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When NRF2 talks, who's listening?

April 2010
Antioxidants and Redox Signaling 13(11):1649-63

April 2010
13(11):1649-63

DOI:10.1089/ars.2010.3216

Source
PubMed

Authors:

Nobunao Wakabayashi

Fred Hutch Cancer Center

Massachusetts Institute of Technology

University of Pittsburgh

Cincinnati Children's Hospital Medical Center

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Activation of the KEAP1-NRF2 signaling pathway is an adaptive response to environmental and endogenous stresses and serves to render animals resistant to chemical carcinogenesis and other forms of toxicity, whereas disruption of the pathway exacerbates these outcomes. This pathway, which can be activated by sulfhydryl-reactive, small-molecule pharmacologic agents, regulates the inducible expression of an extended battery of cytoprotective genes, often by direct binding of the transcription factor to antioxidant response elements in the promoter regions of target genes. However, it is becoming evident that some of the protective effects may be mediated indirectly through cross talk with additional pathways affecting cell survival and other aspects of cell fate. These interactions provide a multi-tiered, integrated response to chemical stresses. This review highlights recent observations on the molecular interactions and their functional consequences between NRF2 and the arylhydrocarbon receptor (AhR), NF-κB, p53, and Notch1 signaling pathways.

The interactions of the NRF2 and NF- j B target proteins HO-1 and iNOS and their products can regulate the activity of each other in macrophages to protect against an overabundance of NO. Excess NO acts as a signal to increase HO-1, which is able to scavenge NO and block the activity of iNOS to prevent further production.

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FORUM REVIEW ARTICLE

When NRF2 Talks, Who’s Listening?

Nobunao Wakabayashi,

1,2

Stephen L. Slocum,

3

John J. Skoko,

4

Soona Shin,

4

and Thomas W. Kensler

1–4

Abstract

Activation of the KEAP1-NRF2 signaling pathway is an adaptive response to environmental and endogenous

stresses and serves to render animals resistant to chemical carcinogenesis and other forms of toxicity, whereas

disruption of the pathway exacerbates these outcomes. This pathway, which can be activated by sulfhydryl-

reactive, small-molecule pharmacologic agents, regulates the inducible expression of an extended battery of

cytoprotective genes, often by direct binding of the transcription factor to antioxidant response elements in the

promoter regions of target genes. However, it is becoming evident that some of the protective effects may be

mediated indirectly through cross talk with additional pathways affecting cell survival and other aspects of cell

fate. These interactions provide a multi-tiered, integrated response to chemical stresses. This review highlights

recent observations on the molecular interactions and their functional consequences between NRF2 and the

arylhydrocarbon receptor (AhR), NF-kB, p53, and Notch1 signaling pathways. Antioxid. Redox Signal. 13, 1649–

1663.

Introduction

NRF2 (NF-E2-related factor 2, NFE2L2) is a transcrip-

tion factor that mediates a broad-based set of adaptive

responses to intrinsic and extrinsic cellular stresses (73). As

such, NRF2 inﬂuences sensitivity to physiologic and patho-

logic processes affected by oxidative and electrophilic stres-

ses, such as those imposed by inﬂammation and exposures to

environmental toxicants. Carcinogenesis, chronic obstructive

pulmonary disease, obesogenesis, and neurodegeneration are

known to be affected by the Nrf2 genotype in murine models

(73).

Molecular details of the NRF2 signaling pathway have

emerged over the past decade, as reviewed elsewhere in this

issue. Notably, deﬁnition of the interacting partners of NRF2,

such as KEAP1 (Kelch-like ECH-associated protein 1), and the

means by which they sense and transduce chemical signals of

stress are under intense investigation in many laboratories

(48). Characterization of the immediate downstream target

genes of NRF2 has also been well enumerated, although

features that deﬁne the tissue and cell-type speciﬁcity in re-

sponses are poorly understood. The gene-expression re-

sponses to activation of NRF2 signaling, mediated through

interactions of NRF2 and the antioxidant response element(s)

(ARE: 50-NTGAG=CNNNGC-30) in the regulatory domains of

its target genes, result in a distinctive cytoprotective response.

Beyond the now-seminal response of catalyzing the detoxiﬁ-

cation of carcinogens and other xenobiotics through conju-

gation and trapping processes, genomic analyses indicated

that gene families affected by Nrf2 (a) provide direct antiox-

idants; (b) encode enzymes that directly inactivate oxidants;

(c) increase levels of glutathione synthesis and regeneration;

(d) stimulate NADPH synthesis; (e) enhance toxin export

through the multidrug-response transporters; (f ) enhance the

recognition, repair, and removal of damaged proteins; (g)

elevate nucleotide excision repair; (h) regulate expression of

other transcription factors, growth factors and receptors, and

molecular chaperones; and (i) inhibit cytokine-mediated in-

ﬂammation (48, 73).

It also is clear, though, that these direct genomic responses

do not account for all of the actions of pharmacologic agents

that activate NRF2 signaling, nor do they explain the range of

stress-response phenotypes observed when components of

the pathway, notably, Nrf2 or Keap1, have been genetically

disrupted in mice (162). Analyses of global transcriptional

responses have hinted toward interactions between NRF2

signaling and other prominent signaling pathways. These

interactions can occur in multiple forms. Post-translational

modiﬁcations such as phosphorylation play a major role in the

regulation of gene expression and function. These covalent

modiﬁcations control intracellular distribution, transcrip-

tional activity, and stability of transcription factors, including

NRF2 (81, 143). Some transcription factors can antagonize

NRF2 either by competing for binding to AREs or by

1

Department of Pharmacology & Chemical Biology, University of Pittsburgh, Pittsburgh, Pennsylvania.

2

Department of Environmental Health Sciences and

3

Department of Biochemistry and Molecular Biology, Bloomberg School of Public

Health,

4

Department of Pharmacology and Molecular Sciences, School of Medicine, Johns Hopkins University, Baltimore, Maryland.

ANTIOXIDANTS & REDOX SIGNALING

Volume 13, Number 11, 2010

ªMary Ann Liebert, Inc.

DOI: 10.1089=ars.2010.3216

1649

inhibiting NRF2 through a physical association. Small MAF

proteins, BACH1, and the immediate early proteins c-FOS

and FRA1 can compete with NRF2 for binding to AREs (103,

151). Furthermore, several transcription factors, including

activating transcription factor 3, proliferator-activated recep-

tor (PPAR)g, and retinoic acid receptor ahave been reported

to form inhibitory complexes with NRF2 (6, 13, 59, 155, 168).

In this review, we focus on recent evidence for transcriptional

cross-talk between NRF2 and the arylhydrocarbon receptor

(AhR), NF-kB, p53, and Notch pathways, as summarized

schematically in Fig. 1.

NRF2 Interactions with AhR Signaling

The aryl hydrocarbon receptor is a ligand-activated mem-

ber of the bHLH=PAS (basic helix-loop-helix=Per-Arnt-Sim)

family of transcription factors that mediates the biologic and

toxic effects of its xenobiotic ligands (44). When bound by

polycyclic aromatic hydrocarbons such as dioxins (TCDD),

AhR translocates from the cytoplasm to the nucleus, hetero-

dimerizes with AhR nuclear translocator (ARNT), and

activates transcription through the xenobiotic-responsive

element (XRE). The XRE consists of a canonic motif of

50-TNGCGTG-30. After nuclear export, AhR is degraded through

the 26S proteasome pathway. It is well recognized that both

AhR and NRF2 signaling regulate the expression of genes

affecting the metabolism of xenobiotics. In some instances, the

response elements recognizing these transcription factors can

be found in the regulatory domains of the same target genes,

such as Nqo1 (104). Ma et al. (95) reported that the inducible

expression of NQO1 by the potent AhR ligand TCDD de-

pends on both AhR and NRF2. Genetic experiments using

Ahr-, Arnt-, or Nrf2-deﬁcient cells revealed that induction of

NQO1 by TCDD depended on the presence of AhR and

ARNT, whereas the basal and inducible expression required a

functional NRF2 pathway. Yaeger et al. (163) extended these

ﬁndings by using Nrf2-deﬁcient mice to demonstrate the re-

quirement of NRF2 for the induction of Nqo1,Ugt1a6, and

Gsta1 transcripts, among other genes, by TCDD in mouse

liver. AhR–Nrf2 compound null mutant mice respond to nei-

ther AhR ligands nor prototypical NRF2 activators with target

genes, such as Nqo1 (105). A functional collaboration between

these pathways is also highlighted by the earlier ﬁndings of

Talalay and colleagues (115), in which they described the roles

of ‘‘monofunctional’’ and ‘‘bifunctional’’ activators of NRF2

signaling. ‘‘Monofunctional’’ inducers affect only NRF2 sig-

naling. ‘‘Bifunctional inducers,’’ such as b-napthoﬂavone,

may interact with the AhR directly to activate AhR-regulated

genes, such as Cyp1a1,Cyp1a2, and Cyp1b1, and then undergo

transformation by these enzymes to reactive intermediates

that then trigger NRF2 signaling (see Fig. 2). In this way, a

comprehensive set of responses to xenobiotic challenges can

be mounted (79).

