The transcription factor nuclear factor-erythroid 2 p45-related factor 2 (Nrf2, with gene called NFE2L2) is a master regulator of the antioxidant response. In the last decade, interest has intensified in this research area as its importance in several physiological and pathological processes has become widely recognized; these include redox signalling and redox homoeostasis, drug metabolism and disposition, intermediary metabolism, cellular adaptation to stress, chemoprevention and chemoresistance, toxicity, inflammation, neurodegeneration, lipogenesis and aging. Regulation of Nrf2 is complex and although much attention has focussed on its repression by Kelch-like ECH-associated protein-1 (Keap1), recently it has become increasingly apparent that it is also controlled by cross-talk with other signalling pathways including the glycogen synthase kinase-3 (GSK-3)−β-transducin repeat-containing protein (β-TrCP) axis, ERAD (endoplasmic reticulum-associated degradation)-associated E3 ubiquitin-protein ligase (Hrd1, also called synoviolin), nuclear factor-kappa B (NF-κB), Notch and AMP kinase. Due to its beneficial role in several diseases, Nrf2 has become a major therapeutic target, with novel natural, synthetic and targeted small molecules currently under investigation to modulate the pathway and in clinical trials.

Background

Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) is a cap’n’collar basic-region leucine zipper transcription factor that plays a central role in cellular defence, particularly in response to oxidative stress and toxic insults. It regulates directly approximately 250 genes that are involved in cell homoeostasis, including those for antioxidant proteins, detoxification enzymes, drug transporters and numerous cytoprotective proteins, and it also influences energy metabolism, inflammation and cell growth [13]. Studies using Nrf2 knockout mice and small molecule activators, usually called inducers, have demonstrated beneficial effects in several animal models of chronic age-related and inflammatory diseases [1,35]. In cancer, however, Nrf2 has dual effects, where it may have chemopreventive actions and block the initiation of carcinogenesis by certain chemical carcinogens or, in contrast, promote tumour growth or chemoresistance [4,6]. The wide-ranging functions of Nrf2 in homoeostasis and disease that have been recently discovered, together with increasing knowledge about cross-talk with other signalling pathways, have attracted interest from a broad range of researchers in various fields, including those within the pharmaceutical industry. In December 2011, the Biochemical Society supported a Hot Topic meeting on ‘Nrf2 signalling in health and disease’ to highlight this important subject, which spans a broad range of research areas, to the scientific community, and the meeting brought together scientists in Europe to discuss this emerging field. Following the success of this Hot Topic meeting, the Biochemical Society supported a Focused Meeting on ‘The Keap1 (Kelch-like ECH-associated protein-1)/Nrf2 Signalling Pathway in Health and Disease’ in January 2015, which attracted a number of key research groups from around the world to discuss current emerging issues in the field. The present paper provides a brief synopsis of this meeting.

Nrf2 regulation

Nrf2 activity can be increased by exposure to soft electrophiles, phytochemicals, oxidants, growth factors, drugs, toxins and numerous other stimuli [2]. Due to its broad effects and wide range of agonists, Nrf2 activation is tightly controlled at multiple levels. It is principally controlled at the protein stability level by redox stress-sensitive and metabolism-sensitive proteasomal degradation, as well as by transcriptional regulation. Nrf2 also engages in cross-talk with several other signalling pathways. Nrf2 regulation at the protein level occurs by the ubiquitin proteasome system [1,2]. The ability of Keap1 to direct Nrf2 ubiquitylation by the Cul3 (cullin-3)–Rbx1 (ring-box protein 1) E3 ubiquitin ligase and subsequent proteasomal degradation is well documented and a cycling mechanism by which Keap1 interacts with Nrf2 and only releases the protein once the transcription factor is ubiquitylated has been identified [1]. Whereas much research to date has focused on cysteine modification of Keap1 as a mechanism of derepression of Nrf2 by Keap1, for example by sulforaphane and other electrophiles, Nrf2 regulation at the protein level has recently been recognized as being far more elaborate and includes Keap1-independent mechanisms. Glycogen synthase kinase-3 (GSK-3)β phosphorylation of the Neh6 (Nrf2-ECH homology) domain of Nrf2 leads to ubiquitylation of the transcription factor by β-TrCP (β-transducin repeat-containing protein)–Skp1 (S-phase kinase-associated protein 1)–Cul1–F-box E3 ubiquitin ligase. It seems probable that growth factors activate Nrf2 through stimulation of upstream protein kinases and subsequent inhibitory phosphorylation of GSK-3β, which prevents the degradation of Nrf2 by this pathway [2]. Another novel E3 ubiquitin ligase, Hrd1 (endoplasmic reticulum-associated degradation, ERAD-associated E3 ubiquitin-protein ligase), targets the Neh4–5 domain of Nrf2 for ubiquitination and degradation of the transcription factor under conditions of oxidative stress and thereby accelerates liver fibrosis/cirrhosis [6].

