Oral arsenic administration to humanizedUDP-glucuronosyltransferase1 neonatal mice induces UGT1A1 through a dependence on Nrf2 and PXR.

Inorganic arsenic (iAs) is an environmental toxicant that can lead to severe health consequences, which can be exacerbated if exposure occurs early in development. Here, we evaluated the impact of oral iAs treatment on UDP-glucuronosyltransferase 1A1 (UGT1A1) expression and bilirubin metabolism in humanized UGT1 (hUGT1) mice. We found that oral administration of iAs to neonatal hUGT1 mice that display severe neonatal hyperbilirubinemia leads to induction of intestinal UGT1A1 and a reduction in total serum bilirubin values. Oral iAs administration accelerates neonatal intestinal maturation, an event that is directly associated with UGT1A1 induction. As a reactive oxygen species producer, oral iAs treatment activated the Keap-Nrf2 pathway in the intestinal tract and liver. When Nrf2-deficient hUGT1 mice (hUGT1/Nrf2-/-) were treated with iAs, it was shown that activated Nrf2 contributed significantly toward intestinal maturation and UGT1A1 induction. However, hepatic UGT1A1 was not induced upon iAs exposure. We previously demonstrated that the nuclear receptor PXR represses liver UGT1A1 in neonatal hUGT1 mice. When PXR was deleted in hUGT1 mice (hUGT1/Pxr-/-), derepression of UGT1A1 was evident in both liver and intestinal tissue in neonates. Furthermore, when neonatal hUGT1/Pxr-/- mice were treated with iAs, UGT1A1 was superinduced in both tissues, confirming PXR release derepressed key regulatory elements on the gene that could be activated by iAs exposure. With iAs capable of generating reactive oxygen species in both liver and intestinal tissue, we conclude that PXR deficiency in neonatal hUGT1/Pxr-/- mice allows greater access of activated transcriptional modifiers such as Nrf2 leading to superinduction of UGT1A1.

RNA demethylase ALKBH5 regulates hypopharyngeal squamous cell carcinoma ferroptosis by posttranscriptionally activating NFE2L2/NRF2 in an m6 A-IGF2BP2-dependent manner.

BACKGROUND: Having emerged as the most abundant posttranscriptional internal mRNA modification in eukaryotes, N6-methyladenosine (m6 A) has attracted tremendous scientific interest in recent years. However, the functional importance of the m6 A methylation machinery in ferroptosis regulation in hypopharyngeal squamous cell carcinoma (HPSCC) remains unclear. METHODS: We herein performed bioinformatic analysis, cell biological analyses, transcriptome-wide m6 A sequencing (m6 A-seq, MeRIP-seq), RNA sequencing (RNA-seq), and RNA immunoprecipitation sequencing (RIP-seq), followed by m6 A dot blot, MeRIP-qPCR, RIP-qPCR, and dual-luciferase reporter assays. RESULTS: The results revealed that ALKBH5-mediated m6 A demethylation led to the posttranscriptional inhibition of NFE2L2/NRF2, which is crucial for the regulation of antioxidant molecules in cells, at two m6 A residues in the 3'-UTR. Knocking down ALKBH5 subsequently increased the expression of NFE2L2/NRF2 and increased the resistance of HPSCC cells to ferroptosis. In addition, m6 A-mediated NFE2L2/NRF2 stabilization was dependent on the m6 A reader IGF2BP2. We suggest that ALKBH5 dysregulates NFE2L2/NRF2 expression in HPSCC through an m6 A-IGF2BP2-dependent mechanism. CONCLUSION: Together, these results have revealed an association between the ALKBH5-NFE2L2/NRF2 axis and ferroptosis, providing insight into the functional importance of reversible mRNA m6 A methylation and its modulators in HPSCC.

The GTPase KRAS suppresses the p53 tumor suppressor by activating the NRF2-regulated antioxidant defense system in cancer cells.

