From the Section of Pulmonary and Critical Care
Medicine, Yale University School of Medicine,
New Haven, Connecticut 06520,
Renal
Electrolyte Division and § Division of Pulmonary, Allergy,
and Critical Care Medicine, Department of Medicine, University of
Pittsburgh, Pittsburgh, Pennsylvania 15231,
Department of
Molecular Genetics, Alton Ochsner Medical Foundation, New
Orleans, Louisiana 70121, and the ** Department of Biochemistry and
Molecular Biology, Louisiana State University Health Sciences Center,
New Orleans, Louisiana 70112
Received for publication, February 7, 2001, and in revised form, March 23, 2001
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ABSTRACT |
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Nrf2 regulates expression of genes encoding
enzymes with antioxidant (e.g. heme oxygenase-1 (HO-1)) or
xenobiotic detoxification (e.g. NAD(P)H:quinone
oxidoreductase, glutathione S-transferase) functions via
the stress- or antioxidant-response elements (StRE/ARE). Nrf2
heterodimerizes with small Maf proteins, but the role of such dimers in
gene induction is controversial, and other partners may exist. By using
the yeast two-hybrid assay, we identified activating transcription
factor (ATF) 4 as a potential Nrf2-interacting protein.
Association between Nrf2 and ATF4 in mammalian cells was
confirmed by co-immunoprecipitation and mammalian two-hybrid assays.
Furthermore, Nrf2·ATF4 dimers bound to an StRE sequence from
the ho-1 gene. CdCl2, a potent inducer of HO-1,
increased expression of ATF4 in mouse hepatoma cells, and detectable
induction of ATF4 protein preceded that of HO-1 (30 min
versus 2 h). A dominant-negative mutant of ATF4
inhibited basal and CdCl2-stimulated expression of a
StRE-dependent/luciferase fusion construct (pE1-luc) in hepatoma cells
but only basal expression in mammary epithelial MCF-7 cells. A dominant
mutant of Nrf2 was equally inhibitory in both cell types in the
presence or absence of CdCl2. These results indicate that
ATF4 regulates basal and CdCl2-induced expression of the ho-1 gene in a cell-specific manner and possibly in a
complex with Nrf2.
Overproduction of oxygen free radicals, attenuation of antioxidant
systems, or both, commonly in response to extracellular stimuli,
disturbs the cellular redox status and leads to oxidative stress. Such
conditions typically elicit an adaptive response aimed at reversing
this imbalance and maintaining redox homeostasis. In part, this
adaptive response includes the activation of specific signaling
pathways and, ultimately, the coordinate induction of a select set of
genes that encode proteins with distinct activities that individually
and collectively manifest antioxidant and cytoprotective functions.
Central to this induction process are redox-sensitive transcription
factors, such as nuclear factor- Recent studies from several laboratories (3-8) have implicated another
transcription regulator, Nrf2, with a potentially significant
role in the adaptive response to oxidative stress. Nrf2 belongs
to the CNC-bZIP subfamily of basic region/leucine zipper (bZIP)
transcription factors. CNC-bZIP proteins are distinguished from other
bZIP subfamilies, including those composed of Jun, Fos, ATF/CREB, or
Maf factors, in that they also contain a Cap'n'Collar (CNC)
structural motif homologous to a region within the
Drosophila homoeotic selector protein encoded by the
cap'n'collar gene (9). bZIP proteins function as obligate
dimers; for example, individual Jun-Jun or Jun-Fos dimers are commonly
and collectively referred to as activator protein-1 transcription
factors. Sequences necessary for both dimerization and DNA binding
reside within the bipartite bZIP domain.
Limited but consistent observations (6, 8) suggest that under normal
conditions, and as is the case for NF- Nrf2, like other CNC/bZIP proteins and Fos family members,
belongs to a sub-class of bZIP factors with leucine zipper motifs incapable of self-dimerization. Consequently, sequence-specific DNA
binding and subsequent induction of target gene transcription requires
association of Nrf2 with other transcription factors. In
accordance with the paradigm established by NF-E2 (10), the first
CNC-bZIP containing mammalian transcription factor isolated, the
most prominent dimerization partners of Nrf2 are the small Maf
proteins, MafF, MafG and MafK (also referred to as p18 (14)). The
precise function of such Nrf2·Maf dimers, however, is
controversial, as they have been proposed to function as both positive
(5) and negative regulators (15) of ARE-dependent gene
transcription. Jun-Nrf2 complexes have also been
implicated as positive effectors of ARE-dependent genes
(16).
Given our incomplete understanding of Nrf2 function, the
propensity of bZIP proteins to form inter- and intra-family dimers (17,
18), and of transcription factors in general to form complexes that
tend to provide both diversity to, and discrimination of, genetic
responses to extracellular stimuli, we reasoned that additional
Nrf2-containing complexes exist intracellularly and that such
complexes would likely regulate Nrf2 target gene expression. Accordingly, we have used the yeast two-hybrid screening procedure to
identify proteins that associate with Nrf2. Herein, we report the
identification of ATF4 as a Nrf2-interacting protein and explore the potential role of ATF4 in the regulation of one Nrf2 target gene, ho-1.
