Pharmacological Activation Of Nrf2 Research Articles

Nrf2 inhibits oxaliplatin-induced peripheral neuropathy via protection of mitochondrial function.

Oxaliplatin-induced peripheral neuropathy (OIPN) is a severe, dose-limiting toxicity associated with cancer chemotherapy. The efficacy of antioxidant administration in OIPN is debatable, as the promising preliminary results obtained with a number of antioxidants have not been confirmed in larger clinical trials. Besides its antioxidant activity, the transcription factor, nuclear factor-erythroid 2 (NF-E2) p45-related factor 2 (Nrf2) plays a crucial role in the maintenance of mitochondrial homeostasis, and mitochondrial dysfunction is a key contributor to OIPN. Here, we have investigated the protective properties of Nrf2 in OIPN. Nrf2-/- mice displayed severe mechanical allodynia and cold sensitivity and thus experienced increased peripheral nervous system injury compared to Nrf2+/+ mice. Furthermore, Nrf2 knockout aggravated oxaliplatin-induced reactive oxygen species production, decreased the mitochondrial membrane potential, led to abnormal intracellular calcium levels, and induced cytochrome c-related apoptosis and overexpression of the TRP protein family. Sulforaphane-induced activation of the Nrf2 signaling pathway alleviated morphological alterations, mitochondrial dysfunction in dorsal root ganglion neurons, and nociceptive sensations in mice. Our findings reveal that Nrf2 may play a critical role in ameliorating OIPN, through protection of mitochondrial function by alleviating oxidative stress and inhibiting TRP protein family expression. This suggests that pharmacological or therapeutic activation of Nrf2 may be used to prevent or slow down the progression of OIPN.

The role of Nrf2 signaling in counteracting neurodegenerative diseases.

The transcription factor Nrf2 (nuclear factor‐erythroid 2 p45‐related factor 2) functions at the interface of cellular redox and intermediary metabolism. Nrf2 target genes encode antioxidant enzymes, and proteins involved in xenobiotic detoxification, repair and removal of damaged proteins and organelles, inflammation, and mitochondrial bioenergetics. The function of Nrf2 is altered in many neurodegenerative disorders, such as Huntington's disease, Alzheimer's disease, amyotrophic lateral sclerosis, and Friedreich's ataxia. Nrf2 activation mitigates multiple pathogenic processes involved in these neurodegenerative disorders through upregulation of antioxidant defenses, inhibition of inflammation, improvement of mitochondrial function, and maintenance of protein homeostasis. Small molecule pharmacological activators of Nrf2 have shown protective effects in numerous animal models of neurodegenerative diseases, and in cultures of human cells expressing mutant proteins. Targeting Nrf2 signaling may provide a therapeutic option to delay onset, slow progression, and ameliorate symptoms of neurodegenerative disorders.

P 087 - NRF2 controls proteostasis through the transcriptional regulation of autophagy

Cells control the abundance and quality of the proteome through a wide network that integrates signaling pathways, gene expression and protein degradation systems. Degradation of cytosolic components inside lysosomes is carried out by specific types of autophagy in mammals: macroautophagy, chaperone mediated autophagy (CMA) and microautophagy. Considering autophagy as a proteostatic and defensive mechanism, we sought to determine if this process was regulated by the transcription factor NRF2, classically considered the master regulator of the antioxidant cell response. A bioinformatics analysis allowed us to identify putative NRF2 binding sequences in macroautophagy and CMA related genes. Several were further validated as NRF2-regulated genes by ChIP assays and quantitative PCR in Nrf2-deficient cells. Consequently, Nrf2-knockout cells exhibited impaired macroautophagy flux in response to H2O2. Oxidative stress also up-regulates CMA through transcriptional induction of Lamp-2a, but to a lesser extent in Nrf2-deficient cells. Moreover, Nrf2-knockout cells showed reduced LAMP-2A lysosomal levels, the limiting step for CMA. Pharmacological activation of NRF2 led to the perinuclear accumulation of LAMP-2A positive lysosomes, indicative of CMA activation. Overall, these results point to a novel role of NRF2 in the regulation of autophagy and suggest a new strategy to combat proteinopathies.

KEAP1-modifying small molecule reveals muted NRF2 signaling responses in neural stem cells from Huntington's disease patients.

