The old free radical theory if aging is dead. And, consuming excessive pure antioxidant supplements can be dangerous rather than health producing – see the blog entry End of the free radical theory of aging and negative consequences of indiscriminante antioxidant supplementation. But a new more sophisticated theory relating to oxidative damage in the body has taken its place, one supported by much empirical evidence. In this theory, the transcription factor Nrf2 plays a key role in activating antioxidant and other protective genes. This is the first of three blog entries that together answer the question “How can you simultaneously warn against indiscriminant antioxidant supplementation and at the same time so enthusiastically endorse consuming foods which have strong antioxidant capabilities, ones like broccoli, coffee, olive oil, chocolate, garlic, green tea and blueberries? And, if antioxidants are bad for you, how can you continue to advocate taking so many antioxidant supplements like curcumin, alpha-lipoic acid, ashwagandha, boswellia, ginger and resveratrol?”
The first discussion on Nrf2 in this blog was in the October 2009 entry Nrf2 and cancer chemoprevention by phytochemicals. Since then hundreds of research papers that deal with aspects of Nrf2 have been published. In these three blog entries, I seek to characterize some of the most important findings. This first blog entry deals with the general mechanisms of operation of Nrf2, with positive effects of Nrf2 in preventing or treating a number of pathological conditions via its ability to turn on genes for the body’s own antioxidant system and genes for combating stress – hundreds of such genes. A second blog entry deals with how a number of substances that are incidentally antioxidants, plant-derived phyto-substances in particular, actually exercise their benefits through promoting the expression of Nrf2 which in turn activates the body’s own antioxidant and hormetic defense systems: The pivotal role of Nrf2. Part 2 – foods, phyto-substances and other substances that turn on Nrf2.The third entry explores whether supplementation with substances that promote Nrf2 might be life-extending: The pivotal role of Nrf2. Part 3– Is promotion of Nrf2 expression a viable strategy for human human healthspan and lifespan extension?
What does Nrf2 do?
Nrf2 is a stress-sensing genetic transcription factor that is part of the cap’n’collar family. As such it appears to be a master regulator of cellular responses to oxidative damage and other stressful conditions. In simplified form, the main theory of how it works is as shown in the diagram(ref):
“Nrf2 is normally bound in the cell’s cytosol in a complex with the cytoskeletal protein Keap1 (Kelch-like ECH-associated protein 1). Under normal conditions, as Nrf2 accumulates it is polyubiquitinated and sent off for destruction by the cell’s proteolysis machinery where it is broken down into simple molecules for recycling. Keap1 contains several reactive cysteine residues that function as sensors of cellular redox changes. If metabolic stress signals are present such as strong change in redox state, proteolysis is blocked, Nrf2 translocates into the cell’s nucleus where it accumulates. “After translocation into nucleus, Nrf2 forms a heterodimer with other transcription factors, such as small Maf, which in turn binds to the 5′-upstream CIS-acting regulatory sequence, termed antioxidant response elements (ARE) or electrophile response elements (EpRE), located in the promoter region of genes encoding various antioxidant and phase 2 detoxifying enzymes(ref).” Hundreds of antioxidant and stress response genes may be affected. “These include NAD(P)H:quinone oxidoreductase-1, heme oxygenase-1, glutamate cysteine ligase, glutathione S-transferase, glutathione peroxidase, thioredoxin, etc. (ref)”
To complete the picture, there are many substances including certain phytochemicals that can block ubiquination and proteolysis as illustrated in the following diagram. Sulforaphane is is an isothiocyanate found in cruciferous vegetables like broccoli. Electrophiles are chemical species that are electron deficient. Electrophilic xenobiotics, statins, and cancer chemopreventive agents are among the substances that block ubiquination and proteolysis of Nrf2.
The above-described process is basic to all of the positive effects of Nrf2 and is described in a great many publications. For example,the Biocart Pathways article Oxidative Stress Induced Gene Expression Via Nrf2relates “Reactive oxygen species (ROS) can damage biological macromolecules and are detrimental to cellular health. Electrophilic compounds, xenobiotics and antioxidants are sources of reactive oxygen species, creating oxidative stress that can harm cells. Enzymes are involved in the Phase II detoxification of xenobiotics to reduce cellular stress include glutathione transferases, quinone reductase, epoxide hydrolase, heme oxygenase, UDP-glucuronosyl transferases, and gamma-glutamylcysteine synthetase. Expression of these genes protects cells from oxidative damage and can prevent mutagenesis and cancer. Transcription of these enzymes is coordinately regulated through antioxidant response elements (AREs). Nrf2 (NF-E2-related factor 2) and Nrf1 are transcription factors that bind to AREs and activate these genes. Inactive Nrf2 is retained in the cytosol by association a complex with the cytoskeletal protein Keap1. Cytosolic Nrf2 is phosphorylated and translocates into the nucleus in response to protein kinase C activation and Map kinase pathways. In the nucleus, Nrf2 activate genes through AREs by interacting with transcription factors in the bZIP family, including CREB, ATF4 and fos or jun. Nrf2 activation of genes is opposed by small maf proteins, including MafG and MafK, maintaining a counterbalance to Nrf2 and balancing the oxidation level of the intracellular environment.”
Put yet again in different terms “Oxidative stress promotes anti-oxidative gene expression via nuclear factor (erythroid-derived 2)-like 2 activation. In a basal state, free Nrf2 level is very low because it forms a complex with Keap1 and the E3 ligase Cul3-Rbx-1, leading to its proteasome degradation. Under the stimulation of oxidative stress, the level of free Nrf2 increases as it is dissociated with Keap1. Free Nrf2 molecules will then enter the nuclei, bind to the cis-element ARE and stimulate the expression of Nrf2 target genes. ARE: Antioxidant response element; Cul3: Cullin 3; E3: Ubiquitin ligase; Keap1: Kelch-like ECH-associated protein 1; Nrf2: Nuclear factor (erythroid-derived 2)-like 2; Rbx-1: RING box protein 1(ref).”
Nrf2 provides a response to stress.