Materials
Tissue culture media were from Life Technologies, Inc., and
fetal bovine serum was obtained from Mediatech. Restriction
endonucleases and other DNA-modifying enzymes were purchased from
either Life Technologies, Inc., or New England Biolabs.
Oligonucleotides were synthesized by IDT, Inc. Radiolabeled nucleotides
were obtained from PerkinElmer Life Sciences. Reagents for luciferase
assays were purchased from Sigma. Anti-mouse Nrf2 was kindly
provided by Dr. M. Yamamoto. Antibodies against other transcription
factors, including anti-human Nrf2, and HO-1 were obtained from
Santa Cruz Biotechnology and StressGen Biotechnologies Corp.,
respectively. All other chemicals were reagent grade.
Plasmids
cDNA Clones--
cDNA clones for mouse Jun D, mouse Fos
B, human ATF3 (I.M.A.G.E. Clone identification number 273190), and
mouse ATF4 (I.M.A.G.E. Clone identification number 1401018) were
obtained from American Type Culture Collection (ATCC).
Mammalian Expression Plasmids and Dominant-negative
Mutants--
Expression plasmids encoding mouse Nrf2
(pEF/Nrf2), p18 (pEF/p18), and the mutant p18 (pEF/p18M) (19)
were kindly provided by Dr. Stuart Orkin. Mouse and rat ATF4 cDNAs
were cloned into pEF/myc/mito and
pcDNA3.1/myc-his (Invitrogen), respectively, to generate
pEF/mATF4 and pCMV/rATF4. A dominant mutant of mouse ATF4 was generated
by overlap extension using PCR resulting in a protein with 6 amino acid
substitutions within the DNA-binding domain
(292RYRQKKR298 to
292GYLEAAA298). The amplification product was
cloned into pEF/myc/mito to generate the plasmid
(pEF/mATF4M). The dominant mutant of Jun D (pCMV/JunDM) was constructed
by cloning the 591-base pair BssHII/BssHII
(blunt-ended) fragment of the mouse Jun D cDNA into the vector
pCMV-Tag2B (Stratagene). This manipulation deletes amino acid residues
1-169 resulting in a trans-activation domain-deficient
mutant of Jun D similar to one described earlier (20). Dominant mutants
of c-Jun and Nrf2 have been described previously (4).
Yeast and Mammalian Two-hybrid (Y2H and M2H) Constructs--
The
"bait" plasmid, pDBLeu-Nrf2, for Y2H was constructed in the
following manner. The mouse Nrf2 cDNA sequence encoding amino acid residues 393-581 (numbering as in Ref. 21) was amplified by PCR
using the primer pair Nrf2-1,
5'-CACGCGTCGACTATGCGTGAATCCCAATG-3', and Nrf2-2,
5'-TCCTCCGGATATCAGTTTTTCTTTGTAT-3'. The amplified product
was digested with SalI and EcoRV restriction
endonucleases (recognition sites underlined) and cloned between the
SalI and StuI sites of the pDBLeu vector (Life
Technologies, Inc.) in-frame with the Gal4 DNA-binding domain (Gdbd).
The integrity of the mouse Nrf2 cDNA and production of the
fusion protein was confirmed by DNA sequencing and Western blotting,
respectively. The mammalian Gdbd vector, pEG, was constructed by
cloning the Gdbd (residues 1-147) into pEF/myc/mito. Mouse
Nrf2 sequences (aa residues 13-581 or 314-581) were
subsequently cloned downstream of, and in-frame with, the Gdbd to
generate pEG/Nrf2 plasmids. The "activation domain" vector
(pAD) was constructed by cloning an 870-base pair BglII/HindIII (blunt-ended) fragment of mouse
Nrf2 (aa 13-302) into pCMV-Tag2B. Full-length ATF3, p18 and rat
ATF4, sequences were subsequently cloned into pAD in-frame with the
Nrf2 sequence.
Reporter Gene Plasmids--
pFRluc, containing 5 tandem copies
of the Gal4-binding site, was obtained from Stratagene. The
construction of pE1-luc, containing the mouse ho-1 gene
distal enhancer 1, and pStREluc, containing three copies of the mouse
ho-1 StRE3, has been described previously (4, 22). Plasmid
pCMV/ Yeast Two-hybrid (Y2H) Screening
Screening was carried out using the Y2H system from Life
Technologies, Inc. Briefly, plasmid pDBLeu-Nrf2 was introduced
into yeast strain MaV203 (MAT Mammalian Cell Culture, Transfection, and Enzyme Assays
COS-7 (African green monkey kidney), Hepa (mouse hepatoma), and
MCF-7 (human mammary epithelial) cells were cultured in Dulbecco's modified Eagle's medium, whereas HeLa (human cervical carcinoma) cells
were cultured in Eagle's minimal essential medium. All media were
supplemented with 0.45% glucose, 10% fetal bovine serum, 50 µg/ml
gentamicin sulfate, and 10 ng/ml insulin (MCF-7 only). Transient
transfection of luciferase constructs was carried out by the calcium
phosphate precipitation technique as described previously (23) or with
Fugene 6 transfection reagent (Roche Molecular Biochemicals) according
to the manufacturer's recommendation. Additional details are provided
in the figure legends. Transfection efficiency was monitored by
co-transfection with pCMV/ Recombinant Protein and Electrophoretic Mobility Shift Assays
(EMSA)
Full-length (p18, Fos B, and ATF3) or nearly full-length (mATF4,
residues 31-349) coding regions were cloned downstream of, and
in-frame with, the hexa-histidine tag in the T7 RNA polymerase-based prokaryotic expression vector series pET30a-c (Novagen Inc.). Recombinant proteins were purified from inclusion bodies by nickel affinity chromatography as per the manufacturer's protocol or according to the protocol of Holzinger et al. (25).
