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John D Hayes

Publications and source records attributed to John D Hayes.

At least 19 recordsLinked to original sources
PubMed2006

Reduction in antioxidant defenses may contribute to ochratoxin A toxicity and carcinogenicity.

Ochratoxin A (OTA) is a renal carcinogen in rodents. Its human health significance is unclear. It likely depends upon the mechanism of carcinogenesis. In a previous microarray study a reduction in nuclear factor-erythroid 2 p45-related factor 2 (Nrf2)-dependent gene expression was observed in the kidney but not in the liver of rats fed OTA up to 12 months. Nrf2 regulates detoxification and antioxidant gene expression. The present report shows that OTA decreased the protein expression of several markers of the Nrf2-regulated gene battery in kidney in vivo indicating that the effects observed at mRNA level may be of biological significance. The OTA-mediated Nrf2 response could be reproduced in an NRK renal cell line and in primary hepatocyte cultures. In in vitro systems, an OTA-mediated inhibition of Nrf2 activity was demonstrated by electrophoretic mobility shift and Antioxidant Regulatory Element-driven luciferase reporter assays. The reduction of Nrf2-regulated gene expression resulted in oxidative DNA damage as evidenced by formation of abasic sites in vitro and confirmed in kidney in vivo. All OTA-mediated effects observed were prevented by pretreatment of cell cultures with inducers of Nrf2 activity. Our data suggest that reduction of cellular defense against oxidative stress by Nrf2 inhibition may be a plausible mechanism of OTA nephrotoxicity and carcinogenicity.

Animals↗
PubMed2006

Generation of a stable antioxidant response element-driven reporter gene cell line and its use to show redox-dependent activation of nrf2 by cancer chemotherapeutic agents.

The NF-E2 p45-related factor 2 (Nrf2) regulates cytoprotective genes that contain an antioxidant response element (ARE) in their promoters. To investigate whether anticancer drugs can induce ARE-driven gene expression, we have developed a stable human mammary MCF7-derived reporter cell line called AREc32, which contains a luciferase gene construct controlled by eight copies of the cis-element. In these cells, luciferase activity was increased up to 50-fold following treatment with 50 mumol/L tert-butylhydroquinone (t-BHQ). Basal and inducible luciferase activities in AREc32 cells were increased by forced overexpression of Nrf2 and reduced by knockdown of endogenous Nrf2 expression with RNA interference. Depletion of cellular reduced glutathione (GSH) by treatment of AREc32 cells with l-buthionine-S,R-sulfoximine (BSO) did not influence basal levels of luciferase activity, but pretreatment with BSO augmented induction of luciferase activity by t-BHQ. Induction of reporter activity by t-BHQ in AREc32 cells was suppressed markedly by the antioxidants N-acetylcysteine and GSH but only modestly by vitamins C or E, suggesting that ARE-luciferase expression is induced primarily by thiol-active electrophiles rather than free radicals. The anticancer drugs cisplatin, etoposide, mitoxantrone, chlorambucil, melphalan, and carmustine [1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)] weakly induced luciferase activity in AREc32 cells. Moreover, treatment of AREc32 cells with BSO immediately before exposure to anticancer drugs enhanced induction of ARE-driven luciferase activity by cisplatin, BCNU, chlorambucil, and melphalan and also induced endogenous AKR1C (AKR1C refers to AKR1C1 and AKR1C2), a target gene of Nrf2. Our findings show that Nrf2 can be activated by certain anticancer agents, and this will influence the effectiveness of chemotherapy.

Antineoplastic Agents↗
PubMed2006

Negative regulation of the Nrf1 transcription factor by its N-terminal domain is independent of Keap1: Nrf1, but not Nrf2, is targeted to the endoplasmic reticulum.

