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9,10-Phenanthrenequinone (9,10-PQ) is a toxicant in diesel exhaust particles and airborne particulate matter ≤2.5 μm in diameter. It is an efficient electron acceptor that readily reacts with dithiol compounds

Space travel burdens health by imposing considerable environmental stress associated with radioactivity and microgravity. In particular, gravity change predominantly impacts blood pressure and bone homeostasis, both of which are controlled mainly by the kidneys. Nuclear factor erythroid-2-related transcription factor 2 (Nrf2) plays essential roles in protecting the kidneys from various environmental stresses and injuries. To elucidate the effects of space travel on mammals in preparation for the upcoming space era, our study investigated the contribution of Nrf2 to kidney function in mice two days after their return from a 31-day stay in the International Space Station using Nrf2 knockout mice. Meaningfully, expression levels of genes regulating bone mineralization, blood pressure and lipid metabolism were found to be significantly altered in the kidneys after space travel in an Nrf2-independent manner. In particular, uridine diphosphate-glucuronosyltransferase 1A (Ugt1a) isoform genes were found to be expressed in an Nrf2-dependent manner and induced exclusively in the kidneys after return to Earth. Since spaceflight elevated the concentrations of fatty acids in the mouse plasma, we suggest that Ugt1a isoform expression in the kidneys was induced to promote glucuronidation of excessively accumulated lipids and excrete them into urine after the return from space. Thus, the kidneys were proven to play central roles in adaptation to gravity changes caused by going to and returning from space by controlling blood pressure and bone mineralization. Additionally, kidney Ugt1a isoform induction after space travel implies a significant role of the kidneys for space travelers in the excretion of excessive lipids.

NRF2 (nuclear factor erythroid 2-related factor 2) is a transcription factor that regulates the expression of many cytoprotective genes. NRF2 activation is mainly regulated by KEAP1 (kelch-like ECH-associated protein 1) through ubiquitination and proteasome degradation. Esophageal cancer is classified histologically into two major types: esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC). ESCC harbors more genetic alterations in the KEAP-NRF2 system than EAC does, which results in NRF2 activation in these cancers. NRF2-addicted ESCC exhibits increased malignancy and acquisition of resistance to chemoradiotherapy. Therefore, it has been recognized that the development of drugs targeting the KEAP1-NRF2 system based on the molecular dissection of NRF2 function is important and urgent for the treatment of ESCC, along with efficient clinical screening for NRF2-addicted ESCC patients. Recently, the fate of NRF2-activated cells in esophageal tissues, which was under the influence of strong cell competition, and its relationship to the pathogenesis of ESCC, was clarified. In this review, we will summarize the current knowledge of the KEAP1-NRF2 system and the treatment of ESCC. We propose three main strategies for the treatment of NRF2-addicted cancer: (1) NRF2 inhibitors, (2) synthetic lethal drugs for NRF2-addicted cancers, and (3) NRF2 inducers of the host defense system.

Nrf2 is a transcription factor that regulates cytoprotective genes in response to oxidative and electrophilic stresses. The activity of Nrf2 is mainly controlled by the protein degradation by Keap1, a ubiquitin E3 ligase adaptor. Most of esophageal squamous cell carcinomas (ESCC) exhibit constitutive Nrf2 activation by Keap1 dysfunction, resulting in the poor prognosis. However, it has not been clarified whether Nrf2 activation serves as the initiation in tumorigenesis of ESCC. We generated tamoxifen-inducible and squamous epithelium-specific Keap1 conditional knockout (Keap1-cKO) mice in which Nrf2 is activated segmentally in esophageal epithelium at the adult stage. Using Nqo1 that is a representative Nrf2 target gene as the marker of Nrf2-acitivated cells, we distinguished Keap1-deleted/Nrf2-activated cells and Nrf2-normal cells in immunohistochemical analysis and gene expression analysis. Single-cell RNA-seq (scRNA-seq) analysis was conducted to compare the gene expression of Nrf2-activated cells and Nrf2-normal cells from Keap1-cKO mice, and control cells from control mice. In Keap1-cKO mice, Nrf2-activated cells accounted for 35% at 1 week and 20% at 4 weeks after induction of Keap1 deletion. This decrease was based on observations that Nrf2-activated cells were eliminated from the epithelium due to the weakened attachment to the basement membrane. In contrast, DNA damage accumulated in Nrf2-normal cells neighboring Nrf2-activated cells. To clarify the cell fate in the esophagus, we compared Nrf2-normal cells to Nrf2-activated cells or control cells in scRNA-seq analysis. Gene sets of G2/M checkpoint and E2F targets, which regulate cell proliferation, were enriched in Nrf2-normal cells. Constitutive Nrf2 activation renders esophageal epithelial cells able to be selectively eliminated via cell competition. Nrf2-normal cells suffered from DNA damage, but not Nrf2-activated cells, have a potency to be an origin of ESCC. These results suggest that Nrf2 activation serves as a tumor promoter of ESCC, but not the initiator. We speculate that the other gene mutations such as Tp53 are needed for the initiation to form ESCC prior to Nrf2 activation.