Nrf2M protein (residues 393-581), containing the DNA binding and
leucine zipper dimerization domains, was synthesized by coupled
in vitro transcription and translation reaction as described
previously (26). EMSA was carried out as described previously (23)
using a double-stranded oligonucleotide containing the sequence
5'-TTTTCTGCTGAGTCAAGGTCCG-3' (core StRE underlined) as
probe. Five µl of Nrf2M synthesis product and/or 100 ng of
recombinant protein were used in EMSA reactions.
Co-immunoprecipitation and Western Blot Analysis
COS-7 cells were transfected 24 h after plating (1 × 106/100-mm plate) with a total of 5 µg of either empty
vector (pEF/myc/mito), pEF/mATF4, pEF/Nrf2, or a
combination of these plasmids using Lipofectin transfection reagent
(Life Technologies, Inc.) as specified by the manufacturer. Cells were
harvested 36 h after transfection and resuspended in 200 µl of
lysis buffer (10 mM Tris-HCl (pH 7.5) containing 0.5%
(v/v) Nonidet P-40, 150 mM NaCl, and 1 mM EDTA). Cell lysates were cleared by centrifugation, and
immunoprecipitation was carried out with 100 µg of cell lysate using
protein G-agarose beads as described (27). Immune complexes were eluted
from the beads with 2× SDS-PAGE sample buffer and subjected to
denaturing polyacrylamide gel electrophoresis. Western blotting was
carried out as described previously (4). All antibodies were used at dilutions recommended by the respective suppliers.
Identification of ATF4 as an Nrf2-interacting
Protein--
To identify proteins that interact with Nrf2, a
cDNA fragment encoding the C-terminal portion of mouse Nrf2
was amplified by PCR and cloned in-frame and downstream of the Gdbd.
The resulting fusion protein was used as "bait" in a yeast
two-hybrid screening assay as described under "Experimental
Procedures." From a total of 2 × 106 yeast
transformants, harboring either rat brain or liver cDNA/Gal4 activation domain fusions, seven independent clones (3 liver and 4 brain) that encoded Nrf2-interacting polypeptides were identified after a series of selection methods.
The nucleotide sequences of the inserts within the positive clones were
determined, and the results indicated that five of the cDNAs were
derived from the same mRNA. A sequence similarity search using
"blastn" revealed significant similarities to sequences encoding mouse ATF4 and human CREB2 (ATF4) (28, 29) suggesting that
these clones encoded the rat homolog of ATF4. Since none of the
isolates contained the initiation codon, the full-length ATF4 cDNA
was obtained by PCR amplification from a rat brain cDNA library,
cloned, and subjected to DNA sequence analysis.
The deduced amino acid sequence of rat ATF4 is aligned with those of
mouse and human ATF4 in Fig. 1. Between
these three species, ATF4 exhibits 84.4% sequence conservation and the
rat protein displays 94.8 and 87.2% sequence identity to mouse and
human ATF4 s, respectively. As expected, the highest degree of
conservation is observed within the basic region (DNA binding) and the
adjacent leucine zipper (dimerization) domains. The second "leucine
zipper" region, to which a function has yet to be assigned, exhibits
greater divergence although the repeating leucine (or corresponding)
residues at every 7th position are completely conserved in the three
proteins.
Interaction between Nrf2 and ATF4 in Mammalian
Cells--
Association between Nrf2 and ATF4 in mammalian cells
was confirmed by co-immunoprecipitation experiments and mammalian
two-hybrid assays. For the former, expression plasmids encoding ATF4 or
Nrf2 were transfected, individually or in combination, into COS-7
cells; the cells were lysed, and the lysates subjected to
immunoprecipitation with anti-ATF4 or anti-Nrf2 antibodies in the
presence or absence of the corresponding blocking peptide.
Immunoprecipitates were subsequently analyzed by Western blotting. As
shown in Fig. 2, Nrf2 was not
detected in lysates of cells transfected with an empty vector
(lane 3) or the ATF4 expression plasmid (lane 6), but was readily observed in lysates of cells transfected with both ATF4
and Nrf2 expression plasmids (lane 4). More
importantly, Nrf2 could be immunoprecipitated with anti-ATF4
antibodies (lane 2), albeit to a lesser extent than that
observed with anti-Nrf2 antibodies (lane 1).