Nrf1 (nuclear factor-erythroid 2 p45 subunit-related factor 1) and Nrf2 regulate ARE (antioxidant response element)-driven genes. At its N-terminal end, Nrf1 contains 155 additional amino acids that are absent from Nrf2. This 155-amino-acid polypeptide includes the N-terminal domain (NTD, amino acids 1-124) and a region (amino acids 125-155) that is part of acidic domain 1 (amino acids 125-295). Within acidic domain 1, residues 156-242 share 43% identity with the Neh2 (Nrf2-ECH homology 2) degron of Nrf2 that serves to destabilize this latter transcription factor through an interaction with Keap1 (Kelch-like ECH-associated protein 1). We have examined the function of the 155-amino-acid N-terminal polypeptide in Nrf1, along with its adjacent Neh2-like subdomain. Activation of ARE-driven genes by Nrf1 was negatively controlled by the NTD (N-terminal domain) through its ability to direct Nrf1 to the endoplasmic reticulum. Ectopic expression of wild-type Nrf1 and mutants lacking either the NTD or portions of its Neh2-like subdomain into wild-type and mutant mouse embryonic fibroblasts indicated that Keap1 controls neither the activity of Nrf1 nor its subcellular distribution. Immunocytochemistry showed that whereas Nrf1 gave primarily cytoplasmic staining that was co-incident with that of an endoplasmic-reticulum marker, Nrf2 gave primarily nuclear staining. Attachment of the NTD from Nrf1 to the N-terminus of Nrf2 produced a fusion protein that was redirected from the nucleus to the endoplasmic reticulum. Although this NTD-Nrf2 fusion protein exhibited less transactivation activity than wild-type Nrf2, it was nevertheless still negatively regulated by Keap1. Thus Nrf1 and Nrf2 are targeted to different subcellular compartments and are negatively regulated by distinct mechanisms.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Dimerization of substrate adaptors can facilitate cullin-mediated ubiquitylation of proteins by a "tethering" mechanism: a two-site interaction model for the Nrf2-Keap1 complex.

The prevalence and mechanistic significance of self-association among substrate adaptors for the Cul-Rbx family of ubiquitin ligases remain unclear. We now report that it is as a homodimer that the substrate adaptor Keap1 interacts with Cul3. The resulting complex facilitates ubiquitylation of the Nrf2 transcription factor but only when this substrate possesses within its Neh2 domain a second cryptic Keap1-binding site, the DLG motif, in addition to its previously described ETGE site. Both motifs recognize overlapping surfaces on Keap1, and the seven lysine residues of Nrf2 that act as ubiquitin acceptors lie between them. Based on these data, we propose a "fixed-ends" model for Nrf2 ubiquitylation in which each binding site becomes tethered to a separate subunit of the Keap1 homodimer. This two-site interaction between Keap1 and Nrf2 constrains the mobility of the target lysine residues in the Neh2 domain, increasing their average concentration in the vicinity of the Rbx-bound ubiquitin-conjugating enzyme, and thus the rate at which the transcription factor is ubiquitylated. We show that self-association is a general feature of Cul3 substrate adaptors and propose that the fixed-ends mechanism is commonly utilized to recruit, orientate, and ubiquitylate substrates upon this family of ubiquitin ligases.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Deficiency of glutathione transferase zeta causes oxidative stress and activation of antioxidant response pathways.

Glutathione S-transferase (GST) zeta (GSTZ1-1) plays a significant role in the catabolism of phenylalanine and tyrosine, and a deficiency of GSTZ1-1 results in the accumulation of maleylacetoacetate and its derivatives maleylacetone (MA) and succinylacetone. Induction of GST subunits was detected in the liver of Gstz1(-/-) mice by Western blotting with specific antisera and high-performance liquid chromatography analysis of glutathione affinity column-purified proteins. The greatest induction was observed in members of the mu class. Induction of NAD(P)H:quinone oxidoreductase 1 and the catalytic and modifier subunits of glutamate-cysteine ligase was also observed. Many of the enzymes that are induced in Gstz1(-/-) mice are regulated by antioxidant response elements that respond to oxidative stress via the Keap1/Nrf2 pathway. It is significant that diminished glutathione concentrations were also observed in the liver of Gstz1(-/-) mice, which supports the conclusion that under normal dietary conditions, the accumulation of electrophilic intermediates such as maleylacetoacetate and MA results in a high level of oxidative stress. Elevated GST activities in the livers of Gstz1(-/-) mice suggest that GSTZ1-1 deficiency may alter the metabolism of some drugs and xenobiotics. Gstz1(-/-) mice given acetaminophen demonstrated increased hepatotoxicity compared with wild-type mice. This toxicity may be attributed to the increased GST activity or the decreased hepatic concentrations of glutathione, or both. Patients with acquired deficiency of GSTZ1-1 caused by therapeutic exposure to dichloroacetic acid for the clinical treatment of lactic acidosis may be at increased risk of drug- and chemical-induced toxicity.

