Sunday, March 25, 2012

Papers about phytochemical detox mechanisms

1. Mol Nutr Food Res. 2011 Feb;55(2):185-97. doi: 10.1002/mnfr.201000204. Epub 2010
Sep 8.

Coffee, broccoli and spices are strong inducers of electrophile response
element-dependent transcription in vitro and in vivo - Studies in electrophile
response element transgenic mice.

Balstad TR, Carlsen H, Myhrstad MC, Kolberg M, Reiersen H, Gilen L, Ebihara K,
Paur I, Blomhoff R.

Department of Nutrition, Faculty of Medicine, University of Oslo, Oslo, Norway.

SCOPE: Cytoprotective gene products, e.g. phase II - and antioxidant enzymes, are
important in cellular redox homeostasis. A common feature of these genes is
binding sites for transcription factor nuclear factor erythroid-2-related factor
2 (Nrf2), named electrophile response elements (EpREs) within their promoters.
METHODS AND RESULTS: To identify dietary bioactive compounds and foods with
Nrf2/EpRE inducing properties in an intact organism, we utilized transgenic mice
encoding luciferase under control of EpRE from the thioredoxin promoter. We found
that 18 of 31 phytochemicals and 10 of 14 dietary plant extracts induced EpRE
activity in liver HepG2 cells. Surprisingly, some dietary plant extracts showed
profound inducing capability as compared to pure compounds indicating
combinatorial effects of compounds found in whole foods. Furthermore,
intraperitoneal injections of carnosol, curcumin and tert benzohydroquinine
induced EpRE-dependent promoter activity in transgenic mice. In further
experiments with curcumin, we found highly induced EpRE activity in intestine,
liver, kidney and spleen. Finally, a combination extract made of coffee, thyme,
broccoli, rosemary, turmeric and red onion fed orally, induced EpRE mediated
luciferase in lung and adipose tissue.
CONCLUSION: These results show that plant-based foods contain compounds that can
be absorbed and induce the antioxidant defence in a living organism in an
organ-specific manner.

PMID: 20827676 [PubMed - in process]

2. Methods Mol Biol. 2010;647:37-74.

Review of molecular mechanisms involved in the activation of the Nrf2-ARE
signaling pathway by chemopreventive agents.

Giudice A, Arra C, Turco MC.

G. Pascale Foundation National Cancer Institute, Naples, Italy.

Human exposures to environmental toxicants have been associated with etiology of
many diseases including inflammation, cancer, and cardiovascular and
neurodegenerative disorders. To counteract the detrimental effect of
environmental insults, mammalian cells have evolved a hierarchy of sophisticated
sensing and signaling mechanisms to turn on or off endogenous antioxidant
responses accordingly. One of the major cellular antioxidant responses is the
induction of antioxidative and carcinogen-detoxification enzymes through the
cytoplasmic oxidative stress system (Nrf2-Keap1) activated by a variety of
natural and synthetic chemopreventive agents. Under normal conditions, Keap1
anchors the Nrf2 transcription factor within the cytoplasm targeting it for
ubiquitination and proteasomal degradation to maintain low levels of Nrf2 that
mediate the constitutive expression of Nrf2 downstream genes. When cells are
exposed to chemopreventive agents and oxidative stress, a signal involving
phosphorylation and/or redox modification of critical cysteine residues in Keap1
inhibits the enzymatic activity of the Keap1-Cul3-Rbx1 E3 ubiquitin ligase
complex, resulting in decreased Nrf2 ubiquitination and degradation. As a
consequence, free Nrf2 translocates into the nucleus and in combination with
other transcription factors (e.g., sMaf, ATF4, JunD, PMF-1) transactivates the
antioxidant response elements (AREs)/electrophile response elements (EpREs) of
many cytoprotective genes, as well as Nrf2 itself. Upon recovery of cellular
redox homeostasis, Keap1 travels into the nucleus to dissociate Nrf2 from the
ARE. Subsequently, the Nrf2-Keap1 complex is exported out of the nucleus by the
nuclear export sequence (NES) in Keap1. Once in the cytoplasm, the Nrf2-Keap1
complex associates with the Cul3-Rbx1 core ubiquitin machinery, resulting in
degradation of Nrf2 and termination of the Nrf2/ARE signaling pathway. The
discovery of multiple nuclear localization signals (NLSs) and nuclear export
signals (NESs) in Nrf2 also suggests that the nucleocytoplasm translocation of
transcription factors is the consequence of a dynamic equilibrium of multivalent
NLSs and NESs. On the other hand, Keap1 may provide an additional regulation of
the quantity of Nrf2 both in basal and inducible conditions. This chapter
summarizes the current body of knowledge regarding the molecular mechanisms
through which ARE inducers (chemopreventive agents) regulate the coordinated
transcriptional induction of genes encoding phase II and antioxidant enzymes as
well as other defensive proteins, via the nuclear factor-erythroid 2
(NF-E2-p45)-related factor 2(Nrf2)/(ARE) signaling pathway.

PMID: 20694660 [PubMed - indexed for MEDLINE]

3. Arch Pharm Res. 2005 Mar;28(3):249-68.

Induction of phase I, II and III drug metabolism/transport by xenobiotics.

Xu C, Li CY, Kong AN.

Department of Pharmaceutics, Ernest Mario School of Pharmacy, Rutgers, The State
University of New Jersey, Piscataway, NJ 08854, USA.

Drug metabolizing enzymes (DMEs) play central roles in the metabolism,
elimination and detoxification of xenobiotics and drugs introduced into the human
body. Most of the tissues and organs in our body are well equipped with diverse
and various DMEs including phase I, phase II metabolizing enzymes and phase III
transporters, which are present in abundance either at the basal unstimulated
level, and/or are inducible at elevated level after exposure to xenobiotics.
Recently, many important advances have been made in the mechanisms that regulate
the expression of these drug metabolism genes. Various nuclear receptors
including the aryl hydrocarbon receptor (AhR), orphan nuclear receptors, and
nuclear factor-erythoroid 2 p45-related factor 2 (Nrf2) have been shown to be the
key mediators of drug-induced changes in phase I, phase II metabolizing enzymes
as well as phase III transporters involved in efflux mechanisms. For instance,
the expression of CYP1 genes can be induced by AhR, which dimerizes with the AhR
nuclear translocator (Arnt), in response to many polycyclic aromatic hydrocarbon
(PAHs). Similarly, the steroid family of orphan nuclear receptors, the
constitutive androstane receptor (CAR) and pregnane X receptor (PXR), both
heterodimerize with the retinoid X receptor (RXR), are shown to transcriptionally
activate the promoters of CYP2B and CYP3A gene expression by xenobiotics such as
phenobarbital-like compounds (CAR) and dexamethasone and rifampin-type of agents
(PXR). The peroxisome proliferator activated receptor (PPAR), which is one of the
first characterized members of the nuclear hormone receptor, also dimerizes with
RXR and has been shown to be activated by lipid lowering agent fibrate-type of
compounds leading to transcriptional activation of the promoters on CYP4A gene.
CYP7A was recognized as the first target gene of the liver X receptor (LXR), in
which the elimination of cholesterol depends on CYP7A. Farnesoid X receptor (FXR)
was identified as a bile acid receptor, and its activation results in the
inhibition of hepatic acid biosynthesis and increased transport of bile acids
from intestinal lumen to the liver, and CYP7A is one of its target genes. The
transcriptional activation by these receptors upon binding to the promoters
located at the 5-flanking region of these CYP genes generally leads to the
induction of their mRNA gene expression. The physiological and the
pharmacological implications of common partner of RXR for CAR, PXR, PPAR, LXR and
FXR receptors largely remain unknown and are under intense investigations. For
the phase II DMEs, phase II gene inducers such as the phenolic compounds
butylated hydroxyanisol (BHA), tert-butylhydroquinone (tBHQ), green tea
polyphenol (GTP), (-)-epigallocatechin-3-gallate (EGCG) and the isothiocyanates
(PEITC, sulforaphane) generally appear to be electrophiles. They generally
possess electrophilic-mediated stress response, resulting in the activation of
bZIP transcription factors Nrf2 which dimerizes with Mafs and binds to the
antioxidant/electrophile response element (ARE/EpRE) promoter, which is located
in many phase II DMEs as well as many cellular defensive enzymes such as heme
oxygenase-1 (HO-1), with the subsequent induction of the expression of these
genes. Phase III transporters, for example, P-glycoprotein (P-gp), multidrug
resistance-associated proteins (MRPs), and organic anion transporting polypeptide
2 (OATP2) are expressed in many tissues such as the liver, intestine, kidney, and
brain, and play crucial roles in drug absorption, distribution, and excretion.
The orphan nuclear receptors PXR and CAR have been shown to be involved in the
regulation of these transporters. Along with phase I and phase II enzyme
induction, pretreatment with several kinds of inducers has been shown to alter
the expression of phase III transporters, and alter the excretion of xenobiotics,
which implies that phase III transporters may also be similarly regulated in a
coordinated fashion, and provides an important mean to protect the body from
xenobiotics insults. It appears that in general, exposure to phase I, phase II
and phase III gene inducers may trigger cellular "stress" response leading to the
increase in their gene expression, which ultimately enhance the elimination and
clearance of these xenobiotics and/or other "cellular stresses" including harmful
reactive intermediates such as reactive oxygen species (ROS), so that the body
will remove the "stress" expeditiously. Consequently, this homeostatic response
of the body plays a central role in the protection of the body against
"environmental" insults such as those elicited by exposure to xenobiotics.

PMID: 15832810 [PubMed - indexed for MEDLINE]

4. Curr Drug Metab. 2002 Oct;3(5):481-90.

Pharmacogenomics, regulation and signaling pathways of phase I and II drug
metabolizing enzymes.

Rushmore TH, Kong AN.

Department of Drug Metabolism, Merck Research Laboratory, West Point, PA, USA.

Drug or xenobiotics metabolizing enzymes (DMEs or XMEs) play central roles in the
biotransformation, metabolism and/or detoxification of xenobiotics or foreign
compounds, that are introduced to the human body. In general, DMEs protect or
defend the body against the potential harmful insults from the environment. Once
in the body, many xenobiotics may induce signal transduction events either
specifically or non-specifically leading to various cellular, physiological and
pharmacological responses including homeostasis, proliferation, differentiation,
apoptosis, or necrosis. For the body to minimize the insults caused by these
xenobiotics, various tissues/organs are well equipped with diverse DMEs including
various Phase I and Phase II enzymes, which are present in abundance either at
the basal level and/or increased/induced after exposure. To better understand the
pharmacogenomic/gene expression profile of DMEs and the underlying molecular
mechanisms after exposure to xenobiotics or drugs, we will review our current
knowledge on DNA microarray technology in gene expression profiling and the
signal transduction events elicited by various xenobiotics mediated by either
specific receptors or non-specific signal transduction pathways. Pharmacogenomics
is the study of genes and the gene products (proteins) essential for
pharmacological or toxicological responses to pharmaceutical agents. In order to
assess the battery of genes that are induced or repressed by xenobiotics and
pharmaceutical agents, cDNA microarray or oligonucleotide-based DNA chip
technology can be a powerful tool to analyze, simultaneously, the gene expression
profiles that are induced or repressed by xenobiotics. The regulation of gene
expression of the various phase I DMEs such as the cytochrome P450 (CYP) as well
as phase II DMEs generally depends on the interaction of the xenobiotics with the
receptors. For instance, the expression of CYP1 genes can be induced via the aryl
hydrocarbon receptor (AhR) which dimerizes with the AhR nuclear translocator
(ARNT), in response to many polycyclic aromatic hydrocarbon (PAHs). Similarly,
the steroid family of orphan receptors, the constitutive androstane receptor
(CAR) and pregnane X receptors (PXR), heterodimerize with the retinoid X receptor
(RXR), transcriptionally activate the promoters of CYP2B and CYP3A gene
expression by xenobiotics such as phenobarbital-like compounds (CAR) and
dexamethasone and rifampin-type of agents (PXR). The peroxisome proliferator
activated receptor (PPAR) which is one of the first characterized members of the
nuclear hormone receptor, also dimerizes with RXR and it has been shown to be
activated by lipid lowering agent fibrate-type of compounds leading to
transcriptional activation of the promoters on the CYP4A genes. The
transcriptional activation of these promoters generally leads to the induction of
their mRNA. The physiological and the pharmacological implications of common
partner of RXR for CAR, PXR, and PPAR receptors largely remain unknown and are
under intense investigations. For the phase II DMEs, phase II gene inducers such
as phenolic compounds butylated hydroxyanisol (BHA), tert-butylhydroquinone
(tBHQ), green tea polyphenol (GTP), (-)-epicatechin-3-gallate (EGCG) and the
isothiocyanates (PEITC, sulforaphane) generally appear to be electrophiles. They
can activate the mitogen-activated protein kinase (MAPK) pathway via
electrophilic-mediated stress response, resulting in the activation of bZIP
transcription factors Nrf2 which dimerizes with Mafs and binds to the
antioxidant/electrophile response element (ARE/EpRE) enhancers which are found in
many phase II DMEs as well as many cellular defensive enzymes such as
thioredoxins, gammaGCS and HO-1, with the subsequent induction of gene expression
of these genes. It appears that in general, exposure to phase I or phase II gene
inducers or xenobiotics may trigger a cellular "stress" response leading to the
increase in the gene expression of these DMEs, which ultimately enhance the
elimination and clearance of the xenobiotics e xenobiotics and/or the "cellular
stresses" including harmful reactive intermediates such as reactive oxygen
species (ROS), so that the body will remove the "stress" expeditiously.
Consequently, this homeostatic response of the body plays a central role in the
protection of the organism against environmental insults such as xenobiotics.
Advances in DNA microarray technologies and mammalian genome sequencing will soon
allow quantitative assessment of expression profiles of all genes in the selected
tissues. The ability to predict phenotypic outcomes from gene expression profiles
is currently in its infancy, however, and will require additional bioinformatic
tools. Such tools will facilitate information gathering from literature and gene
databases as well as integration of expression data with animal physiology
studies. The study of pharmacogenomic/gene expression profile and the
understanding of the regulation and the signal transduction mechanisms elicited
by pharmaceutical agents can be of potential importance during drug discovery and
the drug development.

PMID: 12369894 [PubMed - indexed for MEDLINE]

5. Planta Med. 2008 Oct;74(13):1526-39. Epub 2008 Oct 20.

Nrf2 as a master redox switch in turning on the cellular signaling involved in
the induction of cytoprotective genes by some chemopreventive phytochemicals.

Surh YJ, Kundu JK, Na HK.

National Research Laboratory of Molecular Carcinogenesis and Chemoprevention,
College of Pharmacy, Seoul National University, Seoul, South Korea.

A wide array of dietary phytochemicals have been reported to induce the
expression of enzymes involved in both cellular antioxidant defenses and
elimination/inactivation of electrophilic carcinogens. Induction of such
cytoprotective enzymes by edible phytochemicals largely accounts for their cancer
chemopreventive and chemoprotective activities. Nuclear
factor-erythroid-2-related factor 2 (Nrf2) plays a crucial role in the
coordinated induction of those genes encoding many stress-responsive and
cytoptotective enzymes and related proteins. These include NAD(P)H:quinone
oxidoreductase-1, heme oxygenase-1, glutamate cysteine ligase, glutathione
S-transferase, glutathione peroxidase, thioredoxin, etc. In resting cells, Nrf2
is sequestered in the cytoplasm as an inactive complex with the repressor
Kelch-like ECH-associated protein 1 (Keap1). The release of Nrf2 from its
repressor is most likely to be achieved by alterations in the structure of Keap1.
Keap1 contains several reactive cysteine residues that function as sensors of
cellular redox changes. Oxidation or covalent modification of some of these
critical cysteine thiols would stabilize Nrf2, thereby facilitating nuclear
accumulation of Nrf2. After translocation into nucleus, Nrf2 forms a heterodimer
with other transcription factors, such as small Maf, which in turn binds to the
5'-upstream CIS-acting regulatory sequence, termed antioxidant response elements
(ARE) or electrophile response elements (EpRE), located in the promoter region of
genes encoding various antioxidant and phase 2 detoxifying enzymes. Certain
dietary chemopreventive agents target Keap1 by oxidizing or chemically modifying
one or more of its specific cysteine thiols, thereby stabilizing Nrf2. In
addition, phosphorylation of specific serine or threonine residues present in
Nrf2 by upstream kinases may also facilitate the nuclear localization of Nrf2.
Multiple mechanisms of Nrf2 activation by signals mediated by one or more of the
upstream kinases, such as mitogen-activated protein kinases,
phosphatidylionositol-3-kinase/Akt, protein kinase C, and casein kinase-2 have
recently been proposed. This review highlights the cytoprotective gene expression
induced by some representative dietary chemopreventive phytochemicals with the
Nrf2-Keap1 system as a prime molecular target.

PMID: 18937164 [PubMed - indexed for MEDLINE]

6. Drug Metab Rev. 2001 Aug-Nov;33(3-4):255-71.

Induction of xenobiotic enzymes by the MAP kinase pathway and the antioxidant or
electrophile response element (ARE/EpRE).

Kong AN, Owuor E, Yu R, Hebbar V, Chen C, Hu R, Mandlekar S.

Center for Pharmaceutical Biotechnology, Department of Pharmaceutics and
Pharmacodynamics, College of Pharmacy, University of Illinois at Chicago, USA.

Cellular responses to xenobiotic-induced stress can signal proliferation,
differentiation, homeostasis, apoptosis, or necrosis. To better understand the
underlying molecular mechanisms after exposure to xenobiotics or drugs, we
studied the signal transduction pathways, the mitogen-activated protein kinase
(MAPK), and the basic leucine zipper transcription factor Nrf2, activated by
different agents in the induction of Phase II drug metabolizing enzymes (DMEs).
The MAPKs, characterized as proline-directed serine/threonine kinases, are
essential components of signaling pathways that convert various extracellular
signals into intracellular responses through serial phosphorylation cascades.
Once activated, MAPKs can phosphorylate many transcription factors, such as
c-Jun, ATF-2, and ultimately lead to changes in gene expression. Two classes of
Phase II gene inducers, which are also cancer chemopreventive agents, were
studied: (1) the phenolic antioxidants, namely butylated hydroxyanisole (BHA) and
its active de-methylated metabolite t-butylhydroquinone (tBHQ), and phenolic
flavonoids such as green tea polyphenols (GTP) and (-)-epigallocatechin-3-gallate
(EGCG); and (2) the naturally occurring isothiocyanates, namely phenethyl
isothiocyanate (PEITC), and sulforaphane. BHA and tBHQ are both well-known
phenolic antioxidants used as food preservatives, and strongly activate c-Jun
N-terminal kinase 1 (JNK1), extracellular signal-regulated protein kinase 2
(ERK2), or p38, in a time- and dose-dependent fashion. Free radical scavengers
N-acetyl-L-cysteine (NAC), or glutathione (GSH), inhibited ERK2 activation and,
to a much lesser extent, JNK1 activation by BHA/tBHQ, implicating the role of
oxidative stress. Under conditions where MAPKs were activated, BHA or GTP also
activated ARE/EpRE (antioxidant/electrophile response element), with the
induction of Phase II genes such as NQO. Transfection studies with various cDNAs
encoding wild-type or dominant-negative mutants of MAPKs and/or transcription
factor Nrf2, substantially modulated ARE-mediated luciferase reporter activity in
the presence or absence of phenolic compounds. Other phytochemicals including
PEITC, and sulforaphane, also differentially regulated the activities of MAPKs,
Nrf2, and ARE-mediated luciferase reporter gene activity and Phase II enzyme
induction. A model is proposed where these xenobiotics (BHA, tBHQ, GTP, EGCG,
PEITC, sulforaphane) activate the MAPK pathway via an electrophilic-mediated
stress response, leading to the transcription activation of Nrf2/Maf heterodimers
on ARE/EpRE enhancers, with the subsequent induction of cellular
defense/detoxifying genes including Phase II DMEs, which may protect the cells
against toxic environmental insults and thereby enhance cell survival. The
studies of these signaling pathways may yield insights into the fate of cells
upon exposure to xenobiotics.

PMID: 11768769 [PubMed - indexed for MEDLINE]

7. Physiol Genomics. 2007 Dec 19;32(1):74-81. Epub 2007 Sep 25.

Genetic dissection of the Nrf2-dependent redox signaling-regulated
transcriptional programs of cell proliferation and cytoprotection.

Reddy NM, Kleeberger SR, Yamamoto M, Kensler TW, Scollick C, Biswal S, Reddy SP.

Department of Environmental Health Sciences, The Johns Hopkins Bloomberg School
of Public Health, Baltimore, MD 21205, USA.

The beta zipper (bZip) transcription factor, nuclear factor erythroid 2, like 2
(Nrf2), acting via an antioxidant/electrophile response element, regulates the
expression of several antioxidant enzymes and maintains cellular redox
homeostasis. Nrf2 deficiency diminishes pulmonary expression of several
antioxidant enzymes, rendering them highly susceptible to various mouse models of
prooxidant-induced lung injury. We recently demonstrated that Nrf2 deficiency
impairs primary cultured pulmonary epithelial cell proliferation and greatly
enhances sensitivity to prooxidant-induced cell death. Glutathione (GSH)
supplementation rescued cells from these defects associated with Nrf2 deficiency.
To further delineate the mechanisms by which Nrf2, via redox signaling, regulates
cellular protection and proliferation, we compared the global expression
profiling of Nrf2-deficient cells with and without GSH supplementation. We found
that GSH regulates the expression of various networks of transcriptional programs
including 1) several antioxidant enzymes involved in cellular detoxification of
reactive oxygen species and recycling of thiol status and 2) several growth
factors, growth factor receptors, and integrins that are critical for cell growth
and proliferation. We also found that Nrf2 deficiency enhances the expression
levels of several genes encoding proinflammatory cytokines; however, GSH
supplementation markedly suppressed their expression. Collectively, these
findings uncover an important insight into the nature of genes regulated by
Nrf2-dependent redox signaling through GSH that are involved in cellular
detoxification and proliferation.

PMID: 17895394 [PubMed - indexed for MEDLINE]

8. Mol Med. 2001 Feb;7(2):135-45.

Role of transcription factor Nrf2 in the induction of hepatic phase 2 and
antioxidative enzymes in vivo by the cancer chemoprotective agent, 3H-1,

Kwak MK, Itoh K, Yamamoto M, Sutter TR, Kensler TW.

Department of Environmental Health Sciences, Johns Hopkins School of Hygiene and
Public Health, Baltimore, Maryland 21205, USA.

BACKGROUND: The induction of phase 2 enzymes by dithiolethiones such as oltipraz
is an effective means for achieving protection against environmental carcinogens
in animals and humans. Transcriptional control of the expression of at least some
of these protective enzymes is mediated through the antioxidant response element
(ARE) found in the upstream regulatory region of many phase 2 genes. The
transcription factor Nrf2, which binds to the ARE, appears to be essential for
the induction of proto-typical phase 2 enzymes such as glutathione S-transferase
(GST) Ya, Yp, and NAD(P)H: quinone reductase (NQO1) in vivo.
MATERIALS AND METHODS: In the present study, 3H-1,2-dithiole-3-thione (D3T) was
used as a potent model inducer whose effects on gene expression and
chemopreventive efficacy have been extensively characterized in the rat. Over a
dozen putative D3T-inducible genes were examined in wild-type and nrf2-disrupted
mice by Northern blot hybridization and reverse transcriptase-polymerase chain
reaction (RT-PCR) analysis to elucidate whether loss of Nrf2 function also
affects the induction of a broader representation of phase 2 and antioxidative
enzymes. The effects of D3T on hepatic Nrf2 expression and localization were also
examined in vivo by Northern blot hybridization, electromobility shift assay, and
Western blot analysis.
RESULTS: Specific activities of hepatic GST and NQO1 were increased by D3T in
wild-type mice and were largely blunted in the nrf2-deficient mice. However,
changes in levels of RNA transcripts following D3T treatment of nrf2-disrupted
mice were multidirectional, dependent upon the particular gene examined. Although
elevation of mRNAs for GST Ya, NQO1, microsomal epoxide hydrolase and
gamma-glutamylcysteine synthetase regulatory chain were blocked in the mutant
mice, elevation of GST Yp mRNA was largely unimpeded. Increases in levels of mRNA
for the heavy and light chains of ferritin were only seen in the nrf2-disrupted
mice. Transcript levels of UDP-glucuronyl-transferase 1A6, heme oxygenase-1,
maganese superoxide dismutase, which were inducible in the wild-type mice,
actually decreased in the mutant mice, whereas levels of mRNA for GST Yc,
aflatoxin B1 aldehyde reductase and catalase decreased following D3T treatment in
the mutant mice in the absence of any inductive effect by D3T in the wild-type
mice. In wild-type mice, treatment with D3T lead to 3-fold increases in hepatic
Nrf2 mRNA levels within several hours following dosing as assessed by Northern
blot and RT-PCR analyses. Gel shift analyses with oligonucleotide probes for
human NQO1 ARE, murine GST Ya ARE, and erythroid transcription factor (NF-E2)
binding site showed increased intensity of binding with nuclear extracts prepared
from livers of D3T-treated mice compared to vehicle-treated controls. Antibody to
Nrf2 supershifted the DNA binding bands of these nuclear extracts. Moreover,
immunoblot analysis indicated accumulation of Nrf2 in extracts prepared from
hepatic nuclei of D3T-treated mice at the same time points.
CONCLUSIONS: Nrf2 plays a central role in the regulation of constitutive and
inducible expression of multiple phase 2 and antioxidative enzymes by
chemoprotective dithiolethiones in vivo, although patterns of response vary among
different genes. Knowledge of the factors controlling the specificity of actions
of enzyme inducers will be exceedingly helpful in the design and isolation of
more efficient and selective chemoprotective agents.