The activity of the transcription factor nuclear factor-erythroid 2 p45-derived factor 2 (NRF2) is orchestrated and amplified through enhanced transcription of antioxidant and antiinflammatory target genes. The present study has characterized a triazole-containing inducer of NRF2 and elucidated the mechanism by which this molecule activates NRF2 signaling. In a highly selective manner, the compound covalently modifies a critical stress-sensor cysteine (C151) of the E3 ligase substrate adaptor protein Kelch-like ECH-associated protein 1 (KEAP1), the primary negative regulator of NRF2. We further used this inducer to probe the functional consequences of selective activation of NRF2 signaling in Huntington's disease (HD) mouse and human model systems. Surprisingly, we discovered a muted NRF2 activation response in human HD neural stem cells, which was restored by genetic correction of the disease-causing mutation. In contrast, selective activation of NRF2 signaling potently repressed the release of the proinflammatory cytokine IL-6 in primary mouse HD and WT microglia and astrocytes. Moreover, in primary monocytes from HD patients and healthy subjects, NRF2 induction repressed expression of the proinflammatory cytokines IL-1, IL-6, IL-8, and TNFα. Together, our results demonstrate a multifaceted protective potential of NRF2 signaling in key cell types relevant to HD pathology.

Pathogenic p62/SQSTM1 mutations impair energy metabolism through limitation of mitochondrial substrates

Abnormal mitochondrial function has been found in patients with frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Mutations in the p62 gene (also known as SQSTM1) which encodes the p62 protein have been reported in both disorders supporting the idea of an ALS/FTD continuum. In this work the role of p62 in energy metabolism was studied in fibroblasts from FTD patients carrying two independent pathogenic mutations in the p62 gene, and in a p62-knock-down (p62 KD) human dopaminergic neuroblastoma cell line (SH-SY5Y). We found that p62 deficiency is associated with inhibited complex I mitochondrial respiration due to lack of NADH for the electron transport chain. This deficiency was also associated with increased levels of NADPH reflecting a higher activation of pentose phosphate pathway as this is accompanied with higher cytosolic reduced glutathione (GSH) levels. Complex I inhibition resulted in lower mitochondrial membrane potential and higher cytosolic ROS production. Pharmacological activation of transcription factor Nrf2 increased mitochondrial NADH levels and restored mitochondrial membrane potential in p62-deficient cells. Our results suggest that the phenotype is caused by a loss-of-function effect, because similar alterations were found both in the mutant fibroblasts and the p62 KD model. These findings highlight the implication of energy metabolism in pathophysiological events associated with p62 deficiency.

MP45-05 ACTIVATION OF NRF2 IMPROVES ENDOTHELIAL FUNCTION IN CORPUS CAVERNOSUM FROM AGED RATS AND IN CORPUS CAVERNOSUM AND PENILE ARTERIES FROM ED PATIENTS