In human cancer cells that harbor mutant KRAS and WT p53 (p53), KRAS contributes to the maintenance of low p53 levels. Moreover, KRAS depletion stabilizes and reactivates p53 and thereby inhibits malignant transformation. However, the mechanism by which KRAS regulates p53 is largely unknown. Recently, we showed that KRAS depletion leads to p53 Ser-15 phosphorylation (P-p53) and increases the levels of p53 and its target p21/WT p53-activated fragment 1 (WAF1)/CIP1. Here, using several human lung cancer cell lines, siRNA-mediated gene silencing, immunoblotting, quantitative RT-PCR, promoter-reporter assays, and reactive oxygen species (ROS) assays, we demonstrate that KRAS maintains low p53 levels by activating the NRF2 (NFE2-related factor 2)-regulated antioxidant defense system. We found that KRAS depletion led to down-regulation of NRF2 and its targets NQO1 (NAD(P)H quinone dehydrogenase 1) and SLC7A11 (solute carrier family 7 member 11), decreased the GSH/GSSG ratio, and increased ROS levels. We noted that the increase in ROS is required for increased P-p53, p53, and p21Waf1/cip1 levels following KRAS depletion. Downstream of KRAS, depletion of RalB (RAS-like proto-oncogene B) and IκB kinase-related TANK-binding kinase 1 (TBK1) activated p53 in a ROS- and NRF2-dependent manner. Consistent with this, the IκB kinase inhibitor BAY11-7085 and dominant-negative mutant IκBαM inhibited NF-κB activity and increased P-p53, p53, and p21Waf1/cip1 levels in a ROS-dependent manner. In conclusion, our findings uncover an important role for the NRF2-regulated antioxidant system in KRAS-mediated p53 suppression.

The stress response protein REDD1 promotes diabetes-induced oxidative stress in the retina by Keap1-independent Nrf2 degradation.

The transcription factor nuclear factor erythroid-2-related factor 2 (Nrf2) plays a critical role in reducing oxidative stress by promoting the expression of antioxidant genes. Both individuals with diabetes and preclinical diabetes models exhibit evidence of a defect in retinal Nrf2 activation. We recently demonstrated that increased expression of the stress response protein regulated in development and DNA damage 1 (REDD1) is necessary for the development of oxidative stress in the retina of streptozotocin-induced diabetic mice. In the present study, we tested the hypothesis that REDD1 suppresses the retinal antioxidant response to diabetes by repressing Nrf2 function. We found that REDD1 ablation enhances Nrf2 DNA-binding activity in the retina and that the suppressive effect of diabetes on Nrf2 activity is absent in the retina of REDD1-deficient mice compared with WT. In human MIO-M1 Müller cell cultures, REDD1 deletion prevented oxidative stress in response to hyperglycemic conditions, and this protective effect required Nrf2. REDD1 suppressed Nrf2 stability by promoting its proteasomal degradation independently of Nrf2's interaction with Kelch-like ECH-associated protein 1 (Keap1), but REDD1-mediated Nrf2 degradation required glycogen synthase kinase 3 (GSK3) activity and Ser-351/Ser-356 of Nrf2. Diabetes diminished inhibitory phosphorylation of glycogen synthase kinase 3ß (GSK3ß) at Ser-9 in the retina of WT mice but not in REDD1-deficient mice. Pharmacological inhibition of GSK3 enhanced Nrf2 activity and prevented oxidative stress in the retina of diabetic mice. The findings support a model wherein hyperglycemia-induced REDD1 blunts the Nrf2 antioxidant response to diabetes by activating GSK3, which, in turn, phosphorylates Nrf2 to promote its degradation.

Angiogenin activates the astrocytic Nrf2/antioxidant-response element pathway and thereby protects murine neurons from oxidative stress.