The mechanisms underlying the linkages between AhR and

NRF2 signaling need further investigation. Miao et al. (98)

demonstrated that Nrf2 gene transcription is directly modu-

lated by AhR activation. DNA sequence analyses of the mu-

rine Nrf2 promoter revealed an XRE located at 712 bp and

two additional XRE-like motifs located further upstream.

Direct binding of AhR to the Nrf2 promoter was observed.

Moreover, silencing of AhR expression with siRNA obviated

induction of Nrf2 mRNA by TCDD. NRF2 also autoregulates

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 its target

genes (83).

NRF2 also directly modulates AhR signaling, highlighting

bidirectional interactions of these pathways (138). Constitutive

expression of AhR was affected by Nrf2 genotype. Moreover,

a pharmacologic activator of NRF2 signaling, CDDO-Im

{1-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole}, in-

duced Ahr,Cyp1a1,andCyp1b1 transcription in Nrf2

þ=þ

mouse

embryonic ﬁbroblasts (MEFs) but not in Nrf2

-=-

MEFs. Reporter

analysis and chromatin immunoprecipitation assays revealed

that NRF2 directly binds to one ARE found in the 230 bp

region of the promoter of Ahr. Better understanding of this

bidirectional regulation of AhR and NRF2 pathways should

provide clues on the roles of NRF2 in complex diseases and in

uncharacterized phenotypes.

Cell-culture studies and knockout mouse models have

shown that, in addition to controlling the xenobiotic detoxi-

ﬁcation response, AhR activation leads to G

0

=G

1

arrest, di-

minished capacity for DNA replication, inhibition of cell

proliferation, and impaired cell differentiation. AhR may

function also as a tumor-suppressor gene that becomes si-

lenced during the process of tumor formation (32). Tran-

scriptional responses to ligand-dependent activation of AhR

signaling vary across cell types and by species, but consis-

tently inﬂuence cytochrome P450 and other monooxygenase

activities (17). Ligand-dependent and -independent AhR

signaling inﬂuences (a) positive regulation of transcription,

vascular development, organ morphogenesis, and metabolic

processes; (b) processes related to mRNA processing and

transport; (c) processes related to cell death, proliferation, and

regulation of apoptosis; (d) mitotic cell cycle; and (e) embry-

onic and other developmental processes (132).

FIG. 1. Possible means for regulation of cell survival and

other cell-fate responses through interactions of NRF2 with

additional cell-signaling pathways, including AhR, NF-jB,

p53, and Notch1. (For interpretation of the references to color

in this ﬁgure legend, the reader is referred to the web version

of this article at www.liebertonline.com=ars).

1650 WAKABAYASHI ET AL.

Ahr-null mice and Nrf2-null mice share some common

phenotypes. These transcription factors appear to function as

cell-survival factors; both types of knockout mice exhibit in-

creased sensitivity to infection (75, 147), carcinogenesis (32, 72,

117), altered responses to wounding (12, 18), and metabolic

syndrome (137, 144). Studies on responses to adipogenic

stimuli in vitro and high-fat stress diets in vivo have provided

some opportunities for evaluating the physiological interac-

tions between these pathways.

Shimba et al. (136) observed that TCDD treatment sup-

presses the conversion of 3T3-L1 cells into adipocytes. By

using MEF cell lines derived from Ahr-knockout mice,

Alexander et al. (5) reported that the AhR is a constitutive

inhibitor of triglyceride synthesis and an early regulator of

adipocyte differentiation. In addition, one of the phenotypes

of Ahr-null mice is transient fatty liver (133), implying an

in vivo regulatory role of AhR in the adipogenic process.

Shin et al. (138) postulated that NRF2 would inhibit adipo-

genesis through the interaction with the AhR pathway.

Nrf2

-=-

MEFs showed markedly accelerated adipogenesis on

stimulation, whereas Keap1

-=-

MEFs (which exhibit higher

NRF2 signaling) differentiated slowly compared with their

congenic wild-type MEFs. Ectopic expression of AhR and

dominant-positive Nrf2 in Nrf2

-=-

MEFs also substantially

delayed differentiation. To evaluate how NRF2 regulates

adipogenesis, differentiation markers involved at multiple

stages of adipogenesis were analyzed. CEBPs (CCAAT-

enhancer–binding proteins) and PPARs are the two families

of transcription factors that play critical roles in adipogen-

esis (159). CEBPband CEBPdfunction at an early phase of

the differentiation process by sensing adipogenic stimuli

and initiating expression of CEBPaand PPARg(122). CEBPa

and PPARgplay roles at a later stage by inducing and

maintaining expression of adipocyte-speciﬁc genes, such as

Fabp4. The elevation of CEBPbon adipogenic stimulation is

transient, whereas CEBPaand PPARgremain upregulated

for the duration of adipogenesis. Although the mechanism

by which AhR regulates adipogenesis has not been fully

characterized, recent work suggests that AhR affects dif-

ferentiation stages that follow CEBPbactivation (i.e., CEBPa

or PPARgupregulation. In 3T3-L1 preadipocytes, forced

expression of AhR resulted in lower induction of Fabp4 and

Cebpaupon differentiation than did that in control cells,

whereas the induction of Ppargwas not affected (136). Pparg

expression could be induced by differentiation in Ahr

-=-

MEFs, but levels were lower than those in Ahr

þ=þ

MEFs (5).

Similarly, mRNA levels of Cebpaand Fabp4 were higher in

Nrf2

-=-

MEFs than in Nrf2

þ=þ

MEFs, both before and after

differentiation, whereas induction of Pparg2was not af-

fected by the Nrf2 genotype. mRNA levels of Cebpbwere

lower in Nrf2

-=-

MEFs than in Nrf2

þ=þ

MEFs. In primary

Keap1

-=-

MEFs, disruption of Keap1 resulted in minimal in-

duction of Cebpa,Fabp4, and Pparg2upon differentiation.

CebpbmRNA levels remained higher in Keap1

-=-

MEFs than

in Keap1

þ=þ

MEFs, both before and after induction of adi-

pogenesis. Thus, NRF2 negatively modulates expression of

Cebpaand Pparg2but not Cebpbduring the course of adi-

pogenesis. The stages of differentiation that are affected by

NRF2 directly overlap with those affected by AhR, thereby

supporting the hypothesis that NRF2 inhibits adipogenesis

through cross talk with AhR signaling (see Fig. 3).

Treatment with CDDO-Im effectively prevented high-fat

diet–induced increases in body weight, adipose mass, and

hepatic lipid accumulation in Nrf2 wild-type mice but not in

Nrf2-disrupted mice (137). Wild-type mice on a high-fat diet

and treated with CDDO-Im exhibited higher oxygen con-

sumption and energy expenditure than did vehicle-treated

mice, whereas food intake was lower in CDDO-Im–treated

than in vehicle-treated mice. Levels of gene transcripts for

fatty acid synthesis enzymes were downregulated after

CDDO-Im treatment in the liver of wild-type mice. This in-

hibitory effect of CDDO-Im on lipogenic gene expression was

signiﬁcantly reduced in Nrf2-disrupted mice. However, the

extent to which this pharmacologic activation of Nrf2 to

produce an anti-obesogenic effect is mediated through AhR, if

at all, is unclear.