You have accessJournal of UrologySexual Function/Dysfunction: Basic Research & Pathophysiology I1 Apr 2017MP45-05 ACTIVATION OF NRF2 IMPROVES ENDOTHELIAL FUNCTION IN CORPUS CAVERNOSUM FROM AGED RATS AND IN CORPUS CAVERNOSUM AND PENILE ARTERIES FROM ED PATIENTS Juan Ignacio Martínez-Salamanca, Mariam El Assar, Argentina Fernández, Alberto Sánchez-Ferrer, Agustín Fraile, Leocadio Rodríguez-Mañas, Joaquín Carballido, and Javier Angulo Juan Ignacio Martínez-SalamancaJuan Ignacio Martínez-Salamanca More articles by this author , Mariam El AssarMariam El Assar More articles by this author , Argentina FernándezArgentina Fernández More articles by this author , Alberto Sánchez-FerrerAlberto Sánchez-Ferrer More articles by this author , Agustín FraileAgustín Fraile More articles by this author , Leocadio Rodríguez-MañasLeocadio Rodríguez-Mañas More articles by this author , Joaquín CarballidoJoaquín Carballido More articles by this author , and Javier AnguloJavier Angulo More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.1423AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Adequate antioxidant response is essential for tissue homeostasis and function. However, systems responsible for antioxidant response are down-regulated in some pathological situations and aging. This is the case for Nrf2 that orchestrates cellular response to oxidative stress. The aim was to evaluate pharmacological activation of Nrf2 on the impairment of endothelial relaxation and reactive oxygen species-induced responses in corpus cavernosum (CC) from aged rats and in CC and penile arteries from patients with erectile dysfunction (ED). METHODS Endothelium-dependent relaxations to carbachol and responses to H2O2 were evaluated in CC from 3-months (young) and 20-months (aged) old rats in the absence or the presence of the Nrf2-activators, sulforaphane (10 microM) and oltipraz (30 microM). Upregulation of Nrf2 was assessed by ELISA. The effects of these Nrf2-activators were also evaluated on endothelial relaxation and H2O2-induced responses in human corpus cavernosum (HCC) and penile resistance arteries (HPRA) obtained from patients undergoing penile prosthesis implantation. RESULTS Aged rats CC displayed ED and impaired endothelium-dependent and H2O2-induced relaxation. Ex vivo exposure to either sulforaphane or oltipraz improved endothelial and H2O2-induced relaxation of CC from aged rats. HCC and HPRA were obtained from 19 patients (age: 60.7+/-2.0 years, hypertension: 8, dyslipidemia: 7, diabetes: 6, CVD: 4, obesity: 2). Treatment with sulforaphane improved endothelium-dependent (pD2 for acetylcholine: 5.18+/-0.28 vs 6.34+/-0.37∗) as well as neurogenic relaxation (Emax: 45.7+/-6.4% vs 60.9+/-3.0%∗) in HCC. Sulforaphane also improved endothelial (pD2 for acetylcholine: 5.82+/-0.38 vs 7.21+/-0.32∗) and H2O2-induced vasodilation in HPRA (pD2 for H2O2: 4.13+/-0.13 vs 5.07+/-0.17∗). Positive effects of Nrf2-activation were confirmed by the improvement of endothelial vasodilation driven by oltipraz in HPRA. CONCLUSIONS Pharmacological activation of Nrf2 improves cavernosal function in aged animals with ED. These effects were confirmed in human tissue, including penile arteries, from patients with ED, suggesting that Nrf2 activation could be a reasonable target for the management of ED. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e613 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Juan Ignacio Martínez-Salamanca More articles by this author Mariam El Assar More articles by this author Argentina Fernández More articles by this author Alberto Sánchez-Ferrer More articles by this author Agustín Fraile More articles by this author Leocadio Rodríguez-Mañas More articles by this author Joaquín Carballido More articles by this author Javier Angulo More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Nuclear erythroid 2-related factor 2 activation inhibits house dust mite-induced sinonasal epithelial cell barrier dysfunction.

Dysregulated sinonasal epithelial cell (SNEC) barrier function has been proposed to contribute to the pathogenesis of sinonasal inflammatory conditions such as allergic rhinitis (AR) and chronic rhinosinusitis (CRS). Allergens such as house dust mite (HDM) have been reported to disrupt SNEC barrier integrity. We have recently identified nuclear erythroid 2-related factor 2 (Nrf2) activation via sulforaphane (SFN) stimulation to stabilize SNEC barrier function. The purpose of this study was to explore whether Nrf2 activation could ameliorate HDM-induced SNEC barrier dysfunction. Human SNECs (HSNECs) were grown from patients at the air-liquid interface (ALI). HSNECs were stimulated with HDM with or without pharmacologic activation of Nrf2 with SFN. HSNECs were then stained for the epithelial cell junction protein zonula occludens-1 (ZO-1) and cell surface localization was evaluated by confocal microscopy. Transepithelial electrical resistance (TER) and paracellular fluorescein isothiocyanate (FITC)-dextran permeability was measured in response to stimulation with HDM and SFN. HDM stimulation caused a global disruption of the protein ZO-1 along with an associated decrease in TER (p < 0.001) and increased FITC-dextran paracellular permeability (p < 0.0001). Enhancing Nrf2 activation through treatment with SFN prior to stimulation with HDM was associated with increased localization of ZO-1 at the cell surface and statistically significant increases in TER (p < 0.05) and decrease in paracellular FITC-dextran permeability (p < 0.001). This is the first study to demonstrate that HDM-induced SNEC barrier dysfunction may be preventable by Nrf2 activation. The Nrf2 antioxidant pathway may represent a potential therapeutic target for allergen-induced sinonasal inflammation.