PMCID: PMC1950021
PMID: 11471548 [PubMed - indexed for MEDLINE]

9. Biochem Biophys Res Commun. 1997 Jul 18;236(2):313-22.

An Nrf2/small Maf heterodimer mediates the induction of phase II detoxifying
enzyme genes through antioxidant response elements.

Itoh K, Chiba T, Takahashi S, Ishii T, Igarashi K, Katoh Y, Oyake T, Hayashi N,
Satoh K, Hatayama I, Yamamoto M, Nabeshima Y.

Institute of Basic Medical Sciences and Center for Tsukuba Advanced Research
Alliance, University of Tsukuba, Tennoudai, Japan.

The induction of phase II detoxifying enzymes is an important defense mechanism
against intake of xenobiotics. While this group of enzymes is believed to be
under the transcriptional control of antioxidant response elements (AREs), this
contention is experimentally unconfirmed. Since the ARE resembles the binding
sequence of erythroid transcription factor NF-E2, we investigated the possibility
that the phase II enzyme genes might be regulated by transcription factors that
also bind to the NF-E2 sequence. The expression profiles of a number of
transcription factors suggest that an Nrf2/small Maf heterodimer is the most
likely candidate to fulfill this role in vivo. To directly test these questions,
we disrupted the murine nrf2 gene in vivo. While the expression of phase II
enzymes (e.g., glutathione S-transferase and NAD(P)H: quinone oxidoreductase) was
markedly induced by a phenolic antioxidant in vivo in both wild type and
heterozygous mutant mice, the induction was largely eliminated in the liver and
intestine of homozygous nrf2-mutant mice. Nrf2 was found to bind to the ARE with
high affinity only as a heterodimer with a small Maf protein, suggesting that
Nrf2/small Maf activates gene expression directly through the ARE. These results
demonstrate that Nrf2 is essential for the transcriptional induction of phase II
enzymes and the presence of a coordinate transcriptional regulatory mechanism for
phase II enzyme genes. The nrf2-deficient mice may prove to be a very useful
model for the in vivo analysis of chemical carcinogenesis and resistance to
anti-cancer drugs.

PMID: 9240432 [PubMed - indexed for MEDLINE]

10. Pharm Res. 2010 Jun;27(6):999-1013. Epub 2010 Mar 31.

Nrf2-Keap1 signaling as a potential target for chemoprevention of
inflammation-associated carcinogenesis.

Kundu JK, Surh YJ.

College of Pharmacy, Seoul National University, 599 Kwanak-ro, Kwanak-ku, Seoul
151-742, South Korea.

Persistent inflammatory tissue damage is causally associated with each stage of
carcinogenesis. Inflammation-induced generation of reactive oxygen species,
reactive nitrogen species, and other reactive species not only cause DNA damage
and subsequently mutations, but also stimulate proliferation of initiated cells
and even metastasis and angiogenesis. Induction of cellular cytoprotective
enzymes (e.g., heme oxygenase-1, NAD(P)H:quinone oxidoreductase, superoxide
dismutase, glutathione-S-transferase, etc.) has been shown to mitigate
aforementioned events implicated in inflammation-induced carcinogenesis. A unique
feature of genes encoding these cytoprotective enzymes is the presence of a
cis-acting element, known as antioxidant response element (ARE) or electrophile
response element (EpRE), in their promoter region. A stress-responsive
transcription factor, nuclear factor erythroid-2-related factor-2 (Nrf2),
initially recognized as a key transcriptional regulator of various cytoprotective
enzymes, is known to play a pivotal role in cellular defense against inflammatory
injuries. Activation of Nrf2 involves its release from the cytosolic repressor
Kelch-like ECH-associated protein-1 (Keap1) and subsequent stabilization and
nuclear localization for ARE/EpRE binding. Genetic or pharmacologic inactivation
of Nrf2 has been shown to abolish cytoprotective capability and to aggravate
experimentally induced inflammatory injuries. Thus, Nrf2-mediated cytoprotective
gene induction is an effective strategy for the chemoprevention of
inflammation-associated carcinogenesis.

PMID: 20354764 [PubMed - indexed for MEDLINE]

11. Chem Biol Interact. 2001 Jan 30;130-132(1-3):247-60.

Three different stable human breast adenocarcinoma sublines that overexpress
ALDH3A1 and certain other enzymes, apparently as a consequence of constitutively
upregulated gene transcription mediated by transactivated EpREs (electrophile
responsive elements) present in the 5'-upstream regions of these genes.

Sreerama L, Sládek NE.

Department of Pharmacology, Medical School, Academic Health Center, University of
Minnesota, 6-120 Jackson Hall, 321 Church Street SE, 55455, Minneapolis, MN, USA.

ALDH3A1 catalyzes the detoxification of cyclophosphamide, mafosfamide,
4-hydroperoxycyclophosphamide and other oxazaphosphorines. Constitutive ALDH3A1
levels, as well as those of certain other drug-metabolizing enzymes, e.g. NQO1
and CYP1A1, are relatively low in cultured, relatively
oxazaphosphorine-sensitive, human breast adenocarcinoma MCF-7 cells. However,
transient cellular insensitivity to the oxazaphosphorines can be brought about in
these cells by transiently elevating ALDH3A1 levels in them as a consequence of
transient exposure to: (1) electrophiles such as catechol that induce the
transcription of a battery of genes, e.g. ALDH3A1 and NQO1, having in common an
electrophile responsive element (EpRE) in their 5'-upstream regions; or (2)
Ah-receptor agonists, e.g. indole-3-carbinol and polycyclic aromatic hydrocarbons
such as 3-methylcholanthrene, that induce the transcription of a battery of
genes, e.g. ALDH3A1, NQO1 and CYP1A1, having in common a xenobiotic responsive
element (XRE) in their 5'-upstream regions. Further, MCF-7 sublines that are
constitutively, i.e. when grown in the absence of the original selecting
pressure, relatively oxazaphosphorine-insensitive as a consequence of
constitutively relatively elevated cellular ALDH3A1 levels evolved when MCF-7
cells were: (1) continuously exposed for several months to gradually increasing
concentrations of 4-hydroperoxycyclophosphamide or benz(a)pyrene; or (2) briefly
exposed (once for 30 min) to a high concentration (1 mM) of mafosfamide. Each of
these three stable sublines is constitutively relatively cross-insensitive to
benz(a)pyrene and other polycyclic aromatic hydrocarbons. Cellular levels of
NQO1, but not of CYP1A1, are also constitutively relatively elevated in each of
the three sublines. RT-PCR-based experiments established that ALDH3A1 mRNA levels
are constitutively elevated ( approximately 5- to 8-fold) in each of the three
sublines. The elevated ALDH3A1 mRNA levels are not the consequence of gene
amplification, hypomethylation of a relevant regulatory element, or ALDH3A1 mRNA
stabilization. Collectively, these observations suggest that constitutively
elevated levels of ALDH3A1 and certain other enzymes in the three stable sublines
are probably the consequence of a constitutive change in the cellular
concentration of a key component of the EpRE signaling pathway, such that the
cellular concentration of the relevant ultimate transactivating factor is
constitutively elevated, i.e. gene transcription promoted by transactivated EpREs
is constitutively upregulated. Further, constitutively upregulated gene
transcription mediated by transactivated EpREs can be relatively easily induced,
whereas that mediated by transactivated XREs cannot, at least in MCF-7 cells.
Still further, the three sublines may facilitate study of the signaling pathway
that leads to transactivation of the EpREs present in the 5'-upstream regions of
ALDH3A1, NQO1 and other gene loci.

PMID: 11306049 [PubMed - indexed for MEDLINE]

12. Sheng Li Xue Bao. 2007 Apr 25;59(2):117-27.

Nrf2/ARE regulated antioxidant gene expression in endothelial and smooth muscle
cells in oxidative stress: implications for atherosclerosis and preeclampsia.

Mann GE, Niehueser-Saran J, Watson A, Gao L, Ishii T, de Winter P, Siow RC.

Cardiovascular Division, School of Medicine, King's College London, London, UK.

Increased generation of reactive oxygen species (ROS) in vascular diseases such
as atherosclerosis, diabetes, chronic renal failure and preeclampsia readily
leads to impaired endothelium-dependent relaxation and vascular injury. To
counteract ROS- and electrophile-mediated injury, cells can induce a number of
genes encoding phase II detoxifying enzymes and antioxidant proteins. A
cis-acting transcriptional regulatory element, designated as antioxidant response
element (ARE) or electrophile response element (EpRE), mediates the
transcriptional activation of genes such as heme oxygenase-1,
gamma-glutamylcysteine synthethase, thioredoxin reductase,
glutathione-S-transferase and NAD(P)H:quinone oxidoreductase. Other antioxidant
enzymes such as superoxide dismutase and catalase and non-enzymatic scavengers
such as glutathione are also involved in scavenging ROS. Nuclear factor-erythroid
2-related factor 2 (Nrf2), a member of the Cap nno Collar family of basic
region-leucine zipper (bZIP) transcription factors, plays an important role in
ARE-mediated antioxidant gene expression. Kelch-like ECH-associated protein-1
(Keap1) normally sequesters Nrf2 in the cytoplasm in association with the actin
cytoskeleton, but upon oxidation of cysteine residues Nrf2 dissociates from
Keap1, translocates to the nucleus and binds to ARE sequences leading to
transcriptional activation of antioxidant and phase II detoxifying genes. Protein
kinase C (PKC), mitogen-activated protein kinases (MAPKs) and
phosphotidylinositol 3-kinase (PI3K) have been implicated in the regulation of
Nrf2/ARE signaling. We here review the evidence that the Nrf2/ARE signaling
pathway plays an important role in vascular homeostasis and the defense of
endothelial and smooth muscle cells against sustained oxidative stress associated
with diseases such as atherosclerosis and preeclampsia.

PMID: 17437032 [PubMed - in process]

13. Free Radic Res. 1999 Oct;31(4):319-24.

Regulatory mechanisms of cellular response to oxidative stress.

Itoh K, Ishii T, Wakabayashi N, Yamamoto M.

Center for TARA and Institute for Basic Medical Sciences, University of Tsukuba,

An antioxidant responsive element (ARE) or electrophile responsive element (EpRE)
mediates the transcriptional activation of genes encoding phase II drug
metabolizing enzymes. The ARE consensus sequence shows high similarity to an
erythroid gene regulatory element, and based on the observation, we have recently
found that transcription factor Nrf2 is essential for the coordinate induction of
phase II detoxifying enzymes. The expression of anti-oxidative stress enzyme
genes is also regulated by Nrf2. Detailed analysis of the regulatory mechanisms
of Nrf2 activity has ultimately led us to the identification of a new protein,
which we have named Keap1, that suppresses Nrf2 activity by specific binding to
its evolutionarily-conserved N-terminal Neh2 regulatory domain.

PMID: 10517536 [PubMed - indexed for MEDLINE]

14. Curr Pharm Des. 2004;10(8):879-91.

Induction of cytoprotective genes through Nrf2/antioxidant response element
pathway: a new therapeutic approach for the treatment of inflammatory diseases.

Chen XL, Kunsch C.

Discovery Research, AtheroGenics, Inc., 8995 Westside Parkway, Alpharetta, GA
30004, USA.

In the last decade, it has become recognized that reactive oxygen species (ROS)
play important roles in the multiple biological processes involved in the
pathophysiology of chronic inflammation such as cell proliferation, adhesion
molecule expression, cytokine and chemoattractant production and matrix
metalloproteinase generation. Intracellular redox homeostasis is maintained by
balancing the production of ROS with their removal through cellular antioxidant
defense systems. The antioxidant response element (ARE) is a cis-acting DNA
regulatory element located in the regulatory regions of multiple genes including
phase II detoxification enzymes as well as antioxidant proteins including
glutathione-S-transferases, NAD(P)H:quinone oxidoreductase-1,
gamma-glutamylcysteine synthase, ferritin, and heme oxygenase-1. Nrf2 is the
primary transcription factor that binds to the ARE, and through
heterodimerization with other leucine-zipper containing transcription factors,
activates the expression of these genes. It is evident that activation of
ARE-regulated genes contributes to the regulation of cellular antioxidant defense
systems. More importantly, there is a growing body of evidence suggesting that
modulation of these cytoprotective genes has profound effects on immune and
inflammatory responses. Activation of cytoprotective Nrf2/ARE-regulated genes can
suppress inflammatory responses, whereas decreased expression of these genes
results in autoimmune disease and enhanced inflammatory responses to oxidant
insults. Thus, coordinate induction of cytoprotective genes through Nrf2/ARE
pathway may represent a novel therapeutic approach for the treatment of immune
and inflammatory diseases.

PMID: 15032691 [PubMed - indexed for MEDLINE]

15. Mol Pharmacol. 2006 May;69(5):1662-72. Epub 2006 Feb 14.

Induction of AKR1C2 by phase II inducers: identification of a distal consensus
antioxidant response element regulated by NRF2.

Lou H, Du S, Ji Q, Stolz A.

Division of Gastrointestinal and Liver Diseases, Department of Medicine, Keck
School of Medicine of the University of Southern California, Hoffman Medical
Research Room 101A, 2011 Zonal Ave., Los Angeles, CA 90033, USA.

AKR1C2, also referred to as the human bile acid binder and 3alpha-hydroxysteroid
dehydrogenase type III, is a multifunctional oxidoreductase able to
stereoselectively reduce steroids as well as oxidize or reduce polyaromatic
hydrocarbons. Previously, this same protein was also identified by its robust
induction by phase II inducers in HT29 cells. In HepG2 cells, both AKR1C2 and
AKR1C1 (97% sequence homology) were induced by phase II inducers but not the
highly related AKR1C3 and AKR1C4 family members (84% sequence homology). We now
report the initial characterization of the proximal promoter of AKR1C2 in HepG2
cell line and the identification of a potent enhancer-like element responsive to
phase II inducers located approximately 5.5 kilobases upstream from the
transcription start site. DNA sequence analysis of this enhancer element revealed
that it contained a consensus antioxidant response element (ARE), which was
confirmed by mutation analysis. Treatment with phase II inducers leads to
increased accumulation of nuclear factor-erythroid 2 p45-related factor (NRF) 2
in the nucleus, which was associated with increased binding to this ARE as
determined by electrophoretic mobility shift assay. Transient transfection with
Nrf2 increased the transcriptional activity of the ARE of AKR1C2 comparable with
that observed with phase II inducers. Chromatin immunoprecipitation (ChIP)
analysis also confirmed increased NRF2 binding to the ARE after induction by a
phase II inducer. The AKR1C1 promoter also harbored this same ARE element in a
highly homologous region, which was also bound by NRF2 in a ChiP analysis. No
induction of the ARE of AKR1C2 was detected in Nrf2-/- fibroblasts. The
regulation of AKR1C2 by this distal ARE suggests that AKR1C2 detoxifies products
of reactive oxidant injury, which has important implications for both hormone and
xenobiotic metabolism.

PMID: 16478829 [PubMed - indexed for MEDLINE]

16. J Nutr. 2004 Dec;134(12 Suppl):3499S-3506S.

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.

McWalter GK, Higgins LG, McLellan LI, Henderson CJ, Song L, Thornalley PJ, Itoh
K, Yamamoto M, Hayes JD.

Biomedical Research Centre, Ninewells Hospital and Medical School, University of
Dundee, Dundee DD1 9SY, Scotland, United Kingdom.

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.

PMID: 15570060 [PubMed - indexed for MEDLINE]

17. Biochem Biophys Res Commun. 2001 Nov 23;289(1):212-9.

Functional characterization of transcription regulators that interact with the
electrophile response element.

Zhu M, Fahl WE.

McArdle Laboratory for Cancer Research, University of Wisconsin, Madison,
Wisconsin 53706, USA.

The electrophile response element (EpRE), also referred to as the antioxidant
responsive element (ARE), is found in the 5'-regulatory region of a number of
genes encoding phase II, drug-metabolizing enzymes. Gene knockout studies have
demonstrated the primary regulatory role that an Nrf2:Maf dimer plays by binding
to nucleotides within the EpRE consensus sequence. Current models of
transcription regulation have also shown the involvement of higher-order
transcriptional coactivators, proteins that nucleate around DNA sequence-specific
transcription factors, enhancing transcription of the target gene by interacting
with components of the basal transcriptional apparatus and by enabling chromatin
remodeling. Here, we hypothesized that multiple transcriptional regulators,
including: (i) a primary Nrf2-Maf heterodimer, (ii) a proposed secondary,
EpRE-specific, p160 family coactivator, ARE-binding protein-1, and (iii) a
tertiary coactivator, CBP/p300, nucleate to form a complex at the EpRE that
regulates transcription of the dependent gene. To test this hypothesis, we
constructed a HepG2 cell line which contains a stably integrated green
fluorescent protein (GFP) gene; its inducible expression is regulated by a
synthetic TK promoter containing a linked EpRE. To identify the involvement of
specific, primary and higher-order transcriptional regulators in the
EpRE-mediated regulation of the GFP reporter gene, we microinjected antibodies
directed against specific transcription factors into the HepG2/GFP cells and
determined their effect upon tBHQ-induced expression of the GFP gene. The results
demonstrate that microinjected antibodies directed against Nrf2, MafK, CBP and
p300 could each, individually, significantly inhibit tBHQ-induced GFP expression.
This directly demonstrates the role that the tertiary regulators, CBP or p300,
play in mediating EpRE activation of phase II genes, and also implicates the
involvement of secondary, p160 family coactivators. Moreover, we found that the
same anti-MafK antibody that blocked induction of the EpRE-regulated GFP gene
completely ablated the gel-shift complex that we hypothesize contains an Nrf2:Maf
dimer, ARE-binding protein-1, and CBP or p300.

PMID: 11708801 [PubMed - indexed for MEDLINE]

18. Fish Physiol Biochem. 2010 Sep;36(3):347-53. Epub 2009 Jan 1.

Induction of phase II enzymes and hsp70 genes by copper sulfate through the
electrophile-responsive element (EpRE): insights obtained from a transgenic
zebrafish model carrying an orthologous EpRE sequence of mammalian origin.

Almeida DV, Nornberg BF, Geracitano LA, Barros DM, Monserrat JM, Marins LF.

Instituto de Ciências Biológicas, Universidade Federal do Rio Grande-FURG, Rio
Grande, RS, Brazil.

We have evaluated the homology of the electrophile-responsive element (EpRE) core
sequence, a binding site for the Nrf2 transcription factor, in the proximal
promoters of the mouse and zebrafish glutathione-S-transferase (gst), glutamate
cysteine ligase catalytic subunit (gclc) and heat shock protein 70 (hsp70) genes.
The EpRE sites identified for both species in the three analyzed genes showed a
high similarity with the putative EpRE core sequence. We also produced a
transgenic zebrafish model carrying a transgene comprised of the luciferase (luc)
reporter gene under transcriptional control of a mouse EpRE sequence. This
transgenic model was exposed to copper sulfate, and the reporter gene was
significantly activated. The endogenous gst, gclc and hsp70 zebrafish genes were
analyzed in the EpRE-Luc transgenic zebrafish and showed an expression pattern
similar to that of the reporter transgene used. Our results demonstrate that EpRE
is conserved between mouse and zebrafish for detoxification-related genes and
that the development of genetically modified models using this responsive element
to drive the expression of reporter genes can be an important tool in
understanding the action mechanism of aquatic pollutants.

PMID: 19116768 [PubMed - indexed for MEDLINE]

19. Mutat Res. 2001 Sep 1;480-481:231-41.

Signal transduction events elicited by cancer prevention compounds.

Kong AN, Yu R, Hebbar V, Chen C, Owuor E, Hu R, Ee R, Mandlekar S.

Department of Pharmaceutics and Pharmacodynamics, Center for Pharmaceutical
Biotechnology, MC 870, College of Pharmacy, University of Illinois at Chicago,
900 S. Ashland Ave, Chicago, IL 60607, USA.

Many chemopreventive agents have been shown to modulate gene expression including
induction of phase II detoxifying enzymes, such as glutathione S-transferases
(GST) and quinone reductases (QR). Induction of phase II enzymes in general leads
to protection of cells/tissues against exogenous and/or endogenous carcinogenic
intermediates. The antioxidant or electrophile response element (ARE/EpRE) found
at the 5'-flanking region of these phase II genes may play important role in
mediating their induction by xenobiotics including chemopreventive agents.
Members of the basic leucine zipper (bZIP) transcription factor, Nrf2 which
heterodimerizes with Maf G/K, are found to bind to the ARE, and
transcriptionally-activated ARE. Recently, we showed that the mitogen-activated
protein kinases (MAPK) were activated by phase II gene inducers such as phenolic
antioxidant butylated hydroxyanisol (BHA) and isothiocyanate sulforaphane (SUL),
and involved in the transcription activation of ARE-mediated reporter gene.
Transfection studies with wild-type and dominant negative mutants of Nrf2 and
MAPK showed synergistic response during co-transfection as well as to phase II
gene inducers. However, increasing the concentrations of these compounds such as
BHA, the activities of cell death signaling molecules, caspases, were stimulated
and resulted in apoptotic cell death. At these concentrations, BHA stimulated
loss of mitochondrial membrane potential, cytochrome c release, and activation of
caspase 3, 8 and 9 preceding apoptosis. Further increase in concentrations led to
rapid cell necrosis. A model is proposed for BHA and SUL, in that at low
concentrations, these potential chemopreventive agents may modulate MAPK pathway
leading to transcription activation of Nrf2 and ARE with subsequent induction of
cellular defensive enzymes including phase II detoxifying enzymes as well as
other defensive genes, which may protect the cells against cellular injury, which
is a homeostatic response. At higher concentrations, these agents may activate
the caspase pathways, leading to apoptosis, a potential beneficial effect if
occurs at preneoplastic/neoplastic tissues, but a potential cytotoxic response if
occurs in normal tissues. On the other hand, some phenolic compounds such as
resveratrol inhibits TPA- or UV-induced AP-1-mediated activity through the
inhibition of c-Src non-receptor tyrosine kinase and MAPK pathways. It is
possible that in proliferating or stimulated cells, these chemopreventive
compounds may block proliferation by inhibiting these signaling kinases, whereas
in non-proliferating or quiescent cells, some of these compounds may activate
these signaling kinases leading to gene expression of cellular defensive enzymes
such as phase II detoxifying enzymes. The studies of these and other signaling
pathways may yield insights into the development of potential chemopreventive

PMID: 11506817 [PubMed - indexed for MEDLINE]

20. Antioxid Redox Signal. 2010 Dec 1;13(11):1665-78. Epub 2010 Jul 13.

Discovery of the negative regulator of Nrf2, Keap1: a historical overview.

Itoh K, Mimura J, Yamamoto M.

Department of Stress Response Science, Hirosaki University Graduate School of
Medicine, Japan.

An antioxidant response element (ARE) or an electrophile responsive element
(EpRE) regulate the transcriptional induction of a battery of drug-detoxifying
enzymes that are protective against electrophiles. Based on the high similarity
of the ARE consensus sequence to an erythroid gene regulatory element NF-E2
binding site, we have found that the transcription factor Nrf2 is indispensable
for the ARE-mediated induction of drug-metabolizing enzymes. Recent genome-wide
analysis demonstrated that Nrf2 regulates hundreds of genes that are involved in
the cytoprotective response against oxidative stress. In-depth analysis of Nrf2
regulatory mechanisms has led us to the discovery of a novel protein, which we
have named Keap1. Keap1 suppresses Nrf2 activity by specifically binding to its
evolutionarily conserved N-terminal Neh2 regulatory domain. In this review
article, we summarize the findings and observations that have lead to the
discovery of the Nrf2-Keap1 system. Furthermore, we briefly discuss the function
of the Nrf2-Keap1 system under the regulation of the endogenous electrophilic
compound 15-deoxy-Δ¹²(,)¹⁴-prostaglandin J₂. We propose that Nrf2-Keap1 plays a
significant physiological role in the response to endogenous, environmental, and
pharmacological electrophiles.

PMID: 20446768 [PubMed - in process]

21. Mutat Res. 2010 Aug 7;690(1-2):12-23. Epub 2009 Sep 30.

A protective role of nuclear factor-erythroid 2-related factor-2 (Nrf2) in
inflammatory disorders.

Kim J, Cha YN, Surh YJ.