Immunoprecipitation of Nrf2 by both antibodies was abrogated in
the presence of the corresponding blocking peptides (lanes 5 and 7).
The results from co-immunoprecipitation experiments were corroborated
by mammalian two-hybrid assays. In these experiments, nearly
full-length mouse Nrf2 (aa 13-581; Fig.
3A) or the C-terminal portion
of Nrf2 (aa 314-581; Fig. 3B) was fused to the Gdbd,
and these fusions served as interaction targets. Sequences encoding test proteins were fused in-frame to an N-terminal region of Nrf2 (amino acids 13-302) that contains a potent transcription activation domain (AD). Gdbd-Nrf2-(13-581) strongly
trans-activated a luciferase reporter gene under the control
of Gal4-binding sites, pFRluc. Co-expression of Nrf2 AD further
increased luciferase activity by 4-5-fold suggesting self-interaction
between Nrf2 proteins. AD fusions containing full-length rat ATF4
or mouse ATF3 exhibited even greater trans-activation
capabilities, ~35-fold above control and 7-fold above AD alone. The
MafK (p18) fusion served as a positive control and exhibited the
highest interaction activity. Gdbd-Nrf2-(314-581) contains the
DNA interaction and dimerization (i.e. bZIP) domains but is
transcriptionally inactive. Co-expression of AD did not stimulate
luciferase activity suggesting that Nrf2 self-interaction is
limited to the N-terminal portion of Nrf2. ATF3, ATF4, and p18
interacted with Gdbd-Nrf2-(314-581) with the following rank order: p18 ATF4, in Association with Nrf2, Binds to the
Stress-responsive Element (StRE)--
To understand the consequence of
ATF4/Nrf2 interaction on gene regulation, we initially examined
the binding of ATF4 and Nrf2, individually or as a mixture, to
the StRE, a cis-acting element known to regulate
inducer-mediated ho-1 gene activation in an Nrf2-dependent manner (4, 22, 26). As expected,
Nrf2 did not bind to the StRE (Fig.
4). ATF4 alone also did not bind to the
StRE but, in the presence of Nrf2, exhibited significant binding. Presumably, this DNA-protein interaction reflects the activity of
ATF4·Nrf2 dimers. p18, which can form homodimers and highly stable heterodimers with Nrf2 was used as a positive control for protein dimerization and DNA binding. p18 homodimers, which are known
to interact with MARE and NF-E2 binding sites, also bound to the StRE,
but the strongest binding was observed with p18·Nrf2 heterodimers. The relative affinities (and/or stability) of
ATF4·Nrf2 dimers and p18·Nrf2 dimers for the StRE
correlated with the relative affinities of protein-protein interactions
observed in the mammalian two-hybrid assays. FosB, which cannot form
homodimers and would not be expected to dimerize with Nrf2,
served as a negative control and did not exhibit specific binding in
the absence or presence of Nrf2. Unlike ATF4, recombinant ATF3,
presumably ATF3 homodimers, bound weakly to the StRE, but the binding
was decreased in the presence of Nrf2.
ATF4 Enhances, whereas p18 Inhibits, Nrf2-mediated
Trans-activation of the ho-1 Enhancer--
Binding of
ATF4·Nrf2 dimers to the StRE suggested a role for ATF4 in
ho-1 gene regulation. This potential function was
investigated further by examining the ability of ATF4 to
trans-activate the ho-1 enhancer, E1, in the
reporter construct pE1-luc. In Hepa cells, co-transfection of an ATF4
expression plasmid, up to the maximum level tested, decreased basal
pE1-luc expression by 20-25% (Fig. 5).
ATF4, however, had a synergistic effect on
Nrf2-dependent pE1-luc expression, increasing
luciferase activity up to 2-fold. In contrast, co-expression of p18
dramatically inhibited Nrf2-mediated trans-activation
of E1. This inhibition may, at least in part, be attributed to p18
homodimers as overexpression of p18 alone also inhibited basal pE1-luc
expression.
Cadmium Stimulates ATF4 Expression--
Previous studies from our
laboratory (4, 26) and other laboratories (7) have demonstrated the
requirement for Nrf2 in inducer-dependent
ho-1 gene regulation. To determine the role, if any, of ATF4
in this process, we first examined the effect of cadmium, a known HO-1
stimulant, on ATF4 expression as earlier reports had suggested that
ATF4 is a stress-response protein (30, 31). As shown in Fig.
6, treatment of Hepa cells with 100 µM CdCl2 increased ATF4 levels in a
time-dependent manner to greater than 10-fold above basal
values. Of the other transcription factors tested, only expression of
ATF3 and c-Jun, both documented stress-responsive proteins (32, 33),
was enhanced by cadmium. Interestingly, the temporal profile of c-Jun
and ATF3 induction matched that of HO-1 accumulation with the earliest
detectable enhancement observed at 2 h post-treatment. Increased
expression of ATF4, on the other hand, was detected within 30 min after
treatment of cells with CdCl2, even prior to the detectable
accumulation of ho-1 mRNA by this agent in Hepa cells
(34).