Nrf2 activates cytoprotective gene expression, and Nrf2 activity is regulated through at least two protein degradation pathways: the Keap1-mediated and β-TrCP-mediated pathways. To address the relative contributions of these pathways, we generated knock-in mouse lines expressing an Nrf2(SA) mutant that harbored two substitution mutations of serine residues interacting with β-TrCP. The homozygous (Nrf2(SA/SA)) mice grew normally, with Nrf2 levels comparable to those of wild-type (WT) mice under unstressed conditions. However, when Keap1 activity was suppressed, high levels of Nrf2 accumulated in Nrf2(SA/SA) macrophages compared with that in WT macrophages. We crossed Nrf2(SA/SA) mice with mice in which Keap1 was knocked down to two different levels. We found that the Nrf2(SA/SA) mutation induced higher Nrf2 activity when the Keap1 level was strongly reduced, and these mice showed severe growth retardation. However, activation and growth retardation were not evident when Keap1 was moderately suppressed. These increases in Nrf2 activity induced by the Nrf2(SA) mutation caused severe hyperplasia and hyperkeratosis in the esophageal epithelium but did not cause abnormalities in the other tissues/organs examined. These results indicate that the β-TrCP-mediated pathway cooperates with the Keap1-mediated pathway to regulate Nrf2 activity, which is apparent when the Keap1-mediated pathway is profoundly suppressed.

Radiation-induced oral mucositis is one of the most common adverse events in radiation therapy for head and neck cancers, but treatments for oral mucositis are limited to palliative and supportive care. New approaches are required to prevent radiation-induced mucositis and to improve treatments. The Keap1-Nrf2 system regulates cytoprotection against oxidative and electrophilic stresses. Nrf2 also regulates keratin layer thickness in mouse tongues. Therefore, we hypothesized that Nrf2 may protect the tongue epithelium against radiation-induced mucositis via elimination of reactive oxygen species and induction of keratin layer thickening. To test this hypothesis, we prepared a system for γ-ray exposure of restricted areas and irradiated the tongues of model mice with Nrf2 and Keap1 loss-of-function. We discovered that loss of Nrf2 expression indeed sensitized the tongue epithelium to radiation-induced ulcer formation with inflammation. Constitutive Nrf2 activation by genetic Keap1 knockdown alleviated radiation-induced DNA damage by increasing antioxidation. In agreement with the genetic Nrf2 activation model, the Nrf2 inducer CDDO-Im prevented irradiation damage to the tongue epithelium. These results demonstrate that Nrf2 activation has the potential to prevent the development of radiation-induced mucositis and that Nrf2 inducers are an important therapeutic drug for protection of the upper aerodigestive tract from radiation-induced mucositis.

Space flight produces an extreme environment with unique stressors, but little is known about how our body responds to these stresses. While there are many intractable limitations for in-flight space research, some can be overcome by utilizing gene knockout-disease model mice. Here, we report how deletion of Nrf2, a master regulator of stress defense pathways, affects the health of mice transported for a stay in the International Space Station (ISS). After 31 days in the ISS, all flight mice returned safely to Earth. Transcriptome and metabolome analyses revealed that the stresses of space travel evoked ageing-like changes of plasma metabolites and activated the Nrf2 signaling pathway. Especially, Nrf2 was found to be important for maintaining homeostasis of white adipose tissues. This study opens approaches for future space research utilizing murine gene knockout-disease models, and provides insights into mitigating space-induced stresses that limit the further exploration of space by humans., Using Nrf2 knockout mice, Suzuki, Uruno, Yumoto et al. show that space travel activates Nrf2 signaling, which contributes to the weight gain of mice by regulating fat metabolism of white adipose tissues. This study provides insights into potential interventions to mitigate stresses that accompany space travels.