From the 2010 publication Nrf2: friend or foe for chemoprevention? – “Health reflects the ability of an organism to adapt to stress. Stresses–metabolic, proteotoxic, mitotic, oxidative and DNA-damage stresses–not only contribute to the etiology of cancer and other chronic degenerative diseases but are also hallmarks of the cancer phenotype. Activation of the Kelch-like ECH-associated protein 1 (KEAP1)-NF-E2-related factor 2 (NRF2)-signaling pathway is an adaptive response to environmental and endogenous stresses and serves to render animals resistant to chemical carcinogenesis and other forms of toxicity, whilst disruption of the pathway exacerbates these outcomes.”
NRF2 is protective against multiple types of disorders associated with oxidative insults, for example airways disorders.
The same general pattern of protection appears to apply in the cases of multiple kinds of pathological conditions. For example, the 2010 publication Nrf2 protects against airway disorders reports: “Nuclear factor-erythroid 2 related factor 2 (Nrf2) is a ubiquitous master transcription factor that regulates antioxidant response elements (AREs)-mediated expression of antioxidant enzyme and cytoprotective proteins. In the unstressed condition, Kelch-like ECH-associated protein 1 (Keap1) suppresses cellular Nrf2 in cytoplasm and drives its proteasomal degradation. Nrf2 can be activated by diverse stimuli including oxidants, pro-oxidants, antioxidants, and chemopreventive agents. Nrf2 induces cellular rescue pathways against oxidative injury, abnormal inflammatory and immune responses, apoptosis, and carcinogenesis. Application of Nrf2 germ-line mutant mice has identified an extensive range of protective roles for Nrf2 in experimental models of human disorders in the liver, gastrointestinal tract, airway, kidney, brain, circulation, and immune or nerve system. In the lung, lack of Nrf2 exacerbated toxicity caused by multiple oxidative insults including supplemental respiratory therapy (e.g., hyperoxia, mechanical ventilation), cigarette smoke, allergen, virus, bacterial endotoxin and other inflammatory agents (e.g., carrageenin), environmental pollution (e.g., particles), and a fibrotic agent bleomycin. Microarray analyses and bioinformatic studies elucidated functional AREs and Nrf2-directed genes that are critical components of signaling mechanisms in pulmonary protection by Nrf2. Association of loss of function with promoter polymorphisms in NRF2 or somatic and epigenetic mutations in KEAP1 and NRF2 has been found in cohorts of patients with acute lung injury/acute respiratory distress syndrome or lung cancer, which further supports the role for NRF2 in these lung diseases. In the current review, we address the role of Nrf2 in airways based on emerging evidence from experimental oxidative disease models and human studies.”
Another important form of stress responded to by Nrf2 expression is nitrosative stress.
Although much discussion of Nrf2 is couched in terms of oxidative stress, Nrf2 responds to many other forms of stress including nitrosative stress. The 2009 publication Role of Nrf2-mediated heme oxygenase-1 upregulation in adaptive survival response to nitrosative stress reports: “Nitrosative stress caused by reactive nitrogen species such as nitric oxide and peroxynitrite overproduced during inflammation leads to cell death and has been implicated in the pathogenesis of many human ailments. However, relatively mild nitrosative stress may fortify cellular defense capacities, rendering cells tolerant or adaptive to ongoing and subsequent cytotoxic challenges, a phenomenon known as ‘preconditioning’ or ‘hormesis’. One of the key components of cellular stress response is heme oxygenase-1 (HO-1), the rate limiting enzyme in the process of degrading potentially toxic free heme into biliverdin, free iron and carbon monoxide. HO-1 is upregulated by a wide array of stimuli and has antioxidant, anti-inflammatory and other cytoprotective functions. This review is intended to provide readers with a well-documented account of the research done in the area of cellular adaptive survival response against nitrosative stress with special focus on the role of HO-1 upregulation, especially through activation of the transcription factor, Nrf2.”
Nuclear levels of Nrf2 and its expression generally decline with age
This has been known for some time. The 2004 publication Decline in transcriptional activity of Nrf2 causes age-related loss of glutathione synthesis, which is reversible with lipoic acidrelated “Glutathione (GSH) significantly declines in the aging rat liver. Because GSH levels are partly a reflection of its synthetic capacity, we measured the levels and activity of gamma-glutamylcysteine ligase (GCL), the rate-controlling enzyme in GSH synthesis. With age, both the catalytic (GCLC) and modulatory (GCLM) subunits of GCL decreased by 47% and 52%, respectively (P < 0.005). Concomitant with lower subunit levels, GCL activity also declined by 53% (P < 0.05). Because nuclear factor erythroid2-related factor 2 (Nrf2) governs basal and inducible GCLC and GCLM expression by means of the antioxidant response element (ARE), we hypothesized that aging results in dysregulation of Nrf2-mediated GCL expression. We observed an approximately 50% age-related loss in total (P < 0.001) and nuclear (P < 0.0001) Nrf2 levels, which suggests attenuation in Nrf2-dependent gene transcription. By using gel-shift and supershift assays, a marked reduction in Nrf2/ARE binding in old vs. young rats was noted. To determine whether the constitutive loss of Nrf2 transcriptional activity also affects the inducible nature of Nrf2 nuclear translocation, old rats were treated with (R)-alpha-lipoic acid (LA; 40 mg/kg i.p. up to 48 h), a disulfide compound shown to induce Nrf2 activation in vitro and improve GSH levels in vivo. LA administration increased nuclear Nrf2 levels in old rats after 12 h. LA also induced Nrf2 binding to the ARE, and, consequently, higher GCLC levels and GCL activity were observed 24 h after LA injection. Thus, the age-related loss in GSH synthesis may be caused by dysregulation of ARE-mediated gene expression, but chemoprotective agents, like LA, can attenuate this loss.
