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INTRODUCTION |
Ferritin is a 480-kDa intracellular protein that can store up to
4500 atoms of iron (1). The protein consists of 24 subunits of the H
and L chain type (2). The ratio of subunits within the ferritin protein
varies widely by tissue type; the ratio can be further modulated by
environmental signals, including cytokine stimulation, stress signals,
and disease state (3, 4). The H chain has ferroxidase activity (5),
whereas the L subunit is responsible for iron nucleation and protein
stabilization (6). Because iron functions as a catalyst in the
formation of oxygen free radicals, storage of iron by ferritin may
serve a cytoprotective function (2). Several laboratories have
demonstrated an induction of ferritin by oxidants and pro-oxidant
xenobiotics, including agents such as hydrogen peroxide (7),
t-BHQ1 (7), sodium arsenite
(8), phorone (9), carbon tetrachloride (10), and aqueous extracts of
cigarette smoke (11). Activation of ferritin by these agents is thought
to be a stress response mechanism that contributes to limiting cellular
and organismal damage from these xenobiotics.
The induction of antioxidant and detoxification cytoprotective enzymes
has been proposed as a potential strategy in cancer chemoprevention
(reviewed in Ref. 12). Chemopreventives are natural or synthetic
compounds that have the ability to block or suppress carcinogenesis
(13). Dithiolethiones, including oltipraz
(5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione), a synthetic compound currently in clinical trials, and D3T (1,2-dithiole-3-thione) are prototypical examples of such candidate chemopreventive agents (14). The induction of ferritin in rats treated with the dithiolethione D3T represented an initial indication that ferritin may be among the
cytoprotective proteins induced by chemopreventives (15).
Mechanisms responsible for the induction of cytoprotective proteins in
response to challenge with xenobiotics, including certain chemopreventive agents, are beginning to be clarified. To date, two
mechanisms responsible for the coordinate induction of antioxidant enzymes and phase II detoxification enzymes have been identified (16).
The first mechanism is dependent on an interaction between the inducer
and the cytosolic aromatic hydrocarbon (Ah) receptor (17). Upon binding
of the ligand to the receptor, the receptor-ligand complex translocates
to the nucleus, where it binds to a DNA enhancer element, termed
xenobiotic responsive element (XRE). Most ligands capable of binding to
the Ah receptor are polycyclic hydrocarbons, such as
-NF and
2,3,7,8-tetrachlorodibenzo-p-dioxin (16). Genes regulated by
this mechanism include phase I activating enzymes, such as the
cytochromes P450 1A1 and 1A2 (18, 19), conjugating enzymes such as
glutathione S-transferase (GST) Ya (20), and UDP-glucuronosyltransferases 1A1 and 1A6 (21, 22), and antioxidant enzymes such as NAD(P)H:quinone oxidoreductase 1 (NQO1) (23), and
Cu/Zn-superoxide dismutase (SOD) (24, 25). A second mechanism of
gene activation is Ah receptor-independent and is mediated by an
electrophile-responsive element (EpRE (26); also known as ARE (27),
antioxidant-responsive element) (28). Compounds eliciting this response
are chemically diverse and overlap those that induce the Ah
receptor-dependent pathway. They include oxidants (28, 29),
redoxocycling agents (30), and electrophiles (i.e. Michael
reaction acceptors) (28, 31) as well as polycyclic aromatic
hydrocarbons, such as
-NF (28). Functional EpRE/ARE sequences have
been identified in GST Ya (20, 26), NQO1 (23),
-glutamylcysteine
synthetase heavy and light subunit (GCSh and GCSl, respectively) (32, 33), heme oxygenase (34),
glutathione S-transferase P1 (35), thioredoxin (36), and
NF-E2-related factor 2 (Nrf2) (37).
Regulation of transcription at the EpRE/ARE is incompletely understood.
Early studies focused on members of the AP1 transcription factor family
(38) as regulators of EpRE/ARE-dependent transcription (39-42). More recently, compelling evidence for the involvement of
members of the cap and collar family of transcription factors (43),
particularly Nrf2, has been presented. For example, induction of
cytoprotective enzymes, such as GST and NQO1, by the phenolic antioxidant butylated hydroxyanisole was lost in cells isolated from
Nrf2 knockout mice (44). In addition, forced expression of cap
and collar family members, such as Nrf1 and Nrf2, resulted in
the induction of EpRE/ARE-dependent reporter gene
expression (45-48). Activation of Nrf2 following stimulation of
cells with an inducer requires its dissociation from a cytosolic actin
binding protein, Keap-1, and subsequent translocation to the nucleus
(49, 50). Release of Nrf2 from Keap-1 may be triggered by
modification of reactive cysteine residues in Keap-1 (51) and/or
post-translational modification of Nrf2 by protein kinases
(52).
Our laboratory has identified an EpRE/ARE in the ferritin H gene that
mediates the induction of ferritin H transcription in response to
H2O2 and t-BHQ (7). The ferritin H EpRE/ARE is 75 bp in length and is located ~4.1 kb from the transcription start
site (7). It is comprised of the ferritin H basal enhancer (53), FER-1,
and an AP1/NF-E2 consensus sequence located 8 bp 3' of FER-1. The basal
enhancer, FER-1, is in turn composed of an element with close sequence
similarity to both AP1 and NF-E2 consensus sequences (previously termed
AP1-like element (53, 54) and referred to in this report as
AP1/NF-E2-like element), and a recognition sequence for the SP1/3
transcription factors (53, 54). The AP1/NF-E2-like and the AP1/NF-E2
consensus sequence of the ferritin H EpRE/ARE are arranged in inverse
repeat, and both of these elements are necessary for full induction of
ferritin H by H2O2 and t-BHQ (7). An EpRE/ARE
has also been identified in the murine ferritin L promoter (55).