The activation of the Kelch-like ECH-associated protein 1 (Keap1)-NF-E2-related factor 2 (Nrf2) pathway contributes to cancer progression in addition to oxidative stress responses. Loss-of-function Keap1 mutations were reported to activate Nrf2, leading to cancer progression. We examined the effects of Keap1 deletion in a cholangiocarcinoma mouse model using a mutant K-ras/ p53 mouse. Introduction of the Keap1 deletion into liver-specific mutant K-ras/ p53 expression resulted in the formation of invasive cholangiocarcinoma. Comprehensive analyses of the gene expression profiles identified broad upregulation of Nrf2-target genes such as Nqo1 and Gstm1 in the Keap1-deleted mutant K-ras/ p53 expressing livers, accompanied by upregulation of cholangiocyte-related genes. Among these genes, the transcriptional factor Sox9 was highly expressed in the dysplastic bile duct. The Keap-Nrf2-Sox9 axis might serve as a novel therapeutic target for cholangiocarcinoma. NEW & NOTEWORTHY The Keap1-Nrf2 system has a wide variety of effects in addition to the oxidative stress response in cancer cells. Addition of the liver-specific Keap1 deletion to mice harboring mutant K-ras and p53 accelerated cholangiocarcinoma formation, together with the hallmarks of Nrf2 activation. This process involved the expansion of Sox9-positive cells, indicating increased differentiation toward the cholangiocyte phenotype.

Liver plays essential roles in the metabolism of many endogenous chemicals and exogenous toxicants. Mechanistic studies in liver have been at the forefront of efforts to probe the roles of bioactivation and detoxication of environmental toxins and toxicants in hepatotoxicity. Moreover, idiosyncratic hepatoxicity remains a key barrier in the clinical development of drugs. The now vast NRF2 field emerged in part from biochemical and molecular studies on chemical inducers of hepatic detoxication enzymes and subsequent characterization of the modulation of drug/toxicant induced hepatotoxicities in mice through disruption of either Nrf2 or Keap1 genes. In general, loss of NRF2 increases the sensitivity to such toxic chemicals, highlighting a central role of this transcription factor and its downstream target genes as a modifier to chemical stress. In this review, we summarize the impact of NRF2 on the toxicology of multiple hepatotoxicants, and discuss efforts to utilize the NRF2 response in predictive toxicology.

Skin is constantly exposed to environmental insults, including toxic chemicals and oxidative stress. These insults often provoke perturbation of epidermal homeostasis and lead to characteristic skin diseases. AHR (aryl hydrocarbon receptor) and NRF2 (nuclear factor erythroid 2-related factor 2) are transcription factors that induce a battery of cytoprotective genes encoding detoxication and antioxidant enzymes in response to environmental insults. In addition to their basic functions as key regulators of xenobiotic and oxidant detoxification, recent investigations revealed that AHR and NRF2 also play critical roles in the maintenance of skin homeostasis. In fact, specific disruption of AHR function in the skin has been found to be associated with the pathogenesis of various skin diseases, most prevalently atopic dermatitis (AD). In this review, current knowledge on the roles that AHR and NRF2 play in epidermal homeostasis was summarized. Functional annotations of genetic variants, both regulatory and nonsynonymous SNPs, identified in the AHR and NRF2 loci in the human genome were also summarized. Finally, the possibility that AHR and NRF2 serve as therapeutic targets of AD was assessed.

It was previously identified that systemic Nrf2 deletion attenuates pancreatic cancer progression in a mutant K-ras/p53-expressing mouse model (KPC mouse). In this study, the type of cell that is responsible for the retarded cancer progression was elucidated. Human pancreatic cancers were first examined, and elevated expression of NRF2-target gene products in α-smooth muscle actin-positive cells was found, suggesting that pancreatic stellate cells (PSCs) are involved in this process. Closer examination of primary cultured PSCs from Nrf2-deleted mice revealed that the cells were less proliferative and retained a lower migration capacity. The conditioned medium of Nrf2-deleted PSCs exhibited reduced growth-stimulating effects in pancreatic cancer cells. KPC mouse-derived pancreatic cancer cells coinjected with wild-type PSCs developed significantly larger subcutaneous tumors in immunodeficient mice than those coinjected with Nrf2-deleted PSCs. These results demonstrate that Nrf2 actively contributes to the function of PSCs to sustain KPC cancer progression, thus, suggesting that Nrf2 inhibition in PSCs may be therapeutically important in pancreatic cancer.NEW & NOTEWORTHY This study identified that Nrf2 contributes to PSC activation. Nrf2 deletion in PSCs resulted in attenuation of cancer-promoting role. Nrf2 in PSCs could be an attractive therapeutic target in pancreatic cancer.