A Dominant-negative Mutant of ATF4 Inhibits Basal and
Cadmium-stimulated E1 Activity in Hepa Cells--
To establish further
the role of ATF4 in ho-1 gene regulation, we generated a
dominant-negative mutant of ATF4 and examined its effect on
pE1-luc expression. Overexpression of the mutant ATF4 inhibited both
basal and cadmium-stimulated luciferase activity in a
dose-dependent manner (Fig.
7). This effect was qualitatively and
quantitatively similar to that observed with an Nrf2 dominant mutant. Interestingly, an analogous mutant of p18 inhibited
cadmium-induced but not basal activity. Mutants of c-Jun or Jun D
either enhanced or had no effect on pE1-luc expression.
The ATF4 Dominant-negative Mutant Does Not Inhibit
Cadmium-stimulated pE1-luc Expression in MCF-7 Cells--
We have
recently reported that cadmium is a potent activator of the
ho-1 gene in MCF-7 human mammary epithelial cells,
stimulating ho-1 mRNA accumulation by 300-400-fold, and
that Nrf2 is an important regulator of this response (26).
Consistent with our previous finding, the Nrf2 mutant
significantly inhibited pE1-luc expression in the presence or absence
of CdCl2 (Fig. 8). The p18
mutant also inhibited both basal and cadmium-stimulated activities to
similar levels. The ATF4 mutant, however, inhibited only basal
luciferase activity, revealing cell-dependent differences
in the mechanism of ho-1 gene activation by cadmium and
the role of ATF4 in this process.
In this study we have identified ATF4 as an Nrf2-interacting
protein and have provided evidence implicating ATF4 in basal and
cadmium-induced regulation of the ho-1 gene, the latter
presumably in cooperation with Nrf2. Whereas association between
Nrf2 and ATF/CREB family members has not been previously
documented, such an interaction is not necessarily unexpected as both
classes of factors belong to the bZIP superfamily. In this regard it is
noteworthy that among bZIP protein, ATF4 exhibits relatively
promiscuous interaction activity. For instance, ATF4 forms heterodimers
with c-Jun, c-Fos, and Fra-1 proteins under conditions where ATF2 and ATF3 heterodimerize only with c-Jun and ATF1 does not form any detectable heterodimers at all (17). In addition, ATF4 also forms
heterodimers with CCAAT/enhancer-binding proteins (35), a relatively
more distant subfamily (based on comparison of DNA-binding site
sequences) than the Jun or Fos subfamilies within the bZIP superfamily.
Although not directly tested, it is not unreasonable to assume that
Nrf2 and ATF4 form classical bZIP dimers via their leucine zipper
structures. Indeed, in all likelihood, it is this type of interaction
that will most effectively elicit the proper alignment of the basic
regions necessary for sequence-specific DNA binding as demonstrated
herein. This conclusion is supported by the fact that the C-terminal
portion of Nrf2 containing the bZIP domain was used in the
original Y2H screening, and the observations that ATF4 exhibits
positive interaction with a truncated Nrf2 in M2H assays (Fig. 3)
and in EMSA (Fig. 4). Based on the difference in the apparent relative
affinities of ATF4 for nearly full-length Nrf2 versus
the truncated protein (see Fig. 3) and additional preliminary
observations, we cannot rule out the possibility that ATF4 interacts
with additional domains within the Nrf2 polypeptide. In this
regard, it is interesting that ATF3 interacts very poorly with the
truncated Nrf2 but is as effective as ATF4 in binding to the
larger Nrf2 protein. At present, the precise location of any of
the interaction sites (within Nrf2 or ATF4) is unknown. The
functional significance of any non-leucine zipper associations is also
not obvious. However, we note that ATF4 is known to associate with
non-bZIP factors, including the Tax protein of the human T-cell
leukemia virus type 1 (36, 37) and the The ho-1 gene is activated by a variety of stress-associated
agents including the substrate heme, heavy metals, tumor promoters, UV
irradiation, and inflammatory cytokines. Induction of the mouse gene by
most stimuli is mediated by two 5', distal enhancer regions, E1 and E2,
each containing multiple StREs. The StREs are sufficient and necessary
for inducer-dependent gene activation (reviewed in Ref.
12). Our recent analyses (4, 26) have implicated Nrf2 in the
mechanism of ho-1 gene activation by several agents and in
particular by cadmium. For instance, stable expression of a
dominant-negative mutant of Nrf2, but not of c-Jun, diminishes cadmium-induced ho-1 mRNA accumulation by 75-90% in
L929 fibroblasts and MCF-7 cells. Similarly, in MCF-7 cells,
overexpression of the Nrf2 mutant inhibits induction of an
E1-regulated luciferase reporter gene by cadmium, and mutants of E1
that are not trans-activated by Nrf2 are also
unresponsive to cadmium.