Acetaminophen↗
PubMed2005

Utility of siRNA against Keap1 as a strategy to stimulate a cancer chemopreventive phenotype.

A duplex 21 nucleotide small interfering RNA (siRNA) against human Keap1 is described that represents a unique class of cancer chemopreventive agent. This siRNA can knockdown Keap1 mRNA and thereby relieve negative regulation of nuclear factor erythroid 2 p45-related factor 2 (Nrf2)-mediated gene expression. The siRNA lowered endogenous Keap1 mRNA to <30% of control levels between 24 and 72 h after transfection in human HaCaT keratinocyte cells and was capable of blocking ectopic expression of FLAG-tagged human Keap1 protein but not that of ectopic V5-tagged mouse Keap1 protein. Transfection of human HaCaT cells with Keap1 siRNA markedly enhanced endogenous levels of nuclear factor erythroid 2 p45-related factor 2 (Nrf2) protein and increased transcription of an antioxidant response element-driven reporter gene by 2.3-fold. Furthermore, 48 h after transfection of these cells with Keap1 siRNA, expression of aldo-keto reductase 1C1/2 and the glutamate cysteine ligase catalytic and modifier subunits was elevated between 5- and 14-fold. A modest increase of 3-fold in NAD(P)H:quinone oxidoreductase 1 was also observed. The Keap1 siRNA produced a 1.75-fold increase in intracellular glutathione 48 h after transfection. Thus, antagonism of Keap1 by siRNA can be used to preadapt human cells to oxidative stress without the need to expose them to redox stressors.

Alcohol Oxidoreductases↗
PubMed2005

Evolutionary conserved N-terminal domain of Nrf2 is essential for the Keap1-mediated degradation of the protein by proteasome.

Under homeostatic conditions, Nrf2 activity is constitutively repressed. This process is dependent on Keap1, to which Nrf2 binds through the Neh2 domain. Since the N-terminal subdomain of Neh2 (Neh2-NT) contains evolutionarily conserved motifs, we examined the roles they play in the degradation of Nrf2. In Neh2-NT, we defined a novel motif that is distinct from the previously characterized DIDLID motif and designated it DLG motif. Deletion of Neh2-NT or mutation of the DLG motif largely abolished the Keap1-mediated degradation of Nrf2. These mutations were found to enfeeble the binding affinity of Nrf2 to Keap1. The Neh2-NT subdomain directed DLG-dependent, Keap1-independent, degradation of a reporter protein in the nucleus. By contrast, mutation of DLG did not affect the half-life of native Nrf2 protein in the nucleus under oxidative stress conditions. These results thus demonstrate that DLG motif plays essential roles in the Keap1-mediated proteasomal degradation of Nrf2 in the cytoplasm.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Nomenclature for mammalian soluble glutathione transferases.

The nomenclature for human soluble glutathione transferases (GSTs) is extended to include new members of the GST superfamily that have been discovered, sequenced, and shown to be expressed. The GST nomenclature is based on primary structure similarities and the division of GSTs into classes of more closely related sequences. The classes are designated by the names of the Greek letters: Alpha, Mu, Pi, etc., abbreviated in Roman capitals: A, M, P, and so on. (The Greek characters should not be used.) Class members are distinguished by Arabic numerals and the native dimeric protein structures are named according to their subunit composition (e.g., GST A1-2 is the enzyme composed of subunits 1 and 2 in the Alpha class). Soluble GSTs from other mammalian species can be classified in the same manner as the human enzymes, and this chapter presents the application of the nomenclature to the rat and mouse GSTs.