FIG. 2. Regulation of xenobiotic me-

tabolism by AhR and NRF2. Xenobiotics

are metabolized by cytochrome P450s

(CYPs) into intermediates that are reactive

or nonreactive. Reactive intermediates can

be metabolized by cytoprotective enzymes

into less-toxic and often readily excretable

products. When activated by a ligand,

AhR in the cytoplasm complexes with

ARNT and translocates into the nucleus

and induces the transcription of CYPs by

binding to the xenobiotic response ele-

ments (XREs) in the promoters of target

genes. NRF2 bound to KEAP1 in the cy-

toplasm translocates into the nucleus

when the pathway is activated by exoge-

nous or endogenous electrophilic or free

radical stresses, and binds to the antioxi-

dant response elements (AREs) to induce

transcription of cytoprotective genes.

NRF2 CROSS TALK 1651

As with many transcription factors, the actions of the direct

target genes of AhR alone do not fully explain its toxicologic

and physiologic effects. In addition to interactions with NRF2

signaling, AhR extends its regulatory sweep by modulating

the function of other transcription factors, including estrogen

receptor (ERaand ERb) and androgen receptor (AR). These

cross-talk pathways are important mediators of the functions

of endogenous and exogenous AhR ligands. The liganded

AhR recently was shown to promote the ubiquitination and

proteasomal degradation of ERs and AR by assembling a

ubiquitin ligase complex, CUL4B

AHR

(108). Thus, complexes

of the AhR with ERs or AR appear to regulate transcription as

functional units by multiple mechanisms. As depicted in

Fig. 4, KEAP1 also serves as an adaptor protein for an E3

ubiquitin ligase complex (78). Thus, broad similarities exist in

the ways these pathways sense and respond to stresses. In-

deed, other stress response–signaling pathways such as

HIF-1aand NF-kB also interact with ubiquitin ligase systems

to facilitate proteolytic turnover of signaling components (109).

Direct and Indirect NRF2–NF-kB Interactions

Cross talk between NRF2 and nuclear factor k-light-chain-

enhancer of activated B cells (NF-kB) is an area of extensive

interest. NF-kB proteins are a family of transcription factors

involved in several processes such as inﬂammation, immune

response, apoptosis, development, and cell growth. Targets of

NF-kB include genes classiﬁed as chemokines, cytokines,

immunoreceptors, cell-adhesion molecules, stress-response

genes, regulators of apoptosis, growth factors, and tran-

scription factors, among many others. Tumor necrosis factor a

(TNF-a), inducible NOS (iNOS), interleukin-1 (IL-1), intra-

cellular adhesion molecule-1, and cyclooxygenase (COX-2)

have all been shown to be induced by NF-kB (112). Patholo-

gies related to alterations of the NF-kB pathway include al-

lergies (25), Alzheimer disease (97), autoimmunity (46),

obesity (40), atherosclerosis (125), arthritis (123), cancer (41),

Crohn’s disease (114), diabetes (164), and stroke (51).

All NF-kB proteins contain a conserved Rel homology do-

main responsible for dimerization and DNA binding to the

consensus sequence GGGRNNYYCC, (R ¼purine, N ¼any

base, Y ¼pyrimidine). The NF-kB family of proteins can be

divided into two distinct groups based on the presence of a

transactivation domain. RelA (p65), RelB, and c-Rel all con-

tain transactivation domains, whereas p50 and p52 do not,

and they require heterodimerization with the Rel proteins to

activate transcription. Inactive NF-kB is located in the cyto-

plasm associated with the negative regulator IkB, which can

conceal the nuclear-localization sequence or DNA-binding

domain of NF-kB to prevent transcription (148, 157, 165).

Activation of NF-kB is accomplished through phosphoryla-

tion of IkBbyIkB kinases (IKKs), which leads to the release

and nuclear translocation of NF-kB (22, 28, 150). A wide range

of agents have the ability to stimulate IKK to activate NF-kB,

including H

2

O

2

, TNF-a, IL-1, phorbol esters, hypoxia fol-

lowed by reoxygenation, ultraviolet radiation, or microbial

infection (112).

The NRF2 and NF-kB signaling pathways interface at

several points to control the transcription or function of

downstream target proteins. Many examples exist in which

activation and repression occur between members of the two

pathways through mechanisms of regulation ranging from

direct effects on the transcription factors themselves to pro-

tein–protein interactions and second-messenger effects on

target genes. Several cancer chemopreventive agents trigger

NRF2 signaling with a concomitant repression of NF-kB

FIG. 3. Schematic representation of stages of adipogen-

esis regulated by AhR and NRF2. Adipogenic stimuli such

as dexamethasone and isobutylmethylxanthine induce ex-

pression of the early markers CEBPband CEBPd, which

leads to induction of PPARgand CEBPa. NRF2 induces ex-

pression of Ahr mRNA by directly binding to the promoter

of the Ahr gene. AhR inhibits differentiation stages that fol-

low CEBPbactivation (i.e., CEBPaor PPARgupregulation).

FIG. 4. E3 ubiquitin ligase activities of KEAP1 and AHR.

KEAP1 binds with CUL3, RBX1, and E2 to facilitate the

ubiquitination of NRF2, leading its enhanced degradation by

the proteasome. Ligand-bound AhR assembles a CUL4B-

based E3 ubiquitin ligase complex to mediate a nongenomic

signaling pathway for inﬂuencing estrogen and androgen

actions. ER, estrogen receptor; AR, androgen receptor.

1652 WAKABAYASHI ET AL.

and its target genes. As examples, sulforaphane [(-)-1-

isothiocyanato-(4R)-methylsulﬁnyl)butane] reduces DNA

binding of NF-kB and decreases generation of NO, PGE

2

, and

TNF-ain Raw 264.7 macrophages without affecting IkBor

nuclear translocation of the transcription factor (50). 3H-1,2-

Dithiole-3-thione reduces the nuclear translocation and DNA

binding of NF-kB, along with changes in phosphorylation of

IkB in the hepatocytes of lipopolysaccharide (LPS)-treated

rats (69). Epigallocatechin-3-gallate induces NRF2 and re-

duces NF-kB, TNF-aand IL-1bin the lungs of bleomycin-

treated rats (140). Chalcone has been shown to induce NRF2

and inhibit the activation of NF-kB in endothelial cells (94).

The synthetic triterpenoid CDDO-Me can inhibit NF-kB ac-

tivity through repression of IKKb(1). Curcumin is able to

inhibit NF-kB by blocking phosphorylation and degradation

of IkB in macrophages challenged with LPS (113).

NRF2 and NF-kB can interact to have direct effects on gene

expression. NRF2 and NF-kB mediate the activation of

IkB and ARE sites, respectively, through antagonism of

transcription-factor binding to DNA. NF-kB was recently

shown to prevent the transcription of NRF2-dependent genes

by reducing available co-activator levels and promoting re-

cruitment of a co-repressor. p65 and Nrf2 both bind to the

CH1-KIX domain of CREB-binding protein (CBP), and after

phosphorylation of p65 at Ser276, NF-kB is able to suppress

transcription of ARE-dependent genes by preventing CBP

from binding to Nrf2 (93). A second mechanism of p65 tran-

scriptional repression of the ARE involving histone deacety-

lase 3 (HDAC3) has also been described. Overexpression of

p65 causes the recruitment of HDAC3 to the ARE by binding

to CBP or MafK. HDAC3 was shown to bind MafK in the

NRF2 dimerization region and to prevent the acetylation of

MafK by CBP (93). Further experiments showed that p65 is

able to affect NRF2-dependent transcription through the re-

cruitment of HDAC3 to the ARE by binding to CBP. HDAC3

was previously shown to bind and deacetylate CBP, resulting

in reduced CBP-mediated transcriptional coactivation (24). A

model for effects of reduced coactivator and recruitment of a

co-repressor is depicted in Fig. 5. Phosphorylation of p65 on

Ser276 reduces the available coactivator levels of CBP from

NRF2, which causes decreased transcription through the ARE

and also allows HDAC3 to bind CBP. The loss of CBP from the

MafK-NRF2 heterodimer may then cause destabilization and

allow HDAC3 to bind and deacetylate MafK. The HDAC

could be then be recruited to the ARE through the action of the

CBP-bound HDAC or a MafK homodimer–bound HDAC3

(93). Interestingly, this mechanism may be countered by in-

duction of NRF2, which leads to increased cytoplasmic-to-

nuclear translocation of the transcription factor. Experiments

have shown that repression of NRF2-dependent transcription

was eliminated by transfection of a dominant negative NRF2

that does not undergo cytoplasmic sequestration (93).