Ligation of this element to a luciferase reporter gene demonstrated
that the ferritin L EpRE is functional as an enhancer element in HepG2
cells treated with t-BHQ (55).
Collectively, these results suggest that ferritin may constitute a
component of the cytoprotective response induced by xenobiotics (electrophiles or polycyclic aromatic hydrocarbons) and candidate chemopreventive agents. However, the mechanism of ferritin induction by
these agents is unknown. Here, we demonstrate that ferritins H and L
are induced by oltipraz, D3T, and
-NF in fibroblasts and hepatic
cells. Furthermore, we show that induction of ferritin occurs via an
EpRE/ARE-dependent mechanism that requires Nrf2. These results link ferritin induction mechanistically to the
chemopreventive response.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
NIH3T3, Hepa1-6, and HepG2 cell lines and
Nrf2 wild type (+/+) and Nrf2 knockout (
/
) primary
mouse embryo fibroblasts were maintained at 37 °C in a humidified
atmosphere containing 5% CO2. NIH3T3 (ATCC) cells were
grown in Dulbecco's modified eagle medium (DMEM, Invitrogen)
supplemented with 10% bovine calf serum (HyClone), 100 units/ml
penicillin G sodium, and 100 µg/ml streptomycin sulfate. Hepa1-6
(ATCC) and HepG2 (ATCC) cells were grown in DMEM supplemented with 10%
fetal bovine serum (Gemini Bioproducts), 100 units/ml penicillin G
sodium, and 100 µg/ml streptomycin sulfate. The Nrf2+/+ and
Nrf2
/
primary mouse embryo fibroblasts were grown in
DMEM/F-12 (Invitrogen) supplemented with 15% fetal bovine serum, 2 mM glutamine, 0.1 mM non-essential amino acids,
150 µM
-mercaptoethanol, and 100 units/ml penicillin G
sodium, and 100 µg/ml streptomycin sulfate.
Chemicals--
Oltipraz and D3T were provided by the Division of
Cancer Prevention Repository of NCI, National Institutes of Health, and
-NF was obtained from Sigma. All compounds were dissolved in Me2SO and final Me2SO concentration in
all treatment conditions was 0.4%.
Plasmids--
The following ferritin H-human growth hormone
reporter gene constructs have been previously described (7):
4.8kbFH-hGH,
4.13kbFH-hGH (referred to as
4.0kbAP1+
FH-hGH in
Ref. 7),
4.0kbFH-hGH,
0.32kbFH-hGH, and 107bpEpREFH-hGH (referred
to as 107bpFER-1+AP1 FH-hGH in Ref. 7). To construct ferritin H-luciferase reporter gene constructs
4.8kbFH-CAT (53) was digested
with HindIII to release a fragment that contains the 5'-promoter region of the ferritin H gene, from nucleotide
4819 to
+24. The HindIII fragment was ligated into the
HindIII multiple cloning site of pGL3 (Promega) to create
4.8kbFH-Luc. To construct
4.2kbFH-Luc,
4.0kbFH-Luc, and
0.225kbFH-Luc,
4.8kbFH-Luc was digested with SacI,
BglII, and SmaI to release 616-, 789-, and 4618-bp fragments, respectively. The remaining 9.0-, 8.9-, and 5.1-kb
fragments were gel-purified and religated to form
4.2kbFH-Luc,
4.0kbFH-Luc, and
0.225kbFH-Luc, respectively. The
4.8kb
3.5kbFH-Luc internal deletion construct was made by removing
an internal EcoRI fragment from
4477 to
941 from
4.8kb
FH-Luc by digestion with EcoRI and religation. The
75bpEpREFH-Luc insertion construct was made as follows. Sense and
antisense oligonucleotides corresponding to the 75-bp ferritin H
EpRE/ARE were synthesized by the Wake Forest University School of
Medicine Comprehensive Cancer Center DNA synthesis laboratory,
PAGE-purified, phosphorylated using T4 Polynucleotide kinase, annealed,
and ligated into the SmaI site of
0.225kbFH-Luc. The
Nrf2 dominant negative mutant expression plasmid
(pEF/Nrf2dnm) as well as the empty expression plasmid (pEF) were
kind gifts of Dr. Jawed Alam (56). The Nrf2 expression plasmid
(pEF/Nrf2) has been described previously (57).
Northern Blot Analysis--
Total RNA was isolated from cells
treated for 24 h with vehicle, oltipraz, D3T, and/or
-NF as
described by Chirgwin et al. (58) or utilizing the TRIzol
reagent (Invitrogen) according to the manufacturer's protocol. 10-15
µg of RNA were size-fractionated on 1.1% agarose/6.6% formaldehyde
gels and transferred to an Immobilon Ny+ nylon membrane (Millipore) by
capillary transfer. DNA probes for both ferritin H (59) and L (60) were
generated by random prime labeling and subsequently hybridized to the
UV-cross-linked RNA blot in Quick Hyb solution (Stratagene) according
to the manufacturer's protocol. Membranes were subjected to
autoradiography; quantitation was performed using a PhosphorImager
analyzer (model 445SI, Amersham Biosciences).