The angiogenin (ANG) gene is mutated frequently in individuals with amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease characterized by the progressive loss of motor neurons. Delivering human ANG to mice that display ALS-like symptoms extends their lifespan and improves motor function. ANG is a secretory vertebrate RNase that enters neuronal cells and cleaves a subset of tRNAs, leading to the inhibition of translation initiation and the assembly of stress granules. Here, using murine neuronal and astrocytic cell lines, we find that ANG triggers the activation of the Nrf2 (nuclear factor erythroid 2-related factor 2) pathway, which provides a critical cellular defense against oxidative stress. This activation, which occurred in astrocytes but not in neurons, promoted the survival of proximal neurons that had oxidative injury. These findings extend the role of ANG as a neuroprotective agent and underscore its potential utility in ALS management.

Differential and overlapping targets of the transcriptional regulators NRF1, NRF2, and NRF3 in human cells.

The nuclear factor (erythroid 2)-like (NRF) transcription factors are a subset of cap'n'collar transcriptional regulators. They consist of three members, NRF1, NRF2, and NRF3, that regulate the expression of genes containing antioxidant-response elements (AREs) in their promoter regions. Although all NRF members regulate ARE-containing genes, each is associated with distinct roles. A comprehensive study of differential and overlapping DNA-binding and transcriptional activities of the NRFs has not yet been conducted. Here, we performed chromatin immunoprecipitation (ChIP)-exo sequencing, an approach that combines ChIP with exonuclease treatment to pinpoint regulatory elements in DNA with high precision, in conjunction with RNA-sequencing to define the transcriptional targets of each NRF member. Our approach, done in three U2OS cell lines, identified 31 genes that were regulated by all three NRF members, 27 that were regulated similarly by all three, and four genes that were differentially regulated by at least one NRF member. We also found genes that were up- or down-regulated by only one NRF member, with 84, 84, and 22 genes that were regulated by NRF1, NRF2, and NRF3, respectively. Analysis of the ARE motifs identified in ChIP peaks revealed that NRF2 prefers binding to AREs flanked by GC-rich regions and that NRF1 prefers AT-rich flanking regions. Thus, sequence preference, likely in combination with upstream signaling events, determines NRF member activation under specific cellular contexts. Our analysis provides a comprehensive description of differential and overlapping gene regulation by the transcriptional regulators NRF1, NRF2, and NRF3.

HDAC5 catalytic activity suppresses cardiomyocyte oxidative stress and NRF2 target gene expression.

Histone deacetylase 5 (HDAC5) and HDAC9 are class IIa HDACs that function as signal-responsive repressors of the epigenetic program for pathological cardiomyocyte hypertrophy. The conserved deacetylase domains of HDAC5 and HDAC9 are not required for inhibition of cardiac hypertrophy. Thus, the biological function of class IIa HDAC catalytic activity in the heart remains unknown. Here we demonstrate that catalytic activity of HDAC5, but not HDAC9, suppresses mitochondrial reactive oxygen species generation and subsequent induction of NF-E2-related factor 2 (NRF2)-dependent antioxidant gene expression in cardiomyocytes. Treatment of cardiomyocytes with TMP195 or TMP269, which are selective class IIa HDAC inhibitors, or shRNA-mediated knockdown of HDAC5 but not HDAC9 leads to stimulation of NRF2-mediated transcription in a reactive oxygen species-dependent manner. Conversely, ectopic expression of catalytically active HDAC5 decreases cardiomyocyte oxidative stress and represses NRF2 activation. These findings establish a role of the catalytic domain of HDAC5 in the control of cardiomyocyte redox homeostasis and define TMP195 and TMP269 as a novel class of NRF2 activators that function by suppressing the enzymatic activity of an epigenetic regulator.

Hyperactivity of the transcription factor Nrf2 causes metabolic reprogramming in mouse esophagus.