Several experiments have shown increased NF-kB activa-

tion in Nrf2

-=-

mice when compared with wild-type after

stimuli such as traumatic brain injury (65), LPS (147), TNF-a

(147), ovalbumin (119), and respiratory syncytial virus (23).

NRF2 has been implicated in NF-kB control through attenu-

ation of phosphorylated IkB, which causes NF-kB degrada-

tion. Nrf2

-=-

MEFs have higher levels of phosphorylated IkB

and greater IKK kinase activity when compared with Nrf2

þ=þ

MEFs after challenge with LPS or TNF-a(147). Interestingly,

overexpression of NRF2 seems to change only downstream

NF-kB targets, not NF-kBorIkB (21, 68, 90). Overexpression

of NRF2 in human aortic endothelial cells by using an ade-

noviral vector did not inhibit TNF-a–induced NF-kB activity

or IkB degradation, but induction of monocyte chemoat-

tractant protein-1 and VCAM-1 with TNF-awere inhibited

(20). These inconsistencies could be due to the loss of NRF2,

creating an environment with a diminished capacity to scav-

enge reactive oxygen species (ROS). The impaired expression

of antioxidative genes could potentially lead to increased ac-

tivation of the NF-kB pathway. Alternatively, enhancement of

NF-kB due to direct transcriptional action of NRF2 may occur.

In genetic studies, Nrf2

-=-

ﬁbroblasts have shown reduced

steady-state protein levels of p50 and p65 when compared

with wild-type, whereas in vivo studies revealed that livers of

Nrf2

-=-

mice exhibited greatly reduced transcript levels of p65

(161). By contrast, TNF-aor LPS stimulation of Nrf2

-=-

ﬁbro-

blasts caused increased activation of NF-kB (147). Enhance-

ment of NF-kB by NRF2 may be coordinated in a subset of the

NF-kB family members to drive expression of speciﬁc genes.

In Nrf2

-=-

ﬁbroblasts p50 and p65 show reduced steady-state

protein levels to 30% of wild type, whereas c-Rel protein levels

were increased 400% (161). It is unknown whether NRF2 is

able speciﬁcally to control expression of NF-kB proteins that

lack a transactivation domain to repress transcription of NF-

kB target proteins (88, 91). The enhancement of NRF2 due to

direct transcriptional action of NF-kB may also be possible.

In silico transcription factor–binding site analysis has identi-

ﬁed an NF-kB consensus binding site in the murine promoter

of NRF2 (102), which may increase transcription of NRF2 to

blunt prolonged NF-kB activity. As multiple agents and

stimuli such as ROS, LPS, ﬂow shear stress, oxidized low-

density lipoprotein, and cigarette smoke have been shown to

induce both NRF2 and NF-kB activity (2, 7, 16, 42, 56, 77, 127),

a completely antagonistic effect of the two transcription fac-

tors against one another in all situations is unlikely.

Interactions between downstream targets of NRF2 and NF-

kB that ultimately lead to modulation of transcription factor

activity have been discovered, which adds complexity to the

two pathways. Three examples of NRF2 target genes that are

able to inﬂuence NF-kB activity are heme oxygenase-1 (HO-

1), NAD(P)H:quinone oxidoreductase (NQO1), and thior-

edoxin (TRX). HO-1, which breaks down heme to produce

biliverdin, CO, and free iron, is able to modulate activation of

NF-kB through the action of bilirubin and free iron (3, 66, 111,

135, 139, 145) (see Fig. 6). HO-1 has been shown speciﬁcally to

prevent phosphorylation of RelA through the action of free

iron at a site required for TNF-a–dependent NF-kB activation

(135). Induction of HO-1 can inhibit IkB degradation, whereas

inhibition of HO-1 increases p65 activity in HT-29 cells and

colonic mucosa after treatment with TNF-aor IL-1ß (66).

NQO1 has both positive and negative effects on NF-kB.

NQO1 overexpression reduces LPS-induced expression of the

downstream inﬂammatory genes TNF-aand IL-1 in THP-1

cells independent of NF-kB (128). Mice deﬁcient of NQO1 in

bone marrow, spleen, and thymus showed reduced NF-kB

DNA binding under basal or LPS-treated conditions (61). TRX

also has both positive and negative effects on NF-kB. TRX can

modulate NF-kB activity through the reduction of cysteine

residues (26, 36, 47, 74, 96, 116). Nuclear TRX is required for

the reduction of cysteine residues on p50 to allow binding to

DNA, whereas cytoplasmic TRX is able to block the degra-

dation of IkB (54).

NRF2 CROSS TALK 1653

Target proteins of the NRF2 and NF-kB pathways and their

products can also interact to regulate enzyme activity. The

NF-kB target IL-10 (15) has been shown to induce HO-1 in

murine macrophages and in vivo (87). A feedback loop is

present between HO-1 and iNOS in macrophages to protect

the cell against injury due to excess NO. Expression of iNOS,

which leads to overproduction of NO, causes induction of

HO-1 (34, 60). HO-1 is then able to inhibit iNOS through the

action of free iron (156) and CO (131), as well as through the

overall reduction of heme (4) to prevent further production of

NO (141). Interestingly, NO can be scavenged by biliverdin

and bilirubin to further entangle the signaling pathways (71)

(Fig. 6). HO-1 has also been shown negatively to affect

VCAM-1 expression (9). Additional interactions between HO-

1 and the NF-kB pathway can be seen with the HO-1 product

CO. CO can repress the formation of TNF-a, IL-1b, and

macrophage inﬂammatory protein-1band increase IL-10 after

LPS stimulation (110).

An NF-kB target that has been shown to inﬂuence NRF2

activity is COX-2 and its downstream product 15-deoxy

D(12,14) prostaglandin J2 (15d-PGJ2). Induction of COX-2 by

shear stress has been shown to inhibit phosphatidylinositol 3-

kinase activity, causing reduction of the transcription of

NRF2, NQO1, HO-1, GCLR, and GSTM1 in human chon-

drocytes. Restoration of NRF2-dependent induction of the

genes was seen on treatment with the COX-2–speciﬁc inhib-

itors CAY10404 and NS398 (49). 15d-PGJ2 has been shown to

play a role in NRF2 modulation by binding to KEAP1 and

causing nuclear translocation of NRF2 in macrophages (63).

Further evidence of a direct effect of 15d-PGJ2 on NRF2 acti-

vation was found in vivo by using Nrf2

-=-

mice and the COX-2–

selective inhibitor NS-398 (99). 15d-PGJ2 has also been shown

to provide negative feedback through its own pathway by

binding IKK to prevent the activation of NF-kB in HeLa cells

and appears to do the same in human peripheral blood

monocytes (126).

