Role of Nrf2 in the regulation of the Mrp2 (ABCC2) gene

The Nrf2 (nuclear factor-erythroid 2 p45-related factor 2) transcription factor regulates gene expression of the GCLC (glutamate–cysteine ligase catalytic subunit), which is a key enzyme in glutathione synthesis, and GSTs (glutathione S-transferases) via the ARE (antioxidant-response element). The Mrp2 (multidrug-resistance protein 2) pump mediates the excretion of GSH and GSSG excretion as well as endo- and xeno-biotics that are conjugated with GSH, glucuronate or sulphate. Considering that Mrp2 acts synergistically with these enzymes, we hypothesized that the regulation of Mrp2 gene expression is also dependent on Nrf2. Using BHA (butylated hydroxyanisole), which is a classical activator of the ARE–Nrf2 pathway, we observed an increase in the transcriptional activity of Mrp2, GCLC and Gsta1/Gsta2 genes in the mouse liver. A similar pattern of co-induction of Mrp2 and GCLC genes was also observed in mouse (Hepa 1-6) and human (HepG2) hepatoma cells treated with BHA, β-NF (β-naphthoflavone), 2,4,5-T (trichlorophenoxyacetic acid) or 2AAF (2-acetylaminofluorene), suggesting that these genes share common mechanism(s) of transcriptional activation in response to exposure to xenobiotics. To define the mechanism of Mrp2 gene induction, the 5′-flanking region of the mouse Mrp2 gene (2.0 kb) was isolated, and two ARE-like sequences were found: ARE-2 (−1391 to −1381) and ARE-1 (−95 to −85). Deletion analyses demonstrated that the proximal region (−185 to +99) contains the elements for the basal expression and xenobiotic-mediated induction of the Mrp2 gene. Gel-shift and supershift assays indicated that Nrf2–protein complexes bind ARE sequences of the Mrp2 promoter, preferentially to the ARE-1 sequence. Overexpression of Nrf2 increased ARE-1-mediated CAT (chloramphenicol acetyltransferase) gene activity, while overexpression of mutant Nrf2 protein repressed the activity. Thus Nrf2 appears to regulate Mrp2 gene expression via an ARE element located at the proximal region of its promoter in response to exposure to xenobiotics.

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A direct role for C/EBP and the AP-I-binding site in gene expression linked to adipocyte differentiation.

Molecular and Cellular Biology ◽

10.1128/mcb.9.12.5331 ◽

1989 ◽

Vol 9 (12) ◽

pp. 5331-5339 ◽

Cited By ~ 104

Author(s):

R Herrera ◽

H S Ro ◽

G S Robinson ◽

K G Xanthopoulos ◽

B M Spiegelman

Keyword(s):

Gene Expression ◽

Transcriptional Activation ◽

Adipocyte Differentiation ◽

Binding Protein ◽

Point Mutations ◽

Chloramphenicol Acetyltransferase ◽

Lipid Binding ◽

Direct Role ◽

New Genes ◽

Chloramphenicol Acetyltransferase Gene

Adipocyte differentiation is accompanied by the transcriptional activation of many new genes, including the gene encoding adipocyte P2 (aP2), an intracellular lipid-binding protein. Using specific deletions and point mutations, we have shown that at least two distinct sequence elements in the aP2 promoter contribute to the expression of the chloramphenicol acetyltransferase gene in chimeric constructions transfected into adipose cells. An AP-I site at -120, shown earlier to bind Jun- and Fos-like proteins, serves as a positive regulator of chloramphenicol acetyltransferase gene expression in adipocytes but is specifically silenced by adjacent upstream sequences in preadipocytes. Sequences upstream of the AP-I site at -140 (termed AE-1) can function as an enhancer in both cell types when linked to a viral promoter but can stimulate expression only in fat cells in the intact aP2 promoter. The AE-1 sequence binds an adipocyte protein identical or very closely related to an enhancer-binding protein (C/EBP) that has been previously implicated in the regulation of several liver-specific genes. A functional role for C/EBP in the regulation of the aP2 gene is indicated by the facts that C/EBP mRNA is induced during adipocyte differentiation and the aP2 promoter is transactivated by cotransfection of a C/EBP expression vector into preadipose cells. These results indicate that sequences that bind C/EBP and the Fos-Jun complex play major roles in the expression of the aP2 gene during adipocyte differentiation and demonstrate that C/EBP can directly regulate cellular gene expression.

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Cks1 Activates Transcription by Binding to the Ubiquitylated Proteasome

Molecular and Cellular Biology ◽

10.1128/mcb.00655-09 ◽

2010 ◽

Vol 30 (15) ◽

pp. 3894-3901 ◽

Cited By ~ 5

Author(s):

Roman Holic ◽

Alexander Kukalev ◽

Sophie Lane ◽

Edward J. Andress ◽

Ivy Lau ◽

...