Abstract WP98: Role of Hydrogen Peroxide in Preconditioning of Microglia to Achieve Improved Hematoma Cleanup After ICH

Background: Red blood cells ( RBC ) and other blood components deposited in brain parenchyma during intracerebral hemorrhage ( ICH ) are the source of secondary brain injury, inflammation, and oxidative stress. Therefore, a fast and efficient removal of the blood from the brain is essential for ameliorating secondary injury and for recovery and repair process. Microglia/macrophages ( MΦ )-mediated phagocytosis is a key component of hematoma clearance after ICH. However, the high levels of pro-oxidative molecules (including H 2 O 2 ) generated by MΦ during phagocytosis could be highly cytotoxic not only to brain cells, but also to MΦ themselves. Thus, an efficient coupling between MΦ-mediated phagocytosis and anti-oxidative processes is essential for the safe and efficient hematoma cleanup. Methods and Results: H 2 O 2 at higher concentrations is known to be neurotoxic. In this study, we established that H 2 O 2 at submicromolar, biologically relevant concentrations, acts as a pro-survival factor for MΦ. By activating transcription factor nuclear factor-erythroid 2 p45-related factor 2 ( Nrf2 ), a master regulator of anti-oxidative regulation, H 2 O 2 stimulates the expression of many antioxidant proteins, which protects MΦ from oxidative injury and damaging components of inflammation. We established that Nrf2-deficient MΦ are more susceptible to H 2 O 2 -mediated or toxic blood components-mediated damage (“ICH-like” injury). Nrf2-KO mice subjected to ICH experienced more severe brain edema and delayed hematoma resolution. In addition, the phagocytosis of RBCs (in vitro model of hematoma clearance) was significantly diminished in MΦ treated with Nrf2 decoy or in MΦ that were harvested from Nrf2-KO mice. On the other hand, pharmacologic activation of Nrf2 or administration of low dosages of H 2 O 2 improved phagocytic capacity of MΦ toward RBC. Furthermore, low levels of H 2 O 2 protected MΦ from ICH-like injury, suggesting that improved phagocytic function could involve preservation of MΦ’ integrity by activating Nrf2. Conclusion: H 2 O 2 at low doses could protect MΦ for oxidative injury and facilitate their phagocytic function, which could benefit hematoma clearance, inflammation resolution, and improve neurological recovery after ICH.

Nuclear Factor (Erythroid-Derived 2)-Like 2 and Thioredoxin-1 in Atherosclerosis and Ischemia/Reperfusion Injury in the Heart.

Redox signaling is one of the key elements involved in cardiovascular diseases. Two important molecules are the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) and the oxidoreductase thioredoxin-1 (Trx-1). Recent Advances: During the previous years, a lot of studies investigated Nrf2 and Trx-1 as protective proteins in cardiovascular disorders. Moreover, post-translational modifications of those molecules were identified that play an important role in the cardiovascular system. This review will summarize changes in the vasculature in atherosclerosis and ischemia reperfusion injury of the heart and the newest findings achieved with Nrf2 and Trx-1 therein. Interestingly, Nrf2 and Trx-1 can act together as well as independently of each other in protection against atherosclerosis and ischemia and reperfusion injury. In principle, pharmacological activation of a transcription factor-like Nrf2 can be dangerous, since a transcription regulator has multiple targets and the pleiotropic effects of such activation should not be ignored. Moreover, overactivation of Nrf2 as well as long-term treatment with Trx-1 could be deleterious for the cardiovascular system. Therefore, the length of treatment with Nrf2 activators and/or Trx-1 has first to be studied in more detail in cardiovascular disorders. Moreover, a combination of Nrf2 activators and Trx-1 should be investigated and taken into consideration. Antioxid. Redox Signal. 26, 630-644.

Expression of the NRF2 Target Gene NQO1 Is Enhanced in Mononuclear Cells in Human Chronic Kidney Disease.

Reduced nuclear factor erythroid 2-related factor 2 (NRF2) pathway activity was reported in models of chronic kidney disease (CKD). Pharmacological activation of NRF2 is supposed to improve renal function, but data concerning the NRF2 activity in human CKD are lacking. We investigated the NRF2 target NAD(P)H:quinone oxidoreductase 1 (NQO1) as a readout parameter for NRF2 activity in monocytes of CKD patients (n = 63) compared to those of healthy controls (n = 16). The NQO1 gene expression was quantified using real-time PCR and the protein content by in-cell Western assays. We found a 3-4-fold increase in NQO1 gene expression in CKD 1–5 (n = 29; 3.5 for NQO1/ribosomal protein L41; p < 0.001). This was accompanied by a 1.1-fold increase in NQO1 protein (p = 0.06). Cardiovascular disease prevalence was higher in CKD 1–5 patients with higher compared to those with lower NQO1 gene expression (p = 0.02). In advanced uremia, in dialysis patients (n = 34), NQO1 gene expression was less robustly upregulated than that in CKD 1–5, while NQO1 protein was not upregulated. We conclude that in mononuclear cells of CKD patients, the NRF2 pathway is activated by coexisting pathogenic mechanisms, but in advanced uremia, the effectiveness of this upregulation is reduced. Both processes could interfere with pharmacological NRF2 activation.