NRF2 and NF-kB have a subset of target genes that are

regulated through the actions of both transcription factors on

ARE and kB sites. HO-1 has been shown to have a functional

ARE that can be activated by NRF2 (3), as well as a functional

NF-kB site, by using EMSA and DNase I footprinting analysis

(86). Glutamate-cysteine ligase catalytic subunit (GCLC) can

be activated by NRF2 through an ARE (101) or by NF-kB

through a kB site after ethanol exposure (76). The inhibitory G

protein, Gai2, is another example of a gene activated by both

NF-kB and NRF2 (8). Expression of the chemokine IL-8 can be

increased by both NRF2 and NF-kB, but not through the usual

mechanisms. NF-kB activates transcription of IL-8 by binding

to the kB site (82), whereas NRF2 instead uses a mechanism

that stabilizes IL-8 mRNA (166).

p53 and NRF2: Cooperation and Antagonism

in Protection Against Cancer

The examination of p53 after its discovery in 1979 (92) has

been extensive, with approximately 52,000 articles acces-

sioned when ‘‘p53’’ is searched on PubMed. This investigative

interest is not without reason, inasmuch as p53 is mutated in

upward of 50% of human cancers through single, compound,

or deletion mutations (55). This common frequency of muta-

tions makes p53 an attractive target for chemotherapeutics. If

p53 is to be targeted, however, the body of knowledge sur-

rounding the protein should be expanded to provide full

understanding of the interactions of p53 with other signaling

molecules and networks.

p53 regulates a host of processes within the cell, both di-

rectly, including the induction of apoptosis through the per-

meabilization of the outer mitochondrial membrane through

interaction with proapoptotic factors (43, 89, 100), and indi-

rectly, through the transcription of genes that effect changes

on the cell. Examples include both pro- and antiapoptotic

responses, the induction of cellular senescence, and the repair

of genotoxic damage (39). These various activities of p53 make

it an integral part of the cellular defense against transforma-

FIG. 5. Repression of NRF2

signaling by members of the

NF-jB pathway. p65 represses

NRF2-dependent transcription at

the ARE when phosphorylated

on S276 by either competition for

coactivator binding proteins or

by recruiting HDAC to the ARE,

which can deacetylate histone H4

and MafK.

FIG. 6. The interactions of the NRF2 and NF-jB target

proteins HO-1 and iNOS and their products can regulate

the activity of each other in macrophages to protect against

an overabundance of NO. Excess NO acts as a signal to

increase HO-1, which is able to scavenge NO and block the

activity of iNOS to prevent further production.

1654 WAKABAYASHI ET AL.

tion: p53 controls cell fates by regulating the activation of

apoptosis to remove a heavily damaged cell, induces terminal

differentiation to remove the threat of a moderately damaged

cell, or induces the transcription of cellular scavenging pro-

teins to reestablish cellular homeostasis.

Iida et al. (58) showed that Nrf2 and p53 collaborate to

protect against carcinogenesis (58), with the demonstrations

that, compared with wild-type mice, either Nrf2 knockout

mice or p53 heterozygotic mice are more susceptible to

nitrosamine-induced bladder carcinogenesis, and that the

Nrf2

-=-

::p53

þ=-

compound knockout mice are even more sus-

ceptible. This interaction is not unexpected, as when either the

cytoprotective response system of NRF2, or p53, a cell-fate

decision molecule is removed, the incidence of cancer in-

creases after carcinogen challenge (117, 146). Although broad

explanations based on general understandings of pathways

can be posited, the actual interactions between NRF2 and p53

in this setting and the collective effects on the expression and

function of their downstream target genes remain to be

established.

In a more-direct examination of the interaction between

p53 and NRF2, p53 has been shown to inﬂuence NRF2-based

transcription by Cimino and colleagues (33). Speciﬁcally, p53

suppresses the transcription of three NRF2 target genes dri-

ven through the ARE. These genes, x-ct,Nqo1, and Gst1a1, are

all involved in mounting an antioxidant response within the

cell, speciﬁcally neutralizing ROS. In the study, various cell

lines were transfected with expression vectors containing Nrf2

or p53. In all circumstances, the transformation with both Nrf2

and p53 results in a reduction of transcript levels of the target

genes when compared with cells transformed with Nrf2 alone.

This, in combination with experiments in which cells were

exposed to etoposide (a topoisomerase II–inhibiting chemo-

therapeutic drug) also shows that p53 has the ability to reg-

ulate negatively the NRF2 target gene transcripts. When p53 is

highly upregulated, through either overexpression or a strong

DNA-damaging agent, antioxidant response genes are

downregulated through p53-mediated disruption. Whether

or to what extent NRF2 regulates p53 signaling is not clear.

Transcriptome analyses, along with unpublished observa-

tions (M.K. Kwak and N. Wakabayashi), suggest that NRF2

contributes to the basal expression of MDM2, an inhibitor of

p53 (85). The potential additional level of p53 regulation

through NRF2 allows the dampening of MDM2-based pro-

teasomal degradation of p53 in circumstances in which p53

may be modulating the expression of NRF2 target genes.

Dampening of Mdm2 would allow an enhanced p53 signal.

This effect makes intuitive sense, as if p53 were mediating an

apoptotic response through ROS production, NRF2-driven

gene expression of anti-ROS enzymes would be counterpro-

ductive. This observation, however, is confounded by a recent

article examining the relation between p21, a direct down-

stream target of p53, and NRF2 (19).

p21 is involved with many different cellular processes, in-

cluding cell-cycle arrest, cell differentiation, senescence, apo-

ptosis and DNA replication and repair, and oxidative stress

(29, 31, 38, 86, 107). Chen et al. (19) recently showed a direct

interaction between p21 and NRF2, which may explain the

cytoprotective properties of p21 when faced with oxidative

stress. These investigators demonstrated in vitro that p21 is

able to interact with the DLG motif within NRF2, thereby

attenuating KEAP1-based ubiquitination and subsequent

proteasomal degradation. The antioxidative properties of p21

rely on the presence of NRF2, as an increase in p21 concen-

tration leads to increased transcript levels of ARE-regulated

genes, and the presence of p21 within cells yields an increase

in the half-life of NRF2. Additionally, it was observed in vivo

that both basal and induced levels of NRF2 protein are higher

in p21 wild-type mice than in p21-null mice, with induced

levels of NRF2 target gene proteins NQO1 and HO-1 being

higher in p21 wild-type mice. This discovery contributes sig-

niﬁcantly to appreciation of the emerging network of signal-

ing between p53 and NRF2 by demonstrating that a direct

target of p53 upregulates NRF2 levels and activity.

The three puzzle pieces presented here come together in an

intricate fashion, demonstrating a tunable response by p53 to

induction by any number of stimuli, as shown in Fig. 7. When

p53 is strongly induced, the induction of apoptosis through

the expression of proapoptotic proteins through the genera-

tion of intracellular ROS is secured by the suppression of

antioxidant gene expression regulated by Nrf2, as well as the

suppression of basal levels of Mdm2, to ensure the strongest

possible cell-death signal both through direct levels of p53

and by its direct activities remaining uncompromised. The

upregulation of proapoptotic factors by p53 may rely on in-

creased levels of intracellular ROS production for signaling. In

this setting, ROS is not neutralized by NRF2 target genes such

as Nqo1 and Gst1a1because of transcriptional interference by

p53 (33), and the proteasomal degradation of p53 is hampered

because of the reduction in MDM2 levels.

On the other extreme, in the face of weak p53 induction, p21

is activated, stalling the cell cycle at the G

1

=S-phase check-

point (30, 45, 160) This stalling allows the induction of DNA

damage-repair functions by p53, the expression of proteins in

an attempt to lower intracellular ROS (10, 129), and the sta-

bilization of NRF2 by p21, thereby disrupting proteasomal

degradation of NRF2 by KEAP1. This disruption of NRF2

turnover allows the nuclear translocation of NRF2, and the

subsequent transcription of ARE-driven genes, leading to a

general cytoprotective response. The collective actions of p53

and NRF2 lead to the repair of the cell and evasion of apo-

ptosis. Links between NRF2 and the oxidative-stress–induced

inhibition of proliferation have been well described (120, 121);

in some instances, they may operate independent of p53.