Animals↗
PubMed2005

Glutathione transferases.

This review describes the three mammalian glutathione transferase (GST) families, namely cytosolic, mitochondrial, and microsomal GST, the latter now designated MAPEG. Besides detoxifying electrophilic xenobiotics, such as chemical carcinogens, environmental pollutants, and antitumor agents, these transferases inactivate endogenous alpha,beta-unsaturated aldehydes, quinones, epoxides, and hydroperoxides formed as secondary metabolites during oxidative stress. These enzymes are also intimately involved in the biosynthesis of leukotrienes, prostaglandins, testosterone, and progesterone, as well as the degradation of tyrosine. Among their substrates, GSTs conjugate the signaling molecules 15-deoxy-delta(12,14)-prostaglandin J2 (15d-PGJ2) and 4-hydroxynonenal with glutathione, and consequently they antagonize expression of genes trans-activated by the peroxisome proliferator-activated receptor gamma (PPARgamma) and nuclear factor-erythroid 2 p45-related factor 2 (Nrf2). Through metabolism of 15d-PGJ2, GST may enhance gene expression driven by nuclear factor-kappaB (NF-kappaB). Cytosolic human GST exhibit genetic polymorphisms and this variation can increase susceptibility to carcinogenesis and inflammatory disease. Polymorphisms in human MAPEG are associated with alterations in lung function and increased risk of myocardial infarction and stroke. Targeted disruption of murine genes has demonstrated that cytosolic GST isoenzymes are broadly cytoprotective, whereas MAPEG proteins have proinflammatory activities. Furthermore, knockout of mouse GSTA4 and GSTZ1 leads to overexpression of transferases in the Alpha, Mu, and Pi classes, an observation suggesting they are part of an adaptive mechanism that responds to endogenous chemical cues such as 4-hydroxynonenal and tyrosine degradation products. Consistent with this hypothesis, the promoters of cytosolic GST and MAPEG genes contain antioxidant response elements through which they are transcriptionally activated during exposure to Michael reaction acceptors and oxidative stress.

Animals↗
PubMed2004

Contribution of NAD(P)H:quinone oxidoreductase 1 to protection against carcinogenesis, and regulation of its gene by the Nrf2 basic-region leucine zipper and the arylhydrocarbon receptor basic helix-loop-helix transcription factors.

NAD(P)H:quinone oxidoreductase 1 (NQO1) is a key enzyme involved in defence against reactive forms of oxygen and inhibition of neoplasia. Under conditions of oxidative stress, expression of NQO1 is induced, and the resulting increase in oxidoreductase protein provides the cell with multiple layers of protection against environmental insults. Firstly, the catalytic activity of NQO1 is directed towards the complete reduction and detoxication of highly reactive quinones. Secondly, the oxidoreductase maintains the endogenous lipid-soluble antioxidants, alpha-tocopherol-hydroquinone and ubiquinol in their reduced and active forms. Thirdly, NQO1 is required for the stabilisation of p53 protein in response to DNA-damaging stimuli, and it thereby influences cell fate decisions. In view of the anticarcinogenic actions of NQO1, an understanding of the mechanisms that govern its expression is desirable. The redox sensitivity of NQO1 transcription occurs through a cis-acting antioxidant response element (ARE) located within the regulatory region of the mouse, rat and human genes. This element recruits the positively acting basic leucine zipper (bZip) transcription factor NF-E2 p45-related factor 2 (Nrf2). Under normal constitutive conditions, Nrf2 associates with the cytoskeletal-binding protein Keap1, which regulates the subcellular distribution of the bZip factor and also targets it for proteasome-dependent degradation. Oxidative stress inhibits the Nrf2-Keap1 interaction, thus promoting nuclear accumulation of the transcription factor and transactivation of NQO1 and other ARE-driven genes. Mouse, rat and human NQO1 can also be induced by planar aromatic hydrocarbons through a cis-acting xenobiotic response element (XRE) located in their gene promoters. The XRE recruits the arylhydrocarbon receptor (AhR) and AhR nuclear translocator. Cross-talk may occur between Nrf2 and AhR, but the details of this process remain to be elucidated.