As the central regulator of the oxidative stress response, nuclear factor erythroid 2–related factor 2 (Nrf2) is attracting great interest as a therapeutic target for various cancers, and the possible clinical applications of novel Nrf2 inhibitors have been explored in Nrf2-activated cancers. In the present study, we specifically investigated halofuginone, which is derived from a natural plant alkaloid. We found that halofuginone administration decreased the number of pancreatic intraepithelial neoplasias in pancreas-specific Kras and p53 mutant (KPC) mice. In Nrf2-activated pancreatic cancer cell lines established from KPC mice, halofuginone rapidly depleted Nrf2 in Nrf2-activated cancer cells. Both in vitro and in vivo, it sensitized Nrf2-activated pancreatic cancer cells to gemcitabine, which is the first-line chemotherapy in clinical practice. In our mechanistic study, we found that halofuginone downregulated aldehyde dehydrogenase 3a1 (ALDH3A1) in mouse pancreatic cancer cells. The Nrf2 inducer diethyl maleate upregulated ALDH3A1, and knockdown of Aldh3a1 sensitized Nrf2-activated cancer cells to gemcitabine, strongly suggesting that ALDH3A1 is regulated by Nrf2 and that it contributes to gemcitabine resistance. The current study demonstrated the therapeutic benefits of halofuginone in Nrf2-activated pancreatic cancers. SIGNIFICANCE STATEMENT We identified nuclear factor erythroid 2–related factor 2 (Nrf2) and its downstream target aldehyde dehydrogenase 3a1 (ALDH3A1) as novel therapeutic targets in pancreatic cancer. They negatively affect the efficacy of a conventional chemotherapeutic agent, gemcitabine. We confirmed that Nrf2 plays a pivotal role in the induction of ALDH3A1.

Pancreatic cancer remains intractable owing to the lack of effective therapy for unresectable cases. Activating mutations of K-ras are frequently found in pancreatic cancers, but these have not yet been targeted by cancer therapies. The Keap1-Nrf2 system plays a crucial role in mediating the oxidative stress response, which also contributes to cancer progression. Nrf2 activation reprograms the metabolic profile to promote the proliferation of cancer cells. A recent report suggested that K-ras- and Nrf2-active lung cancer cells are sensitive to glutamine depletion. This finding led to the recognition of glutaminase inhibitors as novel anticancer agents. In the current study, we used murine pancreatic cancer tissues driven by mutant K-ras and p53 to establish cell lines expressing constitutively activated Nrf2. Genetic or pharmacological Nrf2 activation in cells via Keap1 deletion or Nrf2 activation sensitized cells to glutaminase inhibition. This phenomenon was confirmed to be dependent on K-ras activation in human pancreatic cancer cell lines harboring mutant K-ras, i.e., Panc-1 and MiaPaCa-2 in response to DEM pretreatment. This phenomenon was not observed in BxPC3 cells harboring wildtype K-ras. These results indicate the possibility of employing Nrf2 activation and glutaminase inhibition as novel therapeutic interventions for K-ras mutant pancreatic cancers.

Simple Summary Nuclear factor erythroid-derived 2-like 2 (encoded by the Nfe2l2 gene; NRF2) is a transcription factor that regulates a variety of cytoprotective genes, including antioxidant enzymes, detoxification enzymes, inflammation-related proteins, drug transporters and metabolic enzymes. NRF2 is regulated by unique molecular mechanisms that stem from Kelch-like ECH-associated protein 1 (KEAP1) in response to oxidative and electrophilic stresses. It has been shown that disturbance or perturbation of the NRF2 activation causes and/or exacerbates many kinds of diseases. On the contrary, aberrant activations of NRF2 also provoke intriguing pathologic features, especially in cancers. Cancer cells with high NRF2 activity have been referred to as NRF2-addicted cancers, which are frequently found in lung cancers. In this review, we summarize the current accomplishments of the KEAP1–NRF2 pathway analyses in special reference to the therapeutic target of cancer therapy. The concept of synthetic lethality provides a new therapeutic approach for NRF2-addicted cancers. Abstract The Kelch-like ECH-associated protein 1 (KEAP1)—Nuclear factor erythroid-derived 2-like 2 (encoded by the Nfe2l2 gene; NRF2) system attracts extensive interest from scientists in basic and clinical cancer research fields, as NRF2 exhibits activity as both an oncogene and tumor suppressor, depending on the context. Especially unique and malignant, NRF2-addicted cancers exhibit high levels of NRF2 expression. Somatic mutations identified in the NRF2 or KEAP1 genes of NRF2-addicted cancers cause the stabilization and accumulation of NRF2. NRF2-addicted cancers hijack the intrinsic roles that NRF2 plays in cytoprotection, including antioxidative and anti-electrophilic responses, as well as metabolic reprogramming, and acquire a marked advantage to survive under severe and limited microenvironments. Therefore, NRF2 inhibitors are expected to have therapeutic effects in patients with NRF2-addicted cancers. In contrast, NRF2 activation in host immune cells exerts significant suppression of cancer cell growth, indicating that NRF2 inducers also have the potential to be therapeutics for cancers. Thus, the KEAP1–NRF2 system makes a broad range of contributions to both cancer development and suppression. These observations thus demonstrate that both NRF2 inhibitors and inducers are useful for the treatment of cancers with high NRF2 activity.