The theme of age-related decline has been picked up in subsequent publications. For example, the December 2011 publication Identification of age-specific Nrf2 binding to a novel antioxidant response element locus in the Gclc promoter: a compensatory means for the loss of glutathione synthetic capacity in the aging rat liver? “NFE2-related factor 2 (Nrf2) transcriptionally governs the cellular response to harmful electrophiles, xenobiotics, and reactive oxygen species. Its nuclear levels decline with age (Suh et al., 2004a), which in part explains the age-related loss of phase II detoxification. However, little work has yet characterized how age affects Nrf2 DNA binding or the role that alterations to the Nrf2 transcriptional apparatus plays in modulating Nrf2-mediated gene expression. In this study, we used immunoprecipitation assays to show that Nrf2bound to the active antioxidant response element (ARE) of the catalytic subunit of glutamate cysteine ligase (GCLC) is significantly lower in hepatic chromatin from aged vs. young rats. Moreover, the activity at this ARE locus is diminished during aging because of the presence of Bach1 and the absence of CREB-binding protein (CBP), a transcriptional repressor and co-activator, respectively. Further analysis reveals that Nrf2 occupies an alternate ARE site located -2.2 kb downstream from the normally active ARE binding site in livers of old rats, indicating an age-specific adaptation to maintain gene expression. Our results, thus, show that the conversion of Nrf2 binding from an active ARE to an alternative ARE element is not adequate to maintain basal expression of hepatic Gclc in old rats, which provides a potential mechanism for the age-related loss of glutathione synthetic and other phase II enzymes.”
Some important positive biological effects of AMPK such as its anti-apoptotic capabilities are modulated by Nrf2.
Both Victor and I have discussed AMPK, “The Master Metabolic Sensor and Regulator” in previous blog entries. See for example Victor’s latest post Circadian Regulation, NMN, Preventing Diabetes, and Longevity. “In the end, we see that AMPK, once again, plays the primary role of sensing metabolic status and regulating cellular and organismal responses through multiple pathways. In fact, AMPK regulates circadian function by direct phosphorylation of clock components completely independent of sirt1.” Among the multiple pathways affected by AMPK expression, some are mediated by AMPK’s effects on Nrf2. The December 2011 publication AMP-activated protein kinase (AMPK) controls the aging process via an integrated signaling network reports: “Efficient control of energy metabolic homeostasis, enhanced stress resistance, and qualified cellular housekeeping are the hallmarks of improved healthspan and extended lifespan. AMPK signaling is involved in the regulation of all these characteristics via an integrated signaling network. Many studies with lower organisms have revealed that increased AMPK activity can extend the lifespan. Experiments in mammals have demonstrated that AMPK controls autophagy through mTOR and ULK1 signaling which augment the quality of cellular housekeeping. Moreover, AMPK-induced stimulation of FoxO/DAF-16, Nrf2/SKN-1, and SIRT1 signaling pathways improves cellular stress resistance. Furthermore, inhibition of NF-κB signaling by AMPK suppresses inflammatory responses. Emerging studies indicate that the responsiveness of AMPK signaling clearly declines with aging. The loss of sensitivity of AMPK activation to cellular stress impairs metabolic regulation, increases oxidative stress and reduces autophagic clearance. These age-related changes activate innate immunity defence, triggering a low-grade inflammation and metabolic disorders.Metf”
The January 2011 publication Activation of AMPK stimulates heme oxygenase-1 gene expression and human endothelial cell survivalreports “In conclusion, AMPK stimulates HO-1 (heme oxygenase) gene expression in human ECs via the Nrf2/antioxidant responsive element signaling pathway. The induction of HO-1 mediates the antiapoptotic effect of AMPK, and this may provide an important adaptive response to preserve EC viability during periods of metabolic stress.”
Nrf2 appears to stimulate the same pathways that extend life via calorie restriction or alternative day fasting.
From the 2011 publication The role of the antioxidant and longevity-promoting Nrf2 pathway in metabolic regulation: “Recent evidence identifies Nrf2 signaling as a mediator of the salutary effects of caloric restriction. — CR, i.e. restriction of food intake without malnutrition, is a regimen that has been shown to extend the lifespan of organisms across the evolutionary spectrum. In addition to its effect on longevity, CR can confer diverse health benefits, which include decreased risk of cancer, lower blood pressure, higher insulin sensitivity, and improved neuronal function (reviewed in . These broad health effects might be perceived as a result of the switch from “reproduction mode” to “longevity mode”, discussed above. At least some of the benefits of CR correlate with increased resistance against oxidative stress and may involve a system like the Nrf2 pathway, which is known for its antioxidant, cancer preventive and lifespan-extending functions. For example, aging is associated with a decline in the abundance of antioxidant proteins; this has been shown to be reversible with pharmacological activation of Nrf2 [20,21]. — Evidence for a function of Nrf2 as a CR effector has been provided by a number of studies in mice and worms. Pearson et al.  showed that CR induces antioxidant gene expression in mice and that this response is suppressed in nrf2−/− animals, arguing for an upregulation of Nrf2 function in response to CR. Importantly, the previously described cancer protective function of CR [23,24] was diminished in nrf2−/− mice. Thus, CR decreased the cancer incidence and tumor load elicited by a chemical carcinogenesis regimen in an Nrf2-dependent manner. These experiments established that CR increased Nrf2-dependent gene expression, and that Nrf2 was a required factor for several of the health benefits afforded by CR. — Some CR-mediated health benefits have also been reported in humans [26,27]. However, adhering to a strict CR regimen (as low as 50 % of ad libitum food intake) is not a realistic option for the vast majority of the population. Much effort has therefore been devoted to identify “CR mimetics”, drugs that induce metabolic and physiological changes akin to the shift to “longevity mode” induced by CR without the discomfort of a stringent diet . Several such drugs are being evaluated, and two of the best known among them are resveratrol and metformin.”
There appears to be significant cross-talk between the insulin signaling, NF-kappaB, and Nrf2 pathways.
The February 2012 publication Redox-regulating role of insulin: The essence of insulin effect reports: “It is well-known that insulin acts as an important hormone, controlling energy metabolism, cellular proliferation and biosynthesis of functional molecules to maintain a biological homeostasis. Over the past few years, intensive insulin therapy has been believed to be benefit for the outcome of diabetic patients, in which the suppression of oxidative stress plays a role. Moreover, insulin is accepted as a key component of glucose-insulin-potassium, a treatment which has been believed to exert significant cardiovascular protective effect via the reduction of oxidative stress. Furthermore, accumulating evidence has suggested that insulin exerts important redox-regulating actions in various insulin-sensitive target organs, implying the systematic antioxidative role of insulin as a hormone. It is time for us to revisit insulin effects, through summarizing and evaluating the novel functions of insulin and their mechanisms. This review focuses on the antioxidative effect of insulin and highlights insulin-induced regulation of various antioxidant enzymes via insulin signaling pathways and the cross talk between key transcription factors, including nuclear factor erythroid 2-related factor 2 (Nrf2) and nuclear factor κB (NF-κB) which are responsible for the transcription of antioxidant enzymes, leading to reduced generation of reactive oxygen species (ROS) and the enhancement of the elimination of ROS.”