The circumstance of a moderate induction of p53 allows

some speculation that a middle ground exists between the

promotion of programmed cell death and full cell repair and

restoration of homeostasis, namely, that of cellular senescence

through terminal differentiation. This balance is achieved

through p21 activation after p53 induction through signaling.

p21 blocks the cell cycle by stopping progression through the

G

1

=S checkpoint (30, 45, 160), and allows the stabilization of

NRF2 and an increased level of detoxiﬁcation enzymes (19).

p53 almost certainly plays a role in activating additional

proteins that contribute to the process of rescuing a damaged

cell that may be useful in a state of terminal differentiation but

has experienced some insult that renders the cell a liability

when used for mitotic purposes.

This system of dynamic response to cellular stresses by p53,

p21, and NRF2 demonstrates a powerful mechanism for cel-

lular and organismal maintenance, through the repair, dif-

ferentiation, or destruction of damaged cells. In the case of

mild damage, NRF2-mediated detoxiﬁcation of carcinogens

combined with a p21-mediated pause in the cell cycle and a

NRF2 CROSS TALK 1655

p53=p21-mediated induction of DNA damage-repair en-

zymes salvages cells from damage, whereas in the case of

extensive damage to a cell, p53 mediates the disruption of

NRF2-mediated antioxidant response proteins, allowing p53-

driven apoptosis, in toto reﬂecting a tunable response to en-

vironmental stresses.

NRF2 and NOTCH Signaling:

Cell-Survival Factor Meets Cell-Fate Factor

Several lines of differential microarray analyses using

RNAs isolated from MEFs prepared from Nrf2

-=-

and wild-

type mice have shown that Notch1 and its direct downstream

genes (Hes, Herp) (62) displayed decreased expression in

Nrf2

-=-

MEF. Studies from our laboratory demonstrate that

Notch1 signaling triggered through its activating ligands,

DLL1 and JAG1, is dependent on Nrf2 genotype. Moreover,

one or more functional ARE sequences exist in the promoter of

Notch1. Therefore, it appears that NRF2 directly regulates

Notch1 gene expression (153). It is now well established that

signals through the NOTCH receptor are involved in the de-

velopment of several cell types and that the modulation of

these signals can markedly affect differentiation, prolifera-

tion, and apoptotic events. Genetic ablation studies indicate

that Notch1 is crucial for early development and regrowth in a

variety of tissues (57, 154). Activation of the pathway has been

shown to be a potent inhibitor of differentiation in different

developmental contexts and has been associated with the

ampliﬁcation of certain somatic stem cells (27, 35, 64, 80, 134,

142). Considering the signiﬁcance of the NOTCH1 signal

cascade in developmental biology, this indicated the possi-

bility that NRF2 could be a key molecule affecting both em-

bryonic and adult tissue stem cell renewal, as well as cell fate.

Of course, this cross talk might not only be mediated by

transcriptional machinery, as NOTCH signaling is regulated

by various dynamic mechanisms, including posttranslational

modiﬁcations (149). Critical to the evaluation of cross talk

between transcription factors are assessments of their func-

tional consequences. Several physiological lines of evidence

support functional interactions between NRF2 and Notch1:

liver regeneration, keratinogenesis, osteoblastogenesis, and

adipogenesis (Fig. 8).

Nrf2

-=-

mice display an interesting phenotype in the process

of liver regeneration (11). Nrf2

-=-

mice have been challenged

with a two-thirds partial hepatectomy to view the impact of

genotype on the kinetics of liver regeneration. These knockout

mice showed a signiﬁcant delay in the recovery of liver mass

after partial hepatectomy. We have observed that this regen-

eration-defective phenotype is rescued by hepatocyte-speciﬁc

expression of the NOTCH1 intracellular domain (NICD) in

Nrf2

-=-

mice. NICD functions as a transcription co-factor of

RBPJ. This ﬁnding provides clear evidence for the existence

of NRF2-Notch1 cross talk within the damaged liver, and

within the process of liver regeneration (153). In a rat model

in which siRNAs against NOTCH1 and JAG1 (a NOTCH1

ligand) were injected into the superior mesenteric vein before

the two-thirds partial hepatectomy, signiﬁcantly suppressed

proliferation of hepatocytes was reported at days 2 to 4 of the

regenerative response (80). Apparently, the NRF2-NOTCH1

signaling pathway may be activated during liver regeneration

and is potentially contributing to signals affecting cell growth

and differentiation.

Keap1

-=-

mice, which exhibit hyperactivated NRF2 signal-

ing, have a thickened corniﬁed layer on the suprabasal layer of

the esophagus (152), with a huge keratin mass derived from

the corniﬁed layer around the limiting ridge and cardia. All

Keap1-null mice die within 3 weeks of malnutrition caused

from esophageal and forestomach obstruction. Through mi-

croscopic observation, a clear difference is noted between the

FIG. 7. p53 modulates a

gradient of responses to cel-

lular stress. When the cell is

exposed to a high level of

stress, p53 acts in a proa-

poptotic fashion, modulating

multiple pathways to secure a

full commitment to cell death,

whereas with a low level of

cellular stress, p53 modiﬁes

gene expression to ensure cy-

toprotection after removal of

thestress.Theintermediatere-

sponse is also possible, with p53

retaining the ability to induce

cellular senescence through exit

of the cell cycle and induction

of terminal differentiation with

a concurrent cytoprotective

response.

FIG. 8. Possible effects of NRF2–Notch interactions on

cell fate.

1656 WAKABAYASHI ET AL.

processes of corniﬁed layer thickening in the esophagus ver-

sus the foregut. In the case of the esophagus, cell numbers in

the basal layer seem to be unchanged, with the corniﬁed layer

exhibiting thickening. This appears to be a prodifferentiation

mode of hyperkeratosis. Within the limiting ridge of the

greater curvature and the cardiac portion of the forestomach,

however, a proliferative form of hyperkeratosis is observed,

with proliferation of undifferentiated cells. This phenotype

might be explained by the excess Notch signaling produced by

excessive NRF2 activation. NOTCH signaling is expressed in

the gastrointestinal tract. Speciﬁcally, in both the esophagus

and forestomach, NOTCH1 and JAG2 display the highest

levels of expression within their families in the basal layer (70),

where stem and progenitor cells are located (130). Progenitor

cells are differentiated to keratinocytes in the suprabasal layer

and denucleated in the corniﬁed layer, with Notch signaling

being weakly expressed in the suprabasal layer. Interestingly,

NOTCH signaling induces terminal differentiation to activate

p21 gene expression directly in keratinocytes (118, 158). This

function of the NOTCH signal might be stronger in the

esophagus and forestomach of Keap1-null mice.

Hinoi et al. (52) demonstrated that NRF2 negatively regu-

lates osteoblast differentiation in the MC3T3-E1 cell system.

They also showed that NRF2 might inhibit cellular differen-

tiation and maturation in chondrocytes by using the pre-

chondrogenic cell line ATDC5, derived from the mouse

teratocarcinoma AT805 (53). As a proposed mechanism of

these phenomena, they conjectured that the inhibition of

RUNX2 (one of the essential factors for bone formation and

chondral maturation) was caused by either direct or indirect

interaction with NRF2. Conversely, Notch-signaling directly

inﬂuences osteogenesis and chondrogenesis (14). Interestingly,

these phenomena are all related to bHLH-transcription factors,

especially HES and HERP, which are primary target genes of

NOTCH signaling. These proteins function as negative regu-

lators of target gene expression through direct cis-element

(E-box, N-box)–binding machinery. They also facilitate pro-

teasomal degradation through heterodimerization with other

bHLH transcription factors. The heterodimer partners of HES

or HERP primarily promote differentiation. Thus, they can lead

to maintenance of current cell status. HES1 directly binds to the

osteocalcin promoter and represses osteocalcin gene expression

(167). Additionally, HES1=HERP2 physically interacts with

RUNX2 in osteoblasts (37). It is also possible that NOTCH1

expression in osteoblasts is inﬂuenced by the NRF2 transcrip-

tion factor, much as NOTCH1 is in MEF.

As discussed earlier, AhR and its downstream gene ex-

pression is reduced in Nrf2

-=-

MEF (138). This dampened gene

expression impairs adipocyte differentiation from MEF. Nrf

-=-

MEF show markedly accelerated adipogenesis on stimula-

tion, whereas Keap1

-=-

MEF, which demonstrate higher levels

of NRF2 signaling, differentiate slowly compared with their

congenic wild-type MEF. Within this system, ectopic expres-

sion of AhR rescues adipogenesis seen in Nrf2

-=-

MEF.