National Research Laboratory, College of Pharmacy, Graduate School of Convergence
Science and Technology, Seoul National University, Seoul 151-742, Republic of

Nuclear factor-erythroid 2-related factor-2 (Nrf2) is a key transcription factor
that plays a central role in cellular defense against oxidative and electrophilic
insults by timely induction of antioxidative and phase-2 detoxifying enzymes and
related stress-response proteins. The 5'-flanking regions of genes encoding these
cytoprotective proteins contain a specific consensus sequence termed antioxidant
response element (ARE) to which Nrf2 binds. Recent studies have demonstrated that
Nrf2-ARE signaling is also involved in attenuating inflammation-associated
pathogenesis, such as autoimmune diseases, rheumatoid arthritis, asthma,
emphysema, gastritis, colitis and atherosclerosis. Thus, disruption or loss of
Nrf2 signaling causes enhanced susceptibility not only to oxidative and
electrophilic stresses but also to inflammatory tissue injuries. During the
early-phase of inflammation-mediated tissue damage, activation of Nrf2-ARE might
inhibit the production or expression of pro-inflammatory mediators including
cytokines, chemokines, cell adhesion molecules, matrix metalloproteinases,
cyclooxygenase-2 and inducible nitric oxide synthase. It is likely that the
cytoprotective function of genes targeted by Nrf2 may cooperatively regulate the
innate immune response and also repress the induction of pro-inflammatory genes.
This review highlights the protective role of Nrf2 in inflammation-mediated
disorders with special focus on the inflammatory signaling modulated by this
redox-regulated transcription factor.

PMID: 19799917 [PubMed - indexed for MEDLINE]

22. Am J Respir Cell Mol Biol. 2006 Feb;34(2):174-81. Epub 2005 Sep 29.

4-Hydroxynonenal induces rat gamma-glutamyl transpeptidase through
mitogen-activated protein kinase-mediated electrophile response element/nuclear
factor erythroid 2-related factor 2 signaling.

Zhang H, Liu H, Iles KE, Liu RM, Postlethwait EM, Laperche Y, Forman HJ.

Department of Environmental Health Science, School of Public Health, University
of Alabama at Birmingham, USA.

Gamma-glutamyl transpeptidase (GGT) plays critical roles in glutathione
homeostasis and metabolism. Rat GGT is a single-copy gene from which seven types
of GGT mRNA with a common protein encoding sequence, but different
5'-untranslated regions, may be transcribed. We previously showed that type V-2
was the predominant form of GGT mRNA in rat L2 epithelial cells, and that it
could be induced by 4-hydroxynonenal (HNE) through the electrophile response
element (EpRE) located in GGT promoter 5 (GP5). Here, we report transcription
factors binding to GP5 EpRE and the involved signaling pathways. Immunodepletion
gel shift assays demonstrated that GP5 EpRE bound JunB, c-Jun, FosB, and Fra2
from unstimulated cells, and that after exposure to HNE, EpRE binding complexes
contained nuclear factor erythroid 2-related factor (Nrf) 1, Nrf2, JunB, c-Jun,
FosB, c-Fos, Fra1, and Fra2. HNE-induced binding of Nrf2 and c-Jun in GP5 EpRE
was confirmed by chromatin immunoprecipitation assays. Using reporter assays and
specific inhibitors, we found that HNE induction of rat GGT mRNA V-2 was
dependent on activation of extracellular signal-regulated kinase (ERK) and p38
mitogen-activated protein kinase (MAPK), but not protein kinase C or
phosphatidylinositol 3-kinase. Pretreatment with ERK and p38MAPK inhibitors also
blocked HNE-increased EpRE binding. HNE-increased nuclear content of Nrf1, Nrf2,
and c-Jun in L2 cells was partially blocked by inhibition of either ERK1/2 or
p38MAPK and completely blocked by simultaneous inhibition of both MAPKs. In
conclusion, HNE induces GGT mRNA V-2 through altered EpRE transcription factor
binding mediated by both ERK and p38MAPK.

PMCID: PMC2696200
PMID: 16195535 [PubMed - indexed for MEDLINE]

23. J Neurochem. 2006 Sep;98(6):1852-65.

Neuronal sensitivity to kainic acid is dependent on the Nrf2-mediated actions of
the antioxidant response element.

Kraft AD, Lee JM, Johnson DA, Kan YW, Johnson JA.

University of Wisconsin, Madison, WI, USA.

The transcription factor, nuclear factor E2 (erythroid-derived 2)-related factor
2 (Nrf2), is essential for the induction of a battery of phase II detoxification
genes through the antioxidant response element (ARE) that lies in their promoter
region. Genes driven by the ARE are up-regulated in response to various stressors
of the cellular environment. These genetic changes to the cellular reducing
potential may reflect an intrinsic damage response to harmful toxicants. Analysis
of transgenic reporter mice following kainate injection revealed selective ARE
activation within the damaged hippocampus. Further, 2 x 2 microarray analyses
comparing Nrf2 knockout versus wild-type hippocampi unmasked gene changes
associated with ion movement and myelination, in addition to alterations to
detoxification-related genes. Nrf2 knockout mice were more sensitive to kainate
toxicity, as evidenced by elevated seizure severity, seizure duration,
hippocampal neuron damage and mortality. Knockout mice injected with kainate
displayed altered glial fibrillary acidic protein immunoreactivity and increased
microglial infiltration. The wild-type to knockout damage differential was not
dependent on the peripheral metabolism of the excitotoxin, was well correlated
with increased seizure susceptibility, and was therefore not necessarily the
neuroprotective effects of Nrf2. These results combine to support a role for Nrf2
in the neural cell defense response of the adult brain.

PMID: 16945104 [PubMed - indexed for MEDLINE]

24. Expert Rev Mol Med. 2009 Jun 3;11:e17.

The Nrf2-ARE cytoprotective pathway in astrocytes.

Vargas MR, Johnson JA.

Division of Pharmaceutical Sciences, University of Wisconsin, Madison, WI 53705,

The expression of phase-II detoxification and antioxidant enzymes is governed by
a cis-acting regulatory element named the antioxidant response element (ARE).
ARE-containing genes are regulated by the nuclear factor erythroid-2-related
factor 2 (Nrf2), a member of the Cap'n'Collar basic-leucine-zipper family of
transcription factors. ARE-regulated genes are preferentially activated in
astrocytes, which consequently have more efficient detoxification and antioxidant
defences than neurons. Astrocytes closely interact with neurons to provide
structural, metabolic and trophic support, as well as actively participating in
the modulation of neuronal excitability and neurotransmission. Therefore,
functional alterations in astrocytes can shape the interaction with surrounding
cells, such as neurons and microglia. Activation of Nrf2 in astrocytes protects
neurons from a wide array of insults in different in vitro and in vivo paradigms,
confirming the role of astrocytes in determining the vulnerability of neurons to
noxious stimuli. Here, we review the current data supporting Nrf2 activation in
astrocytes as a viable therapeutic approach, not only in acute neuronal damage,
but also in chronic neurodegeneration related to oxidative stress.

PMID: 19490732 [PubMed - indexed for MEDLINE]

25. Proc Natl Acad Sci U S A. 2005 Jan 4;102(1):244-9. Epub 2004 Dec 20.

Protection from mitochondrial complex II inhibition in vitro and in vivo by
Nrf2-mediated transcription.

Calkins MJ, Jakel RJ, Johnson DA, Chan K, Kan YW, Johnson JA.

School of Pharmacy, University of Wisconsin, 777 Highland Avenue, Madison, WI
53705, USA.

Complex II inhibitors 3-nitropropionic acid (3NP) and malonate cause striatal
damage reminiscent of Huntington's disease and have been shown to involve
oxidative stress in their pathogenesis. Because nuclear factor erythroid
2-related factor 2 (Nrf2)-dependent transcriptional activation by means of the
antioxidant response element is known to coordinate the up-regulation of
cytoprotective genes involved in combating oxidative stress, we investigated the
significance of Nrf2 in complex II-induced toxicity. We found that Nrf2-deficient
cells and Nrf2 knockout mice are significantly more vulnerable to malonate and
3NP and demonstrate increased antioxidant response element (ARE)-regulated
transcription mediated by astrocytes. Furthermore, ARE preactivation by means of
intrastriatal transplantation of Nrf2-overexpressing astrocytes before lesioning
conferred dramatic protection against complex II inhibition. These observations
implicate Nrf2 as an essential inducible factor in the protection against complex
II inhibitor-mediated neurotoxicity. These data also introduce Nrf2-mediated ARE
transcription as a potential target of preventative therapy in neurodegenerative
disorders such as Huntington's disease.

PMCID: PMC538748
PMID: 15611470 [PubMed - indexed for MEDLINE]

26. Oncogene. 2003 Mar 27;22(12):1860-5.

Thioredoxin-dependent redox regulation of the antioxidant responsive element
(ARE) in electrophile response.

Kim YC, Yamaguchi Y, Kondo N, Masutani H, Yodoi J.

Department of Biological Responses, Institute for Virus Research, Kyoto
University, Japan.

Thioredoxin is a redox-regulating protein, the expression of which is induced by
various forms of oxidative stress. Thioredoxin controls the interactions of
various transcription factors through redox regulation. In K562 cells, we have
previously reported that hemin induces activation of the thioredoxin gene by
regulating NF-E2-related factor (Nrf2) through the antioxidant responsive element
(ARE). We showed here that tert-butylhydroquinone (tBHQ), an electrophile
stressor, activates the thioredoxin gene through the ARE. In an electrophoretic
mobility shift assay, a specific Nrf2/small Maf binding complex was induced by
tBHQ and bound to the ARE. Overexpression of Nrf2 increased the tBHQ-induced
thioredoxin gene activation through the ARE, whereas that of Jun and Fos
suppressed the activation. The tBHQ-induced ARE binding activity was completely
abrogated by an oxidizing agent, diamide, whereas 2-mercaptoethanol (2-ME)
reversibly recovered the inhibitory effects of diamide, suggesting that ARE
binding activity is redox-dependent. Moreover, overexpression of thioredoxin
enhanced the ARE-mediated thioredoxin gene activation by tBHQ. Therefore,
ARE-mediated induction of thioredoxin expression is a mechanism of enhancing
signal transduction through the ARE in electrophile-induced stress responses.

PMID: 12660821 [PubMed - indexed for MEDLINE]

27. Nucleic Acids Res. 2010 Jan;38(1):48-59. Epub 2009 Oct 28.

Expression of stress-response ATF3 is mediated by Nrf2 in astrocytes.

Kim KH, Jeong JY, Surh YJ, Kim KW.

NeuroVascular Coordination Research Center, College of Pharmacy and Research
Institute of Pharmaceutical Sciences, Seoul National University, Seoul, Korea.

Activating Transcription Factor 3 (ATF3), a member of the ATF/CREB family, is
induced rapidly by various stresses. Its induction mechanism and role in response
to changes in cellular redox status, however, have not been elucidated. Here, we
found that NF-E2-related factor 2 (Nrf2), a transcription factor known to bind to
antioxidant response element (ARE) in promoters, transcriptionally upregulated
ATF3 expression in astrocytes. Treatment with Nrf2 activators and oxidants
provoked ATF3 induction in astrocytes, whereas its expression was reduced in
Nrf2-depleted cells. We further demonstrated that the consensus ARE in the ATF3
promoter is critical for Nrf2-mediation by promoter analyses using an ATF3
promoter-driven luciferase construct and a chromatin immunoprecipitation assay.
In addition, we found that Nrf2-dependent ATF3 induction contributed to the
antioxidative and cytoprotective functions of Nrf2 in astrocytes. Taken together,
our findings suggest that ATF3 is a new target for Nrf2 and has a cytoprotective
function in astrocytes.

PMCID: PMC2800224
PMID: 19864258 [PubMed - indexed for MEDLINE]

28. J Biol Chem. 2010 Aug 20;285(34):26190-8. Epub 2010 May 28.

Nuclear matrix protein (NRP/B) modulates the nuclear factor (Erythroid-derived
2)-related 2 (NRF2)-dependent oxidative stress response.

Seng S, Avraham HK, Birrane G, Jiang S, Avraham S.

Division of Experimental Medicine, Department of Medicine, Beth Israel Deaconess
Medical Center and Harvard Medical School, Boston, Massachusetts 02215, USA.

Reactive molecules have diverse effects on cells and contribute to several
pathological conditions. Cells have evolved complex protective systems to
neutralize these molecules and restore redox homeostasis. Previously, we showed
that association of nuclear factor (NF)-erythroid-derived 2 (E2)-related factor 2
(NRF2) with the nuclear matrix protein NRP/B was essential for the
transcriptional activity of NRF2 target genes in tumor cells. The present study
demonstrates the molecular mechanism by which NRP/B, via NRF2, modulates the
transcriptional activity of antioxidant response element (ARE)-driven genes.
NRP/B is localized in the nucleus of primary brain tissue and human neuroblastoma
(SH-SY5Y) cells. Treatment with hydrogen peroxide (H(2)O(2)) enhances the nuclear
colocalization of NRF2 and NRP/B and induces heme oxygenase 1 (HO1). Treatment of
NRP/B or NRF2 knockdowns with H(2)O(2) induced apoptosis. Co-expression of NRF2
with members of the Kelch protein family, NRP/B, MAYVEN, or MAYVEN-related
protein 2 (MRP2), revealed that the NRF2-NRP/B complex is important for the
transcriptional activity of ARE-driven genes HO1 and NAD(P)H:quinine
oxidoreductase 1 (NQO1). NRP/B interaction with Nrf2 was mapped to NRF2 ECH
homology 4 (Neh4)/Neh5 regions of NRF2. NRP/B mutations that resulted in low
binding affinity to NRF2 were unable to activate NRF2-modulated transcriptional
activity of the ARE-driven genes, HO1 and NQO1. Thus, the interaction of NRP/B
with the Neh4/Neh5 domains of NRF2 is indispensable for activation of
NRF2-mediated ARE-driven antioxidant and detoxifying genes that confer cellular
defense against oxidative stress-induced damage.

PMCID: PMC2924028 [Available on 2011/8/20]
PMID: 20511222 [PubMed - indexed for MEDLINE]

29. Free Radic Biol Med. 2009 Feb 15;46(4):443-53. Epub 2008 Nov 5.

Nrf2-regulated glutathione recycling independent of biosynthesis is critical for
cell survival during oxidative stress.

Harvey CJ, Thimmulappa RK, Singh A, Blake DJ, Ling G, Wakabayashi N, Fujii J,
Myers A, Biswal S.

Department of Environmental Health Science, Bloomberg School of Public Health,
School of Medicine, Johns Hopkins University, Baltimore, MD 21205, USA.

Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) is the primary
transcription factor protecting cells from oxidative stress by regulating
cytoprotective genes, including the antioxidant glutathione (GSH) pathway. GSH
maintains cellular redox status and affects redox signaling, cell proliferation,
and death. GSH homeostasis is regulated by de novo synthesis as well as GSH redox
state; previous studies have demonstrated that Nrf2 regulates GSH homeostasis by
affecting de novo synthesis. We report that Nrf2 modulates the GSH redox state by
regulating glutathione reductase (GSR). In response to oxidants, lungs and
embryonic fibroblasts (MEFs) from Nrf2-deficient (Nrf2(-/-)) mice showed lower
levels of GSR mRNA, protein, and enzyme activity relative to wild type
(Nrf2(+/+)). Nrf2(-/-) MEFs exhibited greater accumulation of glutathione
disulfide and cytotoxicity compared to Nrf2(+/+) MEFs in response to
t-butylhydroquinone, which was rescued by restoring GSR. Microinjection of
glutathione disulfide induced greater apoptosis in Nrf2(-/-) MEFs compared to
Nrf2(+/+) MEFs. In silico promoter analysis of the GSR gene revealed three
putative antioxidant-response elements (ARE1, -44; ARE2, -813; ARE3, -1041).
Reporter analysis, site-directed mutagenesis, and chromatin immunoprecipitation
assays demonstrated binding of Nrf2 to two AREs distal to the transcription start
site. Overall, Nrf2 is critical for maintaining the GSH redox state via
transcriptional regulation of GSR and protecting cells against oxidative stress.

PMCID: PMC2634824
PMID: 19028565 [PubMed - indexed for MEDLINE]

30. Cardiovasc Toxicol. 2008 Summer;8(2):71-85. Epub 2008 May 8.

Nuclear factor E2-related factor 2-dependent myocardiac cytoprotection against
oxidative and electrophilic stress.

Zhu H, Jia Z, Misra BR, Zhang L, Cao Z, Yamamoto M, Trush MA, Misra HP, Li Y.

Division of Biomedical Sciences, Edward Via Virginia College of Osteopathic
Medicine, Virginia Tech Corporate Research Center, Blacksburg, VA, 24060, USA.

Nuclear factor E2-related factor 2 (Nrf2) is a critical regulator of
cytoprotective gene expression. However, the role of this transcription factor in
myocardiac cytoprotection against oxidative and electrophilic stress remains
unknown. This study was undertaken to investigate if Nrf2 signaling could control
the constitutive and inducible expression of antioxidants and phase 2 enzymes in
primary cardiomyocytes as well as the susceptibility of these cells to oxidative
and electrophilic injury. The basal expression of a series of antioxidants and
phase 2 enzymes was significantly lower in cardiomyocytes from Nrf2(-/-) mice
than those from wild-type littermates. Incubation of wild-type cardiomyocytes
with 3H-1,2-dithiole-3-thione (D3T) led to significant induction of various
antioxidants and phase 2 enzymes, including catalase, glutathione, glutathione
peroxidase (GPx), glutathione reductase, glutathione S-transferase,
NAD(P)H:quinone oxidoreductase 1, and heme oxygenase-1. The inducibility of the
above cellular defenses except GPx by D3T was abolished in Nrf2(-/-)
cardiomyocytes. As compared to wild-type cells, Nrf2(-/-) cardiomyocytes were
much more susceptible to cell injury induced by H(2)O(2), peroxynitrite, and
4-hydroxy-2-nonenal. Treatment of wild-type cardiomyocytes with D3T, which
upregulated the cellular defenses, resulted in increased resistance to the above
oxidant- and electrophile-induced cell injury, whereas D3T treatment of Nrf2(-/-)
cardiomyocytes provided no cytoprotection. This study demonstrates that Nrf2 is
an important factor in controlling both constitutive and inducible expression of
a wide spectrum of antioxidants and phase 2 enzymes in cardiomyocytes and is
responsible for protecting these cells against oxidative and electrophilic
stress. These findings also implicate Nrf2 as an important signaling molecule for
myocardiac cytoprotection.

PMID: 18463988 [PubMed - indexed for MEDLINE]

31. Drug Metab Dispos. 2007 May;35(5):787-94. Epub 2007 Feb 15.

Regulation of CYP2A5 gene by the transcription factor nuclear factor
(erythroid-derived 2)-like 2.

Abu-Bakar A, Lämsä V, Arpiainen S, Moore MR, Lang MA, Hakkola J.

National Research Centre for Environmental Toxicology, University of Queensland,
Brisbane, QLD, Australia.

We have previously shown that cadmium, a metal that alters cellular redox status,
induces CYP2A5 expression in nuclear factor (erythroid-derived 2)-like 2
wild-type (Nrf2+/+) mice but not in the knockout (Nrf2-/-) mice. In the present
studies, the potential role of Nrf2 in cadmium-mediated regulation of Cyp2a5 gene
was investigated in mouse primary hepatocytes. Cadmium chloride (CdCl2) caused a
time-dependent induction of the CYP2A5 at mRNA, protein, and activity levels,
with a substantial increase observed within 3 h of exposure. Immunoblotting
showed cadmium-dependent nuclear accumulation of Nrf2 within 1 h of exposure.
Cotransfection of mouse primary hepatocytes with Cyp2a5 promoter-luciferase
reporter plasmids and Nrf2 expression plasmid resulted in a 3-fold activation of
Cyp2a5 promoter-mediated transcription relative to the control. Deletion analysis
of the promoter localized the Nrf2 responsive region to an area from -2656 to
-2339 base pair. Computer-based sequence analysis identified two putative stress
response elements (StRE) within the region at positions -2514 to -2505 and -2386
to -2377. Chromatin immunoprecipitation and electrophoretic mobility shift assays
showed that interaction of the more proximal StRE with Nrf2 was stimulated by
CdCl2. Finally, site-directed mutagenesis of the proximal StRE in Cyp2a5
promoter-luciferase reporter plasmids abolished Nrf2-mediated induction.
Collectively, the results indicate that Nrf2 activates Cyp2a5 transcription by
directly binding to the StRE in the 5'-flanking region of the gene. This
acknowledges Cyp2a5 as the first phase I xenobiotic-metabolizing gene identified
under the control of the StRE-Nrf2 pathway with a potential role in adaptive
response to cellular stress.

PMID: 17303623 [PubMed - indexed for MEDLINE]

32. Toxicol Appl Pharmacol. 1995 Nov;135(1):45-57.

Induction of phase I and phase II drug-metabolizing enzyme mRNA, protein, and
activity by BHA, ethoxyquin, and oltipraz.

Buetler TM, Gallagher EP, Wang C, Stahl DL, Hayes JD, Eaton DL.

Department of Environmental Health, University of Washington, Seattle 98195, USA.

Various natural and synthetic compounds are known to protect against cancer by
elevating phase II detoxification enzymes. Generally classified as
monofunctional, these inducers are believed to trigger cellular signal(s) that
activate gene transcription through an antioxidant or electrophile response
element (ARE/EpRE) in responsive genes. In contrast, the phase I enzymes of drug
metabolism (cytochrome P450s) are not believed to be induced by monofunctional
inducers and P450 genes have not been found to contain functional ARE/EpREs. In
this study, rats were treated with the monofunctional inducers tert-butylated
hydroxyanisole, ethoxyquin, and oltipraz to study the inducibility of individual
glutathione S-transferase isozymes, NADP(H):quinone oxidoreductase,
gamma-glutamylcysteine synthetase, UDP-glucuronosyl transferase, and cytochrome
P450 enzymes. Hepatic mRNAs were analyzed on Northern blots using gene-specific
oligonucleotide probes for GST Ya1, Ya2, Yc1, Yc2, Yb1, Yb2, and Yf, for UGT
1*06, and for P450 1A1, 1A2, 2B1, 2C11, 3A2, and 4A1. NADP(H):quinone
oxidoreductase and gamma-glutamylcysteine synthetase mRNAs were detected using
cDNA probes. All the phase II detoxification enzymes analyzed, except GST Yf,
were induced by the three monofunctional inducers, suggesting that these genes
may be regulated by a mechanism involving an ARE/EpRE element in their promoter
region. Interestingly, it was found that ethoxyquin was a particularly good
inducer for both members of the P450 2B family, 2B1 and 2B2, and both ethoxyquin
and oltipraz were also capable of modestly inducing P450 1A2 and 3A2. Oltipraz
was found to slightly induce P450 2B2, but not 2B1, at the dose and time
analyzed. Induction of mRNA generally correlated well with induction of protein
levels determined by Western blot and/or enzyme activity measurements for
selected enzymes. The results of this study suggest that many phase II enzymes
may contain ARE/EpRE elements in addition to those confirmed to be regulated by a
mechanism involving ARE/EpRE elements. In addition, it was found that several
P450 enzymes were induced by monofunctional inducers, suggesting a possibility
that some phase I enzymes may also be regulated by a mechanism involving ARE/EpRE

PMID: 7482539 [PubMed - indexed for MEDLINE]

33. Proc Natl Acad Sci U S A. 2004 Feb 17;101(7):2040-5. Epub 2004 Feb 5.

Protection against electrophile and oxidant stress by induction of the phase 2
response: fate of cysteines of the Keap1 sensor modified by inducers.

Wakabayashi N, Dinkova-Kostova AT, Holtzclaw WD, Kang MI, Kobayashi A, Yamamoto
M, Kensler TW, Talalay P.

The Lewis B. and Dorothy Cullman Cancer Chemoprotection Center, Department of
Pharmacology and Molecular Sciences, School of Medicine, The Johns Hopkins
University, Baltimore, MD 21205, USA.

Induction of a family of phase 2 genes encoding for proteins that protect against
the damage of electrophiles and reactive oxygen intermediates is potentially a
major strategy for reducing the risk of cancer and chronic degenerative diseases.
Many phase 2 genes are regulated by upstream antioxidant response elements (ARE)
that are targets of the leucine zipper transcription factor Nrf2. Under basal
conditions, Nrf2 resides mainly in the cytoplasm bound to its cysteine-rich,
Kelch domain-containing partner Keap1, which is itself anchored to the actin
cytoskeleton and represses Nrf2 activity. Inducers disrupt the Keap1-Nrf2 complex
by modifying two (C273 and C288) of the 25 cysteine residues of Keap1. The
critical role of C273 and C288 was established by (i) their high reactivity when
purified recombinant Keap1 was treated with dexamethasone mesylate and the
dexamethasone-modified tryptic peptides were analyzed by mass spectrometry, and
(ii) transfection of keap1 and nrf2 gene-deficient mouse embryonic fibroblasts
with constructs expressing cysteine to alanine mutants of Keap1, and measurement
of the ability of cotransfected Nrf2 to repress an ARE-luciferase reporter.
Reaction of Keap1 with inducers results in formation of intermolecular disulfide
bridges, probably between C273 of one Keap1 molecule and C288 of a second.
Evidence for formation of such dimers was obtained by 2D PAGE of extracts of
cells treated with inducers, and by the demonstration that whereas C273A and
C288A mutants of Keap1 alone could not repress Nrf2 activation of the
ARE-luciferase reporter, an equal mixture of these mutant constructs restored
repressor activity.