Associated with its interactions with insulin, treatment of diabetes may be based on targeting Nrf2.
The April 2011 report Oxidative stress and Nrf2 in the pathophysiology of diabetic neuropathy: old perspective with a new angle reported: “Long-standing diabetes and complications thereof particularly, neuropathy stands for one of the major causes of morbidity across the globe. It is postulated that excessive production of reactive oxygen species is a key component in the development and progression of diabetic neuropathy. Oxidative damage is the most common concluding pathway for various pathogenetic mechanisms of neuronal injury in diabetic neuropathy. However despite optimistic preclinical data, it is still very ambiguous that why antioxidants have failed to demonstrate significant neuroprotection in humans. A growing body of evidences now suggests that strategies utilizing a more targeted approach like focusing on Nrf2 (a transcription factor modulating oxidative stress) may provide an enthralling avenue to optimize neuroprotection in diabetes and diabetic neuropathy.”
The January 2012 publication Role of nuclear factor (erythroid-derived 2)-like 2 in metabolic homeostasis and insulin action: A novel opportunity for diabetes treatment?reports: “Redox balance is fundamentally important for physiological homeostasis. Pathological factors that disturb this dedicated balance may result in oxidative stress, leading to the development or aggravation of a variety of diseases, including diabetes mellitus, cardiovascular diseases, metabolic syndrome as well as inflammation, aging and cancer. Thus, the capacity of endogenous free radical clearance can be of patho-physiological importance; in this regard, the major reactive oxygen species defense machinery, the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) system needs to be precisely modulated in response to pathological alterations. While oxidative stress is among the early events that lead to the development of insulin resistance, the activation of Nrf2 scavenging capacity leads to insulin sensitization. Furthermore, Nrf2 is evidently involved in regulating lipid metabolism. Here we summarize recent findings that link the Nrf2 system to metabolic homeostasis and insulin action and present our view that Nrf2 may serve as a novel drug target for diabetes and its complications.”
The February 2011 publication Diabetic downregulation of Nrf2 activity via ERK contributes to oxidative stress-induced insulin resistance in cardiac cells in vitro and in vivo reported: “Objective: Oxidative stress is implicated in cardiac insulin resistance, a critical risk factor for cardiac failure, but the direct evidence remains missing. This study explored a causal link between oxidative stress and insulin resistance with a focus on a regulatory role of redox sensitive transcription factor NF-E2-related factor 2 (Nrf2) in the cardiac cells in vitro and in vivo. Conclusions: ERK-mediated suppression of Nrf2 activity leads to the oxidative stress-induced insulin resistance in adult cardiomyocytes and downregulated glucose utilization in the diabetic heart.”
P62 protein appears to play an important role in oxidative defense through facilitating release of Nrf2 from Keap1.
P62 is involved in autophagy. “Autophagy is an intracellular degradation process by which cytoplasmic contents are degraded in the lysosome. In addition to nonselective engulfment of cytoplasmic materials, the autophagosomal membrane can selectively recognize specific proteins and organelles. It is generally believed that the major selective substrate (or cargo receptor) p62 is recruited to the autophagosomal membrane through interaction with LC3. In this study, we analyzed loading of p62 and its related protein NBR1 and found that they localize to the endoplasmic reticulum (ER)–associated autophagosome formation site independently of LC3 localization to membranes. p62 colocalizes with upstream autophagy factors such as ULK1 and VMP1 even when autophagosome formation is blocked by wortmannin or FIP200 knockout. Self-oligomerization of p62 is essential for its localization to the autophagosome formation site. These results suggest that p62 localizes to the autophagosome formation site on the ER, where autophagosomes are nucleated. This process is similar to the yeast cytoplasm to vacuole targeting pathway(ref).”
Also, “Allelic loss of the essential autophagy gene beclin1 occurs in human cancers and renders mice tumor-prone suggesting that autophagy is a tumor-suppression mechanism. While tumor cells utilize autophagy to survive metabolic stress, autophagy also mitigates the resulting cellular damage that may limit tumorigenesis. In response to stress, autophagy-defective tumor cells preferentially accumulated p62/SQSTM1 (p62), endoplasmic reticulum (ER) chaperones, damaged mitochondria, reactive oxygen species (ROS), and genome damage. Moreover, suppressing ROS or p62 accumulation prevented damage resulting from autophagy defects indicating that failure to regulate p62 caused oxidative stress. Importantly, sustained p62 expression resulting from autophagy defects was sufficient to alter NF-kappaB regulation and gene expression and to promote tumorigenesis. Thus, defective autophagy is a mechanism for p62 upregulation commonly observed in human tumors that contributes directly to tumorigenesis likely by perturbing the signal transduction adaptor function of p62-controlling pathways critical for oncogenesis(ref).”
The 2012 publication p62 at the Interface of Autophagy, Oxidative Stress Signaling, and Cancer reports: “ Significance: Sequestosome 1 (p62/SQSTM1) is a multifunctional adapter protein implicated in selective autophagy, cell signaling pathways, and tumorigenesis. Recent Advances: Recent evidence has revealed that p62/SQSTM1 has a critical role in an oxidative stress response pathway by its direct interaction with the ubiquitin ligase adaptor Kelch-like ECH-associated protein 1 (KEAP1), which results in constitutive activation of the transcription factor NF-E2-related factor 2 (NRF2). Critical Issues: Both NRF2 and KEAP1 are frequently mutated in cancer. The findings just cited uncover a link between p62/SQSTM1, autophagy, and the KEAP1-NRF2 stress response pathway in tumorigenesis and shed light on the interplay between autophagy and cancer. Future Directions: Here, we review the mechanisms by which p62/SQSTM1 implements its multiple roles in the regulation of tumorigenesis with emphasis on the KEAP1-NRF2 stress response signaling pathway.”
