelch-like ECH-associated protein 1 (Keap1) in PC 12 cells. In addition, CB and FE promoted the translation of Nrf2 into nuclear and enhanced the activity of superoxide dismutase (SOD)/catalase, which confirmed cytoprotection against Aβ25-35-induced oxidative damage. Moreover, CB and FE could increase Bcl-2 expression level, decrease the level of Bax and Cyt-c in Aβ25-35-treated PC12 cells. Ultimately, the neuroprotective effect of CB and FE provides a pharmacological basis for its clinical use in prevention and treatment of AD.

Myocardial infarction-induced microRNA-enriched exosomes contribute to cardiac Nrf2 dysregulation in chronic heart failure.

The imbalance between the synthesis of reactive oxygen species and their elimination by antioxidant defense systems results in macromolecular damage and disruption of cellular redox signaling, affecting cardiac structure and function, thus contributing to contractile dysfunction, myocardial hypertrophy, and fibrosis in chronic heart failure [chronic heart failure (CHF)]. The Kelch-like ECH-associated protein 1-nuclear factor erythroid 2-related factor 2 (Nrf2) pathway is an important antioxidant defense mechanism and is closely associated with oxidative stress-mediated cardiac remodeling in CHF. In the present study, we investigated the regulation of myocardial Nrf2 in the postmyocardial infarction (post-MI) state. Six weeks post-MI, Nrf2 protein was downregulated in the heart, resulting in a decrease of Nrf2-targeted antioxidant enzymes, whereas paradoxically the transcription of Nrf2 was increased, suggesting that translational inhibition of Nrf2 may contribute to the dysregulation in CHF. We therefore hypothesized that microRNAs may be involved in the translational repression of Nrf2 mRNA in the setting of CHF. Using quantitative real-time PCR analysis, we found that three microRNAs, including microRNA-27a, microRNA-28-3p, and microRNA-34a, were highly expressed in the left ventricle of infarcted hearts compared with other organs. Furthermore, in vitro analysis revealed that cultured cardiac myocytes and fibroblasts expressed these three microRNAs in response to TNF-α stimulation. These microRNAs were preferentially incorporated into exosomes and secreted into the extracellular space in which microRNA-enriched exosomes mediated intercellular communication and Nrf2 dysregulation. Taken together, these results suggest that increased local microRNAs induced by MI may contribute to oxidative stress by the inhibition of Nrf2 translation in CHF. NEW & NOTEWORTHY The results of this work provide a novel mechanism mediated by microRNA-enriched exosomes, contributing to the nuclear factor erythroid 2-related factor 2 dysregulation and subsequent oxidative stress. Importantly, these new findings will provide a promising strategy to improve the therapeutic efficacy through targeting nuclear factor erythroid 2-related factor 2-related microRNAs in the chronic heart failure state, which show potentially clinical applications.

Pharmacological stimulation of nuclear factor (erythroid-derived 2)-like 2 translation activates antioxidant responses

Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is the master regulator of the antioxidant response, and its function is tightly regulated at the transcriptional, translational, and post-translational levels. It is well-known that Nrf2 is regulated at the protein level by proteasomal degradation via Kelch-like ECH-associated protein 1 (Keap1), but how Nrf2 is regulated at the translational level is less clear. Here, we show that pharmacological stimulation increases Nrf2 levels by overcoming basal translational repression. We developed a novel reporter assay that enabled identification of natural compounds that induce Nrf2 translation by a mechanism independent of Keap1-mediated degradation. Apigenin, resveratrol, and piceatannol all induced Nrf2 translation. More importantly, the pharmacologically induced Nrf2 overcomes Keap1 regulation, translocates to the nucleus, and activates the antioxidant response. We conclude that translational regulation controls physiological levels of Nrf2, and this can be modulated by apigenin, resveratrol, and piceatannol. Also, targeting this mechanism with novel compounds could provide new insights into prevention and treatment of multiple diseases in which oxidative stress plays a significant role.