PMCID: PMC357048
PMID: 14764894 [PubMed - indexed for MEDLINE]

34. Antioxid Redox Signal. 2011 Feb 1;14(3):469-87. Epub 2010 Aug 20.

Impaired redox signaling and antioxidant gene expression in endothelial cells in
diabetes: a role for mitochondria and the nuclear factor-E2-related factor
2-Kelch-like ECH-associated protein 1 defense pathway.

Cheng X, Siow RC, Mann GE.

Cardiovascular Division, School of Medicine, King's College London, London,
United Kingdom.

Type 2 diabetes is an age-related disease associated with vascular pathologies,
including severe blindness, renal failure, atherosclerosis, and stroke. Reactive
oxygen species (ROS), especially mitochondrial ROS, play a key role in regulating
the cellular redox status, and an overproduction of ROS may in part underlie the
pathogenesis of diabetes and other age-related diseases. Cells have evolved
endogenous defense mechanisms against sustained oxidative stress such as the
redox-sensitive transcription factor nuclear factor E2-related factor 2 (Nrf2),
which regulates antioxidant response element (ARE/electrophile response
element)-mediated expression of detoxifying and antioxidant enzymes and the
cystine/glutamate transporter involved in glutathione biosynthesis. We
hypothesize that diminished Nrf2/ARE activity contributes to increased oxidative
stress and mitochondrial dysfunction in the vasculature leading to endothelial
dysfunction, insulin resistance, and abnormal angiogenesis observed in diabetes.
Sustained hyperglycemia further exacerbates redox dysregulation, thereby
providing a positive feedback loop for severe diabetic complications. This review
focuses on the role that Nrf2/ARE-linked gene expression plays in regulating
endothelial redox homeostasis in health and type 2 diabetes, highlighting recent
evidence that Nrf2 may provide a therapeutic target for countering oxidative
stress associated with vascular disease and aging.

PMID: 20524845 [PubMed - in process]

35. Mol Cell Biol. 2002 May;22(9):2883-92.

Enhanced expression of the transcription factor Nrf2 by cancer chemopreventive
agents: role of antioxidant response element-like sequences in the nrf2 promoter.

Kwak MK, Itoh K, Yamamoto M, Kensler TW.

Department of Environmental Health Sciences, Johns Hopkins Bloomberg School of
Public Health, Baltimore, Maryland 21205, USA.

Induction of phase 2 enzymes, which neutralize reactive electrophiles and act as
indirect antioxidants, is an important mechanism for protection against
carcinogenesis. The transcription factor Nrf2, which binds to the antioxidant
response element (ARE) found in the upstream regulatory region of many phase 2
genes, is essential for the induction of these enzymes. We have investigated the
effect of the potent enzyme inducer and anticarcinogen 3H-1,2-dithiole-3-thione
(D3T) on the fate of Nrf2 in murine keratinocytes. Both total and nuclear Nrf2
levels increased rapidly and persistently after treatment with D3T but could be
blocked by cotreatment with cycloheximide. Nrf2 mRNA levels increased
approximately 2-fold 6 h after D3T treatment. To examine the transcriptional
activation of Nrf2 by D3T, the proximal region (1 kb) of the nrf2 promoter was
isolated. Deletion and mutagenesis analyses demonstrated that nrf2
promoter-luciferase reporter activity was enhanced by treatment with D3T and that
ARE-like sequences were required for this activation. Gel shift assays with
nuclear extracts from PE cells indicated that common factors bind to typical AREs
and the ARE-like sequences of the nrf2 promoter. Direct binding of Nrf2 to its
own promoter was demonstrated by chromatin immunoprecipitation assay.
Overexpression of Nrf2 increased the activity of the nrf2 promoter-luciferase
reporter, while expression of mutant Nrf2 protein repressed activity. Thus, Nrf2
appears to autoregulate its own expression through an ARE-like element located in
the proximal region of its promoter, leading to persistent nuclear accumulation
of Nrf2 and protracted induction of phase 2 genes in response to chemopreventive

PMCID: PMC133753
PMID: 11940647 [PubMed - indexed for MEDLINE]

36. J Neurosci. 2004 Feb 4;24(5):1101-12.

Nuclear factor E2-related factor 2-dependent antioxidant response element
activation by tert-butylhydroquinone and sulforaphane occurring preferentially in
astrocytes conditions neurons against oxidative insult.

Kraft AD, Johnson DA, Johnson JA.

School of Pharmacy, University of Wisconsin, Madison, Wisconsin 53705-2222, USA.

Binding of the transcription factor nuclear factor E2-related factor 2 (Nrf2) to
the antioxidant response element (ARE) in neural cells results in the induction
of a battery of genes that can coordinate a protective response against a variety
of oxidative stressors. In this study, tert-butylhydroquinone (tBHQ) and
sulforaphane were used as activators of this pathway. Consistent with previous
studies, treatment of primary cortical cultures from ARE reporter mice revealed
selective promoter activity in astrocytes. This activation protected neurons from
hydrogen peroxide and nonexcitotoxic glutamate toxicity. tBHQ treatment of
cultures from Nrf2 knock-out animals resulted in neither ARE activation nor
neuroprotection. By reintroducing Nrf2 via infection with a replication-deficient
adenovirus (ad), both the genetic response and neuroprotection were rescued.
Conversely, infection with adenovirus encoding dominant-negative (DN) Nrf2
(ad-DN-Nrf2) or pretreatment with the selective phosphatidylinositol-3 kinase
inhibitor LY294002 inhibited the tBHQ-mediated promoter response and
corresponding neuroprotection. Interestingly, the adenoviral infection showed a
high selectivity for astrocytes over neurons. In an attempt to reveal some of the
cell type-specific changes resulting from ARE activation, cultures were infected
with adenovirus encoding green fluorescent protein (GFP) (ad-GFP) or ad-DN-Nrf2
(containing GFP) before tBHQ treatment. A glia-enriched population of
GFP-infected cells was then isolated from a population of uninfected neurons
using cell-sorting technology. Microarray analysis was used to evaluate potential
glial versus neuron-specific contributions to the neuroprotective effects of ARE
activation and Nrf2 dependence. Strikingly, the change in neuronal gene
expression after tBHQ treatment was dependent on Nrf2 activity in the astrocytes.
This suggests that Nrf2-dependent genetic changes alter neuron-glia interactions
resulting in neuroprotection.

PMID: 14762128 [PubMed - indexed for MEDLINE]

37. J Biol Chem. 2000 May 19;275(20):15466-73.

Transcriptional regulation of the antioxidant response element. Activation by
Nrf2 and repression by MafK.

Nguyen T, Huang HC, Pickett CB.

Schering-Plough Research Institute, Kenilworth, New Jersey 07033, USA.

The antioxidant response element (ARE) mediates the transcriptional activation of
many genes encoding phase II drug-metabolizing enzymes in response to oxidative
stress. Recent studies using knockout mice suggest that NF-E2-related factor 2
(Nrf2), along with small Maf proteins, binds and activates the ARE. In this
study, using in vitro binding assays, Nrf2/MafK heterodimers were found to
interact with high affinity to the ARE. However, distinct differences were
observed when this interaction was compared with that formed with nuclear
proteins from H4II EC3 or HepG2 cells. Overexpression of Nrf2 activated
ARE-mediated transcription in HepG2 cells, and this activation was further
increased by tert-butylhydroquinone. In HeLa cells, overexpression of Nrf2
resulted in activation of the ARE, but this activation was no longer induced by
tert-butylhydroquinone. Using ARE constructs with point mutations in the core
sequence, we found that only mutations at the T or G nucleotides within the core
(TGAC) render the ARE unresponsive to Nrf2. Overexpression of MafK led to
dose-dependent repression of ARE activity. Activation of the ARE by Nrf2 was
similarly antagonized by MafK. These data suggest that Nrf2 plays an important
role mediating basal activity of the ARE but that small Maf proteins are
repressors and not activators of ARE-mediated transcription.

PMID: 10747902 [PubMed - indexed for MEDLINE]

38. Cancer Res. 2005 Jun 1;65(11):4789-98.

The synthetic triterpenoids, CDDO and CDDO-imidazolide, are potent inducers of
heme oxygenase-1 and Nrf2/ARE signaling.

Liby K, Hock T, Yore MM, Suh N, Place AE, Risingsong R, Williams CR, Royce DB,
Honda T, Honda Y, Gribble GW, Hill-Kapturczak N, Agarwal A, Sporn MB.

Dartmouth Medical School and Dartmouth College, Hanover, New Hampshire 03755,

The synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid
(CDDO) and its derivative
1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im) are
multifunctional molecules with potent antiproliferative, differentiating, and
anti-inflammatory activities. At nanomolar concentrations, these agents rapidly
increase the expression of the cytoprotective heme oxygenase-1 (HO-1) enzyme in
vitro and in vivo. Transfection studies using a series of reporter constructs
show that activation of the human HO-1 promoter by the triterpenoids requires an
antioxidant response element (ARE), a cyclic AMP response element, and an E Box
sequence. Inactivation of one of these response elements alone partially reduces
HO-1 induction, but mutations in all three sequences entirely eliminate promoter
activity in response to the triterpenoids. Treatment with CDDO-Im also elevates
protein levels of Nrf2, a transcription factor previously shown to bind ARE
sequences, and increases expression of a number of antioxidant and detoxification
genes regulated by Nrf2. The triterpenoids also reduce the formation of reactive
oxygen species in cells challenged with tert-butyl hydroperoxide, but this
cytoprotective activity is absent in Nrf2 deficient cells. These studies are the
first to investigate the induction of the HO-1 and Nrf2/ARE pathways by CDDO and
CDDO-Im, and our results suggest that further in vivo studies are needed to
explore the chemopreventive and chemotherapeutic potential of the triterpenoids.

PMID: 15930299 [PubMed - indexed for MEDLINE]

39. Cancer Res. 2008 Jan 15;68(2):364-8.

Activating transcription factor 3 is a novel repressor of the nuclear factor
erythroid-derived 2-related factor 2 (Nrf2)-regulated stress pathway.

Brown SL, Sekhar KR, Rachakonda G, Sasi S, Freeman ML.

Department of Radiation Oncology and Vanderbilt-Ingram Cancer Center, Vanderbilt
University School of Medicine, Nashville, Tennessee 37232, USA.

The transcription factor nuclear factor erythroid-derived 2-related factor 2
(Nrf2) regulates induction of an extensive cellular stress response network when
complexed with the cAMP-responsive element binding protein (CBP) at antioxidant
response elements (ARE) located in the promoter region of target genes.
Activating transcription factor 3 (ATF3) can repress Nrf2-mediated signaling in a
manner that is not well understood. Here, we show that ATF3-mediated suppression
is a consequence of direct ATF3-Nrf2 protein-protein interactions that result in
displacement of CBP from the ARE. This work establishes ATF3 as a novel repressor
of the Nrf2-directed stress response pathway.

PMID: 18199529 [PubMed - indexed for MEDLINE]

40. Am J Physiol Heart Circ Physiol. 2010 Jul;299(1):H18-24. Epub 2010 Apr 23.

Resveratrol confers endothelial protection via activation of the antioxidant
transcription factor Nrf2.

Ungvari Z, Bagi Z, Feher A, Recchia FA, Sonntag WE, Pearson K, de Cabo R, Csiszar

Dept. of Geriatric Medicine, Reynolds Oklahoma Center on Aging, University of
Oklahoma Health Sciences Center, 975 NE 10th St., Oklahoma City, OK 73104, USA.

Comment in:
    Am J Physiol Heart Circ Physiol. 2010 Jul;299(1):H10-2.

Epidemiological studies suggest that Mediterranean diets rich in resveratrol are
associated with reduced risk of coronary artery disease. Resveratrol was also
shown to confer vasoprotection in animal models of type 2 diabetes and aging.
However, the mechanisms by which resveratrol exerts its antioxidative
vasculoprotective effects are not completely understood. Using a nuclear
factor-E(2)-related factor-2 (Nrf2)/antioxidant response element-driven
luciferase reporter gene assay, we found that in cultured coronary arterial
endothelial cells, resveratrol, in a dose-dependent manner, significantly
increases transcriptional activity of Nrf2. Accordingly, resveratrol
significantly upregulates the expression of the Nrf2 target genes NAD(P)H:quinone
oxidoreductase 1, gamma-glutamylcysteine synthetase, and heme oxygenase-1.
Resveratrol treatment also significantly attenuated high glucose (30 mM)-induced
mitochondrial and cellular oxidative stress (assessed by flow cytometry using
MitoSox and dihydroethidine staining). The aforementioned effects of resveratrol
were significantly attenuated by the small interfering RNA downregulation of Nrf2
or the overexpression of Kelch-like erythroid cell-derived protein 1, which
inactivates Nrf2. To test the effects of resveratrol in vivo, we used mice fed a
high-fat diet (HFD), which exhibit increased vascular oxidative stress associated
with an impaired endothelial function. In HFD-fed Nrf2(+/+) mice, resveratrol
treatment attenuates oxidative stress (assessed by the Amplex red assay),
improves acetylcholine-induced vasodilation, and inhibits apoptosis (assessed by
measuring caspase-3 activity and DNA fragmentation) in branches of the femoral
artery. In contrast, the aforementioned endothelial protective effects of
resveratrol were diminished in HFD-fed Nrf2(-/-) mice. Taken together, our
results indicate that resveratrol both in vitro and in vivo confers endothelial
protective effects which are mediated by the activation of Nrf2.

PMCID: PMC2904129 [Available on 2011/7/1]
PMID: 20418481 [PubMed - indexed for MEDLINE]

41. Cancer Res. 2010 Nov 1;70(21):8886-95. Epub 2010 Oct 12.

Ionizing radiation activates the Nrf2 antioxidant response.

McDonald JT, Kim K, Norris AJ, Vlashi E, Phillips TM, Lagadec C, Della Donna L,
Ratikan J, Szelag H, Hlatky L, McBride WH.

Division of Molecular and Cellular Oncology, Department of Radiation Oncology,
and Pasarow Mass Spectrometry Laboratory, David Geffen School of Medicine at
University of California, Los Angeles, California 90095-1714, USA.

The transcription factor NF-E2-related factor 2 (Nrf2) binds the antioxidant DNA
response element (ARE) to activate important cellular cytoprotective defense
systems. Recently several types of cancers have been shown to overexpress Nrf2,
but its role in the cellular response to radiation therapy has yet to be fully
determined. In this study, we report that single doses of ionizing radiation from
2 to 8 Gy activate ARE-dependent transcription in breast cancer cells in a
dose-dependent manner, but only after a delay of five days. Clinically relevant
daily dose fractions of radiation also increased ARE-dependent transcription, but
again only after five days. Downstream activation of Nrf2-ARE-dependent gene and
protein markers, such as heme oxygenase-1, occurred, whereas Nrf2-deficient
fibroblasts were incapable of these responses. Compared with wild-type
fibroblasts, Nrf2-deficient fibroblasts had relatively high basal levels of
reactive oxygen species that increased greatly five days after radiation
exposure. Further, in vitro clonogenic survival assays and in vivo sublethal
whole body irradiation tests showed that Nrf2 deletion increased radiation
sensitivity, whereas Nrf2-inducing drugs did not increase radioresistance. Our
results indicate that the Nrf2-ARE pathway is important to maintain resistance to
irradiation, but that it operates as a second-tier antioxidant adaptive response
system activated by radiation only under specific circumstances, including those
that may be highly relevant to tumor response during standard clinical
dose-fractionated radiation therapy.

PMCID: PMC2970706 [Available on 2011/11/1]
PMID: 20940400 [PubMed - indexed for MEDLINE]

42. Am J Pathol. 2006 Jun;168(6):1960-74.

Multiorgan autoimmune inflammation, enhanced lymphoproliferation, and impaired
homeostasis of reactive oxygen species in mice lacking the antioxidant-activated
transcription factor Nrf2.

Ma Q, Battelli L, Hubbs AF.

Receptor Biology Laboratory, Toxicology and Molecular Biology Branch/Health
Effects Laboratory Division/National Institute for Occupational Safety and
Health/Centers for Disease Control and Prevention, Morgantown, WV 26505, USA.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is an antioxidant-activated
cap "n" collar basic leucine zipper transcription factor. To assess the function
of Nrf2 in the antioxidant response, we examined mice with targeted disruption of
the Nrf2 gene. Nrf2-null mice developed complex disease manifestations, with a
majority exhibiting a lupus-like autoimmune syndrome characterized by multiorgan
inflammatory lesions with a marked female predominance, appearance of
anti-double-stranded DNA antibodies in young adulthood, intravascular deposition
of immunoglobulin complexes in blood vessels, and premature death due to rapidly
progressing membranoproliferative glomerular nephritis. Mechanistic analyses
revealed that the null mice showed enhanced proliferative response of CD4+ T
cells, altered ratios of CD4+ and CD8+ cells, and increased oxidative lesions in
tissues. Analyses of antioxidant-induced gene expression showed that the knockout
mice were devoid of the basal and inducible expression of certain phase 2
detoxification enzymes and antioxidant genes in hepatic and lymphoid cells in
vivo. Our findings suggest that Nrf2 mediates important antioxidant functions
involved in the control of peripheral lymphocyte homeostasis and autoimmune

PMCID: PMC1606627
PMID: 16723711 [PubMed - indexed for MEDLINE]

43. Biochem Pharmacol. 2007 Jun 15;73(12):1853-62. Epub 2007 Jan 7.

Coordinate regulation of Phase I and II xenobiotic metabolisms by the Ah receptor
and Nrf2.

Köhle C, Bock KW.

Department of Toxicology, Institute of Pharmacology and Toxicology, University of
Tübingen, Germany.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor
with important roles in metabolic adaptation, normal physiology and dioxin
toxicology. Metabolic adaptation is based on coordinate regulation of a set of
xenobiotic-metabolizing enzymes (XMEs), termed AhR battery. Coordination is
achieved by AhR/Arnt-binding to XREs (xenobiotic response elements), identified
in the 5' upstream region of AhR target genes. The AhR battery encodes Phase I
and II enzymes. Interestingly, these Phase II genes are linked to the Nrf2 gene
battery that encodes enzymes that are essential in protection against
oxidative/electrophile stress. Nrf2 binds to AREs (antioxidant response elements)
in the regulatory region of a large and distinct set of target genes.
Functionally characterized response elements such as XREs and AREs in the
regulatory region of target genes may provide a genetic basis to understand AhR-
and Nrf2-induced genes. Linkage between AhR and Nrf2 batteries is probably
achieved by multiple mechanisms, including Nrf2 as a target gene of the AhR,
indirect activation of Nrf2 via CYP1A1-generated reactive oxygen species, and
direct cross-interaction of AhR/XRE and Nrf2/ARE signaling. Linkage appears to be
species- and cell-dependent. However, mechanisms linking XRE- and ARE-controlled
Phase II genes need further investigation. Tightened coupling between Phases I
and II by AhR- and Nrf2-induced XMEs may greatly attenuate health risks posed by
CYP1A1-generated toxic intermediates and reactive oxygen species. Better
recognition of coordinate Phase I and II metabolisms may improve risk assessment
of reactive toxic intermediates in the extrapolation to low level endo- and
xenobiotic exposure.

PMID: 17266942 [PubMed - indexed for MEDLINE]

44. Free Radic Biol Med. 2005 Dec 1;39(11):1438-48. Epub 2005 Aug 19.

Characterization of Nrf2 activation and heme oxygenase-1 expression in NIH3T3
cells exposed to aqueous extracts of cigarette smoke.

Knörr-Wittmann C, Hengstermann A, Gebel S, Alam J, Müller T.

PHILIP MORRIS Research Laboratories GmbH, Fuggerstr. 3, D-51149 Köln, Germany.

Cigarette smoke (CS) is a complex chemical mixture estimated to be composed of up
to 5000 different chemicals, many of which are prooxidant. Here we show that, at
least in vitro, the cellular response designed to combat oxidative stress
resulting from CS exposure is primarily controlled by the transcription factor
Nrf2, a principal inducer of antioxidant and phase II-related genes. The
prominent role of Nrf2 in the cellular response to CS is substantiated by the
following observations: In NIH3T3 cells exposed to aqueous extracts of CS (i)
Nrf2 is strongly stabilized and becomes detectable in nuclear extracts. (ii)
Nuclear localization of Nrf2 coincides with increased DNA binding of a putative
Nrf2/MafK heterodimer to its cognate cis-regulatory site, i.e., the
antioxidant-responsive element (ARE). (iii) Studies on the regulatory elements of
the oxidative stress-inducible gene heme oxygenase-1 (hmox1) using various hmox1
promoter/luciferase reporter constructs revealed that the strong CS-dependent
expression of this gene is primarily governed by the distal enhancers 1 ("E1")
and 2 ("E2"), which both contain three canonical ARE-like stress-responsive
elements (StREs). Notably, depletion of Nrf2 levels caused by RNA interference
significantly compromised CS-induced hmox1 promoter activation, based on the
distinct Nrf2 sensitivity exhibited by E1 and E2. Finally, (iv) siRNA-dependent
knock-down of Nrf2 completely abrogated CS-induced expression of phase II-related
genes. Taken together, these results confirm the outstanding role of Nrf2 both in
sensing (oxidant) stress and in orchestrating an efficient transcriptional
response aimed at resolving the stressing conditions.

PMID: 16274879 [PubMed - indexed for MEDLINE]

45. J Biol Chem. 2002 Nov 8;277(45):42769-74. Epub 2002 Aug 26.

Phosphorylation of Nrf2 at Ser-40 by protein kinase C regulates antioxidant
response element-mediated transcription.

Huang HC, Nguyen T, Pickett CB.

Schering-Plough Research Institute, Kenilworth, New Jersey 07033, USA.

Nrf2, a basic leucine zipper transcription factor, is an essential activator of
the coordinated transcription of genes encoding antioxidant enzymes and phase II
detoxifying enzymes through the regulatory sequence termed antioxidant response
element (ARE). Recently we reported evidence for the involvement of protein
kinase C (PKC) in phosphorylating Nrf2 and triggering its nuclear translocation
in response to oxidative stress. We show here that phosphorylation of purified
rat Nrf2 by the catalytic subunit of PKC was blocked by a synthetic peptide
mimicking one of the potential PKC sites. Accordingly, Nrf2 bearing a Ser to Ala
mutation at amino acid 40 (S40A) could not be phosphorylated by PKC. The S40A
mutation did not affect in vitro binding of Nrf2/MafK to the ARE. However, it
partially impaired Nrf2 activation of ARE-driven transcription in a reporter gene
assay when Keap1 was overexpressed. In vitro transcribed/translated Keap1 could
be coimmunoprecipitated with Nrf2. Phosphorylation of wild-type Nrf2 by PKC
promoted its dissociation from Keap1, whereas the Nrf2-S40A mutant remained
associated. These findings together with our prior studies suggest that the
PKC-catalyzed phosphorylation of Nrf2 at Ser-40 is a critical signaling event
leading to ARE-mediated cellular antioxidant response.

PMID: 12198130 [PubMed - indexed for MEDLINE]

46. Biochem Pharmacol. 2008 Dec 1;76(11):1485-9. Epub 2008 Jul 23.

Activation of Nrf2-antioxidant signaling attenuates NFkappaB-inflammatory
response and elicits apoptosis.

Li W, Khor TO, Xu C, Shen G, Jeong WS, Yu S, Kong AN.

Department of Pharmaceutics, Ernest-Mario School of Pharmacy, Rutgers, the State
University of New Jersey, 160 Frelinghuysen Road, Piscataway, NJ 08854, USA.

Oxidative stress has been implicated in the etiology of neurodegenerative
disease, cancer and aging. Indeed, accumulation of reactive oxygen and nitrogen
species generated by inflammatory cells that created oxidative stress is thought
to be one of the major factor by which chronic inflammation contributes to
neoplastic transformation as well as many other diseases. We have recently
reported that mice lacking nuclear factor-erythroid 2-related factor 2 (Nrf2) are
more susceptible to dextran sulfate sodium (DSS)-induced colitis and colorectal
carcinogenesis. Nrf2 is a basic leucine zipper redox-sensitive transcriptional
factor that plays a center role in ARE (antioxidant response element)-mediated
induction of phase II detoxifying and antioxidant enzymes. We found that
increased susceptibility of Nrf2 deficient mice to DSS-induced colitis and
colorectal cancer was associated with decreased expression of antioxidant/phase
II detoxifying enzymes in parallel with upregulation of pro-inflammatory
cytokines/biomarkers. These findings suggest that Nrf2 may play an important role
in defense against oxidative stress possibly by activation of cellular
antioxidant machinery as well as suppression of pro-inflammatory signaling
pathways. In addition, in vivo and in vitro data generated from our laboratory
suggest that many dietary compounds can differentially regulate Nrf2-mediated
antioxidant/anti-inflammatory signaling pathways as the first line defense or
induce apoptosis once the cells have been damaged. In this review, we will
summarize our thoughts on the potential cross-talks between Nrf2 and NFkappaB
pathways. Although the mechanisms involved in the cross-talk between these
signaling pathways are still illusive, targeting Nrf2-antioxidative stress
signaling is an ideal strategy to prevent or treat oxidative stress-related

PMCID: PMC2610259
PMID: 18694732 [PubMed - indexed for MEDLINE]

47. Biochem Biophys Res Commun. 2000 Jan 7;267(1):12-6.

Activation of nuclear protein binding to the antioxidant/electrophile response
element in vascular smooth muscle cells by benzo(a)pyrene.