Antioxidant activities of ginsenoside Rg1 against cisplatin-induced hepatic injury through Nrf2 signaling pathway in mice

Oxidative stress is mainly caused by reactive oxygen species (ROS). The damage causes a net stress on normal organs, leading to a gradual loss of vital physiological function. ROS, such as free radicals, represent a class of molecules which are derived from the metabolism of oxygen and exist inherently. However, excessive produced ROS can damage all aerobic organisms. Ginseng is one of the most commonly used alternative herbal medicines, also as a traditional Chinese medicine. The aim of this study is to investigate the antioxidant potential function of ginsenoside Rg1 against cisplatin-caused hepatic damage. Male mice were treated with cisplatin to induce oxidative stress to mimic the side effect of anti-cancer drug cisplatin. Ginsenoside Rg1 effectively prevented against cisplatin-induced hepatotoxicity, alleviating histological lesions. Antioxidant functions of Rg1 were restrained by the activation of p62–Keap1–Nrf2 signaling pathway, simultaneously accompanied with expression of protein products. Accumulative p62 and increased activation of JNK in hepatocytes promoted the activation of Nrf2. For the other, degradation of Nrf2 was guided by tyrosine phosphorylation, ubiquitin, and Keap1. In summary, Rg1 prevents hepatotoxicity mainly by inhibiting the binding of Keap1 and Nrf2, partly by p62 accumulation, and more importantly by increasing the production of antioxidative proteins associated to Nrf2. Pharmacological activation of Nrf2 is an effective way in combating against liver injury.

Reversal of cigarette smoke extract-induced sinonasal epithelial cell barrier dysfunction through Nrf2 Activation.

Environmental factors such as inhaled pollutants like cigarette smoke may play a significant role in diseases of the upper airway including chronic rhinosinusitis (CRS). Recent studies have shown that cigarette smoke causes impaired airway epithelial cell barrier function likely through environmental oxidative stress related pathways. The purpose of this study is to explore whether enhancing nuclear factor erythroid 2 [NF-E2]-related factor 2 [Nrf2], the body's master antioxidant system, can ameliorate cigarette smoke-induced sinonasal epithelial cell (SNEC) barrier dysfunction. Human SNECs (HSNECs) were grown from control patients at the air-liquid interface (ALI). HSNECs were stimulated with cigarette smoke extract (CSE) with and without pharmacologic activation of Nrf2. HSNECs were then stained for the epithelial cell junctional proteins zonula occludens 1 (ZO-1) and junctional adhesion molecule A (JAM-A) using confocal microscopy. In addition, transepithelial electrical resistance (TER) was measured in cultures before and after stimulation with CSE. CSE stimulation caused a global disruption of the epithelial junctional proteins ZO-1 and JAM-A along with an associated decrease in TER levels. Enhancing Nrf2 levels prior to stimulation with CSE was associated with increased localization of ZO-1 and JAM-A levels at the cell surface and statistically significant increases in TER levels. This is the first study to demonstrate that cigarette smoke induced SNEC barrier dysfunction is reversible by Nrf2 activation. The Nrf2 antioxidant pathway may represent a potential therapeutic target for cigarette smoke-associated sinonasal inflammation.

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

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

Oxidative stress and dysfunctional NRF2 underlie pachyonychia congenita phenotypes.

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

Therapeutic Potential for Modulation of Nrf2-Keap-1 Signaling Pathway as Treatment for Diabetes and Other Disorders.