Mutations in the genes encoding nuclear factor (erythroid-derived 2)-like 2 (NRF2), Kelch-like ECH-associated protein 1 (KEAP1), and cullin 3 (CUL3) are commonly observed in human esophageal squamous cell carcinoma (ESCC) and result in activation of the NRF2 signaling pathway. Moreover, hyperactivity of the transcription factor Nrf2 has been found to cause esophageal hyperproliferation and hyperkeratosis in mice. However, the underlying mechanism is unclear. In this study, we aimed to understand the molecular mechanisms of esophageal hyperproliferation in mice due to hyperactive Nrf2. Esophageal tissues were obtained from genetically modified mice that differed in the status of the Nrf2 gene and genes in the same pathway (Nrf2-/-, Keap1-/-, K5Cre;Pkm2fl/fl;Keap1-/-, and WT) and analyzed for metabolomic profiles, Nrf2 ChIP-seq, and gene expression. We found that hyperactive Nrf2 causes metabolic reprogramming and up-regulation of metabolic genes in the mouse esophagus. One of the glycolysis genes encoding pyruvate kinase M2 (Pkm2) was not only differentially up-regulated, but also glycosylated and oligomerized, resulting in increased ATP biosynthesis. However, constitutive knockout of Pkm2 failed to inhibit this esophageal phenotype in vivo, and this failure may have been due to compensation by Pkm1 up-regulation. Transient inhibition of NRF2 or glycolysis inhibited the growth of human ESCC cells in which NRF2 is hyperactive in vitro In summary, hyperactive Nrf2 causes metabolic reprogramming in the mouse esophagus through its transcriptional regulation of metabolic genes. Blocking glycolysis transiently inhibits cell proliferation and may therefore have therapeutically beneficial effects on NRF2high ESCC in humans.

Mini-GAGR, an intranasally applied polysaccharide, activates the neuronal Nrf2-mediated antioxidant defense system.

Oxidative stress triggers and exacerbates neurodegeneration in Alzheimer's disease (AD). Various antioxidants reduce oxidative stress, but these agents have little efficacy due to poor blood-brain barrier (BBB) permeability. Additionally, single-modal antioxidants are easily overwhelmed by global oxidative stress. Activating nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2) and its downstream antioxidant system are considered very effective for reducing global oxidative stress. Thus far, only a few BBB-permeable agents activate the Nrf2-dependent antioxidant system. Here, we discovered a BBB-bypassing Nrf2-activating polysaccharide that may attenuate AD pathogenesis. Mini-GAGR, a 0.7-kDa cleavage product of low-acyl gellan gum, increased the levels and activities of Nrf2-dependent antioxidant enzymes, decreased reactive oxygen species (ROS) under oxidative stress in mouse cortical neurons, and robustly protected mitochondria from oxidative insults. Moreover, mini-GAGR increased the nuclear localization and transcriptional activity of Nrf2 similarly to known Nrf2 activators. Mechanistically, mini-GAGR increased the dissociation of Nrf2 from its inhibitor, Kelch-like ECH-associated protein 1 (Keap1), and induced phosphorylation and nuclear translocation of Nrf2 in a protein kinase C (PKC)- and fibroblast growth factor receptor (FGFR1)-dependent manner. Finally, 20-day intranasal treatment of 3xTg-AD mice with 100 nmol of mini-GAGR increased nuclear p-Nrf2 and growth-associated protein 43 (GAP43) levels in hippocampal neurons, reduced p-tau and ß-amyloid (Aß) peptide-stained neurons, and improved memory. The BBB-bypassing Nrf2-activating polysaccharide reported here may be effective in reducing oxidative stress and neurodegeneration in AD.

Kelch-like ECH-associated protein 1 (KEAP1) differentially regulates nuclear factor erythroid-2-related factors 1 and 2 (NRF1 and NRF2).