Western Blotting of Ferritin Induction--
To assess ferritin H
and
-actin protein levels, cytosolic extracts were prepared as
previously described by Schreiber et al. (61). 50 µg of
protein was fractionated on 12%SDS-polyacrylamide gels, transferred to
nitrocellulose, blocked with 5% nonfat dry milk in phosphate-buffered
saline, washed, and incubated with a 1:1000 dilution of polyclonal
rabbit anti-ferritin H peptide antibody (BIOSOURCE
International) followed by a 1:200 dilution of goat anti-rabbit IgG
conjugated to horseradish peroxidase (Bio-Rad). The blots were
developed using the Enhanced Chemiluminescence System (Amersham
Biosciences). To demonstrate equivalent protein loading, blots were
washed and re-blotted using a 1:20,000 dilution of anti-
actin
antibody (Sigma) followed by a 1:5,000 dilution of goat anti-mouse IgG
conjugated to horseradish peroxidase (Calbiochem).
Transfection of Ferritin H-human Growth Hormone Reporter Gene
Constructs and RNase Protection Assay--
NIH3T3 cells were
transfected in duplicate with 2 or 3 µg of FH-hGH reporter gene
constructs using LipofectAMINE Reagent (Invitrogen) according to the
manufacturer's protocol. Cells were allowed to recover for 20-24 h
and treated with 70 µM Oltipraz, 70 µM D3T, or vehicle (Me2SO). RNA was isolated after 24 h, and
RNase protection analysis (RPA) was performed as described previously
(3). The -fold induction was calculated based on means and standard
errors of three to eight independent experiments.
Transfection of Ferritin H-luciferase Reporter Gene Constructs
and Luciferase Assay--
Hepa1-6 cells were transfected for 4 h
with a total of 500 ng of DNA (FH-Luc reporter gene constructs,
-galactosidase transfection control plasmid, and pUC18 as detailed
in the respective figure legends) using the LipofectAMINE reagent
(Invitrogen) according to the manufacturer's procedures. Cells were
allowed to recover for 20-24 h and subsequently were treated with 25 µM
-NF or vehicle (Me2SO) for 24 h.
Cells were harvested, lysed in 1× reporter lysis buffer (Promega), and
assayed for luciferase activity. Luciferase activity was assessed using
the Luciferase Assay kit (Promega) according to the manufacturer's
protocol. Expression of the
-galactosidase transfection control was
measured as previously described (62).
Isolation of Nuclear Extracts and Electrophoretic Mobility Shift
Assay--
Nuclear extracts were isolated as described previously
(53). The oligonucleotides used in electrophoretic mobility shift assays correspond to the ferritin H AP1/NF-E2-like element
(5'-CTCCATGACAAAGCATTT-3') present in the ferritin H basal enhancer,
FER-1, and the ferritin H AP1/NF-E2 consensus element
(5'-AGAATGCTGAGTCACGGTG-3'). The double-stranded oligonucleotides were
end-labeled with [
-32P]ATP (ICN) using T4
Polynucleotide kinase. Competition experiments were performed using
100-fold molar excess of unlabeled oligonucleotides. Normal rabbit
serum or Nrf2 antibody (Santa Cruz Biotechnology, sc-722x) were
used in supershift experiments. DNA-protein complexes were isolated on
a native 4% polyacrylamide gel (80:1, acrylamide:bisacrylamide). Gels
were dried, and DNA-protein complexes were visualized by autoradiography.
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RESULTS |
Xenobiotics and Chemopreventive Agents Induce Ferritin
mRNA--
-NF is a polycyclic aromatic hydrocarbon that has
been used to study activation of phase 2 enzymes by both XRE- and
EpRE/ARE-dependent mechanisms (23, 24, 32, 63). Oltipraz is
an electrophilic dithiolethione that represents a widely studied class
of candidate chemopreventive agents. To create a model system in which
to explore the mechanism of ferritin induction by these agents, we
treated cultured liver cells for varying periods of time with
-NF or oltipraz. As shown in Fig. 1, both
-NF
and oltipraz induced ferritin H and L mRNA in Hepa1-6 cells in a
time-dependent manner. We also performed Northern blot
analysis of NIH3T3 fibroblasts treated with oltipraz and a second
dithiolethione, D3T. As shown in Fig. 2,
both these agents induced ferritin H and L mRNA in NIH3T3 cells. Induction of ferritin H as well as ferritin L mRNA by oltipraz in
NIH3T3 cells was time-dependent and occurred as early as
3 h after treatment, with induction peaking at 24 h. mRNA
induction was accompanied by an increase in ferritin protein of similar magnitude (Fig. 3). Induction of ferritin
mRNA by oltipraz, D3T, and
-NF was also seen in HepG2 cells
(data not shown). Taken together, these results demonstrate that
-NF, oltipraz, and D3T induce both ferritin H and L in a variety of
cells, including murine and human hepatocytes and fibroblasts.

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Fig. 1.
Induction of ferritin H and L mRNA by
oltipraz and -NF. Total RNA was isolated
from Hepa1-6 cells treated with (A) 25 µM
-NF or (B) 70 µM oltipraz for 8 and 24 h. RNA was size-fractionated on denaturing agarose gels, transferred to
nylon membranes, and allowed to hybridize with cDNA probes specific
for ferritin H and L. Ethidium bromide staining was done to assure
equal RNA loading. Shown are the average ± S.E. values for two
experiments.
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Fig. 2.