Keyword(s):

Gene Expression ◽

Saccharomyces Cerevisiae ◽

Transcriptional Activation ◽

Protein Complexes ◽

Cyclin Dependent Kinase ◽

Transcription Activator ◽

Large Protein ◽

Ubiquitin Binding ◽

Ubiquitin Binding Domain ◽

Proteasome Subunits

ABSTRACT Cyclin-dependent kinase-associated protein 1 (Cks1) is involved in the control of the transcription of a subset of genes in addition to its role in controlling the cell cycle in the budding yeast Saccharomyces cerevisiae. By directly ligating Cks1 onto a GAL1 promoter-driven reporter, we demonstrated that Cks1 acts as a transcription activator. Using this method, we dissected the downstream events from Cks1 recruitment at the promoter. We showed that subsequent to promoter binding, Cdc28 binding is required to modulate the level of gene expression. The ubiquitin-binding domain of Cks1 is essential for implementing downstream transcription events, which appears to recruit the proteasome via ubiquitylated proteasome subunits. We propose that the selective ability of Cks1 to bind ubiquitin allows this small molecule the flexibility to bind large protein complexes with specificity and that this may represent a novel mechanism of regulating transcriptional activation.

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Curcumin Protects Human Keratinocytes against Inorganic Arsenite-Induced Acute Cytotoxicity through an NRF2-Dependent Mechanism

Oxidative Medicine and Cellular Longevity ◽

10.1155/2013/412576 ◽

2013 ◽

Vol 2013 ◽

pp. 1-11 ◽

Cited By ~ 34

Author(s):

Rui Zhao ◽

Bei Yang ◽

Linlin Wang ◽

Peng Xue ◽

Baocheng Deng ◽

...

Keyword(s):

Inorganic Arsenic ◽

Human Keratinocytes ◽

Antioxidant Response ◽

Nuclear Accumulation ◽

Related Factor ◽

High Concentration ◽

Low Concentrations ◽

Acute Cytotoxicity ◽

Nrf2 Protein ◽

Dependent Mechanism

Human exposure to inorganic arsenic leads to various dermal disorders, including hyperkeratosis and skin cancer. Curcumin is demonstrated to induce remarkable antioxidant activity in a variety of cells and tissues. The present study aimed at identifying curcumin as a potent activator of nuclear factor erythroid 2-related factor 2 (NRF2) and demonstrating its protective effect against inorganic arsenite- (iAs3+-) induced cytotoxicity in human keratinocytes. We found that curcumin led to nuclear accumulation of NRF2 protein and increased the expression of antioxidant response element- (ARE-) regulated genes in HaCaT keratinocytes in concentration- and time-dependent manners. High concentration of curcumin (20 μM) also increased protein expression of long isoforms of NRF1. Treatment with low concentrations of curcumin (2.5 or 5 μM) effectively increased the viability and survival of HaCaT cells against iAs3+-induced cytotoxicity as assessed by the MTT assay and flow cytometry and also attenuated iAs3+-induced expression of cleaved caspase-3 and cleaved PARP protein. Selective knockdown ofNRF2orKEAP1by lentiviral shRNAs significantly diminished the cytoprotection conferred by curcumin, suggesting that the protection against iAs3+-induced cytotoxicity is dependent on the activation of NRF2. Our results provided a proof of the concept of using curcumin to activate the NRF2 pathway to alleviate arsenic-induced dermal damage.

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Ezetimibe Prevents Ischemia/Reperfusion-Induced Oxidative Stress and Up-Regulates Nrf2/ARE and UPR Signaling Pathways

Antioxidants ◽

10.3390/antiox9040349 ◽

2020 ◽

Vol 9 (4) ◽

pp. 349 ◽

Cited By ~ 1

Author(s):

Denise Peserico ◽

Chiara Stranieri ◽

Ulisse Garbin ◽

Chiara Mozzini C ◽

Elisa Danese ◽

...