Nrf2 is essential for cytoprotection against carcinogens, and through systemic Nrf2 knockout mice, Nrf2-deficient cells were shown to be susceptible to chemical carcinogens and prone to developing cancers. However, the oncogenic potential of Nrf2-deficient epithelial cells surrounded by normal cells in the esophagus could not be assessed by previous models, and the fate of Nrf2-deficient cells in such situations remains elusive. In this study, therefore, we generated mice that harbor almost equal levels of cells with Nrf2 deleted and those with Nrf2 intact in the basal layer of the esophageal epithelium, utilizing inducible Cre-mediated recombination of Nrf2 alleles in adults through moderate use of tamoxifen. In this mouse model, epithelial cells with Nrf2 deleted were maintained with no obvious decrease or phenotypic changes for 12 weeks under unstressed conditions. Upon exposure to the carcinogen 4-nitroquinoline-1-oxide (4NQO), the cells with Nrf2 deleted accumulated DNA damage and selectively disappeared from the epithelium, so almost all 4NQO-induced tumors originated from cells with Nrf2 intact and not from those with Nrf2 deleted. We propose that cells with Nrf2 deleted do not undergo carcinogenesis due to selective elimination upon exposure to 4NQO, indicating that cellular Nrf2 abundance and the epithelial environment determine the cell fate or oncogenic potential of esophageal epithelial cells in 4NQO-induced carcinogenesis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Transcription factor Nrf2 protects hepatocytes against various toxicants by upregulating cytoprotective genes. The heme synthesis inhibitor 3, 5-diethoxycarbonyl-1, 4-dihydrocollidine (DDC) leads to liver injury around the portal vein, unlike other groups of toxicants that cause hemorrhage and necrosis in the centrilobular area. To examine whether and how Nrf2 protects livers from the injury, we fed DDC to Nrf2 knockout (Nrf2KO), wild-type (WT), Keap1flox/flox (Keap1-knockdown; Keap1KD), and liver-specific Keap1 knockout (Keap1-Alb) mice, as these lines of mice exhibit stepwise increases in Nrf2 protein expression levels. Liver-specific Keap1::Nrf2 double-knockout (Keap1::Nrf2-Alb) mice were also exploited to examine the contribution of Nrf2. Two weeks after DDC feeding, Keap1-Alb mice were fully recovered from body weight loss, but the WT and Nrf2KO mice were not. The liver-to-body-weight ratio of Keap1-Alb mice was significantly larger than that of WT and Nrf2KO mice. Two indicators of hepatotoxicity, alanine aminotransferase and bilirubin in plasma, were both elevated in WT mice, but downregulated in Keap1-Alb mice after the DDC-feeding. DDC-induced porphyrin accumulation was reduced in the livers of Keap1-Alb and Keap1KD mice compared with that of WT mice. When assessed by the Nqo1 level, Nrf2 expression was further enhanced by DDC in Keap1-Alb mice, suggesting that DDC may have a Keap1 independent potential to activate Nrf2. Genetic activation of Nrf2 in Keap1-Alb mice increased the extracellular excretion of porphyrins, but contrary to our expectation, hepatic damages in Nrf2KO mice appeared to be similar to that of WT mice. Based on these observations, we conclude that Nrf2 activation protects livers against DDC-elicited hepatotoxicity.

The Kelch-like ECH-associated protein 1 (Keap1)-NF-E2-related factor 2 (Nrf2) system has a wide variety of effects in addition to the oxidative stress response, such as growth promotion and chemoresistance of cancer cells. Nrf2 is constitutively activated in most cancer cells. However, the activation of Nrf2 together with oncogenic mutations does not always result in cancer promotion. K-rasLSL-G12D/+:: p53LSL-R172H/+:: Pdx-1-Cre (KPC) mice are an established model of pancreatic cancer that specifically express mutants of both K-ras and p53 in the pancreas by using Pdx-1-Cre. We here generated Pdx-1-Cre::K-rasLSL-G12D/+:: Keap1fl/fl (KC::Keap1) and KPC:: Keap1fl/fl (KPC::Keap1) mice in which Nrf2 is constitutively activated by Keap1 deletion. KC::Keap1 and KPC::Keap1 mice started to die or showed obvious weakness at approximately around 40 days after birth. Histological examination revealed that KC::Keap1 and KPC::Keap1 mice did not develop pancreatic cancer but, instead, progressive atrophy of the pancreatic parenchyma. In these mice, amylase-positive acinar cells as well as insulin- and glucagon-positive islet cells were decreased and surrounded by fibrotic tissues. KC::Keap1 and KPC::Keap1 mice presented lower body weight and glucose levels than C::Keap1 mice, presumably resulting from pancreatic exocrine insufficiency. Histological changes were not obvious in C::Keap1 and PC::Keap1 mice. The presence of the p53 mutation did not affect the phenotypes in KC::Keap1 mice. Heterologous or homologous Nrf2 deletion ( Nrf2+/− or Nrf2−/−) rescued the pancreatic phenotypes, weight loss, and hypoglycemia in KC::Keap1 mice, suggesting that Nrf2 is a major downstream target of Keap1. In conclusion, simultaneous K-ras activation and Keap1 deletion caused progressive atrophy of the pancreatic parenchyma in mice. NEW & NOTEWORTHY Aberrant activation of the Kelch-like ECH-associated protein 1 (Keap1)-NF-E2-related factor 2 (Nrf2) system usually promotes carcinogenesis, and we assumed that simultaneous activation of K-ras and Nrf2 might promote pancreatic carcinogenesis. Conditional expression of mutant K-ras and Keap1 deletion did not result in pancreatic cancer development. Instead, these mice developed progressive loss of pancreatic parenchyma, accompanied by body weight loss and hypoglycemia, presumably because of pancreatic exocrine insufficiency. Nrf2 activation by Keap1 deletion concomitant with K-ras activation cause pancreatic atrophy.