Decline in levels of p62 and disturbances in signaling of Nrf2 and other pathways can possibly contribute to the pathology of Alzheimer’s disease.
The January 2012 publication Emerging role of p62/sequestosome-1 in the pathogenesis of Alzheimer’s disease reports: “The p62/sequestosome-1 is a multifunctional protein containing several protein-protein interaction domains. Through these interactions p62 is involved in the regulation of cellular signaling and protein trafficking, aggregation and degradation. p62 protein can bind through its UBA motif to ubiquitinated proteins and control their aggregation and degradation via either autophagy or proteasomes. p62 protein has been reported to be seen in association with the intracellular inclusions in primary and secondary tauopathies, α-synucleinopathies and other neurodegenerative brain disorders displaying inclusions with misfolded proteins. In Alzheimer’s disease (AD), p62 protein is associated with neurofibrillary tangles composed primarily of hyperphosphorylated tau protein and ubiquitin. Increasing evidence indicates that p62 has an important role in the degradation of tau protein. The lack of p62 protein expression provokes the tau pathology in mice. Recent studies have demonstrated that the p62 gene expression and cytoplasmic p62 protein levels are significantly reduced in the frontal cortex of AD patients. Decline in the level of p62 protein can disturb the signaling pathways of Nrf2, cyclic AMP and NF-κB and in that way increase oxidative stress and impair neuronal survival.”
Interventions affecting Nrf2 may also play key roles in control if inflammatory diseases.
The 2010 publication A protective role of nuclear factor-erythroid 2-related factor-2 (Nrf2) in inflammatory disorders reports: “Nuclear factor-erythroid 2-related factor-2 (Nrf2) is a key transcription factor that plays a central role in cellular defense against oxidative and electrophilic insults by timely induction of antioxidative and phase-2 detoxifying enzymes and related stress-response proteins. The 5′-flanking regions of genes encoding these cytoprotective proteins contain a specific consensus sequence termed antioxidant response element (ARE) to which Nrf2 binds. Recent studies have demonstrated that Nrf2-ARE signaling is also involved in attenuating inflammation-associated pathogenesis, such as autoimmune diseases, rheumatoid arthritis, asthma, emphysema, gastritis, colitis and atherosclerosis. — Thus, disruption or loss of Nrf2 signaling causes enhanced susceptibility not only to oxidative and electrophilic stresses but also to inflammatory tissue injuries. During the early-phase of inflammation-mediated tissue damage, activation of Nrf2-ARE might inhibit the production or expression of pro-inflammatory mediators including cytokines, chemokines, cell adhesion molecules, matrix metalloproteinases, cyclooxygenase-2 and inducible nitric oxide synthase. It is likely that the cytoprotective function of genes targeted by Nrf2 may cooperatively regulate the innate immune response and also repress the induction of pro-inflammatory genes. This review highlights the protective role of Nrf2 in inflammation-mediated disorders with special focus on the inflammatory signaling modulated by this redox-regulated transcription factor.”
Nrf2 plays an important role in detoxification of drugs and other xenobiotics via upregulating expression of Phase 2 drug metabolizing enzymes.
“Xenobiotic metabolism (from the Greek xenos “stranger” and biotic “related to living beings”) is the set of metabolic pathways that modify the chemical structure of xenobiotics, which are compounds foreign to an organism’s normal biochemistry, such as drugs and poisons. These pathways are a form of biotransformation present in all major groups of organisms, and are considered to be of ancient origin. These reactions often act to detoxify poisonous compounds; however, in some cases, the intermediates in xenobiotic metabolism can themselves be the cause of toxic effects(ref).”
The 2009 publication Role of phase II drug metabolizing enzymes in cancer chemoprevention reports: “Chemical insults, such as environmental or occupational carcinogenic agents, play a major role in the pathogenesis of many cancers. Many carcinogens exert genotoxic and cytotoxic effects via bioactivation into electrophilic species, a process catalyzed primarily by phase I drug metabolizing enzymes, typically cytochrome P450s. These reactive intermediates can induce DNA and RNA damage, and formation of protein adducts. The reactive species are often detoxified by phase II drug metabolizing enzymes, such as glutathione S-transferases (GSTs), UDP-glucuronosyl transferases (UGTs), sulfotransferase (ST) and N-acetyltransferase (NAT). Phase II enzymes classically conjugate these hydrophobic intermediates to a water-soluble group, thus masking their reactive nature, and allowing subsequent excretion. Therefore, strategies that modulate the levels of phase II enzymes by either pharmacological or nutritional means can lead to enhanced elimination of reactive species. Agents that preferentially activate phase II over phase I enzymes can be beneficial as chemopreventives. Compounds, such as isothiocyanates and dithiolthiones have been shown to act as transcriptional activators of phase II enzymes. A consensus enhancer element, known as antioxidant response element (ARE), in the regulatory domains of many phase II genes and an ARE-binding transcription factor nuclear factor E2-related factor 2 (Nrf2) have been implicated in the action of many chemopreventive agents. In this review, we will discuss the mechanisms of regulation of phase II enzymes, including the signal transduction events elicited by chemopreventive agents. We will also summarize the data available for these agents in preclinical models of tumorigenesis. Some chemopreventive agents have progressed to various stages of clinical trials, e.g. biomarker studies in healthy volunteers or in susceptible populations.”
The 2009 publication Nrf2 plays an important role in coordinated regulation of Phase II drug metabolism enzymes and Phase III drug transporters reports: “The nuclear transcription factor E2-related factor 2 (Nrf2) has been shown to play pivotal roles in preventing xenobiotic-related toxicity and carcinogen-induced carcinogenesis. These protective roles of Nrf2 have been attributed in part to its involvement in the induction of Phase II drug conjugation/detoxification enzymes as well as antioxidant enzymes through the Nrf2-antioxidant response element (ARE) signaling pathways. This review summarizes the current research status of the identification of Nrf2-regulated drug metabolism enzymes (DMEs), especially Phase II DMEs, and Phase III drug transporters. In addition, the molecular mechanisms underlying the coordinated regulation of Phase II DMEs and Phase III transporters will also be discussed based on findings published in the literature.”