Animals↗
PubMed2004

Redox-regulated turnover of Nrf2 is determined by at least two separate protein domains, the redox-sensitive Neh2 degron and the redox-insensitive Neh6 degron.

The Nrf2 transcription factor is more rapidly turned over in cells grown under homeostatic conditions than in those experiencing oxidative stress. The variable turnover of Nrf2 is accomplished through the use of at least two degrons and its redox-sensitive interaction with the Kelch-repeat protein Keap1. In homeostatic COS1 cells, the Neh2 degron confers on Nrf2 a half-life of less than 10 min. Analyses of deletion mutants of a Gal4(HA)mNeh2 fusion protein and full-length mNrf2 indicate that full redox-sensitive Neh2 destabilizing activity depends upon two separate sequences within this N-terminal domain. The DIDLID element (amino acids 17-32) is indispensable for Neh2 activity and appears necessary to recruit a ubiquitin ligase to the fusion protein. A second motif within Neh2, the ETGE tetrapeptide (amino acids 79-82), allows the redox-sensitive recruitment of Nrf2 to Keap1. This interaction, which occurs only in homeostatic cells, enhances the capacity of the Neh2 degron to direct degradation by functioning downstream of ubiquitination mediated by the DIDLID element. By contrast with the situation under homeostatic conditions, the Neh2 degron is neither necessary nor sufficient to account for the characteristic half-life of Nrf2 in oxidatively stressed cells. Instead, the previously uncharacterized, redox-insensitive Neh6 degron (amino acids 329-379) is essential to ensure that the transcription factor is still appropriately turned over in stressed cells, albeit with an increased half-life of 40 min. A model can now be proposed to explain how the turnover of this protein adapts in response to alterations in cellular redox state.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Activation of hepatic Nrf2 in vivo by acetaminophen in CD-1 mice.

The transcription factor NF-E2-related factor 2 (Nrf2) plays an essential role in the mammalian response to chemical and oxidative stress through induction of hepatic phase II detoxification enzymes and regulation of glutathione (GSH). Enhanced liver damage in Nrf2-deficient mice treated with acetaminophen suggests a critical role for Nrf2; however, direct evidence for Nrf2 activation following acetaminophen exposure was previously lacking. We show that acetaminophen can initiate nuclear translocation of Nrf2 in vivo, with maximum levels reached after 1 hour, in a dose dependent manner, at doses below those causing overt liver damage. Furthermore, Nrf2 was shown to be functionally active, as assessed by the induction of epoxide hydrolase, heme oxygenase-1, and glutamate cysteine ligase gene expression. Increased nuclear Nrf2 was found to be associated with depletion of hepatic GSH. Activation of Nrf2 is considered to involve dissociation from a cytoplasmic inhibitor, Kelch-like ECH-associated protein 1 (Keap1), through a redox-sensitive mechanism involving either GSH depletion or direct chemical interaction through Michael addition. To investigate acetaminophen-induced Nrf2 activation we compared the actions of 2 other GSH depleters, diethyl maleate (DEM) and buthionine sulphoximine (BSO), only 1 of which (DEM) can function as a Michael acceptor. For each compound, greater than 60% depletion of GSH was achieved; however, in the case of BSO, this depletion did not cause nuclear translocation of Nrf2. In conclusion, GSH depletion alone is insufficient for Nrf2 activation: a more direct interaction is required, possibly involving chemical modification of Nrf2 or Keap1, which is facilitated by the prior loss of GSH.

Acetaminophen↗
PubMed2004

Antioxidant and cytoprotective responses to redox stress.