The Keap1-Nrf2 system contributes to the maintenance of homeostasis by regulating oxidative stress responses in normal tissues and organs, and is exploited in various cancers for proliferation, survival and acquisition of therapy resistance. Pancreatic cancer remains one of the intractable cancers, despite the improved clinical outcomes of other types of cancer, due to its invasive and refractory nature to therapeutic intervention. The current study aimed to clarify the contribution of Nrf2 to pancreatic carcinogenesis using a pancreas-specific mutant K-ras and p53 (KPC) mouse model. Deletion of Nrf2 in KPC mice (KPCN) decreased the formation of precancerous lesions as well as the development of invasive pancreatic cancer. The pancreatic tumor-derived cancer cell lines from KPCN mouse showed decreased expression of glutathione S-transferases (GST), UDP glucuronosyltransferases (UGT) and ABC transporters. Along with these biochemical changes, cell lines from KPCN mice revealed increased sensitivity to oxidative stress and chemotherapeutic agent. The current study revealed that Nrf2 contributes to pancreatic carcinogenesis in a way distinct from the chemoresistance of lung and esophagus, and that Nrf2 could be a novel therapeutic target of pancreatic cancer.

Transgenic mice expressing a constitutively active form of the aryl hydrocarbon receptor in keratinocytes (AhR-CA mice) develop severe dermatitis that substantially recapitulates the pathology of human atopic dermatitis. The neurotrophic factor artemin (Artn) is highly expressed in the epidermis of AhR-CA mice and causes hypersensitivity to itch (alloknesis) by elongating nerves into the epidermis. However, whether the Artn gene is regulated directly by AhR or indirectly through complex regulation associated with AhR remains unclear. To this end, we previously conducted chromatin immunoprecipitation-sequencing analyses of the Artn locus and found a xenobiotic response element (XRE) motif located far upstream (52 kb) of the gene. Therefore, in this study, we addressed whether the XRE actually regulates the Artn gene expression by deleting the region containing the motif. We generated two lines of ArtnΔXRE mice. In the mouse epidermis, inducible expression of the Artn gene by the AhR agonist 3-methylcholanthrene was substantially suppressed compared to that in wild-type mice. Importantly, in AhR-CA::ArtnΔXRE mice, Artn expression was significantly suppressed, and alloknesis was improved. These results demonstrate that the Artn gene is indeed regulated by the distal XRE-containing enhancer, and alloknesis in AhR-CA mice is provoked by the AhR-mediated direct induction of the Artn gene.

Transgenic mice expressing a constitutively active form of the aryl hydrocarbon receptor in keratinocytes (AhR-CA mice) develop severe dermatitis that substantially recapitulates the pathology of human atopic dermatitis. The neurotrophic factor artemin (Artn) is highly expressed in the epidermis of AhR-CA mice and causes hypersensitivity to itch (alloknesis) by elongating nerves into the epidermis. However, whether the Artn gene is regulated directly by AhR or indirectly through complex regulation associated with AhR remains unclear. To this end, we previously conducted chromatin immunoprecipitation-sequencing analyses of the Artn locus and found a xenobiotic response element (XRE) motif located far upstream (52 kb) of the gene. Therefore, in this study, we addressed whether the XRE actually regulates the Artn gene expression by deleting the region containing the motif. We generated two lines of Artn(ΔXRE) mice. In the mouse epidermis, inducible expression of the Artn gene by the AhR agonist 3-methylcholanthrene was substantially suppressed compared to that in wild-type mice. Importantly, in AhR-CA::Artn(ΔXRE) mice, Artn expression was significantly suppressed, and alloknesis was improved. These results demonstrate that the Artn gene is indeed regulated by the distal XRE-containing enhancer, and alloknesis in AhR-CA mice is provoked by the AhR-mediated direct induction of the Artn gene.