However, recently Ross et al. (124) reported that HES1, a

primary downstream gene product of NOTCH1 signaling,

inhibits the differentiation of adipocytes from 3T3-L1 cells.

They also demonstrated that both the bHLH and the WRPW

(67) [which is a co-repressor (GROUCHO=TLE) binding

motif ] domains in HES1 are required for its inhibitory effect.

This study raised the possibility that the enhanced adipo-

genesis shown in Nrf2

-=-

MEF is caused by a weaker output

of both NOTCH1 and AhR signaling than within wild-type

MEF.

Conclusions

The NRF2 pathway has been studied in diverse contexts

because of potential roles in stroke recovery, neurodegen-

eration, chronic obstructive pulmonary disease, and cancer

prevention in multiple organs. The proﬁling of gene-expression

changes mediated by the NRF2 pathway has been well

documented. The interpretation of such studies, however, has

been hampered by the inability to distinguish genes modu-

lated directly by NRF2 from genes induced secondarily by

NRF2-sensitive transcription factors and the mitigating im-

pacts of tissue-speciﬁc responses. Nonetheless, it is clear that

the protective effects of upregulation of NRF2 signaling can

take several forms. Protection can be immediate, reﬂecting in-

duction of genes directly regulated through NRF2 binding to

AREs in target genes (e.g., the innate immune response and

elevated cytoprotective responses to blunt cytokine surges or

detoxify reactive intermediates, respectively) (73). The pro-

tective effects can be secondary through induction of macro-

molecular damage repair=removal systems (proteasome,

DNA repair) (84, 106). Last, the protective effects can be ter-

tiary through activation of tissue repair=regeneration path-

ways. In these latter cases, involvement in cross talk with

additional pathways affecting cell survival and other aspects

of cell fate most certainly play important collaborating roles.

This review has highlighted several such collaborators (and

sometime competitors): AhR, NF-kB, p53, and Notch1. Future

studies evaluating the concordance between global mRNA

expression proﬁles at the transcriptomic levels (i.e., oligonu-

cleotide microarray analyses) and genome-wide NRF2-DNA

binding analysis using high-throughput methods (i.e., chro-

matin-immunoprecipitation with parallel sequencing) should

better establish a systems-level view of NRF2-dependent

processes occurring within cells. Functional importance will

require well-crafted molecular genetic studies in cell culture,

and more important, in vivo, to deﬁne the critical cross-talk

interactions between NRF2 and other transcription factors.

Acknowledgments

This work was supported by NIH grants CA39416 and

CA94076 (TWK) and the Maryland Cigarette Restitution

Fund. SLS was supported by T32 ES07141 and T32 CA009110;

JJS by T32 GM08763; and SS was the recipient of a Samsung

Scholarship (Samsung Foundation of Culture).

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Address correspondence to:

Thomas W. Kensler

Department of Pharmacology & Molecular Sciences

E1352 BST

University of Pittsburgh

Pittsburgh, PA 15261

E-mail: tkensler@pitt.edu

Date of ﬁrst submission to ARS Central, March 26, 2010; date

of acceptance, April 1, 2010.

Abbreviations Used

15d-PGJ2 ¼15-deoxy D(12,14) prostaglandin J2

AhR ¼arylhydrocarbon receptor

ARE ¼antioxidant response element

ARNT ¼AhR nuclear translocator

CBP ¼CREB-binding protein

CDDO-Im ¼1-(2-cyano-3,12-dioxooleana-1,9[11]-

dien-28-oyl)imidazole

CEBPs ¼CCAAT-enhancer-binding proteins

COX-2 ¼cyclooxygenase-2

GST ¼glutathione S-transferase

HO-1 ¼heme oxygenase-1

IKK ¼IkB kinase

IL-1 ¼interleukin-1

iNOS ¼inducible NOS

KEAP1 ¼Kelch-like ECH-associated protein 1

LPS ¼lipopolysaccharide

MEF ¼mouse embryonic ﬁbroblast

NF-kB¼nuclear factor kappa-light-chain-enhancer

of activated B cells

NICD ¼notch1 intracellular domain

NQO1 ¼NAD(P)H: quinone-acceptor 1

NRF2 ¼NF-E2–related factor 2

PPAR ¼peroxisome proliferator-activated

receptor

ROS ¼reactive oxygen species

Sulforaphane ¼(-)-1-isothiocyanato-(4R)-

methylsulﬁnyl)butane

TNF-a¼tumor necrosis factor a

TRX ¼thioredoxin

XRE ¼xenobiotic-responsive element

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Glutathione (GSH) is an abundant cellular non-protein sulfhydryl that functions as an important protectant against reactive oxygen species and electrophiles, is involved in the detoxification of xenobiotics, and contributes to the maintenance of cellular redox balance, The rate-limiting enzyme in the de novo synthesis of glutathione is gamma-glutamylcysteine synthetase (GCS), a heterodimer consisting of heavy and light subunits expressing catalytic and regulatory functions, respectively, Exposure of HepG2 cells to beta-naphthoflavone (beta-NF) resulted in a time- and dose-dependent increase in the steady-state mRNA levels for both subunits, In order to identify sequences mediating the constitutive and induced expression of the heavy subunit gene, a series of deletion mutants created from the 5'-flanking region (-3802 to +465) were cloned into a luciferase reporter vector (pGL3-Basic) and transfected into HepG2 cells, Constitutive expression was maximally directed by sequences between -202 and +22 as well as by elements between -3802 to -2752, The former sequence contains a consensus TATA box, Increased luciferase expression following exposure to 10 mu M beta-NF was only detected in cells transfected with a reporter vector containing the full-length -3802:+465 fragment, Hence, elements directing constitutive and induced expression of the GCS heavy subunit are present in the distal portion of the 5'-flanking region, between positions -3802 and -2752, Sequence analysis revealed the presence of several putative consensus response elements in this region, including two potential antioxidant response elements (ARE3 and ARE4), separated by 34 base pairs. When cloned into the thymidine kinase-luciferase vector, pT81-luciferase, and transfected into HepG2 cells, both ARE3 and ARE4 increased basal luciferase expression approximately 20-fold. When cloned in tandem in their native arrangement the increase in luciferase activity was in excess of 100-fold, suggesting a strong interaction between the two sequences, Luciferase expression was elevated in beta-NF-treated cells transfected with the AREA-th-luciferase vector and all DNA fragments containing ARE4. In contrast, ARE3 did not direct increased luciferase expression in response to beta-NF nor did it significantly modify the magnitude of induction directed by ARE4. The influence of the ARE4 oligonucleotide on constitutive and induced expression was eliminated by introduction of a single base mutation, converting the core ARE sequence in ARE4 fi om 5'-GTGACTCAGCG-3' to 5'-GGGACTCAGCG-3'. When introduced into the full-length -3802:+465 segment, the same single base mutation also eliminated both functions, Collectively the data indicate that the constitutive and P-NF-induced expression of the human GCS heavy subunit gene is mediated by a distal ARE sequence containing an embedded tetradecanoylphorbol-13-acetate-responsive element.

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1. 1. Kimura, 2. et al . 2009. J. Exp. Med. doi: 10.1084/jem.20090560 [OpenUrl][1][Abstract/FREE Full Text][2] [1]: {openurl}?query=rft_id%253Dinfo%253Adoi%252F10.1084%252Fjem.20090560%26rft_id%253Dinfo%253Apmid%252F19703987%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo

The PAS superfamily: sensors of environmental and developmental signals

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Jan 2000

Activators and Target Genes of Rel/NF-кB Transcription Factors

Chapter

Jan 2003

Sixteen years have passed since the description of the nuclear factor-кB (NF-кB) as a regulator of к light-chain gene expression in murine B lymphocytes (Sen & Baltimore, 1986a). During that time, over 4,000 publications have appeared, characterizing the family of Rel/NF-кB transcription factors involved in the control of a large number of normal and pathological cellular processes. The physiological functions of NF-кB proteins include immunological and inflammatory responses, developmental processes, cellular growth and modulating effects on apoptosis. In addition, these factors are activated in a number of diseases, including cancer, arthritis, acute and chronic inflammatory states, asthma, as well as neurodegenerative and heart diseases.