Holderman MT, Miller KP, Ramos KS.

Department of Physiology, College of Veterinary Medicine, College Station, Texas,
77843-4466, USA.

This laboratory has previously shown that binding of nuclear proteins to the
antioxidant/electrophile response element (ARE/EpRE) participates in deregulation
of vascular gene expression by benzo(a)pyrene (BaP), a suspected atherogen. In
the present study, oligonucleotides representing ARE/EpREs within the c-Ha-ras
and glutathione-S-transferase (GST-Ya) promoters were employed to evaluate the
role of flanking sequences in stabilizing protein:DNA interactions in BaP-treated
vascular smooth muscle cells (vSMCs). We also wanted to define promoter-specific
patterns of protein recognition to ARE/EpREs in this cell system. In
electrophoretic mobility shift assays (EMSA), optimal protein binding to a human
Ha-ras ARE/EpRE variant sequence fitted to match the extended mouse(m) GST-Ya
ARE/EpRE core (5'-TMAnnRTGAYnnnGCR-3') was dependent on 5' nucleic acid sequence.
Using immobilized DNA affinity chromatography (IDAC), we identified four nuclear
proteins of M(r) 62, 60, 50, and 30 kDa that associated specifically with the
mGSTYa ARE/EpRE. Photo crosslinking to a BrdU-substituted hHa-ras or mGST
ARE/EpRE probe identified specific proteins of M(r) 80, 60, 55, 25, 23 kDa or 80,
60, 55, 27, 25, 23 kDa, respectively. Protein:DNA complexes detected using IDAC
eluate overlapped with those observed in crude nuclear extracts. Chemical
treatments known to modulate ARE/EpRE protein binding in vSMCs did not alter
overall protein:DNA affinity and/or sequence recognition to either hHa-ras or
mGST-Ya elements. We conclude that nucleotide sequences 5' to the core ARE/EpRE
influence specific binding of nuclear proteins and that multiple proteins bind to
ARE/EpREs in a promoter-specific manner in vSMCs.

PMID: 10623566 [PubMed - indexed for MEDLINE]

48. Toxicol Appl Pharmacol. 2008 Feb 1;226(3):328-37. Epub 2007 Sep 26.

Induction of cancer chemopreventive enzymes by coffee is mediated by
transcription factor Nrf2. Evidence that the coffee-specific diterpenes cafestol
and kahweol confer protection against acrolein.

Higgins LG, Cavin C, Itoh K, Yamamoto M, Hayes JD.

Biomedical Research Centre, Ninewells Hospital and Medical School, University of
Dundee, Dundee, DD1 9SY, Scotland, UK.

Mice fed diets containing 3% or 6% coffee for 5 days had increased levels of mRNA
for NAD(P)H:quinone oxidoreductase 1 (NQO1) and glutathione S-transferase class
Alpha 1 (GSTA1) of between 4- and 20-fold in the liver and small intestine. Mice
fed 6% coffee also had increased amounts of mRNA for UDP-glucuronosyl transferase
1A6 (UGT1A6) and the glutamate cysteine ligase catalytic (GCLC) subunit of
between 3- and 10-fold in the small intestine. Up-regulation of these mRNAs was
significantly greater in mice possessing Nrf2 (NF-E2 p45 subunit-related factor
2) than those lacking the transcription factor. Basal levels of mRNAs for NQO1,
GSTA1, UGT1A6 and GCLC were lower in tissues from nrf2(-/-) mice than from
nrf2(+/+) mice, but modest induction occurred in the mutant animals. Treatment of
mouse embryonic fibroblasts (MEFs) from nrf2(+/+) mice with either coffee or the
coffee-specific diterpenes cafestol and kahweol (C+K) increased NQO1 mRNA up to
9-fold. MEFs from nrf2(-/-) mice expressed less NQO1 mRNA than did wild-type
MEFs, but NQO1 was induced modestly by coffee or C+K in the mutant fibroblasts.
Transfection of MEFs with nqo1-luciferase reporter constructs showed that
induction by C+K was mediated primarily by Nrf2 and required the presence of an
antioxidant response element in the 5'-upstream region of the gene. Luciferase
reporter activity did not increase following treatment of MEFs with 100 mumol/l
furan, suggesting that this ring structure within C+K is insufficient for gene
induction. Priming of nrf2(+/+) MEFs, but not nrf2(-/-) MEFs, with C+K conferred
2-fold resistance towards acrolein.

PMID: 18028974 [PubMed - indexed for MEDLINE]

49. J Biol Chem. 2010 Jul 16;285(29):22576-91. Epub 2010 May 7.

p62/SQSTM1 is a target gene for transcription factor NRF2 and creates a positive
feedback loop by inducing antioxidant response element-driven gene transcription.

Jain A, Lamark T, Sjøttem E, Larsen KB, Awuh JA, Øvervatn A, McMahon M, Hayes JD,
Johansen T.

Molecular Cancer Research Group, Institute of Medical Biology, University of
Tromsø, 9037 Tromsø, Norway.

The p62/SQSTM1 (sequestosome 1) protein, which acts as a cargo receptor for
autophagic degradation of ubiquitinated targets, is up-regulated by various
stressors. Induction of the p62 gene by oxidative stress is mediated by
NF-E2-related factor 2 (NRF2) and, at the same time, p62 protein contributes to
the activation of NRF2, but hitherto the mechanisms involved were not known.
Herein, we have mapped an antioxidant response element (ARE) in the p62 promoter
that is responsible for its induction by oxidative stress via NRF2. Chromatin
immunoprecipitation and gel mobility-shift assays verified that NRF2 binds to
this cis-element in vivo and in vitro. Also, p62 docks directly onto the
Kelch-repeat domain of Kelch-like ECH-associated protein 1 (KEAP1), via a motif
designated the KEAP1 interacting region (KIR), thereby blocking binding between
KEAP1 and NRF2 that leads to ubiquitylation and degradation of the transcription
factor. The KIR motif in p62 is located immediately C-terminal to the
LC3-interacting region (LIR) and resembles the ETGE motif utilized by NRF2 for
its interaction with KEAP1. KIR is required for p62 to stabilize NRF2, and
inhibition of KEAP1 by p62 occurs from a cytoplasmic location within the cell.
The LIR and KIR motifs cannot be engaged simultaneously by LC3 and KEAP1, but
because p62 is polymeric the interaction between KEAP1 and p62 leads to
accumulation of KEAP1 in p62 bodies, which is followed by autophagic degradation
of KEAP1. Our data explain how p62 contributes to activation of NRF2 target genes
in response to oxidative stress through creating a positive feedback loop.

PMCID: PMC2903417 [Available on 2011/7/16]
PMID: 20452972 [PubMed - indexed for MEDLINE]

50. J Biol Chem. 1998 Aug 28;273(35):22409-14.

Sterol regulatory element binding protein-1 activates the cholesteryl ester
transfer protein gene in vivo but is not required for sterol up-regulation of
gene expression.

Chouinard RA Jr, Luo Y, Osborne TF, Walsh A, Tall AR.

Division of Molecular Medicine, Department of Medicine, Columbia University, New
York, New York 10032, USA.

The plasma cholesteryl ester transfer protein (CETP) plays a central role in high
density lipoprotein metabolism and reverse cholesterol transport. Plasma CETP
levels are increased in response to dietary or endogenous hypercholesterolemia as
a result of increased gene transcription in liver and periphery. Deletional
analysis in human CETP transgenic mice localized this response to a region of the
proximal promoter which contains a tandem repeat of the sterol regulatory element
(SRE) of the 3-hydroxy-3-methylglutaryl-CoA reductase gene. The purpose of the
present study was to evaluate the role of the SRE-like element in CETP promoter
activity. Gel shift assays using CETP promoter fragments containing these
elements showed binding of the transcription factors, sterol regulatory
element-binding protein-1 (SREBP-1) and Yin Yang-1 (YY-1). Point mutations in the
SRE-like element, designated MUT1 and MUT2, resulted in decreased binding of
SREBP-1 (MUT1) or SREBP-1 and YY-1 (MUT2). To determine the in vivo significance
of this binding activity, CETP transgenic mice were prepared containing these
promoter point mutations. MUT1 and MUT2 transgenic mice expressed CETP activity
and mass in plasma. In response to high fat, high cholesterol diets, both
MUT1-CETP and MUT2-CETP transgenic mice displayed induction of plasma CETP
activity similar to that observed in natural flanking region (NFR) CETP
transgenic mice. Moreover, in stably transfected adipocyte cell lines, MUT1 and
MUT2 CETP promoter-reporter genes showed significant induction of reporter
activity in response to sterols. To evaluate transactivation by SREBP-1, NFR- and
MUT1-CETP transgenic mice were crossed with SREBP-1 transgenic mice. Induction of
the SREBP transgene in the liver with a low carbohydrate diet resulted in a
3-fold increase in plasma CETP activity in NFR-CETP/SREBP transgenic mice, but
there was no significant change in activity in MUT1-CETP/SREBP transgenic mice.
Thus, SREBP-1 transactivates the NFR-CETP transgene in vivo, as a result of
interaction with the CETP promoter SREs. However, this interaction is not
required for positive sterol induction of CETP gene transcription. The results
suggest independent regulation of the CETP gene by SREBP-1 and a distinct
positive sterol response factor.

PMID: 9712863 [PubMed - indexed for MEDLINE]

51. J Immunol. 2004 Sep 1;173(5):3467-81.

Nrf2 is a key transcription factor that regulates antioxidant defense in
macrophages and epithelial cells: protecting against the proinflammatory and
oxidizing effects of diesel exhaust chemicals.

Li N, Alam J, Venkatesan MI, Eiguren-Fernandez A, Schmitz D, Di Stefano E,
Slaughter N, Killeen E, Wang X, Huang A, Wang M, Miguel AH, Cho A, Sioutas C, Nel

Division of Clinical Immunology and Allergy, Department of Medicine, University
of California, Los Angeles 90095, USA.

The proinflammatory effects of particulate pollutants, including diesel exhaust
particles (DEP), are related to their content of redox cycling chemicals and
their ability to generate oxidative stress in the respiratory tract. An
antioxidant defense pathway, which involves phase II enzyme expression, protects
against the pro-oxidative and proinflammatory effects of DEP. The expression of
enzymes, including heme oxygenase-1 (HO-1) and GST, is dependent on the activity
of a genetic antioxidant response element in their promoters. In this study we
investigated the mechanism by which redox cycling organic chemicals, prepared
from DEP, induce phase II enzyme expression as a protective response. We
demonstrate that aromatic and polar DEP fractions, which are enriched in
polycyclic aromatic hydrocarbons and quinones, respectively, induce the
expression of HO-1, GST, and other phase II enzymes in macrophages and epithelial
cells. We show that HO-1 expression is mediated through accumulation of the bZIP
transcription factor, Nrf2, in the nucleus, and that Nrf2 gene targeting
significantly weakens this response. Nrf2 accumulation and subsequent activation
of the antioxidant response element is regulated by the proteasomal degradation
of Nrf2. This pathway is sensitive to pro-oxidative and electrophilic DEP
chemicals and is also activated by ambient ultrafine particles. We propose that
Nrf2-mediated phase II enzyme expression protects against the proinflammatory
effects of particulate pollutants in the setting of allergic inflammation and

PMID: 15322212 [PubMed - indexed for MEDLINE]

52. J Biol Chem. 2005 May 27;280(21):20340-8. Epub 2005 Mar 23.

Transcriptional regulation of NF-E2 p45-related factor (NRF2) expression by the
aryl hydrocarbon receptor-xenobiotic response element signaling pathway: direct
cross-talk between phase I and II drug-metabolizing enzymes.

Miao W, Hu L, Scrivens PJ, Batist G.

Montreal Center for Experimental Therapeutics in Cancer, Lady Davis Institute for
Medical Research, The Sir Mortimer B. Davis-Jewish General Hospital and
Department of Oncology, McGill University, Montreal, Quebec H3T 1E2, Canada.

The aryl hydrocarbon receptor (AHR) and NF-E2 p45-related factor (NRF2) are two
distinct transcription factors involved in the regulation of drug-metabolizing
enzymes. Increasing evidence from several studies implies that AHR and NRF2 have
direct links, but the molecular mechanism remains unknown. In this work we
demonstrate for the first time that Nrf2 gene transcription is directly modulated
by AHR activation. DNA sequence analyses of the mouse Nrf2 promoter revealed one
xenobiotic response element (XRE)-like element (XREL1) located at -712 and two
additional XRE-like elements located at +755 (XREL2) and +850 (XREL3). Functional
analysis using luciferase assay showed that XREL1, XREL2, and XREL3 are all
inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin treatment, with XREL2 being the
most potent. The functionality of these XRE-like elements was further confirmed
by mutagenesis and gel shift experiments. Finally, we used chromatin
immunoprecipitation assay to show a direct binding of AHR to the Nrf2 promoter.
Cells with silenced AHR expression using siRNA also lost NRF2 mRNA induction by
2,3,7,8-tetrachlorodibenzo-p-dioxin. These new data position NRF2-antioxidant
response element downstream in the AHR-XRE pathway. Moreover, direct regulation
of NRF2 by AHR contributes to couple phase I and II enzymes into an integrated
system facilitating more effective xenobiotic and carcinogen detoxification.

PMID: 15790560 [PubMed - indexed for MEDLINE]

53. Cancer Res. 2002 Sep 15;62(18):5196-203.

Identification of Nrf2-regulated genes induced by the chemopreventive agent
sulforaphane by oligonucleotide microarray.

Thimmulappa RK, Mai KH, Srisuma S, Kensler TW, Yamamoto M, Biswal S.

Department of Environmental Health Sciences, Bloomberg School of Public Health,
Johns Hopkins University, Baltimore, MA 21205-2179, USA.

Electrophiles formed during metabolic activation of chemical carcinogens and
reactive oxygen species generated from endogenous and exogenous sources play a
significant role in carcinogenesis. Cancer chemoprevention by induction of phase
2 proteins to counteract the insults of these reactive intermediates has gained
considerable attention. Nuclear factor E2 p45-related factor 2 (Nrf2), a bZIP
transcription factor, plays a central role in the regulation (basal and or
inducible expression) of phase 2 genes by binding to the "antioxidant response
element" in their promoters. Identification of novel Nrf2-regulated genes is
likely to provide insight into cellular defense systems against the toxicities of
electrophiles and oxidants and may define effective targets for achieving cancer
chemoprevention. Sulforaphane is a promising chemopreventive agent that exerts
its effect by strong induction of phase 2 enzymes via activation of Nrf2. In the
present study, a transcriptional profile of small intestine of wild-type (nrf2
+/+) and knock out (nrf2 -/-) mice treated with vehicle or sulforaphane (9
micromol/day for 1 week, p.o.) was generated using the Murine Genome U74Av2
oligonucleotide array (representing approximately 6000 well-characterized genes
and nearly 6000 expressed sequence tags). Comparative analysis of gene expression
changes between different treatment groups of wild-type and nrf2-deficient mice
facilitated identification of numerous genes regulated by Nrf2 including
previously reported Nrf2-regulated genes such as NAD(P)H:quinone reductase
(NQO1), glutathione S-transferase (GST), gamma-glutamylcysteine synthetase (GCS),
UDP-glucuronosyltransferases (UGT),epoxide hydrolase, as well as a number of new
genes. Also identified were genes encoding for cellular NADPH regenerating
enzymes (glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and
malic enzyme), various xenobiotic metabolizing enzymes, antioxidants (glutathione
peroxidase, glutathione reductase, ferritin, and haptaglobin), and biosynthetic
enzymes of the glutathione and glucuronidation conjugation pathways. The data
were validated by Northern blot analysis and enzyme assays of selected genes.
This investigation expands the horizon of Nrf2-regulated genes, highlights the
cross-talk between various metabolic pathways, and divulges the pivotal role
played by Nrf2 in regulating cellular defenses against carcinogens and other

PMID: 12234984 [PubMed - indexed for MEDLINE]

54. Proc Natl Acad Sci U S A. 1999 Oct 26;96(22):12731-6.

Nrf2 is essential for protection against acute pulmonary injury in mice.

Chan K, Kan YW.

Cardiovascular Research Institute, University of California, San Francisco, CA
94143-0793, USA.

Nrf2 is a member of the "cap 'n' collar" family of transcription factors. These
transcription factors bind to the NF-E2 binding sites (GCTGAGTCA) that are
essential for the regulation of erythroid-specific genes. Nrf2 is expressed in a
wide range of tissues, many of which are sites of expression for phase 2
detoxification genes. Nrf2(-/-) mice are viable and have a normal phenotype under
normal laboratory conditions. The NF-E2 binding site is a subset of the
antioxidant response elements that have the sequence GCNNNGTCA. The antioxidant
response elements are regulatory sequences found on promoters of several phase 2
detoxification genes that are inducible by xenobiotics and antioxidants. We
report here that Nrf2(-/-) mice are extremely susceptible to the administration
of the antioxidant butylated hydroxytoluene. With doses of butylated
hydroxytoluene that are tolerated by wild-type mice, the Nrf2(-/-) mice succumb
from acute respiratory distress syndrome. Gene expression studies show that the
expression of several detoxification enzymes is altered in the Nrf2(-/-) mice.
The Nrf2(-/-) mice may prove to be a good in vivo model for toxicological
studies. As oxidative damage causes DNA breakage, these mice may also be useful
for testing carcinogenic agents.

PMID: 10535991 [PubMed - indexed for MEDLINE]

55. J Biol Chem. 2006 Dec 29;281(52):39776-84. Epub 2006 Oct 31.

p53 suppresses the Nrf2-dependent transcription of antioxidant response genes.

Faraonio R, Vergara P, Di Marzo D, Pierantoni MG, Napolitano M, Russo T, Cimino

Dipartimento di Biochimica e Biotecnologie Mediche, Università di Napoli Federico
II, CEINGE Biotecnologie avanzate, 80131 Napoli, Italy.

Cells respond to the shift of intracellular environment toward pro-oxidant
conditions by activating the transcription of numerous "antioxidant" genes. This
response is based on the activation of the Nrf2 transcription factor, which
transactivates the genes containing in their promoters the antioxidant response
cis-elements (AREs). If the oxidative stress provokes DNA damage, a second
response of the cell takes place, based on the activation of p53, which induces
cell cycle arrest and/or apoptosis. Here we have explored the cross-talk between
these two regulatory mechanisms. The results show that p53 counteracts the
Nrf2-induced transcription of three ARE-containing promoters of the x-CT, NQO1,
and GST-alpha1 genes. Endogenous transcripts of these antioxidant genes
accumulate as a consequence of Nrf2 overexpression or exposure to electrophile
diethylmaleate, but these effects are again blocked by p53 overexpression or
endogenous p53 activation. Chromatin immunoprecipitation experiments support the
hypothesis that this p53-dependent trans-repression is due to the direct
interaction of p53 with the ARE-containing promoters. Considering that
p53-induced apoptosis requires an accumulation of reactive oxygen species, this
negative control on the Nrf2 transactivation appears to be aimed to prevent the
generation of a strong anti-oxidant intracellular environment that could hinder
the induction of apoptosis.

PMID: 17077087 [PubMed - indexed for MEDLINE]

56. Toxicol Lett. 2002 Jun 7;132(1):27-36.

Acrolein causes transcriptional induction of phase II genes by activation of Nrf2
in human lung type II epithelial (A549) cells.

Tirumalai R, Rajesh Kumar T, Mai KH, Biswal S.

Department of Environmental Health Sciences, Division of Toxicological Sciences,
Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD
21205-2179, USA.

Acrolein, an alpha,beta-unsaturated aldehyde, is by far the strongest
electrophile present in cigarette smoke which is involved in several lung
pathophysiological conditions. Acrolein depletes glutathione and creates thiol
imbalance. Acrolein due to thiol imbalance as well as covalent modification of
cysteine is known to inhibit the activity of redox sensitive transcription
factors such as NF-kappaB and AP-1. Exposure of human type II lung epithelial
(A549) cells to non-lethal dose of acrolein (150 fmol/cell for 1 h) depletes 80%
of intracellular glutathione and increases the transcription of
gamma-glutamylcysteine synthetase (gamma-GCS) at 6-12 h post-treatment, which
helps in replenishing the glutathione to normal level. Acrolein treatment
activates transcription of phase II genes in general, as indicated by an increase
in mRNA for NAD (P) H:quinone oxidoreductase (NQO1). Western blot analysis
revealed the increased level of the transcription factor, Nrf2 in the nuclear
extract from acrolein treated cells. Electrophoretic mobility shift assay shows
increased binding of nuclear proteins to human antioxidant response element (ARE)
consensus sequence after treatment with acrolein. The involvement of Nrf2 in ARE
mediated transcriptional activation in response to acrolein exposure has been
confirmed by human NQO1-ARE reporter assay. The ability of acrolein to
transcriptionaly activate genes responsible for phase II enzymes may form the
basis of resistance against cell death and can have implications in cigarette
smoke related lung carcinogenesis.

PMID: 12084617 [PubMed - indexed for MEDLINE]

57. J Biol Chem. 2006 May 26;281(21):14841-51. Epub 2006 Mar 21.

Glycogen synthase kinase-3beta inhibits the xenobiotic and antioxidant cell
response by direct phosphorylation and nuclear exclusion of the transcription
factor Nrf2.

Salazar M, Rojo AI, Velasco D, de Sagarra RM, Cuadrado A.

Instituto de Investigaciones Biomédicas and Departamento de Bioquímica, Facultad
de Medicina, Universidad Autónoma de Madrid, 28029 Madrid, Spain.

The transcription factor Nrf2 (nuclear factor E2-related factor 2) regulates the
expression of antioxidant phase II genes and contributes to preserve redox
homeostasis and cell viability in response to oxidant insults. Nrf2 should be
coordinated with the canonical cell survival pathway represented by
phosphatidylinositol 3-kinase (PI3K) and the Ser/Thr kinase Akt but so far the
mechanistic connections remain undefined. Here we identify glycogen synthase
kinase-3beta (GSK-3beta), which is inhibited by Akt-mediated phosphorylation, as
the link between both processes. Using heme oxygenase-1 (HO-1) as a model phase
II gene, we found that both PI3K and Akt increased mRNA and protein levels of
this enzyme. Pharmacological inhibitors (LiCl and PDZD-8) and genetic variants of
GSK-3beta (constitutively active and dominant negative mutants) indicated that
PI3K/Akt activates and GSK-3beta inhibits the antioxidant response elements of
the ho1 promoter and pointed Nrf2 as directly involved in this process. Indeed,
GSK-3beta phosphorylated Nrf2 in vitro and in vivo. Immunocytochemistry and
subcellular fractionation analyses demonstrated that the effect of
GSK-3beta-mediated phosphorylation of Nrf2 is to exclude this transcription
factor from the nucleus. Nrf2 up-regulated the expression of HO-1, glutathione
peroxidase, glutathione S-transferase A1, NAD(P)H: quinone oxidoreductase and
glutamate-cysteine ligase and protected against hydrogen peroxide-induced
glutathione depletion and cell death, whereas co-expression of active GSK-3beta
attenuated both phase II gene expression and oxidant protection. These results
contribute to clarify the cross-talk between the survival signal elicited by
PI3K/Akt and the antioxidant phase II cell response, and introduce GSK-3beta as
the key mediator of this regulation mechanism.

PMID: 16551619 [PubMed - indexed for MEDLINE]

58. Proc Natl Acad Sci U S A. 2006 Jan 17;103(3):768-73. Epub 2006 Jan 9.

Activation of the Keap1/Nrf2 pathway for neuroprotection by electrophilic
[correction of electrophillic] phase II inducers.

Satoh T, Okamoto SI, Cui J, Watanabe Y, Furuta K, Suzuki M, Tohyama K, Lipton SA.

Center for Neuroscience and Aging, The Burnham Institute for Medical Research,
10901 North Torrey Pines Road, La Jolla, CA 92037, USA.

Electrophilic neurite outgrowth-promoting prostaglandin (NEPP) compounds protect
neurons from oxidative insults. At least part of the neuroprotective action of
NEPPs lies in induction of hemeoxygenase-1 (HO-1), which, along with other phase
II enzymes, serve as a defense system against oxidative stress. Here, we found
that, by using fluorescent tags and immunoprecipitation assays, NEPPs are taken
up preferentially into neurons and bind in a thiol-dependent manner to Keap1, a
negative regulator of the transcription factor Nrf2. By binding to Keap1, NEPPs
prevent Keap1-mediated inactivation of Nrf2 and, thus, enhance Nrf2 translocation
into the nucleus of cultured neuronal cells. In turn, Nrf2 binds to
antioxidant/electrophile-responsive elements of the HO-1 promoter to induce HO-1
expression. Consistent with this notion, NEPP induction of an HO-1 reporter
construct is prevented if the antioxidant-responsive elements are mutated. We
show that NEPPs are neuroprotective both in vitro from glutamate-related
excitotoxicity and in vivo in a model of cerebral ischemia/reperfusion injury
(stroke). Our results suggest that NEPPs prevent excitotoxicity by activating the
Keap1/Nrf2/HO-1 pathway. Because NEPPs accumulate preferentially in neurons, they
may provide a category of neuroprotective compounds, distinct from other
electrophilic compounds such as tert-butylhydroquinone, which activates the
antioxidant-responsive element in astrocytes. NEPPs thus represent a therapeutic
approach for stroke and neurodegenerative disorders.