Keap1 is a negative regulator of Nrf2, a master transcription factor that regulates cytoprotection against oxidative and electrophilic stresses. Although several studies have suggested that the Keap1-Nrf2 system contributes to bone formation besides the maintenance of redox homeostasis, how Nrf2 hyperactivation by Keap1 deficiency affects the bone formation remains to be explored, as the Keap1-null mice are juvenile lethal. To overcome this problem, we used viable Keap1-deficient mice that we have generated by deleting the esophageal Nrf2 in Keap1-null mice (NEKO mice). We found that the NEKO mice exhibit small body size and low bone density. Although nephrogenic diabetes insipidus has been observed in both the NEKO mice and renal-specific Keap1-deficient mice, the skeletal phenotypes are not recapitulated in the renal-specific Keap1-deficient mice, suggesting that the skeletal phenotype by Nrf2 hyperactivation is not related to the renal phenotype. Experiments with primary culture cells derived from Keap1-null mice showed that differentiation of both osteoclasts and osteoblasts was attenuated, showing that impaired differentiation of osteoblasts rather than osteoclasts is responsible for bone hypoplasia caused by Nrf2 hyperactivation. Thus, we propose that the appropriate control of Nrf2 activity by Keap1 is essential for maintaining bone homeostasis.

Cancer cells first adapt to the microenvironment and then propagate. Mutations in tumor suppressor genes or oncogenes are frequently found in cancer cells. Comprehensive genomic analyses have identified somatic mutations and other alterations in the KEAP1 or NRF2 genes and in well-known tumor suppressor genes or oncogenes, such as TP53, CDKN2A, PTEN, and PIK3CA, in various types of cancer. Aberrant NRF2 activation in cancer cells occurs through somatic mutations in the KEAP1 or NRF2 gene as well as through other mechanisms that disrupt the binding of KEAP1 to NRF2. Unregulated NRF2 confers on cancer cells high-level resistance to anti-cancer drugs and reactive oxygen species (ROS) and directs cancer cells toward metabolic reprogramming. Therefore, NRF2 has been studied as a therapeutic target molecule in cancer. Two strategies have been used to target NRF2 via therapeutic drugs: inhibition of NRF2 and induction of NRF2. NRF2 inhibitors may be effective against NRF2-addicted cancer cells in which NRF2 is aberrantly activated. These inhibitors have not yet been established as NRF2-targeted anti-cancer drugs for the treatment of human cancers. Diagnosis of NRF2 activation could facilitate the use of NRF2 inhibitors for the treatment of patients with NRF2-addicted cancers. Conversely, NRF2 inducers have been used or are being developed for non-cancer diseases. In addition, NRF2 inducers may be useful for cancer chemotherapy in combination with conventional anti-cancer agents or even NRF2 inhibitors.