In addition to proteasomal degradation, Nrf2 is transcriptionally regulated, for example by K-Ras and post-transcriptionally regulated by miRNAs. Conversely, Nrf2 regulates the transcription of miRNAs, including miR-29a, miR-29b and miR-125b1 [5]. Recent advances have also revealed cross-talk between Nrf2 and other signalling pathways. For example, Nrf2 interacts with the autophagy pathway via p62/sequestosome-1 [3,6]. In addition, the anti-inflammatory effects of Nrf2 are at least partially due to cross-talk between Nrf2 and the NF-κB (nuclear factor-kappa B) pathway. Several proteins have been identified that are key to both pathways, including Keap1, CBP (CREB-binding protein), MafK (V-maf musculoaponeurotic fibrosarcoma oncogene homologue K), GSK-3 and β-TrCP and therefore both compete for these during signalling. NF-κB also acts directly on Nrf2 by binding to κB response elements in the promoter, which sustains Nrf2-dependent gene expression and both Nrf2 and NF-κB interact with histone deacetylases, affecting transcription [3]. Although it is not thought that nuclear-cytoplasmic shuttling of Nrf2 represents a major regulatory mechanism, evidence was presented that the transcription factor can oscillate between the cytoplasm and nucleus in resting human microvascular endothelial HMEC-1 cells and this may be a previously unrecognized mode of regulation that requires further evaluation [7].

Nrf2 and cellular homoeostasis

The central role of Nrf2 in redox homoeostasis is well established. Nrf2 controls the expression of antioxidants, detoxification enzymes, drug transporters, reductases and other defence proteins to protect cells from various forms of stress [2]. Some of these may then also have feedback effects on the Nrf2 pathway. For example, disulfide reductases such as thioredoxin 1 (Trx1) along with thioredoxin reductase, are induced by Nrf2 and this in turn can then regulate Nrf2 activation through a negative feedback loop that involves activation of Keap1 [8]. Nrf2 also has effects on cellular metabolic pathways and improves mitochondrial function. It induces the expression of enzymes involved in the pentose phosphate pathway and increases availability of NADH and FADH for respiration and accelerating fatty acid oxidation, and suppresses de novo lipogenesis [1,4]. In addition, redox homoeostasis has been recognized recently to play an important role in improving stem cell function and controlling stem cell behaviour and fate, potentially through Nrf2 actions [9].

Nrf2 appears to play a critical role in the restoration of cellular homoeostasis following infection. In this case, Nrf2 prevents exaggerated immune responses, controls the extent of tissue damage caused by infection and results in tolerance to systemic infections, such as severe sepsis or severe malaria, partially through Nrf2 target genes controlling glutathione and haeme metabolism. However, it also appears to have contrasting effects in response to viral infection as viruses activate Nrf2, which promotes their proliferation in the host [10]. Nrf2 also plays an important role in protecting against chemical toxicity and may be useful in pre-clinical testing of chemicals and drugs that may induce toxicity or adverse drug reactions [11].