Identification of ATF4 as an Nrf2-interacting protein led us to
speculate that ATF4, possibly in cooperation with Nrf2, regulates ho-1 gene expression. The transfection experiments
demonstrating the inhibitory effects of the ATF4 dominant-negative
mutant on basal and cadmium-induced E1 activity in Hepa cells provides
support for this idea. It is important to point out that transfection studies of this nature, by themselves, do not conclusively demonstrate the role of a specific transcription factor in gene regulation. This
limitation arises because of the tendency of bZIP proteins to dimerize
with multiple partners. Consequently, inhibition of gene activation
observed with a specific dominant mutant can be attributed not only to
the corresponding endogenous protein (if capable of homodimerization)
but also to any of its dimerization partners, one or more of which may
be the actual effector protein(s). Clearly, under such circumstances,
it is important to obtain corroborative data for the role of a given
factor in gene regulation. For ATF4, such correlative evidence includes
the observations that ATF4·Nrf2 dimers bind to the StRE, that
ATF4 expression is enhanced by cadmium, and that ATF4 has a synergistic
effect on Nrf2 trans-activation of E1. The latter
finding, in particular, distinguishes ATF4 from p18, which exhibits an
inhibitory effect similar to that of MafG on Nrf2
trans-activation of the ARE (5, 15). In the case of
Nrf2, a role in ho-1 gene regulation has been further
substantiated with the use of nrf2-targeted mice (39)
and cells (7). A similar analysis for ATF4 and p18 would of course be
very informative.
Our contention that ATF4 in part regulates ho-1 gene
expression is consistent with emerging data that indicate ATF4 is a
stress-response protein and, consequently, would function as a
regulator of the adaptive response to such stress. For example, anoxia,
which stimulates HO-1 synthesis, strongly increases the expression and
DNA binding activity of ATF4 in fibroblasts (30). Arsenite, another
HO-1 inducer, also stimulates ATF4 DNA binding activity in
pheochromocytoma PC12 cells (31). In addition, ATF4 levels are enhanced
in endothelial (40, 41) and Jurkat (42) cells in response to
homocysteine and the calcium ionophore A23187, respectively, two agents
known to cause endoplasmic reticulum stress. Finally, ATF4 expression is increased in cell lines resistant to various DNA-targeting drugs
(43).
The difference between Hepa and MCF-7 cells with respect to the
ability of the ATF4-dominant mutant to modulate
cadmium-dependent E1 activity is puzzling but certainly
points to cell-specific differences in the induction mechanism.
Perhaps, intracellularly, productive or transcriptionally competent
interaction between ATF4 and Nrf2 requires an additional
cofactor(s) which may be expressed in a cell-specific manner. This
cofactor concept is somewhat similar to one postulated by Venugopal and
Jaiswal (16) who have proposed that formation of Nrf2-Jun
complexes is dependent on one or more presently uncharacterized
cytoplasmic proteins. One consequence of this hypothesis is that it
apparently precludes a role for Nrf2-ATF4 complexes in
cadmium-dependent ho-1 gene activation in MCF-7
cells and, based on our previous studies noted above, requires the use
of other Nrf2-containing complexes. Given the tendency for bZIP
proteins to form multiple and distinct associations, this requirement
is not necessarily insurmountable. Studies are under way to
characterize further the role of ATF4, particularly with respect to
cell and inducer specificity, in the regulation of ho-1 and
other Nrf2 target genes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B
(NF-
B)1 and activator
protein-1, arguably the two most prominent regulators of this cellular
response mechanism (reviewed in Refs. 1 and 2).
B factors, Nrf2 exists
in an inactive, cytoplasm-localized state, in part or fully as a
consequence of binding to the cytoskeleton-associated protein Keap1.
After exposure of cells to electrophiles or oxidative stress-generating
agents, the cytoplasmic retention mechanism is inactivated, and
Nrf2 is transported to the nucleus by an as yet uncharacterized
mechanism(s) but one that may involve protein kinase C-mediated
phosphorylation of Nrf2 (8). Within the nucleus, Nrf2
activates transcription of a select set of target genes by binding to
distinct but very similar DNA elements, individually or alternatively
referred to as the NF-E2-binding site (10), the Maf recognition element
(MARE, 11), the stress-response element (12), or the
antioxidant-response element (13). Many of the Nrf2 target genes
(3-5, 7, 8) encode proteins that play a central role in the adaptive
response to oxidative stress. Among others, these include heme
oxygenase-1 (HO-1), an enzyme that catalyzes the rate-limiting reaction
in heme degradation, a catabolic pathway that leads to the
production of bilirubin, a potent antioxidant; NAD(P)H:quinone
oxidoreductase (NQO), which catalyzes two-electron reduction of
quinones, preventing the participation of such compounds in redox
cycling and oxidative stress;
-glutamylcysteine synthase, which catalyzes the rate-limiting reaction in glutathione biosynthesis; and glutathione S-transferase, which conjugates hydrophobic
electrophiles and reactive oxygen species with glutathione.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-gal, encoding the Escherichia coli
-galactosidase gene, was kindly provided by Dr. Ping Wei.