237 - Activation of Nrf2 Translation by a Keap1 Independent Mechanism

Nuclear factor (erythroid-derived 2)-like 2 (nrf2) is the master regulator of the antioxidant response, and its function is tightly regulated at the transcription, translation, and post-translational level. It is well known that Keap1 regulates Nrf2 signaling but the Nrf2 regulation at the translation level and its chemical modulation is less documented. Here, we show a complementary way to promote an increase in the Nrf2 levels by overcoming its basal translational repression thus opening the possibility for the discovery of novel alternatives for promoting cellular protection against oxidative stress. We developed a novel reporter system that allowed for the identification of natural compounds present in the human diet as inducers of Nrf2 translation by a mechanism independent of Keap1-mediated degradation. Apigenin, resveratrol and piceatannol were identified as inducers of Nrf2 translation. More importantly, the newly synthesized Nrf2 overcomes keap1-regulation, translocates to the nuclei and activates the antioxidant response. We conclude that Nrf2 translational control is physiologically relevant and can be regulated by apigenin. Also, targeting this mechanism could provide new insights into prevention and treatment of multiple diseases where oxidative stress plays a major role.

Noncanonical SQSTM1/p62-Nrf2 pathway activation mediates proteasome inhibitor resistance in multiple myeloma cells via redox, metabolic and translational reprogramming.

Multiple Myeloma (MM) is a B-cell malignancy characterized by the accumulation of clonal plasma cells in the bone marrow, with drug resistance being a major cause of therapeutic failure. We established a carfilzomib-resistant derivative of the LP-1 MM cell line (LP-1/Cfz) and found that the transcription factor NF-E2 p45-related factor 2 (Nrf2; gene symbol NFE2L2) contributes to carfilzomib resistance. The mechanism of Nrf2 activation involved enhanced translation of Nrf2 as well as its positive regulator, the autophagy receptor sequestosome 1 (SQSTM1)/p62. The eukaryotic translation initiation factor gene EIF4E3 was among the Nrf2 target genes upregulated in LP-1/Cfz cells, suggesting existence of a positive feedback loop. In line with this, we found that siRNA knockdown of eIF4E3 decreased Nrf2 protein levels. On the other hand, elevated SQSTM1/p62 levels were due at least in part to activation of the PERK-eIF2α pathway. LP-1/Cfz cells had decreased levels of reactive oxygen species as well as elevated levels of fatty acid oxidation and prosurvival autophagy. Genetic and pharmacologic inhibition of the Nrf2-EIF4E3 axis or the PERK-eIF2α pathway, disruption of redox homeostasis or inhibition of fatty acid oxidation or autophagy conferred sensitivity to carfilzomib. Our findings were supported by clinical data where increased EIF4E3 expression was predictive of Nrf2 target gene upregulation in a subgroup of patients with chemoresistant minimal residual disease and relapsed/refractory MM. Thus, our data offer a preclinical rationale for including inhibitors of the SQSTM1/p62-Nrf2 pathway to the treatment regimens for certain advanced stage MM patients.

Age-related loss of hepatic Nrf2 protein homeostasis: Potential role for heightened expression of miR-146a