Nuclear factor erythroid-2-related factor 1 (NRF1) and NRF2 are essential for maintaining redox homeostasis and coordinating cellular stress responses. They are highly homologous transcription factors that regulate the expression of genes bearing antioxidant-response elements (AREs). Genetic ablation of NRF1 or NRF2 results in vastly different phenotypic outcomes, implying that they play different roles and may be differentially regulated. Kelch-like ECH-associated protein 1 (KEAP1) is the main negative regulator of NRF2 and mediates ubiquitylation and degradation of NRF2 through its NRF2-ECH homology-like domain 2 (Neh2). Here, we report that KEAP1 binds to the Neh2-like (Neh2L) domain of NRF1 and stabilizes it. Consistently, NRF1 is more stable in KEAP1+/+ than in KEAP1-/- isogenic cell lines, whereas NRF2 is dramatically stabilized in KEAP1-/- cells. Replacing NRF1's Neh2L domain with NRF2's Neh2 domain renders NRF1 sensitive to KEAP1-mediated degradation, indicating that the amino acids between the DLG and ETGE motifs, not just the motifs themselves, are essential for KEAP1-mediated degradation. Systematic site-directed mutagenesis identified the core amino acid residues required for KEAP1-mediated degradation and further indicated that the DLG and ETGE motifs with correct spacing are insufficient as a KEAP1 degron. Our results offer critical insights into our understanding of the differential regulation of NRF1 and NRF2 by KEAP1 and their different physiological roles.

Hexokinase 2 and nuclear factor erythroid 2-related factor 2 transcriptionally coactivate xanthine oxidoreductase expression in stressed glioma cells.

A dynamic network of metabolic adaptations, inflammatory responses, and redox homeostasis is known to drive tumor progression. A considerable overlap among these processes exists, but several of their key regulators remain unknown. To this end, here we investigated the role of the proinflammatory cytokine IL-1ß in connecting these processes in glioma cells. We found that glucose starvation sensitizes glioma cells to IL-1ß-induced apoptosis in a manner that depended on reactive oxygen species (ROS). Although IL-1ß-induced JNK had no effect on cell viability under glucose deprivation, it mediated nuclear translocation of hexokinase 2 (HK2). This event was accompanied by increases in the levels of sirtuin 6 (SIRT6), nuclear factor erythroid 2-related factor 2 (Nrf2), and xanthine oxidoreductase (XOR). SIRT6 not only induced ROS-mediated cell death but also facilitated nuclear Nrf2-HK2 interaction. Recruitment of the Nrf2-HK2 complex to the ARE site on XOR promoter regulated its expression. Importantly, HK2 served as transcriptional coactivator of Nrf2 to regulate XOR expression, indicated by decreased XOR levels in siRNA-mediated Nrf2 and HK2 knockdown experiments. Our results highlight a non-metabolic role of HK2 as transcriptional coactivator of Nrf2 to regulate XOR expression under conditions of proinflammatory and metabolic stresses. Our insights also underscore the importance of nuclear activities of HK2 in the regulation of genes involved in redox homeostasis.

NRF2 transcriptionally activates the heat shock factor 1 promoter under oxidative stress and affects survival and migration potential of MCF7 cells.

Functional up-regulation of heat shock factor 1 (HSF1) activity through different posttranslational modifications has been implicated in the survival and proliferation of various cancers. It is increasingly recognized that the HSF1 gene is also up-regulated at the transcriptional level, a phenomenon correlated with poor prognosis for patients with different cancers, including breast cancer. Here, we analyzed the transcriptional up-regulation of HSF1 in human cells upon arsenite- or peroxide-induced oxidative stress. Sequential promoter truncation coupled with bioinformatics analysis revealed that this activation is mediated by two antioxidant response elements (AREs) located between 1707 and 1530 bp upstream of the transcription start site of the HSF1 gene. Using shRNA-mediated down-regulation, ChIP of NRF2, site-directed mutagenesis of the AREs, and DNase I footprinting of the HSF1 promoter, we confirmed that nuclear factor erythroid-derived 2-like 2 (NRF2, also known as NFE2L2) interacts with these AREs and up-regulates HSF1 expression. We also found that BRM/SWI2-related gene 1 (BRG1), a catalytic subunit of SWI2/SNF2-like chromatin remodeler, is involved in this process. We further show that NRF2-dependent HSF1 gene regulation plays a crucial role in cancer cell biology, as interference with NRF2-mediated HSF1 activation compromised survival, migration potential, and the epithelial-to-mesenchymal transition and autophagy in MCF7 breast cancer cells exposed to oxidative stress. Taken together, our findings unravel the mechanistic basis of HSF1 gene regulation in cancer cells and provide molecular evidence supporting a direct interaction between HSF1 and NRF2, critical regulators of two cytoprotective mechanisms exploited by cancer cells.