Nrf2 and disease

The role of Nrf2 in the pathogenesis and progression of chronic age-related diseases has been extensively studied using Nrf2 knockout animal models of various diseases and pharmacological activators in mice. Recent developments suggest that Nrf2 is a promising target in the treatment of neurodegenerative diseases, including Alzheimer's disease, multiple sclerosis and Parkinson's disease [1,3]. Pharmacological activators of Nrf2 are neuroprotective, reducing inflammation, in addition to decreasing numbers of overactive microglia and astrocytes and improving mitochondrial function in animal models of Parkinson's disease [1,3]. In the case of cancer, the role of Nrf2 is more complex. Activation of the Nrf2 pathway by phytochemicals may be of benefit in the prevention of cancer and absence of Nrf2 increases susceptibility of cells to cancer. However, on the flip side, excessive sustained Nrf2 activation is observed in many cancer cell types, particularly the lung, due to loss-of-function somatic mutations in Keap1 or gain-of-function ‘hot spot’ mutations in NFE2L2 that encodes Nrf2 [4,12]. Nrf2 can drive cell proliferation and inhibit apoptosis and enhance tumour development through metabolic reprogramming, including up-regulation of the pentose phosphate pathway. Cancer cells also hijack the Keap1–Nrf2 pathway to enhance chemoresistance and radio-resistance. Large-scale multi-omics projects in various cancer types are underway to identify mechanisms leading to increased Nrf2 activity in cancer [4,12,13].

Nrf2 as a therapeutic target

As Nrf2 can modulate disease outcomes in animal models, it is unsurprising that it has attracted major interest as a therapeutic target for various diseases. Nrf2 activators include phytochemicals such as sulforaphane, which has been well studied [1,4]. Yates et al. [14] originally developed synthetic triterpenoids as anti-inflammatory agents, which are now known to activate Nrf2 by modifying cysteine residues on Keap1. Among the compounds they developed, bardoxolone methyl (CDDO-Me) is currently undergoing clinical trials for various diseases. Dimethyl fumarate, licensed for use in psoriasis and multiple sclerosis, was also recently discovered to be a potent Nrf2 activator [4]. In addition, small molecules that can directly disrupt the Keap 1–Nrf2 protein–protein interaction are in early development and have not yet made pre-clinical trials. These include peptides and other small molecules such as tetrahydrosioquinolines and naphthalene sulfonamides, which are demonstrating potential as lead compounds for new Nrf2 activator drugs [15]. Keap-1-independent Nrf2-activators have also been identified, including nordihydroguaiaretic acid, 4U8C and LS-102. In addition, Nrf2 inhibitors are of interest in cancer therapy, potentially as adjuvants in chemotherapy to overcome chemoresistance, with Brusatol being the best characterized so far and others under development [6].

In conclusion, recent developments that have emerged in the Nrf2 field have given us further insights into the regulation of this important transcription factor, including cross-talk with other signalling pathways and novel roles in homoeostasis. Investigations into its role in diseases, including cancer and neurodegenerative disorders have increased our knowledge and provided further evidence that it is an important therapeutic target. Natural and synthetic molecules that activate Nrf2 are currently under development and in clinical trials for various diseases and Nrf2 inhibitors are under development for adjuvants in cancer chemotherapy.

Biochemical Society Staff at Robinson College.

Abbreviations

     
  • β-TrCP

    β-transducin repeat-containing protein

  •  
  • Cul

    cullin

  •  
  • GSK-3

    glycogen synthase kinase-3

  •  
  • Hrd1

    ERAD (endoplasmic reticulum-associated degradation)-associated E3 ubiquitin-protein ligase

  •  
  • Keap1

    Kelch-like ECH-associated protein-1

  •  
  • NF-κB

    nuclear factor-kappa B

  •  
  • Nrf2

    nuclear factor-erythroid 2 p45-related factor 2

The Keap1/Nrf2 Pathway in Health and Disease: Held at Robinson College, Cambridge, UK, 6–8 Jan 2015.

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