Nrf2 regulates the expression of numerous anti-oxidant, anti-inflammatory, and metabolic genes. We observed that, paradoxically, Nrf2 protein levels decline in the livers of aged rats despite the inflammatory environment evident in that organ. To examine the cause(s) of this loss, we investigated the age-related changes in Nrf2 protein homeostasis and activation in cultured hepatocytes from young (4–6 months) and old (24–28 months) Fischer 344 rats. While no age-dependent change in Nrf2 mRNA levels was observed (p>0.05), Nrf2 protein content, and the basal and anetholetrithione (A3T)-induced expression of Nrf2-dependent genes were attenuated with age. Conversely, overexpression of Nrf2 in cells from old animals reinstated gene induction. Treatment with A3T, along with bortezomib to inhibit degradation of existing protein, caused Nrf2 to accumulate significantly in cells from young animals (p<0.05), but not old, indicating a lack of new Nrf2 synthesis. We hypothesized that the loss of Nrf2 protein synthesis with age may partly stem from an age-related increase in microRNA inhibition of Nrf2 translation. Microarray analysis revealed that six microRNAs significantly increase >2-fold with age (p<0.05). One of these, miRNA-146a, is predicted to bind Nrf2 mRNA. Transfection of hepatocytes from young rats with a miRNA-146a mimic caused a 55% attenuation of Nrf2 translation that paralleled the age-related loss of Nrf2. Overall, these results provide novel insights for the age-related decline in Nrf2 and identify new targets to maintain Nrf2-dependent detoxification with age.

Erythropoietin activates Keap1–Nrf2/ARE pathway in rat brain after ischemia

rhEPO has previously been shown to exert neuroprotective action in focal cerebral ischemia. However, its mechanism is not clear. We established the model of permanent focal cerebral ischemia. rhEPO was administered (5000 IU/kg i.p.) 2 h later after the successful ischemia model in rhEPO group and increased translation of Nrf2 and HO-1 and decreased the H2O2 concentration in the brain confirming activation of the Keap1–Nrf2/ARE pathway. The results show that rhEPO activate Keap1–Nrf2/ARE pathway after ischemia to protect the brain tissue.

Translational control of Nrf2 within the open reading frame

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.

Cap-independent Nrf2 translation is part of a lipoic acid-stimulated detoxification stress response

Little is known about either the basal or stimulated homeostatic mechanisms regulating nuclear tenure of Nf-e2-related factor 2 (Nrf2), a transcription factor that mediates expression of over 200 detoxification genes. Our data show that stress-induced nuclear Nrf2 accumulation is largely from de novo protein synthesis, rather than translocation from a pre-existing cytoplasmic pool. HepG2 cells were used to monitor nuclear Nrf2 24h following treatment with the dithiol micronutrient (R)-α-lipoic acid (LA; 50μM), or vehicle. LA caused a ≥2.5-fold increase in nuclear Nrf2 within 1h. However, pretreating cells with cycloheximide (50μg/ml) inhibited LA-induced Nrf2 nuclear accumulation by 94%. Providing cells with the mTOR inhibitor, rapamycin, decreased basal Nrf2 levels by 84% after 4h, but LA overcame this inhibition. LA-mediated de novo protein translation was confirmed using HepG2 cells transfected with a bicistronic construct containing an internal ribosome entry sequence (IRES) for Nrf2, with significant (P<0.05) increase in IRES use under LA treatment. These results suggest that a dithiol stimulus mediates Nrf2 nuclear tenure via cap-independent protein translation. Thus, translational control of Nrf2 synthesis, rather than reliance solely on pre-existing protein, may mediate the rapid burst of Nrf2 nuclear accumulation following stress stimuli.

Abstract 4022: Antioxidant-induced modification of INrf2C151 and PKCdelta-mediated phosphorylation of Nrf2S40 are both required for stabilization and nuclear translation of Nrf2 and increased drug-resistance

Abstract 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-delta -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-delta+/+ and PKC-delta −/− 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-delta-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- delta+/+ and PKC-delta −/− cells occurred, but Nrf2 failed to be released in PKC-delta −/− cells. The antioxidant activation of Nrf2 reduced etoposide-mediated DNA fragmentation and promoted cell survival in PKC- delta+/+ but not in PKC- delta−/− cells. These data together demonstrate that both modification of INrf2C151 and PKC- delta-mediated phosphorylation of Nrf2S40 play crucial roles in Nrf2 release from INrf2, antioxidant induction of defensive gene expression, promoting cell survival, and increasing drug resistance. J. Cell Science in press 2009. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4022.