Aerobic cells produce reactive oxygen species as a consequence of normal cellular metabolism, and an array of antioxidant systems are in place to maintain the redox balance. When the redox equilibrium of the cell is upset by pro-oxidant environmental stimuli, adaptive responses to the redox stress take place, which can result in up-regulation of antioxidant proteins and detoxification enzymes. Over the past few years, it has become apparent that members of the CNC (cap 'n' collar)-basic leucine zipper family of transcription factors are principal mediators of defensive responses to redox stress. In mammals, the CNC family members nuclear factor-erythroid 2 p45-related factors 1 and 2 (Nrf1 and Nrf2) have been shown to be involved in the transcriptional up-regulation of cytoprotective genes including those encoding glutamate cysteine ligase, NAD(P)H:quinone oxidoreductase, glutathione S-transferases and aldo-keto reductases. An evolutionarily conserved system exists in Caenorhabditis elegans, and it is possible that Drosophila melanogaster may also utilize CNC transcription factors to induce antioxidant genes in response to pro-oxidant chemicals. The advent of microarray and proteomic technologies has advanced our understanding of the gene batteries regulated by oxidative insult, but has highlighted the complexity of gene regulation by environmental factors. This review focuses on the antioxidant response to environmental stress, and the impact that microarrays and proteomics have made in this field.

Animals↗
PubMed2004

Transcription factor Nrf2 is essential for induction of NAD(P)H:quinone oxidoreductase 1, glutathione S-transferases, and glutamate cysteine ligase by broccoli seeds and isothiocyanates.

Cruciferous vegetables contain glucosinolates that, after conversion to isothiocyanates (ITC), are capable of inducing cytoprotective genes. We examined whether broccoli seeds can elicit a chemoprotective response in mouse organs and rodent cell lines and investigated whether this response requires nuclear factor-erythroid 2 p45-related factor 2 (Nrf2). The seeds studied contained glucosinolate at 40 mmol/kg, of which 59% comprised glucoiberin, 19% sinigrin, 8% glucoraphanin, and 7% progoitrin. Dietary administration of broccoli seeds to nrf2(+/+) and nrf2(-/-) mice produced a approximately 1.5-fold increase in NAD(P)H:quinone oxidoreductase 1 (NQO1) and glutathione S-transferase (GST) activities in stomach, small intestine, and liver of wild-type mice but not in mutant mice; increased transferase activity was associated with elevated levels of GSTA1/2, GSTA3, and GSTM1/2 subunits. These seeds also increased significantly the level of glutamate cysteine ligase catalytic (GCLC) subunit in the stomach and the small intestine of nrf2(+/+) mice but not nrf2(-/-) mice. An aqueous broccoli seed extract was prepared for treatment of cultured cells that contained ITC at approximately 600 mumol/L, composed of 61% 3-methylsulfinylpropyl ITC, 30% sulforaphane, 4% allyl ITC, and 4% 3-butenyl ITC. This extract induced GSTA1/2, GSTA3, NQO1, and GCLC between 3-fold and 10-fold in mouse Hepa-1c1c7 and rat liver RL-34 cells. The broccoli seed extract affected increases in GSTA3, GSTM1, and NQO1 proteins in nrf2(+/+) mouse embryonic fibroblasts but not in nrf2(-/-) mouse embryonic fibroblasts. These experiments show that broccoli seeds are effective at inducing antioxidant and detoxication proteins, both in vivo and ex vivo, in an Nrf2-dependent manner.

Animals↗
PubMed2004

Tissue-specific expression and subcellular distribution of murine glutathione S-transferase class kappa.

Class kappa glutathione S-transferases are a poorly characterized family of detoxication enzymes whose localization has not been defined. In this study we investigated the tissue, cellular, and subcellular distribution of mouse glutathione S-transferase class kappa 1 (mGSTK1) protein using a variety of immunolocalization techniques. Western blotting analysis of mouse tissue homogenates demonstrated that mGSTK1 is expressed at relatively high levels in liver and stomach. Moderate expression was observed in kidney, heart, large intestine, testis, and lung, whereas sparse or essentially no mGSTK1 protein was detected in small intestine, brain, spleen, and skeletal muscle. Immunohistochemical (IHC) analysis for mGSTK1 revealed granular staining of hepatocytes throughout the liver, consistent with organelle staining. IHC analysis of murine kidney localized GSTK1 to the straight portion of the proximal convoluted tubule (pars recta). Staining was consistent with regions rich in mitochondria. Electron microscopy, using indirect immunocolloidal gold staining, clearly showed that mGSTK1 was localized in mitochondria in both mouse liver and kidney. These results are consistent with a role for mGST K1-1 in detoxification, and the confirmation of the intramitochondrial localization of this enzyme implies a unique role for GST class kappa as an antioxidant enzyme.