Nuclear factor erythroid 2-related factor 2 enhances carcinogenesis by suppressing apoptosis and promoting autophagy in nickel-transformed cells.

Nickel-containing compounds are widely used in industry. Nickel is a known human carcinogen that primarily affects the lungs. Proposed mechanisms of nickel-induced carcinogenesis include disruption of cellular iron homeostasis, generation of reactive oxygen species (ROS), and induction of hypoxia signaling. However, the precise molecular mechanisms of nickel-induced malignant transformation and tumor development remain unclear. This study shows that the transcription factor Nrf2 is highly expressed in lung tumor tissue and in nickel-transformed human lung bronchial epithelial BEAS-2B cells (NiT cells). Additionally, constitutively high levels of Nrf2 play a critical role in apoptosis resistance in NiT cells. Basal ROS levels were extremely low in NiT cells and were correlated with elevated expression levels of both antioxidant enzymes (e.g. catalase and superoxide dismutases) and antiapoptotic proteins (e.g. Bcl-2 and Bcl-xL). These processes are tightly controlled by Nrf2. Autophagy inhibition, induced pharmacologically or genetically, enhanced Ni2+-induced apoptosis, indicating that the induction of autophagy is the cause of apoptosis resistance in NiT cells. Using similar approaches, we show that in NiT cells the inhibition of apoptosis decreases autophagy. We have shown that Stat3, which is up-regulated by Nrf2, controls autophagy induction in NiT cells. Colony formation and tumor growth were significantly attenuated by knockdown of Nrf2 or Bcl-2. Taken together, this study demonstrates that in NiT cells constitutively high Nrf2 expression inhibits apoptosis by up-regulating antioxidant enzymes and antiapoptotic proteins to increase autophagy via Stat3 signaling. These findings indicate that the Nrf2-mediated suppression of apoptosis and promotion of autophagy contribute to nickel-induced transformation and tumorigenesis.

The nuclear factor-erythroid 2-related factor/heme oxygenase-1 axis is critical for the inflammatory features of type 2 diabetes-associated osteoarthritis.

Epidemiological findings support the hypothesis that type 2 diabetes mellitus (T2DM) is a risk factor for osteoarthritis (OA). Moreover, OA cartilage from patients with T2DM exhibits a greater response to inflammatory stress, but the molecular mechanism is unclear. To investigate whether the antioxidant defense system participates in this response, we examined here the expression of nuclear factor-erythroid 2-related factor (Nrf-2), a master antioxidant transcription factor, and of heme oxygenase-1 (HO-1), one of its main target genes, in OA cartilage from T2DM and non-T2DM patients as well as in murine chondrocytes exposed to high glucose (HG). Ex vivo experiments indicated that Nrf-2 and HO-1 expression is reduced in T2DM versus non-T2DM OA cartilage (0.57-fold Nrf-2 and 0.34-fold HO-1), and prostaglandin E2 (PGE2) release was increased in samples with low HO-1 expression. HG-exposed, IL-1ß-stimulated chondrocytes had lower Nrf-2 levels in vitro, particularly in the nuclear fraction, than chondrocytes exposed to normal glucose (NG). Accordingly, HO-1 levels were also decreased (0.49-fold) in these cells. The HO-1 inducer cobalt protoporphyrin IX more efficiently attenuated PGE2 and IL-6 release in HG+IL-1ß-treated cells than in NG+IL-1ß-treated cells. Greater reductions in HO-1 expression and increase in PGE2/IL-6 production were observed in HG+IL-1ß-stimulated chondrocytes from Nrf-2-/- mice than in chondrocytes from wild-type mice. We conclude that the Nrf-2/HO-1 axis is a critical pathway in the hyperglucidic-mediated dysregulation of chondrocytes. Impairments in this antioxidant system may explain the greater inflammatory responsiveness of OA cartilage from T2DM patients and may inform treatments of such patients.