Induction of peroxisome proliferator-activated receptor gamma during the conversion of 3T3 fibroblasts into adipocytes is mediated by C/EBPbeta, C/EBPdelta, and glucocorticoids.

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Aug 1996

Iron regulates nitric oxide synthase activity by controlling nuclear transcription

Article

Sep 1994

Guenter Weiss

Recently, it was reported that nitric oxide (NO) directly controls intracellular iron metabolism by activating iron regulatory protein (IRP), a cytoplasmic protein that regulates ferritin translation. To determine whether intracellular iron levels themselves affect NO synthase (NOS), we studied the effect of iron on cytokine-inducible NOS activity and mRNA expression in the murine macrophage cell line J774A.1. We show here that NOS activity is decreased by about 50% in homogenates obtained from cells treated with interferon gamma plus lipopolysaccharide (IFN-gamma/LPS) in the presence of 50 microM ferric iron [Fe(3+)] as compared with extracts from cells treated with IFN-gamma/LPS alone. Conversely, addition of the iron chelator desferrioxamine (100 microM) at the time of stimulation with IFN-gamma/LPS increases NOS activity up to 2.5-fold in J774 cells. These effects of changing the cellular iron state cannot be attributed to a general alteration of the IFN-gamma/LPS signal, since IFN-gamma/LPS-mediated major histocompatibility complex class II antigen expression is unaffected. Furthermore, neither was the intracellular availability of the NOS cofactor tetrahydrobiopterin altered by treatment with Fe(3+) or desferrioxamine, nor do these compounds interfere with the activity of the hemoprotein NOS in vitro. We demonstrate that the mRNA levels for NOS are profoundly increased by treatment with desferrioxamine and reduced by Fe(3+). The half-life of NOS mRNA appeared not to be significantly altered by administration of ferric ion, and NOS mRNA stability was only slightly prolonged by desferrioxamine treatment. Nuclear run-off experiments demonstrate that nuclear transcription of cytokine-inducible NOS mRNA is strongly increased by desferrioxamine whereas it is decreased by Fe(3+). Thus, this transcriptional response appears to account quantitatively for the changes in enzyme activity. Our results suggest the existence of a regulatory loop between iron metabolism and the NO/NOS pathway.

Article

Nrf2 Signaling and Cell Survival

July 2009 · Toxicology and Applied Pharmacology

Nrf2:INrf2 acts as a sensor for oxidative/electrophilic stress. INrf2 serves as an adaptor to link Nrf2 to the ubiquitin ligase Cul3-Rbx1 complex that ubiquitinate and degrade Nrf2. Under basal conditions, cytosolic INrf2/Cul3-Rbx1 is constantly degrading Nrf2. When a cell encounters stress Nrf2 dissociates from the INrf2 and translocates into the nucleus. Oxidative/electrophilic stress induced ... [Show full abstract] modification of INrf2Cysteine151 and/or protein kinase C (PKC)-mediated phosphorylation of Nrf2Serine40 controls Nrf2 release from INrf2 followed by stabilization and nuclear translocation of Nrf2. Nrf2 binds to the antioxidant response element (ARE) and activates a myriad of genes that protect cells against oxidative/electrophilic stress and neoplasia. A delayed response of oxidative/electrophilic stress activates GSK-3beta that phosphorylates Fyn at unknown threonine residue(s). Phosphorylated Fyn translocates to the nucleus and phosphorylates Nrf2Tyrosine568 that leads to nuclear export and degradation of Nrf2. Prothymosin-alpha mediated nuclear translocation of INrf2 also degrades nuclear Nrf2. The degradation of Nrf2 both in cytosol and nuclear compartments rapidly brings down its levels to normal resulting in suppression of Nrf2 downstream gene expression. An auto-regulatory loop between Nrf2 and INrf2 controls their cellular abundance. Nrf2 regulates INrf2 by controlling its transcription, and INrf2 controls Nrf2 by degrading it. In conclusion, switching on and off of Nrf2 combined with promoting an auto-regulatory loop between them regulates activation/deactivation of defensive genes leading to protection of cells against adverse effects of oxidative and electrophilic stress and promote cell survival.

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Regulation of Notch1 Signaling by Nrf2: Implications for Tissue Regeneration

July 2010 · Science Signaling

The Keap1-Nrf2-ARE signaling pathway elicits an adaptive response for cell survival after endogenous and exogenous stresses, such as inflammation and carcinogens, respectively. Keap1 inhibits the transcriptional activation activity of Nrf2 (p45 nuclear factor erythroid-derived 2-related factor 2) in unstressed cells by facilitating its degradation. Through transcriptional analyses in Keap1- or ... [Show full abstract] Nrf2-disrupted mice, we identified interactions between the Keap1-Nrf2-ARE and the Notch1 signaling pathways. We found that Nrf2 recognized a functional antioxidant response element (ARE) in the promoter of Notch1. Notch1 regulates processes such as proliferation and cell fate decisions. We report a functional role for this cross talk between the two pathways and show that disruption of Nrf2 impeded liver regeneration after partial hepatectomy and was rescued by reestablishment of Notch1 signaling.

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Nrf2: Control of sensitivity to carcinogens

March 2011 · Archives of Toxicology

Induction of enzymes that enhance the detoxication of chemical carcinogens has been a broadly effective strategy for chemoprevention of experimental carcinogenesis in rodent models. Several inducing agents are now in clinical trials to evaluate utility for prevention of cancers associated with unavoidable high exposures to environmental carcinogens. The successes of these pre-clinical and ... [Show full abstract] clinical interventions lead to studies to define the molecular basis for protection by these agents, which now include phenolic antioxidants, dithiolethiones, isothiocyanates, and triterpenoids. In the mid-1990s, the NF-E2-related factor 2 (Nrf2) transcription factor was identified as a key regulator of the inducible expression of enzymes such as glutathione S-transferases and NAD(P)H: quinone oxidoreductase in catalyzing the detoxication of reactive electrophiles and oxidants that contribute to the formation of mutations and ultimately cancers. Nrf2 is now recognized to regulate a broad cytoprotective, transcriptional response leading to prevention of damage to DNA, proteins and lipids; recognition, repair and removal of macromolecular damage; and tissue renewal following toxic assaults. Highlighting the importance of this pathway as a determinant of susceptibility to carcinogenesis, multiple studies now demonstrate enhanced incidence, multiplicity, and/or tumor burden in Nrf2-disrupted mice compared to wild-type in models of inflammation and colon cancer, bladder cancer, lung disease and cancer, stomach cancer, mammary cancer, skin cancer, and hepatocarcinogenesis.

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Cross-talk between Transcription Factors AhR and Nrf2: Lessons for Cancer Chemoprevention from Dioxi...

August 2009 · Toxicological Sciences

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The Guardian Recruits Cops: The p53-p21 Axis Delegates Prosurvival Duties to the Keap1-Nrf2 Stress P...

July 2009 · Molecular Cell

Michel B Toledano

Cellular oxidative damage occurs continuously from exposure to many sources of reactive oxygen species (ROS), and reactive nitrogen species (RNS), and electrophilic xenobiotics. Nrf2, a cap‘n’collar bZip protein, regulates an adaptive response that increases survival in the face of these stresses (Hayes and McMahon, 2009). Nrf2 is induced by these stresses and functions by activating more than a ... [Show full abstract] hundred genes involved in activities such as chemical detoxification, ROS elimination, and restoration of cellular redox homeostasis. Nrf2-induced factors also include chaperones and proteasome subunits. The importance of Nrf2 in stress adaptation is underscored by the increased susceptibility of Nrf2 knockout (KO) mice to the genotoxic, cytotoxic, and inflammatory effects of environmental stressors.