, leu2-3, 112, trp1-901, his3
200, ade2-101, gal4
, gal80
,
SPAL10::URA3,
GAL1::lacZ,
HIS3UAS GAL1::HIS3@LYS2, can1R, cyh2R), and
transformants were selected and purified on medium lacking leucine.
Subsequently, DNA representing rat brain or liver cDNAs, cloned
into the Gal4 activation domain vector pPC86, was transformed into
MaV203/pDBLeu-Nrf2 strain. Transformants were selected on medium
containing 25 mM 3-amino-1,2,4-triazole but lacking
tryptophan, leucine, uracil, and histidine. Positive colonies were
assayed for activation of the lacZ reporter gene. Plasmids
isolated from the positive colonies were rescued in E. coli
HD10B and re-assayed for interaction activity by transformation into
the MaV203/pDBLeu-Nrf2 strain and growth on selection media. The
5' end of the rat ATF4 cDNA was isolated by PCR amplification
(5'-rapid amplification from cDNA ends) from a rat brain
Marathon-Ready cDNA mixture (CLONTECH) according to the manufacturer's recommendation using two different gene-specific primers ATF4-1 (5'-TAGGACTCAGGGCTCATACAGATGCCA-3') and
ATF4-2 (5'- TTGAAGTGCTTGGCCACCTCCAGATAG-3') and the adaptor primer
provided in the kit. Both amplification products were purified and
cloned into the pT-Adv vector (CLONTECH
Laboratories Inc). Automated DNA sequence analysis was carried out by
the Howard hughes Medical Institute Biopolymer/W. M. Keck Foundation
Biotechnology Resource Laboratory at Yale University. Identical 5'
sequences were obtained from clones derived from both amplification products.
-gal. Preparation of cell extract and
measurement of luciferase activity were carried out as described
previously (24).
-Galactosidase activity was measured using the
Galacto-Light (Tropix, Inc.) chemiluminescent assay kit according to
the manufacturer's protocol.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (64K):
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Fig. 1.
Comparison of rat, mouse, and human ATF4
protein sequences. The amino acid sequence alignment was generated
using the ClustalW algorithm (44). Residues conserved in all sequences
are marked with asterisks. Colons and
periods indicate conservative and semi-conservative
substitutions, respectively. The bZIP domain (basic region and leucine
zipper I) and the second zipper (zipper II) are indicated. The leucine
(or corresponding) residues within the heptad repeat of the zippers are
marked with filled circles above. Mouse ATF4
accession number is M94087; human ATF4 accession number is
XP_010004
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Fig. 2.
Co-immunoprecipitation of Nrf2 by
anti-ATF4 antibody. COS-7 cells were transfected with empty vector
or the indicated transcription factor expression plasmid. Cell lysates
(100 µg of protein) were subjected to immunoprecipitation
(IP) with anti-ATF4 (A) or anti-Nrf2
(N) antibodies (Ab) in the presence or absence of
the corresponding blocking peptide (BP). Cell lysates (25 µg) or immune complexes (total sample from a single precipitation)
were size-fractionated on a denaturing 10% polyacrylamide gel;
proteins were transferred to nitrocellulose, and the Nrf2 protein
(marked by arrow) was detected by Western blotting.
ATF4
ATF3. Presumably these interactions reflect dimerization between leucine zipper domains. Relative to p18, ATF4
exhibits greater association with Gdbd-Nrf2-(13-581) than with
Gdbd-Nrf2 (314) (~40% versus 5%), even though
the latter is produced at higher levels intracellularly (data not
shown).
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Fig. 3.
Interaction between ATF4 and Nrf2 in
mammalian two-hybrid assays. HeLa cells were plated (1 × 105/well of 6-well plates) 48 h prior to transfection
by CaPO4-DNA co-precipitation. Cells in each well were
transfected for 6 h with a DNA mixture consisting of 3 µg of
pFRluc, 1 µg of pCMV/ Gal, 2 µg of the indicated Gdbd plasmid,
and 2 µg of an empty vector (EV) or the indicated
activation domain (AD) plasmid. Cells were cultured for an
additional 48 h and then lysed. Eight and 4% of the cell extracts
were used to measure luciferase and
-galactosidase activities,
respectively.
-Galactosidase-normalized luciferase activities are
presented. A, luciferase activity obtained with the empty
vector (EV), an average of 10,100 light units, was
arbitrarily assigned a value of 1. Such standardization was not
possible in B because no activity was detected in the
absence of plasmids encoding interacting proteins. Each data
bar represents the average ± S.E. from 3 to 6 independent
experiments.
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Fig. 4.
The Nrf2·ATF4 heterodimer binds to
the StRE. Protein synthesis and EMSA reactions were carried out as
described under "Experimental Procedures." EMSA gels were exposed
to x-ray film for 16 h. Lanes designated
" Nrf2" contained in vitro
transcription/translation products from reactions directed by the empty
expression vector pGEM2. Nonspecific complexes resulting from the
transcription/translation extracts are marked with
asterisks.