Nrf2 is protective against the oxidative stress induced by drinking alcohol and mediated by CYP2E1.
As reported in the 2006 publication Cytochrome P450 2E1-dependent oxidant stress and upregulation of anti-oxidant defense in liver cells, “Induction of cytochrome P450 2E1 (CYP2E1) is a central pathway by which ethanol generates oxidative stress. Cytochrome P450 2E1 metabolizes many other toxicologic compounds. Toxicity of these agents is enhanced by ethanol, due to induction of CYP2E1. — These results suggest that Nrf2 is activated and its levels are increased when CYP2E1 is elevated. It is suggested that Nrf2 plays a key role in the adaptive response against increased oxidative stress caused by CYP2E1.” See also (2009) Nrf2 and antioxidant defense against CYP2E1 toxicity, and (2011) Proteasome inhibitor up regulates liver antioxidative enzymes in rat model of alcoholic liver disease.
Nrf2 and Parkinson’s disease
There seems to be a substantial interest in developing therapeutics for Parkinson’s disease that work via activating Nrf2 expression. I mention three relevant 2011 publications. The July 2011 publication NRF2 activation restores disease related metabolic deficiencies in olfactory neurosphere-derived cells from patients with sporadic Parkinson’s disease relates: “BACKGROUND: Without appropriate cellular models the etiology of idiopathic Parkinson’s disease remains unknown. We recently reported a novel patient-derived cellular model generated from biopsies of the olfactory mucosa (termed olfactory neurosphere-derived (hONS) cells) which express functional and genetic differences in a disease-specific manner. Transcriptomic analysis of Patient and Control hONS cells identified the NRF2 transcription factor signalling pathway as the most differentially expressed in Parkinson’s disease. RESULTS: We tested the robustness of our initial findings by including additional cell lines and confirmed that hONS cells from Patients had 20% reductions in reduced glutathione levels and MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] metabolism compared to cultures from healthy Control donors. We also confirmed that Patient hONS cells are in a state of oxidative stress due to higher production of H(2)O(2) than Control cultures. siRNA-mediated ablation of NRF2 in Control donor cells decreased both total glutathione content and MTS metabolism to levels detected in cells from Parkinson’s Disease patients. Conversely, and more importantly, we showed that activation of the NRF2 pathway in Parkinson’s disease hONS cultures restored glutathione levels and MTS metabolism to Control levels. Paradoxically, transcriptomic analysis after NRF2 pathway activation revealed an increased number of differentially expressed mRNAs within the NRF2 pathway in L-SUL treated Patient-derived hONS cells compared to L-SUL treated Controls, even though their metabolism was restored to normal. We also identified differential expression of the PI3K/AKT signalling pathway, but only post-treatment. CONCLUSIONS: Our results confirmed NRF2 as a potential therapeutic target for Parkinson’s disease and provided the first demonstration that NRF2 function was inducible in Patient-derived cells from donors with uniquely varied genetic backgrounds. However, our results also demonstrated that the response of PD patient-derived cells was not coordinated in the same way as in Control cells. This may be an important factor when developing new therapeutics.”
The Sept-Oct 2011 article Genetic activation of Nrf2 signaling is sufficient to ameliorate neurodegenerative phenotypes in a Drosophila model of Parkinson’s disease reports: “Parkinson’s disease (PD) is the most common neurodegenerative movement disorder. Oxidative stress has been associated with the etiology of both sporadic and monogenic forms of PD. The transcription factor Nrf2, a conserved global regulator of cellular antioxidant responses, has been implicated in neuroprotection against PD pathology. However, direct evidence that upregulation of the Nrf2 pathway is sufficient to confer neuroprotection in genetic models of PD is lacking. Expression of the PD-linked gene encoding α-synuclein in dopaminergic neurons of Drosophila results in decreased locomotor activity and selective neuron loss in a progressive age-dependent manner, providing a genetically accessible model of PD. Here we show that upregulation of the Nrf2 pathway by overexpressing Nrf2 or its DNA-binding dimerization partner, Maf-S, restores the locomotor activity of α-synuclein-expressing flies. Similar benefits are observed upon RNA-interference-mediated downregulation of the prime Nrf2 inhibitor, Keap1, as well as in conditions of keap1 heterozygosity. Consistently, the α-synuclein-induced dopaminergic neuron loss is suppressed by Maf-S overexpression or keap1 heterozygosity. Our data validate the sustained upregulation of the Nrf2 pathway as a neuroprotective strategy against PD. This model provides a genetically accessible in vivo system in which to evaluate the potential of additional Nrf2 pathway components and regulators as therapeutic targets.”
The article The Nrf2/ARE Pathway: A Promising Target to Counteract Mitochondrial Dysfunction in Parkinson’s Disease reports: “Mitochondrial dysfunction is a prominent feature of various neurodegenerative diseases as strict regulation of integrated mitochondrial functions is essential for neuronal signaling, plasticity, and transmitter release. Many lines of evidence suggest that mitochondrial dysfunction plays a central role in the pathogenesis of Parkinson’s disease (PD). Several PD-associated genes interface with mitochondrial dynamics regulating the structure and function of the mitochondrial network. Mitochondrial dysfunction can induce neuron death through a plethora of mechanisms. Both mitochondrial dysfunction and neuroinflammation, a common denominator of PD, lead to an increased production of reactive oxygen species, which are detrimental to neurons. The transcription factor nuclear factor E2-related factor 2 (Nrf2, NFE2L2) is an emerging target to counteract mitochondrial dysfunction and its consequences in PD. Nrf2 activates the antioxidant response element (ARE) pathway, including a battery of cytoprotective genes such as antioxidants and anti-inflammatory genes and several transcription factors involved in mitochondrial biogenesis. Here, the current knowledge about the role of mitochondrial dysfunction in PD, Nrf2/ARE stress-response mechanisms, and the evidence for specific links between this pathway and PD are summarized. The neuroprotection of nigral dopaminergic neurons by the activation of Nrf2 through several inducers in PD is also emphasized as a promising therapeutic approach.”