Keyword(s):

Oxidative Stress ◽

Gene Expression ◽

Cultured Cells ◽

Ischemia Reperfusion ◽

Cholesterol Absorption ◽

Antioxidant Response ◽

Related Factor ◽

Cellular Models ◽

Compound C ◽

Cholesterol Absorption Inhibitor

Background: While reperfusion is crucial for survival after an episode of ischemia, it also causes oxidative stress. Nuclear factor-E2-related factor 2 (Nrf2) and unfolded protein response (UPR) are protective against oxidative stress and endoplasmic reticulum (ER) stress. Ezetimibe, a cholesterol absorption inhibitor, has been shown to activate the AMP-activated protein kinase (AMPK)/Nrf2 pathway. In this study we evaluated whether Ezetimibe affects oxidative stress and Nrf2 and UPR gene expression in cellular models of ischemia-reperfusion (IR). Methods: Cultured cells were subjected to simulated IR with or without Ezetimibe. Results: IR significantly increased reactive oxygen species (ROS) production and the percentage of apoptotic cells without the up-regulation of Nrf2, of the related antioxidant response element (ARE) gene expression or of the pro-survival UPR activating transcription factor 6 (ATF6) gene, whereas it significantly increased the pro-apoptotic CCAAT-enhancer-binding protein homologous protein (CHOP). Ezetimibe significantly decreased the cellular ROS formation and apoptosis induced by IR. These effects were paralleled by the up-regulation of Nrf2/ARE and ATF6 gene expression and by a down-regulation of CHOP. We also found that Nrf2 activation was dependent on AMPK, since Compound C, a pan inhibitor of p-AMPK, blunted the activation of Nrf2. Conclusions: Ezetimibe counteracts IR-induced oxidative stress and induces Nrf2 and UPR pathway activation.

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Transcriptional activation of the chicken lysozyme gene by NF-κ Bp65 (RelA) and c-Rel, but not by NF-κ Bp50

Biochemical Journal ◽

10.1042/bj3130039 ◽

1996 ◽

Vol 313 (1) ◽

pp. 39-44 ◽

Cited By ~ 25

Author(s):

Loc VAN PHI

Keyword(s):

Transcription Factors ◽

Transcriptional Activation ◽

Nuclear Factor Kappa B ◽

Protein Complexes ◽

Terminal Differentiation ◽

Binding Activity ◽

Nuclear Factor ◽

Lysozyme Gene ◽

Chloramphenicol Acetyltransferase Gene ◽

Chicken Lysozyme

The lysozyme gene is expressed at a low level in myeloblasts and is progressively activated to constitutively high expression in mature macrophages. The binding activity of the newly defined NF-ĸB/Rel family of transcription factors increases during the terminal differentiation of macrophages. In this study, I show that NF-ĸB/Rel-like proteins bind to the nuclear factor kappa B (ĸB)-like sequence of the lysozyme promoter. These binding activities were induced by treatment of HD11 cells with lipopolysaccharide. Immunomobility shift assays show that c-Rel is possibly a factor in the complexes that bind to the ĸB-like sequence lysĸB. Binding activity to one of the protein complexes seems to be regulated by phosphorylation. In fact, overexpression of p65 and c-Rel stimulates expression of the chloramphenicol acetyltransferase gene controlled by the lysozyme promoter. Furthermore, co-transfection experiments reveal that the ĸB-like sequence within the lysozyme promoter mediates the transactivation by p65 and c-Rel. These results indicate that the p65 and c-Rel could be components of the protein complexes that bind to the ĸB-like sequence and this binding could contribute to the progressively activated expression of the lysozyme gene during the terminal differentiation of macrophages.

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Transcription factor Nrf2 regulates SHP and lipogenic gene expression in hepatic lipid metabolism

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00322.2010 ◽

2010 ◽

Vol 299 (6) ◽

pp. G1211-G1221 ◽

Cited By ~ 107

Author(s):

Jiansheng Huang ◽

Imene Tabbi-Anneni ◽

Viswanath Gunda ◽

Li Wang

Keyword(s):

Gene Expression ◽

Transcriptional Activation ◽

Ketone Bodies ◽

Fatty Acid Content ◽

Liver Weight ◽

Lipid Homeostasis ◽

Related Factor ◽

Lipogenic Gene ◽

Hepatic Lipid ◽

Lipogenic Gene Expression

Nuclear factor erythroid-2 related factor 2 (Nrf2) plays a pivotal role in cytoprotection against both endogenous and exogenous stresses. Here, we establish a novel molecular link between Nrf2, nuclear receptor small heterodimer partner (SHP; NROB2), lipogenic genes, and hepatic lipid homeostasis. Deletion of Nrf2 ( Nrf2−/−) in mice resulted in a reduced liver weight, a decrease in fatty acid content of hepatic triacylglycerol, as well as concomitant increases in the levels of serum VLDL-triglyceride (TG), HDL cholesterol, and ketone bodies at 6 mo of age. Liver weight and hepatic TG content were consistently lower in Nrf2−/− mice upon a high-fat challenge. This phenotype was accompanied by downregulation of genes in lipid synthesis and uptake and upregulation of genes in lipid oxidation in older Nrf2−/− mice. Interestingly, SHP expression was induced with age in Nrf2+/+ mice but decreased by Nrf2 deficiency. Forced expression and activation of Nrf2 by Nrf2 activators consistently induced SHP expression, and Nrf2 was identified as a novel activator of the SHP gene transcription. We also identified PPAR-γ, Fas, Scd1, and Srebp-1 as direct targets of Nrf2 activation. These findings provide evidence for a role of Nrf2 in the modulation of hepatic lipid homeostasis through transcriptional activation of SHP and lipogenic gene expression.