Animals↗
PubMed2003

Expression of the murine glutathione S-transferase alpha3 (GSTA3) subunit is markedly induced during adipocyte differentiation: activation of the GSTA3 gene promoter by the pro-adipogenic eicosanoid 15-deoxy-Delta12,14-prostaglandin J2.

The expression of class alpha, micro, and pi glutathione S-transferases (GSTs) has been examined during the adipose conversion of mouse 3T3-L1 cells. The GSTA4, GSTM1, and GSTP1/2 subunits are expressed constitutively in confluent 3T3-L1 cells, and their levels remain essentially unchanged during adipocyte differentiation. By contrast, the GSTA3 subunit is virtually undetectable in confluent 3T3-L1 cells under basal conditions, but its expression is markedly induced during adipose conversion. Inhibition of the 3T3-L1 adipogenic program demonstrated that GSTA3 expression is associated specifically with acquisition of the adipocytic phenotype. Reporter gene assays demonstrated that the mouse GSTA3 5(')-upstream region is transcriptionally activated by 15-deoxy-Delta(12,14)-prostaglandin J(2) through an antioxidant response element, suggesting that this pro-adipogenic eicosanoid may be involved in regulating GSTA3 expression during adipogenesis. These data suggest a previously unrecognised role for GSTs in mouse adipocytes.

3T3-L1 Cells↗
PubMed2003

Identification of a novel Nrf2-regulated antioxidant response element (ARE) in the mouse NAD(P)H:quinone oxidoreductase 1 gene: reassessment of the ARE consensus sequence.

NQO1 [NAD(P)H:quinone oxidoreductase 1] has an integral role in cellular responses to oxidative stress. The expression of NQO1 is up-regulated in the mouse following challenge with electrophilic chemicals, in an Nrf2 (NF-E2 p45-related factor 2)-dependent fashion, but the molecular basis for this observation remains unexplained. Through characterization of the murine nqo1 5'-upstream region, we now show that Nrf2 regulates this gene directly via an ARE (antioxidant response element) that lies within a 24 bp region spanning nt -444 to -421. A comprehensive mutation study of this ARE revealed that it does not conform to the currently accepted ARE consensus sequence [(5'-TMAnnRTGAYnnnGCRwwww-3', with essential nucleotides shown in capitals); two cytosine residues (shown in bold in the following sequence) that have been designated 'n' previously because they were thought to be redundant (5'-gagTcA C aGTgAGt C ggCAaaatt-3') have now been found to be essential for enhancer activity; two guanines (also shown in bold) previously regarded as essential for ARE function (5'-gagTcACaGT g AGtCg g CAaaatt-3') have proven to be dispensable]. Examination of wild-type and nrf2 (-/-) mouse embryonic fibroblasts demonstrated that Nrf2 is essential for both constitutive expression of NQO1 and its induction by sulphoraphane. Electrophoretic mobility-shift and chromatin immunoprecipitation assays revealed that Nrf2 associates, in low amounts, with the nqo1 ARE under constitutive conditions, and following sulphoraphane challenge of cells, Nrf2 is recruited to the ARE in substantially greater quantities, as a heterodimer with the small Maf (musculoaponeurotic fibrosarcoma virus) protein, MafK. Also, MafK was found to bind the nqo1 ARE in an Nrf2-independent fashion, and may contribute to transcriptional repression of the oxidoreductase gene. These findings allow a model for transcriptional control of nqo1 through the ARE to be proposed. Furthermore, our results indicate that distinct AREs have differential sequence requirements, and a universally applicable consensus sequence cannot be derived.

5' Flanking Region↗