PMCID: PMC1334635
PMID: 16407140 [PubMed - indexed for MEDLINE]

59. J Biol Chem. 2005 Aug 5;280(31):28430-8. Epub 2005 May 23.

Nrf2 Possesses a redox-insensitive nuclear export signal overlapping with the
leucine zipper motif.

Li W, Jain MR, Chen C, Yue X, Hebbar V, Zhou R, Kong AN.

Department of Pharmaceutics, Ernest-Mario School of Pharmacy, Rutgers, the State
University of New Jersey, Piscataway, New Jersey 08854, USA.

Basic leucine zipper (bZIP) protein Nrf2 is a key transcription factor mediating
the antioxidant response. Under homeostatic conditions Nrf2 is anchored to
cysteine-rich Keap1 and sequestered in the cytoplasm. When challenged with
oxidative stress, Keap1 functions as a redox-sensitive switch and releases Nrf2.
Subsequently, Nrf2 translocates into the cell nucleus and binds to a cis-acting
enhancer called the antioxidant response element located in the promoters of a
battery of cytoprotective genes and initiates their transcription. In this study
we identify a canonical nuclear export signal (NES) ((537)LKKQLSTLYL(546))
located in the leucine zipper (ZIP) domain of the Nrf2 protein. The enhanced
green fluorescent protein-tagged ZIP domain of Nrf2 (amino acids 503-589)
exhibited a CRM1-dependent cytosolic distribution that could be abrogated by
site-directed mutations or treatment with the nuclear export inhibitor,
leptomycin B. Ectotopic expression of the Nrf2-NES could also exclude the GAL4
DNA binding domain into the cytoplasm. This NES overlapped with the ZIP motif in
Nrf2, suggesting that the formation of heterodimers between Nrf2 and other bZIP
proteins may simultaneously mask the NES and attenuate Nrf2 nuclear export. The
Nrf2-NES appeared to be redox-insensitive. Neither oxidants (sulforaphane and
diethyl maleate) nor reducing compounds (N-acetyl-l-cysteine and reducing
glutathione) could disrupt the cytosolic distribution of Nrf2zip. Because Nrf2
activation is generally redox-sensitive, the redox insensitivity of this Nrf2-NES
indicates the importance of Keap1 retention as a key rate-limiting step in Nrf2
activation. The characterization of the Nrf2 NES may help decipher the mechanisms
governing nuclear localization and subsequent transcriptional activation of
Nrf2-mediated cytoprotective genes.

PMID: 15917227 [PubMed - indexed for MEDLINE]

60. Free Radic Biol Med. 2004 Aug 15;37(4):433-41.

The pathways and molecular mechanisms regulating Nrf2 activation in response to
chemical stress.

Nguyen T, Yang CS, Pickett CB.

Schering-Plough Research Institute, Kenilworth, NJ 07033, USA.

The induction of many antioxidant and phase II drug-metabolizing enzymes by
phenolic antioxidants and electrophilic compounds is regulated at the
transcriptional level. The response to these compounds is mediated by the
cis-acting antioxidant response element (ARE) found in the promoter of the
encoding genes. The transcription factor NF-E2-related factor 2, or Nrf2, has
emerged as the central protein that binds to the ARE to activate gene
transcription. Data from many studies indicate that Nrf2 is constitutively and
ubiquitously expressed in a number of tissues and cell lines and is thus
responsible for the low-level expression of its target genes observed under
physiological conditions. However, in cells exposed to oxidative stress, Nrf2
activity is increased, further driving the transcriptional activation of genes
whose expression is essential to control cellular redox homeostasis. Recent
studies suggest that the activation of Nrf2 involves a coordinated process and is
regulated at multiple levels. Nrf2 activity is believed to be repressed through
the binding of the cytoskeleton-associated protein Keap1, and its activation
involves mechanisms that interfere with this interaction. Activation of Nrf2 has
also been demonstrated to be dependent on mechanisms that mediate its
stabilization. In this review, the mechanisms controlling this activation process
as reported in recent studies will be examined and discussed, with particular
emphasis on those affecting Nrf2 stability at the molecular level.

PMID: 15256215 [PubMed - indexed for MEDLINE]

61. Mutat Res. 2004 Nov 2;555(1-2):133-48.

Chemoprevention through the Keap1-Nrf2 signaling pathway by phase 2 enzyme

Kwak MK, Wakabayashi N, Kensler TW.

Department of Environmental Health Sciences, Johns Hopkins University Bloomberg
School of Public Health, 615 N. Wolfe St., Baltimore, MD 21205, USA.

One successful strategy for cancer chemoprevention is modulation of drug
metabolizing enzymes, leading to a facilitated elimination of endogenous and
environmental carcinogens. Inducers of phase 2 enzymes such as dithiolethiones
inhibit tumorigenesis of environmental carcinogens in various animal models and
modulate the metabolism of the carcinogen aflatoxin B1 in human clinical trials.
Increasing lines of evidence show that the Keap1-Nrf2 complex is a key molecular
target of chemopreventive phase 2 enzyme inducers. The transcription factor Nrf2
is a member of the basic leucine-zipper NF-E2 family and interacts with the
antioxidant response element (ARE) in the promoter region of phase 2 detoxifying
enzymes. A cytoplasmic actin-binding protein, Keap1, is an inhibitor of Nrf2 that
sequesters it in the cytoplasm. Inducers dissociate this complex, allowing Nrf2
to translocate to the nucleus. Disruption of the nrf2 gene in mice leads to the
loss of chemopreventive efficacy by inducers. This review focuses on (1) the role
of Nrf2 in the regulation of phase 2 and antioxidative genes, (2) the molecular
actions of dithiolethiones on the Keap1-Nrf2 pathway, and (3) the contribution of
Nrf2-regulated gene families to the cytoprotective actions of dithiolethiones and
other inducers. Rapidly accumulating data on this pathway is providing insight
into the coordinated mammalian defense systems against electrophiles and
oxidative stresses and the means by which it may be targeted by small molecules.

PMID: 15476857 [PubMed - indexed for MEDLINE]

62. J Nutr. 2010 Jul;140(7):1211-9. Epub 2010 May 12.

Ajoene, a stable garlic by-product, has an antioxidant effect through
Nrf2-mediated glutamate-cysteine ligase induction in HepG2 cells and primary

Kay HY, Won Yang J, Kim TH, Lee da Y, Kang B, Ryu JH, Jeon R, Kim SG.

College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul
National University, Seoul 151-742, Korea.

Cytoprotective effects of chemopreventive agents may be attributed to the
induction of antioxidant enzymes. Among these, the induction of
glutamate-cysteine ligase (GCL) protects cells from oxidative injury by
increasing glutathione (GSH) content. Nuclear factor erythroid-2-related factor 2
(Nrf2) transcriptionally regulates the expression of genes encoding for GCL and
other cysteine-metabolizing enzymes. Despite extensive studies on the components
in garlic, little information is available on organosulfur by-products made from
garlic. In this study, we investigated whether ajoene, a chemically stable garlic
by-product, has the ability to activate Nrf2 and induce GCL, and, if so, what is
the role of activating Nrf2 in cytoprotection against oxidative stress.
Immunoblottings and reporter gene assays were performed in HepG2 cells. Ajoene
treatment activated Nrf2, as indicated by increased phosphorylation and nuclear
accumulation of Nrf2, decreased interaction with Kelch-like ECH-associated
protein-1, and decreased Nrf2 ubiquitination. Consistently, treatment of ajoene
increased antioxidant response element reporter gene activity and the mRNA and
protein levels of GCL subunits. Ajoene activated protein kinase C-delta
(PKCdelta). Inhibition of PKCdelta activation by rottlerin abrogated its ability
to activate Nrf2 and induce GCL, suggesting that ajoene promotes the
Nrf2-dependent antioxidant defense system via PKCdelta activation. Consequently,
ajoene prevented cell death, GSH depletion, and hydrogen peroxide production
elicited by tert-butylhydroperoxide. The important role of Nrf2 in cytoprotection
was verified by the reversal of ajoene's ability to protect hepatocytes in
Nrf2-knockout mice. Our results demonstrate that ajoene increases
PKCdelta-dependent Nrf2 activation, GCL induction, and the cellular GSH
concentration, which may contribute to protecting cells from oxidative stress.

PMID: 20463144 [PubMed - indexed for MEDLINE]

63. J Biochem Mol Biol. 2004 Mar 31;37(2):139-43.

An important role of Nrf2-ARE pathway in the cellular defense mechanism.

Lee JM, Johnson JA.

School of Pharmacy, University of Wisconsin, Madison, WI 53705, USA.

The antioxidant responsive element (ARE) is a cis-acting regulatory element of
genes encoding phase II detoxification enzymes and antioxidant proteins, such as
NAD(P)H: quinone oxidoreductase 1, glutathione S-transferases, and
glutamate-cysteine ligase. Interestingly, it has been reported that Nrf2
(NF-E2-related factor 2) regulates a wide array of ARE-driven genes in various
cell types. Nrf2 is a basic leucine zipper transcription factor, which was
originally identified as a binding protein of locus control region of beta-globin
gene. The DNA binding sequence of Nrf2 and ARE sequence are very similar, and
many studies demonstrated that Nrf2 binds to the ARE sites leading to
up-regulation of downstream genes. The function of Nrf2 and its downstream target
genes suggests that the Nrf2-ARE pathway is important in the cellular antioxidant
defense system. In support of this, many studies showed a critical role of Nrf2
in cellular protection and anti-carcinogenicity, implying that the Nrf2-ARE
pathway may serve as a therapeutic target for neurodegenerative diseases and
cancers, in which oxidative stress is closely implicated.

PMID: 15469687 [PubMed - indexed for MEDLINE]

64. Mol Cell Endocrinol. 2005 Nov 24;243(1-2):27-34. Epub 2005 Sep 28.

Repression of cancer protective genes by 17beta-estradiol: ligand-dependent
interaction between human Nrf2 and estrogen receptor alpha.

Ansell PJ, Lo SC, Newton LG, Espinosa-Nicholas C, Zhang DD, Liu JH, Hannink M,
Lubahn DB.

Department of Biochemistry, University of Missouri, Columbia, MO, USA.

Repression of cancer-protective phase II enzymes may help explain why estrogen
exposure leads to the development of cancer. In an earlier report we described
the ability of 17beta-estradiol (E(2)) to repress phase II enzyme activity in
vivo. Phase II enzymes are coordinately regulated via the presence of the
antioxidant response element (ARE) in their promoter. We wanted to determine if
estrogen receptors (ER) repress ARE-dependent gene expression through a mechanism
that requires interaction with Nrf2, the transcription factor that regulates
ARE-mediated gene transcription. E(2)-bound ERalpha, but not ERbeta, represses
ARE-regulated gene expression in the presence of exogenously expressed Nrf2 as
well as when the transactivation domain of Nrf2 was fused to a heterologous
DNA-binding domain. Deletion of the activation function-2 (AF-2) and the
ligand-binding domain of ERalpha result in a constitutive repression of
Nrf2-mediated transcription. Finally, E(2)-bound ERalpha co-immunoprecipitates
with Nrf2. Repression of Nrf2-mediated transcription by E(2)-bound ERalpha
expands our knowledge of E(2)-regulated genes and provides a potential
drug-screening target for the development of selective estrogen receptor
modulators with a lower risk of causing cancer.

PMID: 16198475 [PubMed - indexed for MEDLINE]

65. Mol Cell Biol. 2006 Apr;26(7):2845-56.

Hemin-mediated regulation of an antioxidant-responsive element of the human
ferritin H gene and role of Ref-1 during erythroid differentiation of K562 cells.

Iwasaki K, Mackenzie EL, Hailemariam K, Sakamoto K, Tsuji Y.

Department of Environmental and Molecular Toxicology, North Carolina State
University, Campus Box 7633, Raleigh, North Carolina 27695, USA.

An effective utilization of intracellular iron is a prerequisite for erythroid
differentiation and hemoglobinization. Ferritin, consisting of 24 subunits of H
and L, plays a crucial role in iron homeostasis. Here, we have found that the H
subunit of the ferritin gene is activated at the transcriptional level during
hemin-induced differentiation of K562 human erythroleukemic cells. Transfection
of various 5' regions of the human ferritin H gene fused to a luciferase reporter
into K562 cells demonstrated that hemin activates ferritin H transcription
through an antioxidant-responsive element (ARE) that is responsible for induction
of a battery of phase II detoxification genes by oxidative stress. Gel
retardation and chromatin immunoprecipitation assays demonstrated that hemin
induced binding of cJun, JunD, FosB, and Nrf2 b-zip transcription factors to AP1
motifs of the ferritin H ARE, despite no significant change in expression levels
or nuclear localization of these transcription factors. A Gal4-luciferase
reporter assay did not show activation of these b-zip transcription factors after
hemin treatment; however, redox factor 1 (Ref-1), which increases DNA binding of
Jun/Fos family members via reduction of a conserved cysteine in their DNA binding
domains, showed induced nuclear translocation after hemin treatment in K562
cells. Consistently, Ref-1 enhanced Nrf2 binding to the ARE and ferritin H
transcription. Hemin also activated ARE sequences of other phase II genes, such
as GSTpi and NQO1. Collectively, these results suggest that hemin activates the
transcription of the ferritin H gene during K562 erythroid differentiation by
Ref-1-mediated activation of these b-zip transcription factors to the ARE.

PMCID: PMC1430308
PMID: 16537925 [PubMed - indexed for MEDLINE]

66. J Biol Chem. 2005 Mar 18;280(11):9786-95. Epub 2005 Jan 7.

Transcriptional activation of the human Galphai2 gene promoter through nuclear
factor-kappaB and antioxidant response elements.

Arinze IJ, Kawai Y.

Department of Biochemistry, Meharry Medical College, 1005 David B. Todd Jr.,
Blvd, Nashville, Tennessee 37208-3599, USA.

Very little is known regarding molecular mechanism(s) underlying transcriptional
regulation of any G-protein gene despite the importance of G-protein expression
in modulating cellular processes. Here we show that phorbol myristate acetate
(PMA) and tert-butylhydroquinone (tBHQ), which induce oxidative stress in cells,
up-regulate transcription of Galpha(i2) in K562 cells. Redox-sensing chemicals
abrogated this transcriptional effect. A dominant negative I-kappaB double mutant
(S32A/S36A) suppressed PMA-induced transcription by 54-62%, suggesting
involvement of nuclear factor-kappaB (NF-kappaB). SN50, a cell-permeable peptide
that inhibits nuclear import of stress-responsive transcription factors (such as
NF-kappaB), inhibited PMA- and tBHQ-induced transcription. Deletion of an
NF-kappaB-binding motif that maps at +10/+19 in the promoter resulted in 55-60%
suppression of PMA-induced transcription, and 81% suppression of tBHQ-induced
transcription. Mutation of an antioxidant response element (ARE) that maps at
-84/-76 in the promoter resulted in 51 and 86% decrease in PMA- and tBHQ-induced
transcription, respectively. In electrophoretic mobility shift assays, this
element formed complexes with the transcription factors NF-E2p45 and Nrf2 that
are prototypic for binding to the ARE, as well as with c-Fos, which can also
interact with the ARE. Chromatin immunoprecipitation analysis demonstrated
recruitment of these transcription factors to the promoter. Exogenously
transfected Nrf2 transactivated the Galpha(i2) gene promoter; the
cytoskeleton-associated protein, Keap1, abrogated this effect. Taken together,
the present studies reveal that transcription factors that bind NF-kappaB and/or
antioxidant response elements play an activating role in the transcription of the
human Galpha(i2) gene.

PMID: 15640523 [PubMed - indexed for MEDLINE]

67. J Biol Chem. 2010 Dec 24;285(52):41074-86. Epub 2010 Oct 18.

Hepatitis B virus induces expression of antioxidant response element-regulated
genes by activation of Nrf2.

Schaedler S, Krause J, Himmelsbach K, Carvajal-Yepes M, Lieder F, Klingel K,
Nassal M, Weiss TS, Werner S, Hildt E.

Institute of Infection Medicine, Molecular Medical Virology, University of Kiel,
D-24105 Kiel, Germany.

The expression of a variety of cytoprotective genes is regulated by short
cis-acting elements in their promoters, called antioxidant response elements
(AREs). A central regulator of ARE-mediated gene expression is the NF-E2-related
factor 2 (Nrf2). Human hepatitis B virus (HBV) induces a strong activation of
Nrf2/ARE-regulated genes in vitro and in vivo. This is triggered by the
HBV-regulatory proteins (HBx and LHBs) via c-Raf and MEK. The Nrf2/ARE-mediated
induction of cytoprotective genes by HBV results in a better protection of
HBV-positive cells against oxidative damage as compared with control cells.
Furthermore, there is a significantly increased expression of the
Nrf2/ARE-regulated proteasomal subunit PSMB5 in HBV-positive cells that is
associated with a decreased level of the immunoproteasome subunit PSMB5i. In
accordance with this finding, HBV-positive cells display a higher constitutive
proteasome activity and a decreased activity of the immunoproteasome as compared
with control cells even after interferon α/γ treatment. The HBV-dependent
induction of Nrf2/ARE-regulated genes might ensure survival of the infected cell,
shape the immune response to HBV, and thereby promote establishment of the

PMCID: PMC3003406 [Available on 2011/12/24]
PMID: 20956535 [PubMed - indexed for MEDLINE]

68. Biochem Pharmacol. 1995 Dec 22;50(12):2057-68.

Interaction between the Ah receptor and proteins binding to the AP-1-like
electrophile response element (EpRE) during murine phase II [Ah] battery gene

Vasiliou V, Puga A, Chang CY, Tabor MW, Nebert DW.

Department of Environmental Health, University of Cincinnati Medical Center, OH
45267-0056, USA.

We have studied three Phase II genes in the mouse dioxin-inducible [Ah] battery:
Nmo1 [encoding NAD(P)H:menadione oxidoreductase], Ahd4 (encoding the cytosolic
aldehyde dehydrogenase ALDH3c), and Ugt1*06 (a UDP glucuronosyltransferase).
Oxidant-induced Nmo1 gene expression in the c14CoS/c14CoS mouse appears likely to
be caused by homozygous loss of the fumarylacetoacetate hydrolase (Fah) gene on
Chr 7 and absence of the enzyme (FAH), which leads to increased levels of
endogenous tyrosine oxidative metabolites. We show here that increases in [Ah]
Phase II gene expression in the 14CoS/14CoS mouse are correlated with an
AP-1-like DNA motif called the electrophile response element (EpRE), which has
been found in the 5' flanking regulatory regions of all murine (Ah) Phase II
genes. Aromatic hydrocarbon response element (AhREs) are responsible for
dioxin-mediated upregulation of all six [Ah] battery genes, and one or more AhREs
have been found in the 5' flanking regulatory regions of all of these [Ah] genes.
Gel mobility shift assays, with a synthetic oligonucleotide probe corresponding
to the EpRE, show that EpRE-binding proteins are more than twice as abundant in
14CoS/14CoS than in the wild-type ch/ch nuclear extracts. Competition studies of
EpRE-specific binding with an excess of EpRE, mutated EpRE, AP-1, AhRE3, mutated
AhRE3, and C/EBP alpha oligonucleotides suggest that several common
transcriptional factors bind to the EpRE and AhRE3 motifs. Two monospecific
antibodies to the Ah receptor (AHR) protein block formation of an EpRE-specific
complex on gel mobility electrophoresis. These data suggest that AHR (or
AHR-related protein) might be an integral part of the EpRE-binding
transcriptional complex associated with the oxidative stress response. To our
knowledge, this is among the first reports of the same transcription factor
operating at two different response elements upstream of a single gene.

PMID: 8849333 [PubMed - indexed for MEDLINE]

69. Bioessays. 2006 Feb;28(2):169-81.

Activation of the Nrf2-ARE signaling pathway: a promising strategy in cancer

Giudice A, Montella M.

G. Pascale Foundation National Cancer Institute, Unit of Epidemiology and
Prevention, Naples, Italy.

A major protective mechanism against oxidizing substances capable of damaging DNA
integrity and initiating carcinogenesis is the induction of phase II
detoxification and antioxidant enzymes by chemopreventive agents. A key finding
in the field of chemoprevention has been the discovery that the induction of
these enzymes is mediated by the cytoplasmic oxidative stress system
(Nrf2-Keap1). Under basal (reducing) conditions, Keap1 anchors the Nrf2
transcription factor within the cytoplasm, targeting it for ubiquitination and
proteasome degradation, thus repressing its ability to induce phase II genes.
When cells are exposed to chemopreventive agents and oxidative stress, however, a
signal involving phosphorylation and/or redox modification is transmitted to the
Nrf2-Keap1 complex, leading to its dissociation and the nuclear translocation of
Nrf2, which, after hetero-dimerically partnering with other transcription
factors, binds to the AREs/EpREs present within phase II gene promoters,
increasing their transcription. These data should assist in developing new phase
II detoxification enzyme inducers as cancer chemopreventive agents within the
clinical environment.

PMID: 16435293 [PubMed - indexed for MEDLINE]

70. J Biol Chem. 2007 Mar 23;282(12):8749-58. Epub 2007 Jan 26.

Nrf2-Keap1 signaling pathway regulates human UGT1A1 expression in vitro and in
transgenic UGT1 mice.

Yueh MF, Tukey RH.

Laboratory of Environmental Toxicology, Department of Chemistry, University of
California, San Diego, La Jolla, California 92093, USA.

The formation of beta-D-glucopyranosides (glucuronides) by the
UDP-glucuronosyltransferases (UGTs) is a significant metabolic pathway that
facilitates the elimination of small hydrophobic molecules such as drugs, dietary
constituents, steroids, and bile acids. We elucidate here that an anti-oxidative
response leads to induction of UGT1A1 through the Nrf2-Keap1 pathway. When human
HepG2 cells were treated with the prooxidants tert-butylhydroquinone and
beta-naphthoflavone, cellular UGT1A1 glucuronidation activities were increased.
The induction of UGT1A1 proceeded following the overexpression of Nrf2 and was
blocked following overexpression of Keap1, demonstrating that Keap1 suppresses
Nrf2 activation of the UGT1A1 gene. Loss of function analysis for Nrf2 conducted
by small interfering RNA revealed that induction of UGT1A1 was not seen in Nrf2
knock-out cells. To examine the contribution of oxidants toward the regulation of
human UGT1A1 in vivo, transgenic mice bearing the human UGT1 locus (Tg-UGT1) were
treated with tert-butylhydroquinone. Human UGT1A1 was markedly increased in small
and large intestines as well as in liver. Gene mapping experiments including
transfections of UGT1A1 reporter gene constructs into HepG2 cells coupled with
functional analysis of Nrf2 expression and binding to anti-oxidant-response
elements (ARE) resulted in identification of an ARE in the phenobarbital-response
enhancer module region of the UGT1A1 gene. The ARE flanks the recently identified
Ah receptor xenobiotic-responsive element. The results suggest that
Nrf2-Keap1-dependent UGT1A1 induction by prooxidants might represent a key
adaptive response to cellular oxidative stress that defends against a variety of
environmental insults, including electrophile attacks and chemical

PMID: 17259171 [PubMed - indexed for MEDLINE]

71. Cancer Epidemiol. 2010 Oct;34(5):523-33. Epub 2010 Jul 17.

Nrf2-ARE signaling pathway and natural products for cancer chemoprevention.

Zhao CR, Gao ZH, Qu XJ.

Department of Pharmacology, School of Pharmaceutical Sciences, Shandong
University, 44 Wen Hua Xi Road, Jinan 250012, China.

BACKGROUND: One of the potential strategies for preventing cancers is using
food-based natural products to induce cytoprotective enzymes including phase II
and antioxidative enzymes that act in concert to detoxify and eliminate harmful
reactive intermediates formed from carcinogens. The antioxidant response element
(ARE), which is activated upon binding of the nuclear factor E2-related protein 2
(Nrf2) transcription factor protein, has been identified in the regulatory
regions of numerous genes encoding cytoprotective enzymes. Herein, we summarized
the current body of knowledge regarding Nrf2 regulation as well as highlighted
the Nrf2/ARE activators from natural products, which will potentially be used as
chemopreventive agents for cancer patients.
METHODS: Via reviewing Pubmed, we summarized the current progress in the
molecular mechanisms of Nrf2 regulation and the major classes of dietary
components that act as promising chemopreventive agents through evoking Nrf2-ARE
core signaling pathway.
RESULTS: Under basal condition, Nrf2 is at low level, sequestered in the
cytoplasm by being tethered to an actin binding Kelch-like ECH associating
protein 1 (Keap1). Pharmacological and putative chemopreventive agents trigger
the release of Nrf2 from Keap1, allowing it to translocate into the nucleus and
drive the gene expression of detoxifying enzymes to perform cancer
chemopreventive effect.
CONCLUSION: Augmenting both expression and activity of phase II detoxification
and antioxidant enzymes via Nrf2-ARE core signaling pathway would be a rational
approach for cancer chemoprevention and the number of novel Nrf2/ARE activators
from dietary sources is growing.