Anaplastic gliomas and glioblastomas are aggressive brain tumors with median survival times ranging from 72.1 to 82.6 months in anaplastic gliomas and 14.6 months in glioblastomas.1,2 One of the main causes of the poor prognosis is chemo- and radioresistance. Thus, elucidation of mechanisms underlying this resistance is urgent to improve the treatment outcomes of malignant gliomas. The system involving Kelch-like ECH (erythroid cell-derived protein with cap'n'collar homology)-associated protein 1 (KEAP1) and nuclear factor erythroid 2–related factor 2 (NRF2) plays pivotal roles in protecting normal and neoplastic cells from oxidative and electrophilic insults by activating cytoprotective genes. These gene products are involved in glutathione synthesis (eg, glutamate cysteine ligase of catalytic [GCLC] and of modifier subunit [GCLM] genes), reactive oxygen species (ROS) elimination, xenobiotic metabolism (eg, NAD(P)H:quinone oxidoreductase gene [NQO1]), and crossmembrane transport, often resulting in multidrug resistance.3 Under normal conditions, KEAP1 ubiquitinates NRF2, and NRF2 is degraded by the proteosome. In the presence of electrophiles or ROS, KEAP1 is inactivated, and NRF2 is stabilized, binds to antioxidant response elements (AREs), and induces the cytoprotective genes. Recently, constitutive activation of NRF2 was found in various cancers. Multiple causes of the constitutive activation of NRF2 have been described, including somatic mutations of KEAP1 or NRF2, reduced expression of KEAP1, and accumulation of p62.4 Increased NRF2 activity confers cell growth advantage and resistance to chemo- and radiotherapy on lung, prostate, gallbladder, and ovarian cancers5–9 and leads to poor prognosis in lung, esophagus, gallbladder, and breast cancers.10–14 Although similar impacts of NRF2 activation on glioblastoma cell lines and a small cohort of glioblastoma patients have been reported,15,16 the prognostic significance of NRF2 activation in malignant gliomas remains to be elucidated. Somatic mutations in the isocitrate dehydrogenase 1 (IDH1) gene or IDH2 gene have been identified in adult gliomas.17–19 These mutations occur at a very early stage of gliomatogenesis and confer genetic and prognostic differences on anaplastic gliomas and glioblastomas.17–19 Malignant gliomas with IDH1/2 mutations had better prognoses than those with wild-type IDH1/2.17–19 Although the hypermethylated phenotype is frequently found in World Health Organization grades II and III gliomas with IDH1/2 mutations, underlying mechanisms for better prognosis remain unclear. Recently, IDH1 mutations have been shown to sensitize glioma cells to bis-chloroethylnitrosourea (BCNU)–induced oxidative stress by decreasing the amount of the reduced form of glutathione.20 Because the synthesis and reduction of glutathione is often heavily dependent on NRF2 activity,4 we hypothesized that the NRF2 pathway was suppressed in the presence of mutated IDH1, leading to the better prognosis of malignant gliomas with IDH1/2 mutations. To test this hypothesis, we characterized the prevalence, possible causes, and prognostic value of the NRF2 activation in anaplastic gliomas with mutated or wild-type IDH1/2 and glioblastomas. We also evaluated the association between IDH1/2 mutations and NRF2 activity by analyzing the expression of NRF2 and its target genes in surgical specimens, and confirmed the effect of IDH1 mutation on NRF2 activity, using IDH1 mutant-expressing cells.

Keap1-Nrf2 system plays a central role in the stress response. While Keap1 ubiquitinates Nrf2 for degradation under unstressed conditions, this Keap1 activity is abrogated in response to oxidative or electrophilic stresses, leading to Nrf2 stabilization and coordinated activation of cytoprotective genes. We recently found that nuclear accumulation of Nrf2 is significantly increased by simultaneous deletion of Pten and Keap1, resulting in the stronger activation of Nrf2 target genes. To clarify the impact of the cross talk between the Keap1-Nrf2 and Pten-phosphatidylinositide 3-kinase-Akt pathways on the liver pathophysiology, in this study we have conducted closer analysis of liver-specific Pten::Keap1 double-mutant mice (Pten::Keap1-Alb mice). The Pten::Keap1-Alb mice were lethal by 1 month after birth and displayed severe hepatomegaly with abnormal expansion of ductal structures comprising cholangiocytes in a Nrf2-dependent manner. Long-term observation of Pten::Keap1-Alb::Nrf2(+/-) mice revealed that the Nrf2-heterozygous mice survived beyond 1 month but developed polycystic liver fibrosis by 6 months. Gsk3 directing the Keap1-independent degradation of Nrf2 was heavily phosphorylated and consequently inactivated by the double deletion of Pten and Keap1 genes. Thus, liver-specific disruption of Keap1 and Pten augments Nrf2 activity through inactivation of Keap1-dependent and -independent degradation of Nrf2 and establishes the Nrf2-dependent molecular network promoting the hepatomegaly and cholangiocyte expansion.

The disruption of the NRF2 (nuclear factor erythroid-derived 2-like 2)/glutathione-mediated antioxidant defense pathway is a critical step in the pathogenesis of several chronic pulmonary diseases and cancer. While the mechanism of NRF2 activation upon oxidative stress has been widely investigated, little is known about the endogenous signals that regulate the NRF2 pathway in lung physiology and pathology. Here we show that an E-box-mediated circadian rhythm of NRF2 protein is essential in regulating the rhythmic expression of antioxidant genes involved in glutathione redox homeostasis in the mouse lung. Using an in vivo bleomycin-induced lung fibrosis model, we reveal a clock “gated” pulmonary response to oxidative injury, with a more severe fibrotic effect when bleomycin was applied at a circadian nadir in NRF2 levels. Timed administration of sulforaphane, an NRF2 activator, significantly blocked this phenotype. Moreover, in the lungs of the arrhythmic ClockΔ19 mice, the levels of NRF2 and the reduced glutathione are constitutively low, associated with increased protein oxidative damage and a spontaneous fibrotic-like pulmonary phenotype. Our findings reveal a pivotal role for the circadian control of the NRF2/glutathione pathway in combating oxidative/fibrotic lung damage, which might prompt new chronotherapeutic strategies for the treatment of human lung diseases, including idiopathic pulmonary fibrosis.