Summary: Theinventioninthispatentapplicationisrelatedtonaphthylsulfonamidepyrrolidinederivativesrepresentedgenerallybyformula(I). ThesecompoundsareKeap-1modulatorsandmaybeusefulforthepreventionand/ortreatmentofdiabetes,obesity,dyslipidemia, and related disorders as well as neuroinflammatory and neurodegenerative diseases such as multiple sclerosis, Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease. The transcription factor nuclearfactor erythroid2 p45 (NF-E2)-related factor (Nrf2) is a cellular sensor ofoxidative and electrophilic stress. It is a member of the Cap ‘n’ collar family of basic leucine zipper transcription factors, and it is critical for the expression of severalcytoprotectivegenes.Nrf2isregulatedbytheKelch-likeECH-associatedprotein1(Keap-1),whichisanadaptorproteinof the cullin 3-dependent ubiquitin E3 ligase complex. Keap-1 contains functional domains, including a broad complex/tramtrack/ Bric-a-Brac (BTB) domain, an intervening region (IVR) domain, and a Kelch domain. Ubiquitin is the original member of a family of small proteins, which are found in most cellular tissues in the human body that help in regulating the processes of other cellular proteins. Ubiquitin and other relative molecules add covalently to cellular proteins in a process known as ubiquitinylation that alters their activities and the functions. This process plays a critical role in apoptosis of proteinsaswell asin severalothercellularprocesses relatedtothe regulationofproteins.Keap-1mediatesthe ubiquitinylationand subsequentdegradationofNrf2bythe26Sproteasome.Inthisinteraction,theKelchdomainonaKeap-1homodimerbindstothe DLGandETGEsequencemotivesofNrf2molecule.TheKeap-1BTBdomaincontainsacysteineresidueatthe151position,while theIVRdomaincontainstwocysteineresidues,Cys273andCys288.WhileKeap-1hasothercysteineresidues,thesethreeresidues arehighlyreactiveandplaycriticalrolesinstresssensing.Theyaremodifiedinresponsetooxidativestressorelectrophilicreagents, and that alters the conformation of Keap-1, inhibits its function, and prevents the degradation of Nrf2. This leads to the translocation of Nrf2 into the nucleus and its subsequent activation. Nrf2 binds to the antioxidant response element (ARE) in the nucleusanddrivestheexpressionofNrf2targetgenessuchasNAD(P)Hquinoneoxidoreductase1(NQO1)andhemeoxygenase 1 (HMOX1), which are upregulated by the activation of Nrf2. The activation of Nrf2 may provide a potential therapy for the prevention and treatment of several diseases associated with increased oxidative and inflammatory stress. For example, diabetes is associated with impaired activation of Nrf2, which causes a reduced oxidative-stress defense. Diabetes is also associated with low-grade inflammation. Inflammatory processes and excessive oxidative stress induce insulin resistance, endothelial dysfunction, and diabetic nephropathy. Therefore, pharmacological activation of Nrf2 may potentially improve glucose control and lipid handling as well as micro- and macrocardiovascular disease in diabetics. In addition, the activation of Nrf2 reduces cellular stress and damage in response to xenobiotics, which could be useful for the prevention of cancer. Since excessive production of reactive oxidative species is implicated in neurodegenerative diseases such as multiple sclerosis, Huntington’s and Parkinson’s disease; therefore, the activation of Nrf2 may also provide a potential therapeutic strategy for these diseases. Therefore, targeting Nrf2 may also be useful in treating chronic inflammatory diseases such as chronic obstructive pulmonary disease, arthritis, and sepsis. The modulators of KEAP-1, particularly those with antagonistic activities, such as the compounds of formula (I) described in this patent application can lead to activation of Nrf2 and may potentially provide therapies for the prevention and/or treatment of diabetes, obesity, dyslipidemia, and other related disorders.

Nrf2 Activation Inhibits Effects of Thrombin in Human Amnion Cells and Thrombin-Induced Preterm Birth in Mice.

Nrf2 is a key transcription factor that modulates cell defense mechanisms against endogenous and exogenous stress. Previously, we reported that thrombin increased matrix metalloproteinases and prostaglandin synthesis in human amnion mesenchymal cells. We sought to determine whether activation of Nrf2 alters the effect of thrombin on prostaglandin synthesis, protease activation, and cytokine release in human amnion. Furthermore, we analyzed the effect of Nrf2 activation on thrombin-induced preterm labor in mice. Primary human amnion mesenchymal cells and pregnant mice were employed to investigate the effect of Nrf2 on thrombin-induced inflammation and preterm birth. This was a laboratory-based study using cells and mice. As expected, thrombin increased cyclooxygenase-2, IL-1β, IL-6, IL-8, and matrix metalloproteinase-1 in amnion mesenchymal cells. Preincubation with Nrf2 activators, diethyl maleate or 15-deoxy-Δ12, 14-prostaglandin J2 (15d-PGJ2), profoundly repressed thrombin-induced gene expression. In addition, Nrf2 activation inhibited thrombin-induced cyclooxygenase-2 protein levels and secretion of prostaglandin E2, IL-1β, IL-6, IL-8, TNFα, and granulocyte-macrophage colony-stimulating factor in the media. Whereas vehicle and 15d-PGJ2 did not alter gestational length, all pregnant mice treated with thrombin delivered preterm. 15d-PGJ2 delayed thrombin-induced preterm birth significantly. The results indicate that Nrf2 activation represents a key stress response in amnion mesenchyme cells and in pregnant mice to mitigate the adverse proinflammatory effects of thrombin on the fetal membranes. We suggest, therefore, that pharmacological activation of Nrf2 may prevent the increased risk of preterm premature rupture of the membranes associated with thrombin activation that accompanies subchorionic hemorrhage or bleeding during pregnancy.