PMID: 20638930 [PubMed - indexed for MEDLINE]

72. Cancer Lett. 2005 Jun 28;224(2):171-84. Epub 2004 Nov 11.

Nrf2 as a novel molecular target for chemoprevention.

Lee JS, Surh YJ.

National Research Laboratory of Molecular Carcinogenesis and Chemoprevention,
College of Pharmacy, Seoul National University, Shinlim-dong, Kwanak-ku, Seoul
151-742, South Korea.

One of the rational and effective strategies for chemoprevention is the blockade
of DNA damage caused by carcinogenic insult. This can be achieved either by
reducing the formation of reactive carcinogenic species or stimulating their
detoxification. A wide spectrum of xenobiotic metabolizing enzymes catalyze both
phase I (oxidation and reduction) and phase II biotransformation (conjugation)
reactions involved in carcinogen activation and/or deactivation. Several
antioxidant-response element (ARE)-regulated gene products such as glutathione
S-transferase, NAD(P)H:quinone oxidoreductase 1, UDP-glucuronosyltransferase,
gamma-glutamate cysteine ligase, and hemeoxygenase-1 are known to mediate
detoxification and/or to exert antioxidant functions thereby protecting cells
from genotoxic damage. The transcription of ARE-driven genes is regulated, at
least in part, by nuclear transcription factor erythroid 2p45 (NF-E2)-related
factor 2 (Nrf2), which is sequestered in cytoplasm by Kelch-like ECH-associated
protein 1 (Keap1). Exposure of cells to ARE inducers results in the dissociation
of Nrf2 from Keap1 and facilitates translocation of Nrf2 to the nucleus, where it
heterodimerizes with small Maf protein, and binds to ARE, eventually resulting in
the transcriptional regulation of target genes. The Nrf2-Keap1-ARE signaling
pathway can be modulated by several upstream kinases including
phosphatidylinositol 3-kinase, protein kinase C, and mitogen-activated protein
kinases. Selected Nrf2-Keap1-ARE activators, such as oltipraz, anethole
dithiolethione, sulforaphane, 6-methylsulphinylhexyl isothiocyanate, curcumin,
caffeic acid phenethyl ester, 4'-bromoflavone, etc. are potential chemopreventive
agents. This mini-review will focus on a chemopreventive strategy directed
towards protection of DNA and other important cellular molecules by inducing de
novo synthesis of phase II detoxifying or antioxidant genes via the Nrf2-ARE core
signaling pathway.

PMID: 15914268 [PubMed - indexed for MEDLINE]

73. Free Radic Biol Med. 2009 Sep 1;47(5):659-67. Epub 2009 Jun 11.

Structure activity relationship of carotenoid derivatives in activation of the
electrophile/antioxidant response element transcription system.

Linnewiel K, Ernst H, Caris-Veyrat C, Ben-Dor A, Kampf A, Salman H, Danilenko M,
Levy J, Sharoni Y.

Clinical Biochemistry, Faculty of Health Sciences, Ben-Gurion University and
Soroka Medical Center of Kupat Holim, Beer-Sheva, Israel.

Induction of phase II detoxifying enzymes is a major cellular strategy for
reducing the risk of cancer. We previously reported that carotenoids activate the
electrophile/antioxidant response element (EpRE/ARE) transcription system and
induced the expression of phase II enzymes. Various electrophilic phytonutrients
have been shown to induce the EpRE/ARE system by disrupting the inhibitory
activity of Keap1 on Nrf2, the major EpRE/ARE activating transcription factor.
However, hydrophobic carotenoids such as lycopene lack any electrophilic group
and, thus, are unlikely to directly activate Nrf2 and the EpRE/ARE system. Here
we demonstrate that carotenoid oxidation products are the active mediators in the
stimulation of the EpRE/ARE system by carotenoids. Two lines of evidence support
this conclusion. (A) The oxidized derivatives, extracted by ethanol from
partially oxidized lycopene, transactivated EpRE/ARE with a potency similar to
that of the unextracted lycopene mixture, whereas the intact carotenoid showed a
nonsignificant effect. (B) Using a series of characterized mono- and
diapocarotenoids that potentially can be derived from in vivo metabolism of
carotenoids we defined the following structure-activity rules for activation of
EpRE/ARE: (I) aldehydes and not acids are the active molecules; (II) the activity
depends on the relative position of the methyl group to the terminal aldehyde
which determines the reactivity of the conjugated double bond; (III) the optimal
length of a dialdehyde derivative is 12 carbons in the main chain of the
molecule. The apocarotenals inhibited breast and prostate cancer cell growth with
a similar order of potency to the activation of EpRE/ARE. These results may
provide a mechanistic explanation for the cancer preventive activity of

PMID: 19524036 [PubMed - indexed for MEDLINE]

74. Life Sci. 2006 Oct 12;79(20):1944-55. Epub 2006 Jun 20.

Identification of Nrf2-regulated genes induced by chemopreventive isothiocyanate
PEITC by oligonucleotide microarray.

Hu R, Xu C, Shen G, Jain MR, Khor TO, Gopalkrishnan A, Lin W, Reddy B, Chan JY,
Kong AN.

Graduate Program in Pharmaceutical Science, Ernest Mario School of Pharmacy,
Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA.

Electrophiles generated during metabolic activation of carcinogens and reactive
oxygen species formed from endogenous and exogenous sources might play a
significant role in carcinogenesis. Cancer chemoprevention by induction of phase
II detoxifying enzymes to counteract the insults of these reactive intermediates
is under intensive investigation. Nrf2, a bZIP transcription factor, plays a
central role in the regulation of phase II genes by binding to the antioxidant
response element (ARE) in their promoters. Identification of novel Nrf2-regulated
genes is likely to provide insight into cellular defense systems against the
toxicities of electrophiles and oxidants and may define effective targets for
achieving cancer chemoprevention. Phenethyl isothiocyanate (PEITC) is a promising
chemopreventive agent that exerts its effects by induction of phase II enzymes
via activation of Nrf2. In the present study, a transcriptional profile of liver
of the wild-type (Nrf2+/+) and knock-out (Nrf2-/-) mice after treatments with
vehicle or PEITC at 3 h and at 12 h was generated using the Affymetrix Mouse
Genome 430 2.0 Array. Comparative analysis of gene expression changes between
different treatment groups of wild-type and Nrf2-deficient mice facilitated
identification of numerous genes regulated by Nrf2. These Nrf2-dependent and
PEITC-inducible genes include known detoxication enzymes, as well as novel
xenobiotic-metabolizing genes regulated by Nrf2 such as CYP 2c55, CYP 2u1 and
aldehyde oxidase. Unexpected clusters included genes for heat shock proteins,
ubiquitin/26 S proteasome subunits, and lipid metabolism molecules. Collectively,
the identification of these genes not only provides novel insight into the effect
of PEITC on global gene expression and chemoprevention, but also reveals the role
of Nrf2 in those processes, which would confer cancer chemopreventive future.

PMID: 16828809 [PubMed - indexed for MEDLINE]

75. Bioorg Med Chem Lett. 2010 Apr 15;20(8):2675-9. Epub 2010 Jan 20.

Enhanced Nrf2-dependent induction of glutathione in mouse embryonic fibroblasts
by isoselenocyanate analog of sulforaphane.

Emmert SW, Desai D, Amin S, Richie JP Jr.

Department of Public Health Sciences, Penn State Hershey Cancer Institute,
Pennsylvania State University College of Medicine, Hershey, PA 17033, USA.

Epidemiological and laboratory studies have highlighted the potent
chemopreventive effectiveness of both dietary selenium and cruciferous
vegetables, particularly broccoli. Sulforaphane (SFN), an isothiocyanate, was
identified as the major metabolite of broccoli responsible for its anti-cancer
properties. An important mechanism for SFN chemoprevention is through the
enhancement of glutathione (GSH), the most abundant antioxidant in animals and an
important target in chemoprevention. Enhancement of GSH biosynthetic enzymes
including the rate-limiting glutamate cysteine ligase (GCL), as well as other
Phase II detoxification enzymes results from SFN-mediated induction of the
nuclear factor-erythroid 2-related factor 2 (Nrf2)/antioxidant response elements
(ARE) signaling pathway. While isothiocyanate compounds such as SFN are among the
most potent Nrf2 inducers known, we hypothesized that substitution of sulfur with
selenium in the isothiocyanate functional group of SFN would result in an
isoselenocyanate compound (SFN-isoSe) with enhanced Nrf2 induction capability.
Here we report that SFN-isoSe activated an ARE-luciferase reporter in HepG2 cells
more potently than SFN. It was also found that SFN-isoSe induced GCL and GSH in
MEF cells in an Nrf2-dependent manner. Finally, we provide evidence that
SFN-isoSe was more effective in killing HepG2 cancer cells, yet was less toxic to
non-cancer MEF cells, than SFN. These data support our hypothesis, and suggest
that SFN-isoSe and potentially other isoselenocyanates may be highly effective
chemoprotective agents in vivo due to their ability to induce Nrf2 with low
toxicity in normal cells and high efficiency at killing cancer cells.

PMCID: PMC2929643 [Available on 2011/4/15]
PMID: 20304643 [PubMed - indexed for MEDLINE]

76. J Biol Chem. 2005 Sep 9;280(36):31768-75. Epub 2005 Jun 28.

Specific patterns of electrophile adduction trigger Keap1 ubiquitination and Nrf2

Hong F, Sekhar KR, Freeman ML, Liebler DC.

Department of Pharmacology and Toxicology, College of Pharmacy, University of
Arizona, Tucson, Arizona 85721, USA.

Activation of the transcription factor Nrf2 regulates expression of phase II
enzymes and other adaptive responses to electrophile and oxidant stress. Nrf2
concentrations are regulated by the thiol-rich sensor protein Keap1, which is an
adaptor protein for Cul3-dependent ubiquitination and degradation of Nrf2.
However, the links between site specificity of Keap1 modification by
electrophiles and mechanisms of Nrf2 activation are poorly understood. We studied
the actions of the prototypical Nrf2 inducer tert-butylhydroquinone (tBHQ) and
two biotin-tagged, thiol-reactive electrophiles,
N-iodoacetyl-N-biotinylhexylenediamine (IAB) and
1-biotinamido-4-(4'-[maleimidoethyl-cyclohexane]-carboxamido)butane (BMCC). Both
IAB and tBHQ induced antioxidant response element (ARE)-directed green
fluorescent protein (GFP) expression in ARE/thymidine kinase GFP HepG2 cells, and
both initiated nuclear Nrf2 accumulation and induction of heme oxygenase 1 in
HEK293 cells. In contrast, BMCC produced none of these effects. Liquid
chromatography tandem mass spectrometry (MS-MS) analysis of human Keap1 modified
by IAB or BMCC in vitro indicated that IAB adduction occurred primarily in the
central linker domain, whereas BMCC modified other Keap1 domains. Treatment of
FLAG-Keap1-transfected HEK293 with the Nrf2-activating compounds IAB and tBHQ
generated high molecular weight Keap1 forms, which were identified as K-48-linked
polyubiquitin conjugates by immunoblotting and liquid chromatography MS-MS. Keap1
polyubiquitination coincided with Nrf2 stabilization and nuclear accumulation. In
contrast, BMCC did not induce Keap1 polyubiquitination. Our results suggest that
Nrf2 activation is regulated through the polyubiquitination of Keap1, which in
turn is triggered by specific patterns of electrophile modification of the Keap1
central linker domain. These results suggest that Keap1 adduction triggers a
switching of Cul3-dependent ubiquitination from Nrf2 to Keap1, leading to Nrf2

PMID: 15985429 [PubMed - indexed for MEDLINE]

77. Toxicol Appl Pharmacol. 2010 Apr 1;244(1):43-56. Epub 2009 Jul 29.

Nrf2 protects against airway disorders.

Cho HY, Kleeberger SR.

Laboratory of Respiratory Biology, National Institute of Environmental Health
Sciences, National Institutes of Health, Building 101, MD D-201, 111 T.W.
Alexander Drive, Research Triangle Park, NC 27709, USA.

Nuclear factor-erythroid 2 related factor 2 (Nrf2) is a ubiquitous master
transcription factor that regulates antioxidant response elements (AREs)-mediated
expression of antioxidant enzyme and cytoprotective proteins. In the unstressed
condition, Kelch-like ECH-associated protein 1 (Keap1) suppresses cellular Nrf2
in cytoplasm and drives its proteasomal degradation. Nrf2 can be activated by
diverse stimuli including oxidants, pro-oxidants, antioxidants, and
chemopreventive agents. Nrf2 induces cellular rescue pathways against oxidative
injury, abnormal inflammatory and immune responses, apoptosis, and
carcinogenesis. Application of Nrf2 germ-line mutant mice has identified an
extensive range of protective roles for Nrf2 in experimental models of human
disorders in the liver, gastrointestinal tract, airway, kidney, brain,
circulation, and immune or nerve system. In the lung, lack of Nrf2 exacerbated
toxicity caused by multiple oxidative insults including supplemental respiratory
therapy (e.g., hyperoxia, mechanical ventilation), cigarette smoke, allergen,
virus, bacterial endotoxin and other inflammatory agents (e.g., carrageenin),
environmental pollution (e.g., particles), and a fibrotic agent bleomycin.
Microarray analyses and bioinformatic studies elucidated functional AREs and
Nrf2-directed genes that are critical components of signaling mechanisms in
pulmonary protection by Nrf2. Association of loss of function with promoter
polymorphisms in NRF2 or somatic and epigenetic mutations in KEAP1 and NRF2 has
been found in cohorts of patients with acute lung injury/acute respiratory
distress syndrome or lung cancer, which further supports the role for NRF2 in
these lung diseases. In the current review, we address the role of Nrf2 in
airways based on emerging evidence from experimental oxidative disease models and
human studies.

PMID: 19646463 [PubMed - indexed for MEDLINE]

78. Molecules. 2010 Oct 20;15(10):7266-91.

The Nrf2 system as a potential target for the development of indirect

Jung KA, Kwak MK.

College of Pharmacy, Yeungnam University, Gyeongsan, Gyeongsangbuk-do 712-749,

Oxidative stress causes damage to multiple cellular components such as DNA,
proteins, and lipids, and is implicated in various human diseases including
cancer, neurodegeneration, inflammatory diseases, and aging. In response to
oxidative attack, cells have developed an antioxidant defense system to maintain
cellular redox homeostasis and to protect cells from damage. The thiol-containing
small molecules (e.g. glutathione), reactive oxygen species-inactivating enzymes
(e.g. glutathione peroxidase), and phase 2 detoxifying enzymes (e.g. NAD(P)H:
quinine oxidoreductase 1 and glutathione-S-transferases) are members of this
antioxidant system. NF-E2-related factor 2 (Nrf2) is a CNC-bZIP transcription
factor which regulates the basal and inducible expression of a wide array of
antioxidant genes. Following dissociation from the cytosolic protein Keap1, a
scaffolding protein which binds Nrf2 and Cul3 ubiquitin ligase for proteasome
degradation, Nrf2 rapidly accumulates in the nucleus and transactivates the
antioxidant response element in the promoter region of many antioxidant genes.
The critical role of Nrf2 has been demonstrated by various animal studies showing
that mice with a targeted disruption of the nrf2 gene are prone to develop
lesions in response to environmental toxicants/carcinogens, drugs, and
inflammatory insults. In this review, we discuss the role of the Nrf2 system,
with particular focus on Nrf2-controlled target genes and the potential
pleiotropic effects of Nrf2 activation of indirect antioxidants.

PMID: 20966874 [PubMed - indexed for MEDLINE]

79. Free Radic Biol Med. 2004 Oct 15;37(8):1152-9.

Human glutamate cysteine ligase gene regulation through the electrophile response

Dickinson DA, Levonen AL, Moellering DR, Arnold EK, Zhang H, Darley-Usmar VM,
Forman HJ.

Department of Environmental Health Sciences, University of Alabama at Birmingham,
Birmingham, AL 35294, USA.

Glutathione (GSH) is the primary nonprotein thiol in the cell. It has many
important roles in cell function, including regulating redox-dependent signal
transduction pathways. The content of GSH within the cell varies with stress. In
many cases, a process involving GSH synthesis results in adaptation to subsequent
stressors. Sustained increases in GSH content are controlled primarily through
induction of two genes, Gclc and Gclm, leading to the synthesis of the
rate-limiting enzyme for GSH synthesis, glutamate cysteine ligase. Each of these
genes in humans has a number of putative enhancer elements in their promoters.
Overall, the most important element in both Gclc and Gclm expression is the
electrophile response element. We review the evidence that has led to this
conclusion and the implications for the redox-dependent regulation of this
critical intracellular antioxidant.

PMID: 15451055 [PubMed - indexed for MEDLINE]

80. J Biol Chem. 2005 Apr 29;280(17):16891-900. Epub 2005 Feb 24.

Bach1 competes with Nrf2 leading to negative regulation of the antioxidant
response element (ARE)-mediated NAD(P)H:quinone oxidoreductase 1 gene expression
and induction in response to antioxidants.

Dhakshinamoorthy S, Jain AK, Bloom DA, Jaiswal AK.

Department of Pharmacology, Baylor College of Medicine, Houston, Texas 77030,

The antioxidant response element (ARE) and Nrf2 are known to regulate the
expression and coordinated induction of genes encoding detoxifying enzymes
including NAD(P)H:quinone oxidoreductase1 (NQO1) in response to antioxidants. In
this report, we demonstrate that overexpression of the transcription factor Bach1
in Hep-G2 cells negatively regulated NQO1 gene expression and induction in
response to antioxidant t-BHQ. Bandshift and supershift assays revealed that
Bach1 binds to the ARE as a heterodimer with small Maf proteins but not as a
homodimer or heterodimer with Nrf2. The transfection and ChIP assays revealed
that Bach1 and Nrf2 competed with each other to regulate ARE-mediated gene
expression. Heme, a negative regulator of Bach1 relieved the Bach1 repression of
NQO1 gene expression in transfected cells. The transcription of Bach1 and Nrf2
did not change in response to t-BHQ. Immunofluorescence assays and Western blot
analysis revealed that both Bach1 and Nrf2 localized in the cytoplasm and nucleus
of the untreated cells. The treatment of cells with t-BHQ resulted in the nuclear
accumulation of both Bach1 and Nrf2. Interestingly, the t-BHQ-induced nuclear
accumulation of Bach1 was significantly delayed over that of Nrf2. These results
led to the conclusion that a balance of Nrf2 versus Bach1 inside the nucleus
influences up- or down-regulation of ARE-mediated gene expression. The results
further suggest that antioxidant-induced delayed accumulation of Bach1
contributes to the down-regulation of ARE-regulated genes, presumably to reduce
the antioxidant enzymes to normal levels.

PMID: 15734732 [PubMed - indexed for MEDLINE]

81. Biochem J. 2006 May 1;395(3):599-609.

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

Vollrath V, Wielandt AM, Iruretagoyena M, Chianale J.

Department of Gastroenterology, School of Medicine, Catholic University of Chile,
Casilla 114-D, Santiago, Chile.

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, beta-NF
(beta-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.

PMCID: PMC1462684
PMID: 16426233 [PubMed - indexed for MEDLINE]

82. Annu Rev Pharmacol Toxicol. 2003;43:233-60. Epub 2002 Jan 10.

Regulatory mechanisms controlling gene expression mediated by the antioxidant
response element.

Nguyen T, Sherratt PJ, Pickett CB.

Schering-Plough Research Institute, Kenilworth, New Jersey 07033, USA.

The expression of genes encoding antioxidative and Phase II detoxification
enzymes is induced in cells exposed to electrophilic compounds and phenolic
antioxidants. Induction of these enzymes is regulated at the transcriptional
level and is mediated by a specific enhancer, the antioxidant response element or
ARE, found in the promoter of the enzyme's gene. The transcription factor Nrf2
has been implicated as the central protein that interacts with the ARE to
activate gene transcription constitutively or in response to an oxidative stress
signal. This review focuses on the molecular mechanisms whereby the
transcriptional activation mediated by the interaction between the ARE and
NF-E2-related factor 2 (Nrf2) is regulated. Recent studies suggest that the
sequence context of the ARE, the nature of the chemical inducers, and the cell
type are important for determining the activity of the enhancer in a particular

PMID: 12359864 [PubMed - indexed for MEDLINE]

83. Mol Nutr Food Res. 2008 Jun;52 Suppl 1:S128-38.

Direct and indirect antioxidant properties of inducers of cytoprotective

Dinkova-Kostova AT, Talalay P.

Division of Clinical Pharmacology, Department of Medicine, The Johns Hopkins
University School ofMedicine, Baltimore, MD, USA.

Cellular protection against oxidative and electrophile toxicities is provided by
two types of small-molecule antioxidants: (i) direct antioxidants, which are
redox active, short-lived, are sacrificed in the process of their antioxidant
actions and need to be replenished or regenerated, and may evoke pro-oxidant
effects; and (ii) indirect antioxidants, that may or may not be redox active.
Indirect antioxidants activate the Keap1/Nrf2/ARE pathway resulting in
transcriptional induction of a battery of cytoprotective proteins (also known as
phase 2 enzymes) that act catalytically, are not consumed, have long half-lives,
and are unlikely to evoke pro-oxidant effects. These protective systems are
involved in a complex functional interplay, such that many cytoprotective
proteins participate in the synthesis and/or regeneration of direct antioxidants,
whereas some direct antioxidants are required for the catalytic functions of
cytoprotective proteins. Importantly, many inducers of cytoprotective proteins
have been isolated from edible plants, e. g., sulforaphane from broccoli and
curcumin from turmeric. Both are pleiotropic agents with multiple biological
activities that could collectively contribute to their protective effects in
various animal studies, including models of carcinogenesis, hypertension,
neuronal and retinal damage. In addition to inducing cytoprotective proteins,
molecules like curcumin which contain Michael acceptor functionalities (olefins
or acetylenes conjugated to electron withdrawing groups) and phenolic hydroxyl
groups can scavenge directly and potently oxygen- and nitrogen-centered reactive
intermediates. Such bifunctional antioxidants can play a dual protective role by:
(i) scavenging hazardous oxidants directly and instantaneously, and (ii) inducing
cytoprotective enzymes that in turn function to resolve the consequences of
hazardous processes that are already in progress, and to ensure long-term
protection against subsequent challenges.

PMID: 18327872 [PubMed - indexed for MEDLINE]

84. Proc Natl Acad Sci U S A. 2001 Mar 13;98(6):3410-5.

Sensitivity to carcinogenesis is increased and chemoprotective efficacy of enzyme
inducers is lost in nrf2 transcription factor-deficient mice.

Ramos-Gomez M, Kwak MK, Dolan PM, Itoh K, Yamamoto M, Talalay P, Kensler TW.

Department of Environmental Health Sciences, The Johns Hopkins University,
Baltimore, MD 21205, USA.

Comment in:
    Proc Natl Acad Sci U S A. 2001 Mar 13;98(6):2941-3.

Induction of phase 2 enzymes, which neutralize reactive electrophiles and act as
indirect antioxidants, appears to be an effective means for achieving protection
against a variety of carcinogens in animals and humans. Transcriptional control
of the expression of these enzymes is mediated, at least in part, through the
antioxidant response element (ARE) found in the regulatory regions of their
genes. The transcription factor Nrf2, which binds to the ARE, appears to be
essential for the induction of prototypical phase 2 enzymes such as glutathione
S-transferases (GSTs) and NAD(P)H:quinone oxidoreductase (NQO1). Constitutive
hepatic and gastric activities of GST and NQO1 were reduced by 50-80% in
nrf2-deficient mice compared with wild-type mice. Moreover, the 2- to 5-fold
induction of these enzymes in wild-type mice by the chemoprotective agent
oltipraz, which is currently in clinical trials, was almost completely abrogated
in the nrf2-deficient mice. In parallel with the enzymatic changes,
nrf2-deficient mice had a significantly higher burden of gastric neoplasia after
treatment with benzo[a]pyrene than did wild-type mice. Oltipraz significantly
reduced multiplicity of gastric neoplasia in wild-type mice by 55%, but had no
effect on tumor burden in nrf2-deficient mice. Thus, Nrf2 plays a central role in
the regulation of constitutive and inducible expression of phase 2 enzymes in
vivo and dramatically influences susceptibility to carcinogenesis. Moreover, the
total loss of anticarcinogenic efficacy of oltipraz in the nrf2-disrupted mice
highlights the prime importance of elevated phase 2 gene expression in
chemoprotection by this and similar enzyme inducers.

PMID: 11248092 [PubMed - indexed for MEDLINE]

85. Mol Nutr Food Res. 2011 Mar 24. doi: 10.1002/mnfr.201100115. [Epub ahead of

Coffees rich in chlorogenic acid or N-methylpyridinium induce chemopreventive
phase II-enzymes via the Nrf2/ARE pathway in vitro and in vivo.

Boettler U, Volz N, Pahlke G, Teller N, Kotyczka C, Somoza V, Stiebitz H, Bytof
G, Lantz I, Lang R, Hofmann T, Marko D.