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Fig. 5.
ATF4 enhances and p18 represses
Nrf2-mediated trans-activation of pE1-luc.
Hepa cells were plated (5 × 105/well of 6-well
plates) 24 h prior to transfection by CaPO4-DNA
co-precipitation. Cells in each well were transfected for 6 h with
a DNA mixture consisting of 2 µg of pE1-luc, 1 µg of pCMV- -Gal,
1 µg of pEF/Nrf2 or pEF/myc/mito, and the indicated
amount of pCMV/rATF4 or pEF/p18. Total DNA was equalized with an
appropriate empty expression vector. Cells were cultured for an
additional 48 h and then lysed. Eight and 4% of the cell extracts
were used to measure luciferase and
-galactosidase activities,
respectively. Luciferase activity was normalized to
-galactosidase
activity in the same extract and is presented as a percentage of
activity in cells transfected without pCMV/rATF4 or pEF/p18. Each data
point represents the average ± S.E. from three independent
experiments. Average fold trans-activation by Nrf2 (in
the absence of ATF or p18) was 53.4.
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Fig. 6.
Cadmium induces ATF3, ATF4, c-Jun, and HO-1
expression in Hepa cells. Approximately 5 × 105
cells were plated in each well of a 6-well plate. Cells were cultured
in complete medium for 48 h and, subsequently, in serum-free
medium for 24 h. Cells were exposed to 100 µM
CdCl2 for the indicated time (hours). Western blot analyses
were carried out as described under "Experimental Procedures" using
antibodies directed against the indicated proteins.
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Fig. 7.
A dominant-negative mutant of ATF4
inhibits basal and cadmium-dependent pE1-luc expression in
Hepa cells. Cell were plated and transfected as described in the
legend to Fig. 5. The DNA mixtures consisted of 2 µg of pE1-luc, 1 µg of pCMV/ Gal, and the indicated amount of the dominant mutant
expression plasmid. Total DNA was equalized with an appropriate empty
expression vector. Forty hours after transfection, cells were treated
with vehicle or 100 µM CdCl2 for 5 h in
serum-free medium. Eight and 4% of the cell extract were used to
measure luciferase and
-galactosidase activities, respectively.
-Galactosidase-normalized luciferase activity is presented as the
percentage of activity in cells transfected without any dominant mutant
plasmid. Each data point represents the average of two independent
experiments (c-Jun, Jun D) or the average ± S.E. from three
experiments.
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Fig. 8.
The ATF4 dominant-negative mutant only
inhibits basal pE1-luc expression in MCF-7 cells. Cell were plated
(1 × 105/well of a 12-well plate) and transfected 20 h later
using Fugene 6 transfection reagent. Each well was transfected with a
DNA mixture consisting of 50 ng of pE1-luc, 10 ng of pCMV- Gal and
200 ng of the indicated expression plasmid. Forty hours after
transfection, cells were treated with vehicle or 10 µM
CdCl2 for 5 h in serum-free medium. Twelve and 6% of
the cell extract were used to measure luciferase and
-galactosidase
activities, respectively.
-Galactosidase-normalized luciferase
activity is presented as the percentage of activity in cells
transfected without any dominant mutant plasmid. Each data point
represents the average ± S.E. from three experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-aminobutyric acid type B
receptor (38). Interestingly, in both situations the interacting site
within ATF4 was localized to the bZIP domain and, in the case of Tax,
the association leads to enhanced trans-activation capacity
for the Tax protein, similar to that observed here for Nrf2.
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ACKNOWLEDGEMENT |
---|
We thank Margaret Overstreet for assistance in preparation of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by United States Public Health Service Grants DK-43135, HL-55330, HL-60234, and AI-42365.
¶ Both authors contributed equally to this work and should be considered as first authors.
§§ Both laboratories contributed equally to this work.
¶¶ To whom correspondence should be addressed: Dept. of Molecular Genetics, Alton Ochsner Medical Foundation, 1516 Jefferson Hwy., New Orleans, LA 70121. Tel.: 504-842-3314; Fax: 504-842-3381; E-mail: jalam@ochsner.org.
Published, JBC Papers in Press, March 26, 2001, DOI 10.1074/jbc.M101198200
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ABBREVIATIONS |
---|
The abbreviations used are:
NF-B, nuclear
factor-
B;
heme, ferriprotoporphyrin IX;
HO-1, heme oxygenase-1;
ATF/CREB, activating transcription factor/cAMP-response element-binding
protein;
CNC-bZIP, Cap `N' Collar/basic-leucine zipper;
Nrf, NF-E2
related factor;
NF-E2, nuclear factor-erythroid 2;
bZIP, basic
region/leucine zipper;
Gdbd, Gal4 DNA binding domain;
Y2H, yeast
two-hybrid;
M2H, mammalian two-hybrid;
StRE, stress-response element;
ARE, antioxidant-response element;
MARE, Maf recognition element;
EMSA, electrophoretic mobility shift assay;
AD, activation domain;
aa, amino
acid;
Hepa, hepatoma.
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REFERENCES |
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