Induction of ferritin H and L mRNA by
dithiolethiones. NIH3T3 cells were treated with 70 µM D3T for 24 h or 70 µM oltipraz for
0, 3, 6, 9, 12, and 24 h. Total RNA was isolated from cells,
size-fractionated on denaturing agarose gels, transferred to nylon
membranes, and allowed to hybridize with cDNA probes specific for
ferritin H and L. Ethidium bromide staining of the gel was done to
assure equal RNA loading. Quantitatively similar results were obtained
when hybridization to -actin cDNA was used to verify RNA
integrity and loading (data not shown). Average ± S.E. values for
four experiments are shown.
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Fig. 3.
Oltipraz and D3T induce ferritin H protein
levels. Cell lysates were obtained 24 (D3T-treated cells) or 48 (oltipraz-treated cells) h after treatment of NIH3T3 cells with vehicle
(Me2SO), 70 µM oltipraz, or 70 µM D3T. Cell lysates were electrophoresed on a 12%
SDS-polyacrylamide gel and transferred to a nitrocellulose membrane.
The membrane was blotted with antibody to ferritin H and -actin
(loading control) as described under "Experimental
Procedures."
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Induction of Ferritin Is Mediated by the EpRE/ARE--
To assess
the mechanism of ferritin induction, we transiently transfected NIH3T3
cells with a chimeric ferritin H-human growth hormone (FH-hGH) gene
construct that spans 4.8 kb of the murine ferritin H promoter region
fused to the human growth hormone reporter gene (
4.8kbFH-hGH).
Subsequently, cells were treated with 70 µM oltipraz or
70 µM D3T. After 24 h, RNA was isolated and RNase protection analysis was performed to assess the induction of the reporter gene as well as the endogenous ferritin H gene. Fig. 4 demonstrates that both oltipraz and D3T
induce the endogenous ferritin H gene, confirming the Northern blot
analysis shown in Fig. 2. In addition, induction of the
4.8kbFH-hGH
reporter gene construct by oltipraz and D3T was observed. These results
indicate that induction of ferritin H by oltipraz and D3T is mediated
by a transcriptional mechanism.

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Fig. 4.
Increases in ferritin H mRNA by oltipraz
and D3T are due to a transcriptional mechanism. A, a
ferritin H-human growth hormone (FH-hGH) reporter gene construct
spanning 4.8 kb of the murine ferritin H promoter region
( 4.8kbFH-hGH) was transiently transfected into NIH3T3 cells. 20-24 h
after transfection, cells were treated with vehicle
(Me2SO), 70 µM oltipraz, or 70 µM D3T for 24 h. Total RNA was isolated, and 10 µg
of RNA was used for RNase protection analysis (RPA). For the RPA, a
probe was used that encompasses a region of the ferritin H and human
growth hormone gene, thereby allowing for simultaneous detection of the
endogenous ferritin H as well as the transfected hGH gene.
B, the average ± S.E. of three independent experiments
for induction of the endogenous ferritin H and the transfected hGH gene
by oltipraz and D3T.
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To delineate the element responsible for transcriptional activation of
ferritin H, deletion analysis was performed. FH-hGH 5' deletion
constructs containing 4.8, 4.13, or 4.0 kb of the murine ferritin H 5'
flanking region were transiently transfected into NIH3T3 cells, treated
with 70 µM oltipraz, and analyzed by RNase protection
assay. As shown in Fig. 5, a region
located between
4.13 and
4.0 kb of the ferritin H promoter is
responsible for activation of ferritin H transcription. Thus, both the
4.8kbFH-hGH and
4.13kbFH-hGH construct were induced by oltipraz,
whereas the
4.0kbFH-hGH construct was not. Because the 75-bp ferritin H EpRE/ARE is located between
4.13 kb and
4.0 kb of the 5'ferritin H promoter region, this result suggested that the ferritin H EpRE/ARE mediates induction of ferritin H in response to oltipraz. To test this,
we used a chimeric gene in which a 107-bp region containing the
ferritin H EpRE/ARE was inserted into a minimal ferritin H promoter
construct (
0.32kbFH-hGH). As shown in Fig.
6, activity of this promoter was enhanced
by oltipraz, demonstrating that the ferritin H EpRE/ARE is sufficient
to mediate induction of ferritin H in response to oltipraz. Thus,
ferritin H contains a functional EpRE/ARE that mediates responsiveness
to oltipraz.

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Fig. 5.
Ferritin H transcription by dithiolethiones
is mediated by an enhancer element located between 4.13 and 4.0
kb. A, schematic of the ferritin H-human growth
hormone (FH-hGH) 5' deletion constructs used in defining the
dithiolethione-responsive element. The location of the ferritin H
EpRE/ARE is indicated by the black rectangle. B,
FH-hGH reporter gene constructs were transiently transfected into
NIH3T3 cells. 20-24 h after transfection cells were treated with
vehicle (Me2SO) or 70 µM oltipraz for 24 h. Total RNA was isolated, and 10 µg of RNA was used for RNase
protection analysis. C, the average ± S.E. of five to
eight independent experiments for induction of the transfected hGH
gene.
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Fig. 6.
The ferritin H EpRE/ARE mediates
transcriptional activation of ferritin H in response to
dithiolethiones. A, schematic of the insertion
constructs. 0.32kbFH-hGH contains 320 bp of the ferritin H promoter
region fused to the human growth hormone reporter gene. 107bpEpREFH-hGH
is the same as 0.32kbFH-hGH with the exception that a 107-bp region
containing the 75-bp ferritin H EpRE/ARE was inserted in front of the
320-bp ferritin H minimal promoter region. B, FH-hGH
reporter gene constructs were transiently transfected into NIH3T3
cells. 20-24 h after transfection, cells were treated with vehicle
(Me2SO) or 70 µM oltipraz for 24 h.