Nrf2 and cancers
A great deal of research related to Nrf2 has been conducted in the context under the umbrella of cancer research. I present a number of key findings.
Certain cancers work to epigenetically silence the expression of Nrf2. Such may be the case, for example, in prostate cancers.
The 2010 publication Nrf2 Expression Is Regulated by Epigenetic Mechanisms in Prostate Cancer of TRAMP Micereported “Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) is a transcription factor which regulates the expression of many cytoprotective genes. In the present study, we found that the expression of Nrf2 was suppressed in prostate tumor of the Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) mice. Similarly, the expression of Nrf2 and the induction of NQO1 were also substantially suppressed in tumorigenic TRAMP C1 cells but not in non-tumorigenic TRAMP C3 cells. Examination of the promoter region of the mouse Nrf2 gene identified a CpG island, which was methylated at specific CpG sites in prostate TRAMP tumor and in TRAMP C1 cells but not in normal prostate or TRAMP C3 cells, as shown by bisulfite genomic sequencing. Reporter assays indicated that methylation of these CpG sites dramatically inhibited the transcriptional activity of the Nrf2 promoter. — Taken together, these results indicate that the expression of Nrf2 is suppressed epigenetically by promoter methylation associated with MBD2 and histone modifications in the prostate tumor of TRAMP mice. Our present findings reveal a novel mechanism by which Nrf2 expression is suppressed in TRAMP prostate tumor, shed new light on the role of Nrf2 in carcinogenesis and provide potential new directions for the detection and prevention of prostate cancer.”
The 2010 review publication Targeting NRF2 signaling for cancer chemoprevention relates: “Modulation of the metabolism and disposition of carcinogens through induction of cytoprotective enzymes is one of several promising strategies to prevent cancer. Chemopreventive efficacies of inducers such as dithiolethiones and sulforaphane have been extensively studied in animals as well as in humans. The KEAP1-NRF2 system is a key, but not unilateral, molecular target for these chemopreventive agents. The transcription factor NRF2 (NF-E2-related factor 2) is a master regulator of the expression of a subset of genes, which produce proteins responsible for the detoxication of electrophiles and reactive oxygen species as well as the removal or repair of some of their damage products. — It is believed that chemopreventive enzyme inducers affect the interaction between KEAP1 and NRF2 through either mediating conformational changes of the KEAP1 protein or activating phosphorylation cascades targeting the KEAP1-NRF2 complex. These events in turn affect NRF2 stability and trafficking. Recent advances elucidating the underlying structural biology of KEAP1-NRF2 signaling and identification of the gene clusters under the transcriptional control of NRF2 are facilitating understanding of the potential pleiotropic effects of NRF2 activators and discovery of novel classes of potent chemopreventive agents such as the triterpenoids. Although there is appropriately a concern regarding a deleterious role of the KEAP1-NRF2 system in cancer cell biology, especially as the pathway affects cell survival and drug resistance, the development and the use of NRF2 activators as chemopreventive agents still holds a great promise for protection of normal cells from a diversity of environmental stresses that contribute to the burden of cancer and other chronic, degenerative diseases.”
The 2011 publication NRF2, cancer and calorie restriction speaks about the interest in Nrf2 from a cancer therapeutic viewpoint: “The transcription factor NF-E2-related factor (NRF2) is a key regulator of several enzymatic pathways, including cytoprotective enzymes in highly metabolic organs. In this review, we summarize the ongoing research related to NRF2 activity in cancer development, focusing on in vivo studies using NRF2 knockout (KO) mice, which have helped in defining the crucial role of NRF2 in chemoprevention. The lower cancer protection observed in NRF2 KO mice under calorie restriction (CR) suggests that most of the beneficial effects of CR on the carcinogenesis process are likely mediated by NRF2. We propose that future interventions in cancer treatment would be carried out through the activation of NRF2 in somatic cells, which will lead to a delay or prevention of the onset of some forms of human cancers, and subsequently an extension of health- and lifespan.”
Nrf2 activators may be of benefit to patients with chronic renal failure.
The January 2012 publication Dietary and synthetic activators of the antistress gene response in treatment of renal disease reports: “Renal failure is associated with increased vascular inflammation, oxidative stress and dicarbonyl stress linked to development of cardiovascular disease, and other complications. The endogenous defense to inflammatory, oxidative, and dicarbonyl challenge to vascular function is coordinated by nuclear factor E2-related factor 2 (nrf2), kelch-related erythroid cell-derived protein with CNC homology (ECH) protein 1 (keap1), and antioxidant response element-linked gene expression in the antistress gene response. Intervention trials of the synthetic nrf2 activator, bardoloxone methyl, in patients with advanced diabetic nephropathy, showing improvement of renal function and decreased inflammation, suggest that nrf2 activators may have therapeutic benefit in chronic renal failure. Activators of nrf2 are of both synthetic and dietary origin. The aim of this review is to describe the “nrf2/keap1/antioxidant response element” transcriptional system and studies of this system in renal failure, and to assess the current status and future prospects that dietary nrf2 activators may be of benefit to patients with chronic renal failure.”
Protection against the negative effects of strokes is another of the many medical benefits that might be possible through endogenous stimulation of Nrf2.
The September 2011 publication Targeting the Nrf2-Keap1 antioxidant defence pathway for neurovascular protection in stroke reports: “Endogenous defence mechanisms by which the brain protects itself against noxious stimuli and recovers from ischaemic damage are a key target of stroke research. The loss of viable brain tissue in the ischaemic core region after stroke is associated with damage to the surrounding area known as the penumbra. Activation of the redox-sensitive transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) plays a pivotal role in the cellular defence against oxidative stress via transcriptional upregulation of phase II defense enzymes and antioxidant stress proteins. Although recent evidence implicates Nrf2 in neuroprotection, it is not known whether activation of this pathway within the neurovascular unit protects the brain against blood-brain barrier breakdown and cerebrovascular inflammation. Targeting the neurovascular unit should provide novel insights for effective treatment strategies and facilitate translation of experimental findings into clinical therapy. This review focuses on the cytoprotective role of Nrf2 in stroke and examines the evidence that the Nrf2-Keap1 defence pathway may serve as a therapeutic target for neurovascular protection.”