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Drosophila Ada2b Is Required for Viability and Normal Histone H3 Acetylation

Molecular and Cellular Biology ◽

10.1128/mcb.24.18.8080-8089.2004 ◽

2004 ◽

Vol 24 (18) ◽

pp. 8080-8089 ◽

Cited By ~ 37

Author(s):

Dai Qi ◽

Jan Larsson ◽

Mattias Mannervik

Keyword(s):

Gene Expression ◽

Transcriptional Activation ◽

Protein Complexes ◽

P53 Protein ◽

Polytene Chromosomes ◽

Histone H3 ◽

Saga Complex ◽

Histone H3 Acetylation ◽

H3 Acetylation

ABSTRACT Regulation of chromatin through histone acetylation is an important step in gene expression. The Gcn5 histone acetyltransferase is part of protein complexes, e.g., the SAGA complex, that interact with transcriptional activators, targeting the enzyme to specific promoters and assisting in recruitment of the basal RNA polymerase transcription machinery. The Ada2 protein directly binds to Gcn5 and stimulates its catalytic activity. Drosophila contains two Ada2 proteins, Drosophila Ada2a (dAda2a) and dAda2b. We have generated flies that lack dAda2b, which is part of a Drosophila SAGA-like complex. dAda2b is required for viability in Drosophila, and its deletion causes a reduction in histone H3 acetylation. A global hypoacetylation of chromatin was detected on polytene chromosomes in dAda2b mutants. This indicates that the dGcn5-dAda2b complex could have functions in addition to assisting in transcriptional activation through gene-specific acetylation. Although the Drosophila p53 protein was previously shown to interact with the SAGA-like complex in vitro, we find that p53 induction of reaper gene expression occurs normally in dAda2b mutants. Moreover, dAda2b mutant animals show excessive p53-dependent apoptosis in response to gamma radiation. Based on this result, we speculate that dAda2b may be necessary for efficient DNA repair or generation of a DNA damage signal. This could be an evolutionarily conserved function, since a yeast ada2 mutant is also sensitive to a genotoxic agent.

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Gain-of-function genetic screen of the kinome reveals BRSK2 as an inhibitor of the NRF2 transcription factor

Journal of Cell Science ◽

10.1242/jcs.241356 ◽

2020 ◽

Vol 133 (14) ◽

pp. jcs241356 ◽

Cited By ~ 5

Author(s):

Tigist Y. Tamir ◽

Brittany M. Bowman ◽

Megan J. Agajanian ◽

Dennis Goldfarb ◽

Travis P. Schrank ◽

...

Keyword(s):

Transcription Factor ◽

Protein Kinase ◽

Human Cancer ◽

Antioxidant Response ◽

Genetic Screen ◽

Related Factor ◽

Dependent Manner ◽

Gain Of Function ◽

Protein Levels ◽

Nrf2 Protein

ABSTRACTNuclear factor erythroid 2-related factor 2 (NFE2L2, also known as NRF2) is a transcription factor and master regulator of cellular antioxidant response. Aberrantly high NRF2-dependent transcription is recurrent in human cancer, but conversely NRF2 activity diminishes with age and in neurodegenerative and metabolic disorders. Although NRF2-activating drugs are clinically beneficial, NRF2 inhibitors do not yet exist. Here, we describe use of a gain-of-function genetic screen of the kinome to identify new druggable regulators of NRF2 signaling. We found that the under-studied protein kinase brain-specific kinase 2 (BRSK2) and the related BRSK1 kinases suppress NRF2-dependent transcription and NRF2 protein levels in an activity-dependent manner. Integrated phosphoproteomics and RNAseq studies revealed that BRSK2 drives 5′-AMP-activated protein kinase α2 (AMPK) signaling and suppresses the mTOR pathway. As a result, BRSK2 kinase activation suppresses ribosome-RNA complexes, global protein synthesis and NRF2 protein levels. Collectively, our data illuminate the BRSK2 and BRSK1 kinases, in part by functionally connecting them to NRF2 signaling and mTOR. This signaling axis might prove useful for therapeutically targeting NRF2 in human disease.This article has an associated First Person interview with the first author of the paper.

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