Total RNA was isolated, and 10 µg of RNA was used for RNase
protection analysis. C, average ± S.E. of four
independent experiments for induction of the transfected hGH
gene.
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To determine whether the induction of ferritin H by
-NF utilized a
similar mechanism, we first considered the potential role of XRE
sequences.
-NF can activate transcription via both XRE- and
EpRE/ARE-dependent mechanisms (16). The XRE consensus
sequence has been defined as 5'-T(A/T)GCGTG-3' (18), and functional XRE sequences have previously been genetically identified in the
cytochromes P450 1A1 and 1A2 (18, 19, 64), GST Ya (20), UDP-glucoronyl transferase 1A1 and 1A6 (21, 22), NQO1 (23), and Cu/Zn-SOD gene (24,
25). Inspection of the ferritin H 5' promoter sequence revealed five
potential consensus XRE sequences, one on the sense strand and four on
the antisense strand. Their specific locations and sequences are as
follows. The XRE sequence present in the sense strand is located
between
3089 and
3083 (5'-CTGCGTG-3'). The XRE sequences in the
antisense strand are located between
4579 and
4573 (5'-CACGCAC-3'),
2817 and
2811 (5'-CACGCCC-3'),
413 and
407 (5'-CACGCTT-3'), and
351 and
345 (5'-CACGCAC-3'). To determine if these elements were
functional, we prepared ferritin H-luciferase constructs (luciferase
was used to simplify assessment of reporter gene expression) (Fig.
7). These constructs spanned
4.8,
4.2, and
4.0 kb of the murine ferritin H 5' promoter region. In
addition, to determine the involvement of the XRE sequence located from
4579 to
4573 independent of the ferritin H EpRE/ARE sequence
(
4117 to
4043), an internal deletion construct was made in which
3.5 kb of the ferritin H promoter were removed from the full-length
4.8kbFH-Luc construct spanning a region from
4477 to
941. The
ferritin H-luciferase 5' deletion and internal deletion constructs were
transiently transfected into Hepa1-6 cells. Subsequently, cells were
treated with 25 µM
-NF for 24 h, and luciferase
activity was determined. The constructs
4.8kbFH-Luc and
4.2kbFH-Luc
demonstrated luciferase induction in response to
-NF, whereas
4.8kb
3.5kbFH-Luc and
4.0kbFH-Luc did not (Fig. 7). Hence,
induction of ferritin H by
-NF is mediated by an enhancer element
located between
4.2 and
4.0 kb, a region that contains the ferritin
H EpRE/ARE but none of the XRE sequences. To confirm that activation of
ferritin H by
-NF occurred via the EpRE/ARE, the 75-bp EpRE/ARE
element was inserted into a minimal promoter construct, which contains
225 bp of the ferritin H promoter (
0.225kbFH-Luc). As shown in Fig.
7, 75bpEpREFH-Luc mediated induction of luciferase activity, whereas
0.225kbFH-Luc did not. Thus, ferritin H is transcriptionally
activated by
-NF via a mechanism that depends on the EpRE/ARE and
not the XRE sequences.

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Fig. 7.
-NF activates ferritin H
transcriptionally via an EpRE/ARE- and not XRE-dependent
mechanism. A, schematic of the ferritin H-luciferase
(FH-Luc) reporter gene constructs used. The internal deletion in the
4.8kb 3.5kbFH-Luc construct is signified by a dotted line.
The location of the EpRE/ARE and XRE sequences are indicated by
black and gray rectangles, respectively.
B, 50 ng of reporter gene plasmids and 10 ng of
-galactosidase transfection control plasmid were transfected
together with 440 ng of pUC18 to generate a total of 500 ng of DNA.
20-24 h after transfection cells were treated with vehicle
(Me2SO) or 25 µM -NF for 24 h. Cell
lysates were obtained to perform luciferase and -galactosidase
assays as described under "Experimental Procedures." Luciferase
values were normalized to -galactosidase expression, and -fold
induction was calculated with 4.8 kb FH-Luc treated with vehicle
defined as 1. Shown are the average ± S.E. values of three
independent experiments.
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Elements in the Ferritin H EpRE/ARE Bind
Nrf2--
Nrf2 is a member of the NF-E2 family of
transcription factors that has been shown to mediate
EpRE/ARE-dependent transcription of a variety of
cytoprotective genes (36, 47, 56, 65). The ferritin H EpRE/ARE contains
two elements with sequence similarity to the NF-E2 consensus sequence.
The AP1 consensus sequence of the ferritin H EpRE/ARE is embedded in a
canonical NF-E2 site; the AP1-like sequence in the ferritin H EpRE/ARE
also possesses considerable sequence similarity to the NF-E2 consensus
(9/11 residues) (Fig. 8). To examine the
ability of Nrf2 to bind to the ferritin H EpRE/ARE, we performed
electrophoretic mobility shift assays. We assessed binding to both the
AP1/NF-E2-like element and the AP1/NF-E2 element, both of which are
contained in the ferritin H EpRE/ARE and show high homology to the
consensus EpRE/ARE sequence (Fig. 8). These experiments were performed
using nuclear extracts from HepG2 cells, because in our hands
commercially available Nrf2 antibodies did not reliably bind to
mouse Nrf2. As shown in Fig. 9,
nuclear extracts isolated from HepG2 cells treated with 70 µM oltipraz or 25 µM
-NF for 6 h
bound these components of the EpRE/ARE. Fig. 9 also demonstrates that
addition of an antibody to Nrf2 results in a supershift at both
elements; in contrast, addition of normal rabbit serum did not result
in a supershift. Thus, Nrf2 binds to the ferritin H
EpRE/ARE.