Anti-hyperlipidemic and anti-peroxidative role of pterostilbene via Nrf2 signaling in experimental diabetes

Nuclear factor erythroid 2-related factor (Nrf2), a key transcription factor triggers the expression of antioxidant and detoxification genes thereby providing cellular protective functions against oxidative stress-mediated disorders. Recent research has identified that pharmacological activation of Nrf2 also regulates the largest cluster of genes associated with lipid metabolism. With this background, this paper highlights the anti-hyperlipidemic and anti-peroxidative role of pterostilbene (PTS), an Nrf2 activator, in streptozotocin (STZ)-induced diabetic model. PTS administration to diabetic mice for 5 weeks significantly regulated blood glucose levels through the elevation of insulin secretion. The circulatory and liver lipid profiles of total cholesterol (TC), triglycerides (TG) and non-esterified fatty acids (NEFA) were maintained to normal levels upon PTS treatment. Moreover, PTS administration also normalized the circulatory levels of very low-, low- and high density lipoprotein cholesterols (VLDL-, LDL-, HDL-C) and also reduced lipid peroxidation in STZ-induced diabetic mice. In addition, Nrf2 and its downstream targets, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) enzyme activities and glutathione (GSH) levels were significantly elevated in liver tissues of diabetic mice upon PTS administration. Further, H&E staining of diabetic mouse liver showed collapse in hepatic microvesicles due to altered lipid metabolism. Both structural and functional alterations were attenuated by PTS indicating its role in diabetic dyslipidemia through Nrf2-mediated mechanism that could be considered as a promising therapeutic agent.

Role of Nrf2 and Autophagy in Acute Lung Injury.

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

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

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

OP8 - The dark and the bright sides of the transcription factor Nrf2 in skin protection and disease (Nrf2 and epidermal barrier function)

The epidermis is the outermost layer of the body. It serves as a barrier, which prevents excessive water loss and protects from environmental insults, such as UV light and air pollutants. However, these insults can damage the epidermis by formation of reactive oxygen species (ROS). A key player in the protection of cells against ROS damage is the transcription factor Nrf2, which regulates expression of various antioxidant enzymes, cytoprotective proteins and transporters. Therefore, Nrf2 is a promising pharmacological target for skin protection and cancer prevention. We analyzed the consequences of pharmacological and genetic Nrf2 activation on keratinocyte protection, barrier formation and hair follicle regeneration. Nrf2 activation protected murine keratinocytes from UVB damage through enhancement of ROS detoxification. On the other hand, strong Nrf2 activation resulted in thickening and hyperkeratosis of the interfollicular epidermis and hair follicle infundibulum as well as in inflammation. As a consequence, Nrf2 transgenic mice have scaly skin and form cysts upon ageing. This resulted from upregulation of the cornified envelope proteins small proline-rich proteins (Sprr) 2d and 2h and of secretory leukocyte peptidase inhibitor (Slpi), which we identified as novel Nrf2 targets in keratinocytes. Since Sprrs are potent ROS scavengers and since Slpi has antimicrobial activities, their upregulation contributed to Nrf2’s protective function. However, it also caused corneocyte fragility and impaired desquamation, and consequently alterations in the epidermal lipid barrier, inflammation and overexpression of mitogens that induced keratinocyte hyperproliferation. Thus Nrf2 is a central regulator in protection of keratinocytes from oxidative damage but strong and prolonged Nrf2 activation leads to epidermal barrier disturbance, hyperkeratosis and cyst formation. This limits the therapeutic potential of Nrf2 activators for skin protection.