NADPH Oxidase 4 (Nox4) Suppresses Mitochondrial Biogenesis and Bioenergetics in Lung Fibroblasts via a Nuclear Factor Erythroid-derived 2-like 2 (Nrf2)-dependent Pathway.

Mitochondrial bioenergetics are critical for cellular homeostasis and stress responses. The reactive oxygen species-generating enzyme, NADPH oxidase 4 (Nox4), regulates a number of physiological and pathological processes, including cellular differentiation, host defense, and tissue fibrosis. In this study we explored the role of constitutive Nox4 activity in regulating mitochondrial function. An increase in mitochondrial oxygen consumption and reserve capacity was observed in murine and human lung fibroblasts with genetic deficiency (or silencing) of Nox4. Inhibition of Nox4 expression/activity by genetic or pharmacological approaches resulted in stimulation of mitochondrial biogenesis, as evidenced by elevated mitochondrial-to-nuclear DNA ratio and increased expression of the mitochondrial markers transcription factor A (TFAM), citrate synthase, voltage-dependent anion channel (VDAC), and cytochrome c oxidase subunit 4 (COX IV). Induction of mitochondrial biogenesis was dependent on TFAM up-regulation but was independent of the activation of the peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α). The enhancement of mitochondrial bioenergetics as well as the increase in mitochondrial proteins in Nox4-deficient lung fibroblasts is inhibited by silencing of nuclear factor erythroid-derived 2-like 2 (Nrf2), supporting a key role for Nrf2 in control of mitochondrial biogenesis. Together, these results indicate a critical role for both Nox4 and Nrf2 in counter-regulation of mitochondrial biogenesis and metabolism.

Stress-sensing mechanisms and the physiological roles of the Keap1-Nrf2 system during cellular stress.

Transcription factor Nrf2 (NF-E2-related factor 2) is a master regulator of cellular responses against environmental stresses. Nrf2 induces the expression of detoxification and antioxidant enzymes and suppresses the induction of pro-inflammatory cytokine genes. Keap1 (Kelch-like ECH-associated protein 1) is an adaptor subunit of Cullin 3-based E3 ubiquitin ligase. Keap1 regulates the activity of Nrf2 and acts as a sensor for oxidative and electrophilic stresses. In this review, we discuss the molecular mechanisms by which the Keap1-Nrf2 system senses and regulates the cellular response to environmental stresses. In particular, we focus on the multiple stress-sensing mechanisms of Keap1 and novel regulatory functions of Nrf2.

Sustained O-GlcNAcylation reprograms mitochondrial function to regulate energy metabolism.

Dysfunctional mitochondria and generation of reactive oxygen species (ROS) promote chronic diseases, which have spurred interest in the molecular mechanisms underlying these conditions. Previously, we have demonstrated that disruption of post-translational modification of proteins with ß-linked N-acetylglucosamine (O-GlcNAcylation) via overexpression of the O-GlcNAc-regulating enzymes O-GlcNAc transferase (OGT) or O-GlcNAcase (OGA) impairs mitochondrial function. Here, we report that sustained alterations in O-GlcNAcylation either by pharmacological or genetic manipulation also alter metabolic function. Sustained O-GlcNAc elevation in SH-SY5Y neuroblastoma cells increased OGA expression and reduced cellular respiration and ROS generation. Cells with elevated O-GlcNAc levels had elongated mitochondria and increased mitochondrial membrane potential, and RNA-sequencing analysis indicated transcriptome reprogramming and down-regulation of the NRF2-mediated antioxidant response. Sustained O-GlcNAcylation in mouse brain and liver validated the metabolic phenotypes observed in the cells, and OGT knockdown in the liver elevated ROS levels, impaired respiration, and increased the NRF2 antioxidant response. Moreover, elevated O-GlcNAc levels promoted weight loss and lowered respiration in mice and skewed the mice toward carbohydrate-dependent metabolism as determined by indirect calorimetry. In summary, sustained elevation in O-GlcNAcylation coupled with increased OGA expression reprograms energy metabolism, a finding that has potential implications for the etiology, development, and management of metabolic diseases.