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Fig. 8.
Schematic of the 75-bp ferritin H
EpRE/ARE. A, sequences above and
below the schematic indicate the presence of AP1/NF-E2-like
and AP1/NF-E2 consensus elements in this region. Arrows
above and below the sequence denote the orientation of
the transcription factor binding sites. B, listing of the
EpRE/ARE, AP1, and NF-E2 consensus sequence.
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Fig. 9.
Nrf2 binds to the AP1/NF-E2-like and
AP1/NF-E2 consensus elements of the ferritin H EpRE/ARE. Nuclear
extracts were isolated from HepG2 cells that had been treated with
vehicle (Me2SO), 70 µM oltipraz, or 25 µM -NF for 6 h. 10-20 µg of extract was
incubated with 32P-labeled AP1/NF-E2-like or AP1/NF-E2
consensus oligonucleotide (100,000-150,000 cpm), and 100-fold molar
excess of specific (SC) and nonspecific (NC)
competitors where indicated. A NF B binding element (Promega) was
used as a nonspecific competitor. Following a 20-min incubation at room
temperature, an antibody specific for Nrf2 or normal rabbit
serum (c) was added to the indicated samples for a 30-min
room temperature incubation. The DNA-protein complexes were separated
by 4% polyacrylamide gel electrophoresis. Specific bands are pointed
out by the large arrows, and supershifted Nrf2 is
marked by the small arrows.
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Role for Nrf2 in Induction of Ferritin by Oltipraz, D3T, and
-NF--
To test involvement of Nrf2 in ferritin induction,
we compared the ability of Nrf2+/+ and Nrf2
/
primary
mouse embryo fibroblasts to induce ferritin H and L in response to
oltipraz, D3T, and
-NF. As shown in Fig.
10, induction of ferritin H and L
mRNA was blocked in Nrf2
/
cells, whereas both ferritin H
and L were induced in the Nrf2+/+ cells. Basal levels of
ferritin H and L mRNA were also reduced in Nrf2 knockout
cells, suggesting a role for Nrf2 in both basal and induced
ferritin transcription.

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Fig. 10.
Nrf2 is required for activation of
ferritin H and L by oltipraz, D3T, and
-NF. Total RNA was isolated from
Nrf2+/+ and Nrf2 / cells that had been treated with
vehicle (Me2SO), oltipraz, D3T, and -NF for 24 h.
RNA was size-fractionated on denaturing agarose gels, transferred to
nylon membranes, and allowed to hybridize with cDNA probes for
ferritin H and L. Ethidium bromide staining was done to assure equal
RNA loading. The -fold induction was calculated with Nrf2+/+
cells treated with vehicle defined as 1. Shown are the average ± S.E. values for two ( -NF) or four (oltipraz and D3T) independent
experiments.
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|
Nrf2 Mediates Transcription of Ferritin H at the
EpRE/ARE--
To demonstrate that Nrf2 mediates
transcriptional activation of ferritin H via an
EpRE/ARE-dependent mechanism, we transiently cotransfected
a Nrf2 dominant negative mutant expression plasmid and the
75bpEpREFH-Luc reporter gene construct into Hepa1-6 cells. As seen in
Fig. 11, cotransfection of the
75bpEpREFH-Luc reporter gene construct with an empty expression vector
(pEF) and subsequent treatment with 25 µM
-NF for
24 h resulted in induction of luciferase activity. However,
cotransfection of a dominant negative mutant of Nrf2
(pEF/Nrf2dnm) and the 75bpEpREFH-Luc reporter gene construct suppressed
-NF-induced activation of luciferase activity. In addition, pEF/Nrf2dnm decreased basal activity of the
75bpEpREFH-Luc reporter gene construct, supporting the results in Fig.
10 indicating that Nrf2 affects basal as well as inducible
ferritin H expression. Neither pEF/Nrf2dnm nor
-NF had any
effect on basal or inducible expression of a ferritin H minimal
promoter construct lacking the EpRE/ARE (
0.225kbFH-Luc) (Fig. 11). To
confirm these results, 75bpEpREFH-Luc was cotransfected with increasing
amounts of a Nrf2 expression plasmid (pEF/Nrf2). As shown
in Fig. 12, Nrf2 activates luciferase activity in a dose-dependent manner. This
response requires the EpRE/ARE, because
0.225kbFH-Luc was unaffected
by increasing amounts of pEF/Nrf2 (Fig. 12).

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Fig. 11.
Nrf2 mediates transcriptional
activation of ferritin H at the EpRE/ARE. 50 ng of reporter vector
( 0.225kbFH-Luc or 75bpEpREFH-Luc) was cotransfected with 50 ng of pEF
or pEF/Nrf2dnm,10 ng of -galactosidase transfection control
plasmid, and 390 ng of pUC18 to generate a total of 500 ng of DNA.
20-24 h after transfection, cells were treated with vehicle
(Me2SO) or 25 µM -NF for 24 h. Cell
lysates were obtained to perform luciferase and -galactosidase
assays as described under "Experimental Procedures." Luciferase
values were normalized to -galactosidase expression, and -fold
induction was calculated with 75bpEpREFH-Luc cotransfected with pEF and
with vehicle treatment defined as 1. Shown are the average ± S.E.
values of three independent experiments.
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Fig. 12.