Steady laminar blood flow (s-flow) results in activation of Nrf2 and consequent inhibition of oxidative stress and inflammation in the vessel wall and is atheroprotective. By contrast, disturbed blood flow (d-flow) results in the activation of activator protein 1 (AP-1) and nuclear factor kappaB (NF-κB), inflammation, and predisposes to the development of plaques.
The September 2011 publication Flow shear stress and atherosclerosis: a matter of site specificity reports: “It is well accepted that atherosclerosis occurs in a site-specific manner especially at branch points where disturbed blood flow (d-flow) predisposes to the development of plaques. Investigations both in vivo and in vitro have shown that d-flow is pro-atherogenic by promoting oxidative and inflammatory states in the artery wall. In contrast, steady laminar blood flow (s-flow) is atheroprotective by inhibition of oxidative stress and inflammation in the vessel wall. The mechanism for inflammation in endothelial cells (ECs) exposed to d-flow has been well studied and includes redox-dependent activation of apoptosis signal-regulating kinase 1 (ASK1) and Jun NH2-terminal kinase (JNK) that ultimately lead to the expression of adhesive molecules. In contrast, s-flow leads to the activation of the mitogen extracellular-signal-regulated kinase kinase 5/extracellular signal-regulated kinase-5 (MEK5/ERK5) pathway that prevents pro-inflammatory signaling. Important transcriptional events that reflect the pro-oxidant and pro-inflammatory condition of ECs in d-flow include the activation of activator protein 1 (AP-1) and nuclear factor kappaB (NFκB), whereas in s-flow, activation of Krüppel-like factor 2 (KLF2) and nuclear factor erythroid 2-like 2 (Nrf2) are dominant. Recent studies have shown that protein kinase c zeta (PKCζ) is highly activated under d-flow conditions and may represent a molecular switch for EC signaling and gene expression. The targeted modulation of proteins activated in a site-specific manner holds the promise for a new approach to limit atherosclerosis.”
Overexpression of Nrf2 is not always necessarioly a good thing. It can result in protection of cancer cells.
The April 2011 publication Nrf2 is overexpressed in pancreatic cancer: implications for cell proliferation and therapy relates: “Background: Nrf2 is a key transcriptional regulator of a battery of genes that facilitate phase II/III drug metabolism and defence against oxidative stress. Nrf2 is largely regulated by Keap1, which directs Nrf2 for proteasomal degradation. The Nrf2/Keap1 system is dysregulated in lung, head and neck, and breast cancers and this affects cellular proliferation and response to therapy. Here, we have investigated the integrity of the Nrf2/Keap1 system in pancreatic cancer. Results: Keap1, Nrf2 and the Nrf2 target genes AKR1c1 and GCLC were detected in a panel of five pancreatic cancer cell lines. Mutation analysis of NRF2 exon 2 and KEAP1 exons 2-6 in these cell lines identified no mutations in NRF2 and only synonomous mutations in KEAP1. RNAi depletion of Nrf2 caused a decrease in the proliferation of Suit-2, MiaPaca-2 and FAMPAC cells and enhanced sensitivity to gemcitabine (Suit-2), 5-flurouracil (FAMPAC), cisplatin (Suit-2 and FAMPAC) and gamma radiation (Suit-2). The expression of Nrf2 and Keap1 was also analysed in pancreatic ductal adenocarcinomas (n = 66 and 57, respectively) and matching normal benign epithelium (n = 21 cases). Whilst no significant correlation was seen between the expression levels of Keap1 and Nrf2 in the tumors, interestingly, Nrf2 staining was significantly greater in the cytoplasm of tumors compared to benign ducts (P < 0.001). Conclusions: Expression of Nrf2 is up-regulated in pancreatic cancer cell lines and ductal adenocarcinomas. This may reflect a greater intrinsic capacity of these cells to respond to stress signals and resist chemotherapeutic interventions. Nrf2 also appears to support proliferation in certain pancreatic adenocarinomas. Therefore, strategies to pharmacologically manipulate the levels and/or activity of Nrf2 may have the potential to reduce pancreatic tumor growth, and increase sensitivity to therapeutics.”
NRF2 is protective against forms of hearing loss.
The November 2011 publication Protective role of Nrf2 in age-related hearing loss and gentamicin ototoxicity reports: “Expression of antioxidant enzymes is regulated by transcription factor NF-E2-related factor (Nrf2) and induced by oxidative stress. Reactive oxygen species contribute to the formation of several types of cochlear injuries, including age-related hearing loss and gentamicin ototoxicity. In this study, we examined the roles of Nrf2 in age-related hearing loss and gentamicin ototoxicity by measuring auditory brainstem response thresholds in Nrf2-knockout mice. Although Nrf2-knockout mice maintained normal auditory thresholds at 3 months of age, their hearing ability was significantly more impaired than that of age-matched wild-type mice at 6 and 11 months of age. additionally, the numbers of hair cells and spiral ganglion cells were remarkably reduced in Nrf2-knockout mice at 11 months of age. To examine the importance of Nrf2 in protecting against gentamicin-induced ototoxicity, 3-day-old mouse organ of
Corti explants were cultured with gentamicin. Hair cell loss caused by gentamicin treatment was enhanced in the Nrf2-deficient tissues. Furthermore, the expressions of some Nrf2-target genes were activated by gentamicin treatment in wild-type mice but not in Nrf2-knockout mice. The present findings indicate that Nrf2 protects the inner ear against age-related hearing injuries and gentamicin ototoxicity by up-regulating antioxidant enzymes and detoxifying proteins.”
The pubmed database shows 2,358 research publications related to Nrf2, so I have had to be selective in compiling the above. The following blog entry The pivotal role of Nrf2. Part 2 – foods, phyto-substances and other substances that turn on Nrf2 deals with how a number of substances that are incidentally antioxidants, plant-derived phyto-substances in particular, actually exercise their benefits through promoting the expression of Nrf2 which in turn activates the body’s own antioxidant and hormetic defense systems. A third entry The pivotal role of Nrf2. Part 3– Is promotion of Nrf2 expression a viable strategy for human human healthspan and lifespan extension? explores whether supplementation with substances that promote Nrf2 might be life-extending.