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

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

Glucocorticoid receptor signaling represses the antioxidant response by inhibiting histone acetylation mediated by the transcriptional activator NRF2.

NRF2 (nuclear factor erythroid 2-related factor 2) is a key transcriptional activator that mediates the inducible expression of antioxidant genes. NRF2 is normally ubiquitinated by KEAP1 (Kelch-like ECH-associated protein 1) and subsequently degraded by proteasomes. Inactivation of KEAP1 by oxidative stress or electrophilic chemicals allows NRF2 to activate transcription through binding to antioxidant response elements (AREs) and recruiting histone acetyltransferase CBP (CREB-binding protein). Whereas KEAP1-dependent regulation is a major determinant of NRF2 activity, NRF2-mediated transcriptional activation varies from context to context, suggesting that other intracellular signaling cascades may impact NRF2 function. To identify a signaling pathway that modifies NRF2 activity, we immunoprecipitated endogenous NRF2 and its interacting proteins from mouse liver and identified glucocorticoid receptor (GR) as a novel NRF2-binding partner. We found that glucocorticoids, dexamethasone and betamethasone, antagonize diethyl maleate-induced activation of NRF2 target genes in a GR-dependent manner. Dexamethasone treatment enhanced GR recruitment to AREs without affecting chromatin binding of NRF2, resulting in the inhibition of CBP recruitment and histone acetylation at AREs. This repressive effect was canceled by the addition of histone deacetylase inhibitors. Thus, GR signaling decreases NRF2 transcriptional activation through reducing the NRF2-dependent histone acetylation. Consistent with these observations, GR signaling blocked NRF2-mediated cytoprotection from oxidative stress. This study suggests that an impaired antioxidant response by NRF2 and a resulting decrease in cellular antioxidant capacity account for the side effects of glucocorticoids, providing a novel viewpoint for the pathogenesis of hypercorticosteroidism.

Critical Contribution of Nuclear Factor Erythroid 2-related Factor 2 (NRF2) to Electrophile-induced Interleukin-11 Production.

Nuclear factor erythroid 2-related factor 2 (NRF2) is a transcription factor that plays a crucial role in protection of cells from electrophile-induced toxicity through up-regulating phase II detoxifying enzymes and phase III transporters. We previously reported that oxidative stress induces up-regulation of interleukin-11 (IL-11), a member of the IL-6 family that ameliorates acetaminophen-induced liver toxicity. However, a role for IL-11 in protection of cells from electrophile-induced toxicity remains unclear. Here we show that an environmental electrophile, 1,2-naphthoquinone (1,2-NQ), but not 15d-prostaglandin J2 (PGJ2) or tert-butylhydroxyquinone (tBHQ), induced IL-11 production. Consistent with a crucial role for prolonged ERK activation in H2O2-induced IL-11 production, 1,2-NQ, but not 15d-PGJ2 or tBHQ, elicited prolonged ERK activation. Conversely, inhibition of the ERK pathway by a MEK inhibitor completely blocked 1,2-NQ-induced IL-11 production at both protein and mRNA levels, further substantiating an intimate cross-talk between ERK activation and 1,2-NQ-induced IL-11 production. Promoter analysis of the Il11 gene revealed that two AP-1 sites were essential for 1,2-NQ-induced promoter activities. Among various members of the AP-1 family, Fra-1 was up-regulated by 1,2-NQ, and its up-regulation was blocked by a MEK inhibitor. Although NRF2 was not required for H2O2-induced IL11 up-regulation, NRF2 was essential for 1,2-NQ-induced IL11 up-regulation by increasing Fra-1 proteins possibly through promoting mRNA translation of FOSL1 Finally, intraperitoneal administration of 1,2-NQ induced body weight loss in wild-type mice, which was further exacerbated in Il11ra1-/- mice compared with Il11ra1+/- mice. Together, both Fra-1 and NRF2 play crucial roles in IL-11 production that protects cells from 1,2-NQ intestinal toxicity.