Forced expression of Nrf2 drives
expression of 75bpEpREFH-Luc. 50 ng of reporter vector
( 0.225kbFH-Luc or 75bpEpREFH-Luc) was cotransfected with 10 ng of
-galactosidase transfection control plasmid, and varying amounts of
pEF/Nrf2 (0, 5, 10, 25, and 50 ng) and pEF (150, 140, 125, and
100 ng), and 290 ng of pUC18 to generate a total of 500 ng of DNA.
48 h after transfection, cell lysates were obtained to perform
luciferase and -galactosidase assays as described under
"Experimental Procedures." Luciferase values were normalized to
-galactosidase expression, and -fold induction was calculated with
75bpEpREFH-Luc cotransfected with 0 ng of pEF/Nrf2 defined as 1. Shown are the average ± S.E. values of two (5 ng of
pEF/Nrf2) or three (0, 10, 25, and 50 ng of pEF/Nrf2)
independent experiments.
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DISCUSSION |
Several laboratories have demonstrated that ferritin H and L are
induced in response to oxidants and pro-oxidant xenobiotics (7-11),
and a transcriptional mechanism has been implicated (9). Activation of
ferritin in response to these agents is thought to serve a
cytoprotective function from iron-catalyzed oxidative damage.
Previously, we have genetically defined the DNA element responsible for
inducing ferritin H transcription in response to oxidants (Fig. 8).
This element contains a cis-acting DNA-enhancer located ~4.1 kb from
the transcription start site and possesses sequence similarity to the
consensus electrophile/antioxidant-responsive element (EpRE/ARE) (7).
Sequence searches and reporter assays have also identified a functional
EpRE/ARE in the ferritin L gene (55).
In this report we demonstrate that ferritin H and L mRNA are
induced by
-NF and by chemopreventive dithiolethiones in fibroblasts and hepatic cells. Elevation of ferritin H mRNA by the
dithiolethiones oltipraz and D3T is the result of a transcriptional
mechanism. Similarly, Primiano et al. (15) provided evidence
for transcriptional activation of ferritin H and L in the livers of
rats treated with D3T. Using ferritin H deletion constructs fused to
the human growth hormone gene, the results presented in this study
identify the EpRE/ARE as the mediator of transcriptional activation of
ferritin H in response to
-NF, oltipraz, and D3T. These results
implicate ferritin in the chemopreventive response and suggest that
ferritin, together with other EpRE/ARE-mediated cytoprotective
proteins, contributes to the chemoprotected phenotype in cells in which these proteins have been induced. Classes of proteins induced by
chemopreventive agents include both conjugating enzymes (glutathione S-transferases and UDP-glucuronosyltransferase)
involved in detoxification and export and antioxidant enzymes
(superoxide dismutase and NAD(P)H:quinone oxidoreductase 1). Ferritin,
with its ability to sequester iron and prevent the formation of oxygen
free radicals, has some characteristics of this latter class. In fact,
we and others (66, 67) have shown that repression of ferritin synthesis
is correlated with enhanced sensitivity to oxidative stress;
conversely, induction/overexpression of ferritin has been linked to
enhanced cellular protection against oxidant-induced cytotoxicity (68,
69).
A sequence search of 4.8 kb of the murine ferritin H 5'-flanking
sequence revealed the presence of five XRE sequences that were
potential candidates for regulating ferritin H transcription in
response to polycyclic aromatic hydrocarbons, such as
-NF. However,
deletion analysis of the ferritin H 5'-promoter region using ferritin
H-luciferase (FH-Luc) reporter gene constructs demonstrated that these
elements are non-functional in ferritin induction by
-NF, because
reporter constructs containing these putative XRE sequences but lacking
the EpRE/ARE were not induced in response to
-NF (Fig. 7). Rather,
the EpRE/ARE controls ferritin H transcription in response to
-NF
(Fig. 7). Because the five XRE sequences closely resemble the consensus
XRE sequence (5'-T(A/T)GCGTG-3') (18), it was surprising that none of
these elements in the ferritin H promoter region conferred inducibility
of the gene in response to
-NF. In particular, comparison of the
ferritin H XRE sequences to XRE sequences identified previously in
various genes showed that the ferritin H XRE sequences at nucleotides
(nt)
4579 to
4573 and nt
351 to
345 are identical to the
functional XRE identified in the rat UDP-glucoronyl transferase 1A1
(21) and human CYP 1A2 (19) gene. Likewise, the XRE sequence at nt
3089 to
3083 and nt
2817 to
2811 are identical to the XREs
present in the rat and human Cu/Zn-SOD genes, respectively (24, 25). It
has been suggested that the XRE core sequence itself is insufficient to
confer transcriptional induction, and that additional nucleotides flanking the core sequence exert an important influence on the ability
of the Ah receptor to bind to the core XRE sequence (70). These
contextual requirements may contribute to the inactivity of the XRE
elements in the ferritin H promoter.
The electrophile/antioxidant-responsive element consensus sequence
resembles the recognition sequence for transcription factors of the AP1
and NF-E2 family of DNA binding proteins, allowing for a wide variety
of transcription factors to bind to this element and mediate basal as
well as inducible transcription. Several laboratories have demonstrated
the involvement of the transcription factor Nrf2 in inducing
cytoprotective proteins in response to a variety of agents, including
-NF and dithiolethiones (47, 65, 71). Some genes require Nrf2
for basal as well as D3T-inducible transcription, whereas others showed
induction by D3T independent of Nrf2 status (65). Results
presented here demonstrate that treatment of Nrf2+/+ and
Nrf2
/
primary mouse embryo fibroblasts results in induction
of ferritin H and L mRNA in wild type but not Nrf2 knockout
cells, indicating that Nrf2 is necessary for dithiolethione- and
-NF-induced transcription of both ferritin H and L. Gel shift and
transfection experiments indicate that Nrf2-mediated induction
of ferritin H targets the ferritin H EpRE/ARE (Figs. 9, 11, and 12).