University of Vienna, Department of Food Chemistry and Toxicology, Vienna,

Recently, the coffee constituents 5-O-caffeoylquinic acid (CGA) and
N-methylpyridinium (NMP) were identified as inducers of the
Nrf2/antioxidant-response element (ARE) detoxifying pathway under cell-culture
condition. To study the impact of CGA and NMP on the Nrf2-activating properties
of a complex coffee beverage, two different model coffees were generated by
variation of the roasting conditions: a low-roast coffee rich in CGA and a
heavy-roast low in CGA but containing high levels of NMP. Activation of the
Nrf2/antioxidant-response element pathway was monitored in vitro and in vivo.

PMID: 21448860 [PubMed - as supplied by publisher]

86. Am J Pathol. 2008 Jun;172(6):1529-41. Epub 2008 May 5.

Endogenous hydrogen peroxide regulates glutathione redox via nuclear factor
erythroid 2-related factor 2 downstream of phosphatidylinositol 3-kinase during
muscle differentiation.

Ding Y, Choi KJ, Kim JH, Han X, Piao Y, Jeong JH, Choe W, Kang I, Ha J, Forman
HJ, Lee J, Yoon KS, Kim SS.

Department of Biochemistry and Molecular Biology, School of Medicine, Kyung Hee
University, Seoul 130-701, Korea.

We reported previously that endogenous reactive oxygen species (ROS) function as
myogenic signaling molecules. It has also been determined that excess ROS induce
electrophile-response element (EpRE)-driven gene expression via activation of
nuclear factor erythroid 2-related factor 2 (Nrf2). Nonetheless, the relationship
between the metabolism of ROS (eg, H(2)O(2)) through glutathione (GSH)
up-regulation, GSH-dependent reduction of H(2)O(2), and Nrf2-dependent gene
regulation is not well established. Therefore, we attempted to determine whether
H(2)O(2) controls the intracellular GSH redox state via the
Nrf2-glutamate-cysteine ligase (GCL)/glutathione reductase (GR)-GSH signaling
pathway. In our experiments, enhanced H(2)O(2) generation was accompanied by an
increase in both total GSH levels and the GSH/GSSG ratio during muscle
differentiation. Both GCL and GR transcriptional expression levels were markedly
increased during muscle differentiation but reduced by catalase treatment. Nrf2
protein expression and nuclear translocation increased during myogenesis. The
inhibition of GCL, GR, and Nrf2 both by inhibitors and by RNA interference
blocked muscle differentiation. Phosphatidylinositol 3-kinase regulated the
expression of the GCL C (a catalytic subunit) and GR genes via the induction of
Nrf2 nuclear translocation and expression. In conclusion, endogenous H(2)O(2)
generated during muscle differentiation not only functions as a signaling
molecule, but also regulates the GSH redox state via activation of the
Nrf2-GCL/GR-GSH signaling pathway downstream of phosphatidylinositol 3-kinase.

PMCID: PMC2408414
PMID: 18458092 [PubMed - indexed for MEDLINE]

87. Antioxid Redox Signal. 2006 Jan-Feb;8(1-2):88-98.

Role of the Nrf2-mediated signaling pathway as a negative regulator of
inflammation: implications for the impact of particulate pollutants on asthma.

Li N, Nel AE.

Division of Clinical Immunology and Allergy, Department of Medicine, University
of California, Los Angeles, California 90095, USA.

Particulate matter (PM) is an environmental factor that may contribute to the
exacerbation and possibly the development of asthma. PM contain redox-active
chemicals and transition metals which generate reactive oxygen species (ROS).
Excessive ROS can induce oxidative stress, which proceeds in hierarchical fashion
to generate cellular responses. The most sensitive cellular response to mild
oxidative stress is the activation of antioxidant and phase II enzymes (tier 1).
If this protection fails, further increase of oxidative stress can induce
inflammation (tier 2) and cell death (tier 3). Tier 1 antioxidant defenses are
critical for protecting against airway inflammation and asthma. The expression of
these antioxidant enzymes is regulated by the transcription factor, Nrf2. In
response to oxidative stress, Nrf2 escapes from Keap1-mediated proteasomal
degradation resulting in prolonged protein half-life and its nuclear
accumulation. Nrf2 interacts with the antioxidant response element (ARE) in the
promoters of phase II enzyme genes, leading to their transcriptional activation.
Several phase II expression polymorphisms are associated with an increased risk
of asthma. The indispensable role of Nrf2 in tier-1 oxidative stress response
suggests that polymorphisms of Nrf2-regulated genes may be useful susceptibility
markers for asthma. Moreover, chemopreventive Nrf2 inducers may be used for
treating PM-exacerbated asthma.

PMID: 16487041 [PubMed - indexed for MEDLINE]

88. Toxicol Sci. 2007 Mar;96(1):30-9. Epub 2006 Nov 16.

Reduction in antioxidant defenses may contribute to ochratoxin A toxicity and

Cavin C, Delatour T, Marin-Kuan M, Holzhäuser D, Higgins L, Bezençon C, Guignard
G, Junod S, Richoz-Payot J, Gremaud E, Hayes JD, Nestler S, Mantle P, Schilter B.

Quality and Safety Department, Nestlé Research Center, CH-1000 Lausanne 26,

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

PMID: 17110534 [PubMed - indexed for MEDLINE]

89. Mol Nutr Food Res. 2008 Jun;52 Suppl 1:S84-94.

Molecular mechanisms of natural products in chemoprevention: induction of
cytoprotective enzymes by Nrf2.

Eggler AL, Gay KA, Mesecar AD.

The Center for Pharmaceutical Biotechnology and Department of Medicinal Chemistry
and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago,
Chicago, IL 60607, USA.

Cancer chemoprevention involves the use of natural or synthetic compounds to
reduce the risk of developing cancer. One of the potential strategies for
preventing cancer in the human population is to use food-based natural products
to induce cytoprotective enzymes, such as NAD(P)H:quinone oxidoreductase 1,
glutathione S-transferase, superoxide dismutase, and heme oxygenase-1. The
regulatory regions of these inducible genes contain the antioxidant response
element (ARE), which is activated upon binding of the nuclear factor E2-related
protein 2 (Nrf2) transcription factor protein. Nrf2 has been shown to be
essential in the upregulation of these genes in response to oxidative stress and
treatment with certain dietary phytochemicals. This review presents the current
body of knowledge regarding the molecular mechanisms of Nrf2 regulation, and
highlights the need for future investigations into how these mechanisms apply to
natural product inducers of cytoprotective enzymes.

PMID: 18435489 [PubMed - indexed for MEDLINE]

90. Biochemistry. 1993 Sep 7;32(35):9080-93.

Factors participating in the liver-specific expression of the human
apolipoprotein A-II gene and their significance for transcription.

Cardot P, Chambaz J, Kardassis D, Cladaras C, Zannis VI.

Department of Medicine, Housman Medical Research Center, Boston University
Medical Center, Massachusetts 02118.

We have shown previously that the hepatic and intestinal transcription of the
human apolipoprotein A-II (apoA-II) gene in cell cultures is controlled by a
complex set of regulatory elements A-N [Chambaz, et al. (1991) J. Biol. Chem.
266, 11676-11685; Cardot, et al. (1991) J. Biol. Chem. 266, 24460-24470]. In the
present communication, we have assessed the functional importance of each of the
regulatory elements. In addition, we have used DNA binding and competition assays
and protein fractionation to identify the hepatic nuclear activities which are
involved in the regulation of the human apoA-II gene. Such activities may be of
general importance for the regulation of liver-specific genes. The DNA binding
and competition analysis showed that the regulatory elements M, D, and F bind new
activities which have not been identified in apolipoprotein or other
liver-specific promoters. These activities have been designated AIIM1 and AIIM2
for element M, AIID1 and AIID2 for element D, and AIIF2 for element F. The
activity AIIM2 is present in liver, but absent in CaCo-2 cells. A set of
regulatory elements binds activities which resemble liver-enriched or ubiquitous
factors previously shown to play important roles in the regulation of their
target genes. Thus, element I binds to activities related to NF1, and elements L,
C, D, G, AB, and F bind to C/EBP alpha as well as other heat-stable activities.
The affinity of the bacterially expressed C/EBP alpha for the various apoA-II
regulatory regions follows the order: AIIL approximately AIIC > AIID > AIIF >
AIIG > AIIAB. Protein fractionation showed that element J binds at least three
hepatic nuclear activities and is also recognized by members of the nuclear
receptor family, HNF4, EAR2, EAR3, and ARP1. Another liver-enriched factor, HNF1,
was shown previously to bind to element H. Despite the importance of HNF1, HNF4,
NF1, and C/EBP alpha in the regulation of numerous other target genes, deletion
of the HNF1, NF1, and HNF4 and several C/EBP binding sites did not drastically
affect the hepatic transcription of the apoA-II gene. Rather, the hepatic and
intestinal transcription is affected severely by deletion of elements A, B, K, L,
and N. In addition, the intestinal transcription is affected by deletion of
elements C, J, and M. The in vivo physiological importance of these elements will
require analysis of their function in transgenic animals. This analysis
establishes the organization of several nuclear activities on the human apoA-II

PMID: 8369280 [PubMed - indexed for MEDLINE]

91. Mol Cells. 2009 Mar 31;27(3):279-82. Epub 2009 Mar 19.

Transcriptional regulation of the AP-1 and Nrf2 target gene sulfiredoxin.

Soriano FX, Baxter P, Murray LM, Sporn MB, Gillingwater TH, Hardingham GE.

Centre for Integrative Physiology, University of Edinburgh, Edinburgh EH89XD, UK.

"Two-cysteine" peroxiredoxins are antioxidant enzymes that exert a cytoprotective
effect in many models of oxidative stress. However, under highly oxidizing
conditions they can be inactivated through hyperoxidation of their peroxidatic
active site cysteine residue. Sulfiredoxin can reverse this hyperoxidation, thus
reactivating peroxiredoxins. Here we review recent investigations that have shed
further light on sulfiredoxin's role and regulation. Studies have revealed
sulfiredoxin to be a dynamically regulated gene whose transcription is induced by
a variety of signals and stimuli. Sulfiredoxin expression is regulated by the
transcription factor AP-1, which mediates its up-regulation by synaptic activity
in neurons, resulting in protection against oxidative stress. Furthermore,
sulfiredoxin has been identified as a new member of the family of genes regulated
by nuclear factor erythroid 2-related factor (Nrf2) via a conserved Aáë-acting
antioxidant response element (ARE). As such, sulfiredoxin is likely to contribute
to the net antioxidative effect of small molecule activators of Nrf2. As
discussed here, the proximal AP-1 site of the sulfiredoxin promoter is embedded
within the ARE, as is common with Nrf2 target genes. Other recent studies have
shown that sulfiredoxin induction via Nrf2 may form an important part of the
protective response to oxidative stress in the lung, preventing peroxiredoxin
hyperoxidation and, in certain cases, subsequent degradation. We illustrate here
that sulfiredoxin can be rapidly induced in vivo by administration of CDDO-TFEA,
a synthetic triterpenoid inducer of endogenous Nrf2, which may offer a way of
reversing peroxiredoxin hyperoxidation in vivo following chronic or acute
oxidative stress.

PMCID: PMC2837916
PMID: 19326073 [PubMed - indexed for MEDLINE]

92. J Biol Chem. 2007 Aug 24;282(34):24670-8. Epub 2007 Jul 3.

The p65 isoform of Nrf1 is a dominant negative inhibitor of ARE-mediated

Wang W, Kwok AM, Chan JY.

Department of Pathology, University of California, Irvine, Irvine, California
92697, USA.

Oxidative stress-responsive transcription is regulated in part through cis-active
sequences known as antioxidant response elements (ARE). Activation through the
ARE involves members of the CNC-subfamily of basic leucine zipper proteins
including Nrf1 and Nrf2. In particular, Nrf2 has been shown to coordinate
induction of genes encoding antioxidant and phase 2 metabolizing enzymes in
response to stimulation with electrophilic compounds and exposure to xenobiotics.
Here we show that the 65-kDa isoform of the Nrf1 gene functions as a repressor of
Nrf2. Transient expression of p65Nrf1 suppressed Nrf2-mediated activation of
ARE-dependent reporter genes in cells. Induction of endogenous ARE-genes is
blocked in Hepa1c1c7 cells stably expressing p65Nrf1 leading to increased cell
death. Consistent with these findings, electrophilic activation of ARE-gene
expression is augmented by loss of p65Nrf1 function in Nrf1(-/-) fibroblasts, and
the protective effects of oxidative preconditioning and ARE-gene expression are
blocked in Nrf1(-/-) cells stably expressing p65Nrf1. Gel shift experiments
demonstrated that p65Nrf1 binds the antioxidant response element as a heterodimer
with small-Maf protein. Immunoprecipitation studies demonstrated that p65Nrf1
competes with Nrf2 for interaction with small-Maf protein and binding to the
antioxidant response element in vivo. Together, these results demonstrate that
p65Nrf1 has the potential to play an important role in modulating the response to
oxidative stress by functioning as a transdominant repressor of Nrf2-mediated
activation of ARE-dependent gene transcription.

PMID: 17609210 [PubMed - indexed for MEDLINE]

93. J Nutr Biochem. 2010 Jul 22. [Epub ahead of print]

Coffee constituents as modulators of Nrf2 nuclear translocation and ARE
(EpRE)-dependent gene expression.

Boettler U, Sommerfeld K, Volz N, Pahlke G, Teller N, Somoza V, Lang R, Hofmann
T, Marko D.

Department of Food Chemistry and Toxicology, University of Vienna, Währinger Str.
38, A-1090 Vienna, Austria; Institute of Applied Biosciences, Section of Food
Toxicology, Universität Karlsruhe (TH), Adenauerring 20, 76131 Karlsruhe,

Oxidative cellular stress initiates Nrf2 translocation into the nucleus, thus
inducing antioxidant response element (ARE)-mediated expression of Phase II
enzymes involved in detoxification and antioxidant defence. We investigated
whether coffee extracts (CEs) of different proveniences and selected constituents
have an impact on the Nrf2/ARE pathway in human colon carcinoma cells (HT29).
Assessed as increased nuclear Nrf2 protein, Nrf2 nuclear translocation was
modulated by different CEs as observed by Western blot analysis. In addition to
the known Nrf2 activator 5-O-caffeoylquinic acid (CGA), pyridinium derivatives
like the N-methylpyridinium ion (NMP) were identified as potent activators of
Nrf2 nuclear translocation and ARE-dependent gene expression of selected
antioxidative Phase II enzymes in HT29. Thereby, the substitution pattern at the
pyridinium core structure determined the impact on Nrf2-signalling. In contrast,
trigonelline was found to interfere with Nrf2 activation, effectively suppressing
the NMP-mediated induction of Nrf2/ARE-dependent gene expression. In conclusion,
several coffee constituents, partly already present in the raw material as well
as those generated during the roasting process, contribute to the
Nrf2-translocating properties of consumer-relevant coffee. A fine tuning in the
degradation/formation of activating and deactivating constituents of the Nrf2/ARE
pathway during the roasting process appears to be critical for the
chemopreventive properties of the final coffee product.

PMID: 20655719 [PubMed - as supplied by publisher]

94. J Biol Chem. 2008 Jan 25;283(4):2021-30. Epub 2007 Nov 26.

Deletion of the selenocysteine tRNA gene in macrophages and liver results in
compensatory gene induction of cytoprotective enzymes by Nrf2.

Suzuki T, Kelly VP, Motohashi H, Nakajima O, Takahashi S, Nishimura S, Yamamoto

Exploratory Research for Advanced Technology Environmental Response Project,
Center for Tsukuba Advanced Research Alliance, University of Tsukuba, Japan.

The selenocysteine tRNA (tRNA(Sec)) molecule is the sight of synthesis for the
amino acid selenocysteine and the adaptor for its translational insertion into
selenoprotein enzymes, the majority of which contribute to cellular redox
homeostasis. To examine the consequences of selenoprotein depletion on the
oxidative environment of the cell, we generated a conditional knock-out mouse for
the tRNA(Sec) gene (Trsp). Deletion of Trsp in either macrophages or liver
elevated oxidative stress and activated the transcriptional induction of
cytoprotective antioxidant and detoxification enzyme genes, including glutathione
S-transferase P1 and NAD(P)H:quinone oxidoreductase 1, and other well known
target genes of the transcription factor Nrf2 (NF-E2-related factor 2).
Simultaneous disruption of Trsp and Nrf2 severely compromised the cytoprotective
response. Double knock-out macrophages displayed reduced viability, elevated
oxidative stress, and increased susceptible to hydrogen peroxide treatment
compared with deletion of either gene alone. Mice carrying a liver-specific
deletion of Trsp on an Nrf2-null background experienced hepatocellular apoptosis
and displayed a severely reduced survival rate compared with loss of Trsp alone.
Our results thus demonstrate that reduced selenoprotein activity is
counterbalanced by an Nrf2-mediated cytoprotective response, which is essential
for maintaining cellular redox homeostasis and viability.

PMID: 18039655 [PubMed - indexed for MEDLINE]

95. J Biol Chem. 2005 Jun 17;280(24):22925-36. Epub 2005 Apr 19.

Induction of the Nrf2-driven antioxidant response confers neuroprotection during
mitochondrial stress in vivo.

Shih AY, Imbeault S, Barakauskas V, Erb H, Jiang L, Li P, Murphy TH.

Kinsmen Laboratory, Department of Psychiatry, Brain Research Center, Vancouver,

NF-E2 related factor (Nrf2) controls a pleiotropic cellular defense, where
multiple antioxidant/detoxification pathways are up-regulated in unison. Although
small molecule inducers of Nrf2 activity have been reported to protect neurons in
vitro, whether similar pathways can be accessed in vivo is not known. We have
investigated whether in vivo toxicity of the mitochondrial complex II inhibitor
3-nitropropionic acid (3-NP) can be attenuated by constitutive and inducible Nrf2
activity. The absence of Nrf2 function in Nrf2(-/-) mice resulted in 3-NP
hypersensitivity that became apparent with time and increasing dose, causing
motor deficits and striatal lesions on a more rapid time scale than identically
treated Nrf2(+/+) and Nrf2(+/-) controls. Striatal succinate dehydrogenase
activity, the target of 3-NP, was inhibited to the same extent in all genotypes
by a single acute dose of 3-NP, suggesting that brain concentrations of 3-NP were
similar. Dietary supplementation with the Nrf2 inducer tert-butylhydroquinone
attenuated 3-NP toxicity in Nrf2(+/-) mice, but not Nrf2(-/-), confirming the
Nrf2-specific action of the inducer in vivo. Increased Nrf2 activity alone was
sufficient to protect animals from 3-NP toxicity because intrastriatal
adenovirus-mediated Nrf2 overexpression significantly reduced lesion size
compared with green fluorescent protein overexpressing controls. In cultured
astrocytes, 3-NP was found to increase Nrf2 activity leading to antioxidant
response element-dependent gene expression providing a potential mechanism for
the increased sensitivity of Nrf2(-/-) animals to 3-NP toxicity in vivo. We
conclude that Nrf2 may underlie a feedback system limiting oxidative load during
chronic metabolic stress.

PMID: 15840590 [PubMed - indexed for MEDLINE]

96. Cancer Res. 2004 Nov 1;64(21):7893-909.

BRCA1 induces antioxidant gene expression and resistance to oxidative stress.

Bae I, Fan S, Meng Q, Rih JK, Kim HJ, Kang HJ, Xu J, Goldberg ID, Jaiswal AK,
Rosen EM.

Department of Oncology, Lombardi Cancer Center, Georgetown University,
Washington, DC 20057-1469, USA.

Mutations of the breast cancer susceptibility gene 1 (BRCA1), a tumor suppressor,
confer an increased risk for breast, ovarian, and prostate cancers. To
investigate the function of the BRCA1 gene, we performed DNA microarray and
confirmatory reverse transcription-PCR analyses to identify BRCA1-regulated gene
expression changes. We found that BRCA1 up-regulates the expression of multiple
genes involved in the cytoprotective antioxidant response, including glutathione
S-transferases, oxidoreductases, and other antioxidant genes. Consistent with
these findings, BRCA1 overexpression conferred resistance while BRCA1 deficiency
conferred sensitivity to several different oxidizing agents (hydrogen peroxide
and paraquat). In addition, in the setting of oxidative stress (due to hydrogen
peroxide), BRCA1 shifted the cellular redox balance to a higher ratio of reduced
to oxidized glutathione. Finally, BRCA1 stimulated antioxidant response
element-driven transcriptional activity and enhanced the activity of the
antioxidant response transcription factor nuclear factor erythroid-derived 2 like
2 [also called NRF2 (NFE2L2)]. The ability of BRCA1 to stimulate antioxidant
response element-dependent transcription and to protect cells against oxidative
stress was attenuated by inhibition of nuclear factor erythroid-derived 2 like 2.
These findings suggest a novel function for BRCA1, i.e., to protect cells against
oxidative stress. This function would be consistent with the postulated role of
BRCA1 as a caretaker gene in preserving genomic integrity.

PMID: 15520196 [PubMed - indexed for MEDLINE]

97. J Biol Chem. 2008 Nov 28;283(48):33554-62. Epub 2008 Sep 30.

Nrf1 and Nrf2 play distinct roles in activation of antioxidant response
element-dependent genes.

Ohtsuji M, Katsuoka F, Kobayashi A, Aburatani H, Hayes JD, Yamamoto M.

Graduate School of Comprehensive Human Sciences, Japan Science and Technology
Corp., 1-1-1 Tennoudai, Tsukuba 305-8577, Japan.

Nrf1 is a member of the vertebrate Cap'n'Collar (CNC) transcription factor family
that commonly contains a unique basic-leucine zipper domain. Among CNC family
members, Nrf2 is known to regulate a battery of antioxidant and
xenobiotic-metabolizing enzyme genes through the antioxidant response element
(ARE). Although Nrf1 has also been shown to bind the ARE, it is unclear whether
it plays a distinct role from Nrf2 in regulating genes with this element. To
address this issue in vivo, we generated mice bearing a hepatocyte-specific
disruption of the Nrf1 gene. AlthoughNrf2 knock-out mice did not exhibit liver
damage when they were maintained in an unstressed condition, hepatocyte-specific
deletion of Nrf1 caused liver damage resembling the human disease non-alcoholic
steatohepatitis. Gene expression analysis revealed that the disruption of Nrf1
causes stress that activates a number of ARE-driven genes in an Nrf2-dependent
manner, indicating that Nrf2 cannot compensate completely for loss of Nrf1
function in the liver. In contrast, expression of metallothionein-1 and -2 (MT1
and MT2) genes, each of which harbors at least one ARE in its regulatory region,
was decreased in the Nrf1-null mutant mice. Whereas Nrf1 and Nrf2 bound the MT1
ARE with comparable affinity, Nrf1 preferentially activated the reporter gene
expression through the MT1 ARE. This study has, thus, identified the first
ARE-dependent gene that relies exclusively on Nrf1, suggesting that it plays a
distinct functional role in regulating ARE-driven genes.

PMCID: PMC2662273
PMID: 18826952 [PubMed - indexed for MEDLINE]

98. Mol Pharmacol. 2006 May;69(5):1554-63. Epub 2006 Jan 31.

trans-Stilbene oxide induces expression of genes involved in metabolism and
transport in mouse liver via CAR and Nrf2 transcription factors.

Slitt AL, Cherrington NJ, Dieter MZ, Aleksunes LM, Scheffer GL, Huang W, Moore
DD, Klaassen CD.

Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas
Medical Center, 3901 Rainbow Boulevard, Kansas City, KS 66160-7417, USA.

trans-Stilbene oxide (TSO) induces drug metabolizing enzymes in rat and mouse
liver. TSO is considered a phenobarbital-like compound because it induces Cyp2B
mRNA expression in liver. Phenobarbital increases Cyp2B expression in liver via
activation of the constitutive androstane receptor (CAR). The purpose of this
study was to determine whether TSO induces gene expression in mouse liver via CAR
activation. TSO increased CAR nuclear localization in mouse liver, activated the
human Cyp2B6 promoter in liver in vivo, and activated a reporter plasmid that
contains five nuclear receptor 1 (NR1) binding sites in HepG2 cells. TSO
administration increased expression of Cyp2b10, NAD(P)H:quinone oxidoreductase
(Nqo1), epoxide hydrolase, heme oxygenase-1, UDP-glucuronosyl-transferase (Ugt)
1a6 and 2b5, and multidrug resistance-associated proteins (Mrp) 2 and 3 mRNA in
livers from male mice. Cyp2b10 and epoxide hydrolase induction by TSO was
decreased in livers from CAR-null mice, compared with wild-type mice, suggesting
CAR involvement. In contrast, TSO administration induced Nqo1 and Mrp3 mRNA
expression equally in livers from wild-type and CAR-null mice, suggesting that
TSO induces expression of some genes through a mechanism independent of CAR. TSO
increased nuclear staining of the transcription factor Nrf2 in liver, and
activated an antioxidant/electrophile response element luciferase reporter
construct that was transfected into HepG2 cells. In summary, in mice, TSO
increases Cyp2b10 and epoxide hydrolase expression in mice via CAR, and
potentially induces Nqo1 and Mrp3 expression via Nrf2. Moreover, our data
demonstrate that a single compound can activate both CAR and Nrf2 transcription
factors in liver.

PMID: 16449384 [PubMed - indexed for MEDLINE]


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