Although we did not quantitate nuclear Nrf2 following treatment
with oltipraz, D3T, or
-NF, the increasing intensity in band shift
seen in Fig. 9 is consistent with increased binding of Nrf2 to
the ferritin H EpRE/ARE following stimulation with xenobiotics. This
would be concordant with demonstrations of nuclear translocation of
Nrf2 following treatment with inducing agents (36, 49).
Our experiments showed not only that Nrf2 is involved in
activating transcription of ferritin H and L in response to
dithiolethiones and
-NF, but also suggested the involvement of
Nrf2 in basal transcription of these genes, since expression of
ferritin H and L mRNA was decreased in Nrf2
/
cells when
compared with the wild type cells (Fig. 10). Effects on ferritin H
basal transcription were confirmed by cotransfecting 75bpEpREFH-Luc
with a dominant negative mutant of Nrf2, which both suppressed
EpRE/ARE-dependent induction of ferritin H in response to
an inducer and decreased basal expression. A similar involvement of
Nrf2 in basal transcription has been reported for other genes,
including glutathione S-transferases (72) and
-glutamylcysteine
synthetase (57).
The ferritin H EpRE/ARE has two sites with which Nrf2 interacts,
namely the AP1/NF-E2 consensus sequence and the AP1/NF-E2-like element
of FER-1 (Fig. 9). Although we did not examine the interaction of the
ferritin L EpRE/ARE with Nrf2, inspection of the ferritin L
promoter reveals a NF-E2 consensus sequence embedded in the ferritin L
EpRE/ARE, suggesting that Nrf2 may be involved in the coordinate
regulation of both ferritin subunits through targeting of the
EpRE/ARE.
Nrf2 has been demonstrated to be important in the regulation of
several EpRE/ARE-dependent genes. However,
Nrf2-independent mechanisms of gene regulation at the EpRE/ARE
also exist. For example, it was recently reported that the ability of
the model chemopreventive agent sulforaphane to induce some but not all EpRE/ARE-dependent genes was abrogated in the intestine of
Nrf2 knockout mice (73). Kwak et al. (65) reported
that in the livers of Nrf2 knockout mice treated with D3T,
induction of ferritin was enhanced rather than suppressed. These
findings differ from results presented here that indicate that
Nrf2 is required for induction of ferritin by oltipraz, D3T, and
-NF. However, our experiments were performed in knockout cells
derived from a different genetic background (74) than those used by
Kwak et al. Given the existence of alternative pathways of
regulation at the EpRE/ARE, and the potential of AP1 and Maf proteins
and other transcription factors to modulate Nrf2 activity both
positively and negatively, it is possible that the relative abundance
and/or activity of such factors may influence gene inducibility. Thus,
cellular context and genotype may determine the contribution of
Nrf2 to the regulation of target genes. For example, in the
GCSh gene, higher concentrations of c-Jun repressed
expression, presumably due to formation of c-Jun/c-Fos complexes
that interfered with binding of the Nrf2/c-Jun complex to the
EpRE/ARE (75). Genetic variation, including polymorphisms in
Nrf2 itself, may add an additional level of complexity to such interactions (76). Collectively, these observations may point to the
existence of additional regulatory pathways that permit fine-tuning of
the cellular response to xenobiotics, perhaps allowing the coordinate
induction of subsets of antioxidant and detoxification genes in
different cell types dependent on cellular context, genotype, and
xenobiotic challenge.
We have previously identified a number of transcription factors that
assemble at the FER-1 component of the ferritin H EpRE/ARE. These
include members of the AP1 family such as JunD and FosB, and the
ATF/CREB family member ATF1 (54). Results presented here implicate
Nrf2 as an additional participant in this transcription factor
complex, conferring added potential for both positive and negative
regulation at this element. Nrf2 has been shown to bind to DNA
as a heterodimer with small Maf (MafG and MafK) proteins, an
interaction that can be negatively regulated by large Maf proteins such
as c-Maf (77). Nrf2 may also interact directly with other transcription factors, such as AP1 (45, 47) and ATF4 (78). Because the
DNA recognition sequence for AP1 and Nrf2 family members overlaps considerably, it has also been suggested that under selected circumstances Nrf2 function may be modulated indirectly through displacement of Nrf2 from DNA by AP1 family members (75).
Nrf2 can also interact with global adaptor and chromatin
remodeling factors such as p300/CBP (79). We have previously
demonstrated that the p300/CBP transcriptional adaptor proteins are
involved in mediating basal ferritin H transcription through the FER-1 element, possibly due to an interaction between p300/CBP and FER-1 binding proteins (80). Indirect evidence indicates that p300/CBP may be
necessary for EpRE/ARE-mediated induction of ferritin H, because NIH3T3
cells stably transfected with E1A lose the ability to induce ferritin H
mRNA in response to t-BHQ, a classic inducer of antioxidant and
phase II enzymes (67). These results suggest that Nrf2 may
mediate some of its effects on basal and inducible ferritin H
expression through interaction with p300/CBP. Further studies will be
required to test how the assembly of this complex array of
transcription factors, cofactors, and chromatin remodeling factors is
regulated at the ferritin EpRE/ARE.