From the Institutes of Molecular Biology and
§ Zoology, University of Zürich, Winterthurerstrasse
190, CH-8057 Zürich, Switzerland
Received for publication, October 6, 2000, and in revised form, March 20, 2001
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ABSTRACT |
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The metal-regulatory transcription factor 1 (MTF-1) is a key regulator of heavy metal-induced transcription of
metallothionein I and II and other genes in mammals and other
metazoans. Transcriptional activation of genes by MTF-1 is mediated
through binding to metal-responsive elements of consensus TGCRCNC in
the target gene promoters. In an attempt to further clarify the
mechanisms by which certain external signals activate MTF-1 and in turn
modulate gene transcription, we show here that human MTF-1 has a dual
nuclear and cytoplasmic localization in response to diverse stress
stimuli. MTF-1 contains a consensus nuclear localization signal located
just N-terminal to the first zinc finger that contributes to but is not
essential for nuclear import. MTF-1 also harbors a leucine-rich,
nuclear export signal. Under resting conditions, the nuclear export
signal is required for cytoplasmic localization of MTF-1 as indicated by mutational analysis and transfer to the heterologous green fluorescent protein. Export from the nucleus was inhibited by leptomycin B, suggesting the involvement of the nuclear export protein
CRM1. Our results further show that in addition to the heavy metals
zinc and cadmium, heat shock, hydrogen peroxide, low extracellular pH
(pH 6.0), inhibition of protein synthesis by cycloheximide, and serum
induce nuclear accumulation of MTF-1. However, heavy metals alone (and
not the other stress conditions) induce a significant
transcriptional response via metal-responsive element promoter
sequences, implying that nuclear import of MTF-1 is necessary but not
sufficient for transcriptional activation. Possible roles for
nuclear import under non-metal stress conditions are discussed.
Gene regulation in response to cellular stress is mediated through
a variety of signaling pathways. One type of stress response is
triggered by heavy metals, such as zinc, cadmium, and copper, and
induces transcription of metallothionein genes. Metallothioneins (MTs)1 are a group of low
molecular weight metal-binding proteins that are represented by four
isoforms in mammals (1). MT gene transcription is also induced by many
different conditions other than heavy metals, including oxidative
stress, hypoxia, hormones, and viral infection (2, 3). Most, if not
all, other eukaryotes and some prokaryotes also contain
metallothioneins, but their primary sequence and their transcriptional
regulation may deviate from the situation in vertebrates (4, 5).
The mammalian metal-regulatory transcription factor 1 (MTF-1) is a zinc
finger transcription factor that activates the transcription of target
genes in response to heavy metal exposure via binding to MREs (6).
These sequence motifs of consensus TGCRCNC are present in the promoter
regions of MT-I, MT-II, and a number of other genes, many of which are
involved in stress (7-9). Recently, targeted gene disruption of MTF-1
in the mouse revealed that MTF-1 is essential for liver development
during embryogenesis because the null mutant embryos died in
utero at embryonic day 14 due to liver decay. MTF-1-deficient
cells in culture are more susceptible to cadmium and
H2O2 treatments in comparison with their wild
type counterparts (7). Another implication of MTF-1 in cellular stress
response came from the work of Dalton et al. (10), who found
that MTF-1 is activated in response to reactive oxygen species. Recently, hypoxia has also been reported as a condition by which MTF-1
activates MT gene expression through MRE sequences (11). However, the
mechanism(s) of MTF-1 activation by heavy metals is only partially
understood, and the activation by non-metal stimuli remains to be elucidated.
The rapid cellular response to a multitude of external and internal
stress-associated agents is orchestrated by multiple signal transduction pathways. Several transcription factors involved in
stress-regulated rapid events, including p53, nuclear factor Plasmid Construction--
The human MTF-1 expression vector was
described previously (21). An 11-mer VSV epitope from vesicular
stomatitis virus G protein (22) was inserted at amino acid position 744 in the C-terminal end. The NES and NLS mutants (designated
MTF-1NESmut and LLI(133-135)MTF-1-VSV,
respectively) were created by site-directed mutagenesis. The
oligonucleotides used were as follows (the sense strand is
shown; lowercase letters represent mutated nucleotides): (a)
NESmut,
5'-GGATACAAATCACTCACTTTGTgcAAGTGACgctAGCgcTgcGTCCACAGATTCTG-3'; (b) NES'mut,
5'-CCACAGGAAATTCAGCATCTgcATCTgcTCCAgcTGTAgcGCAACCTGGCCTCTCCG-3'; (c) LLI(133-135)MTF-1-VSV,
5'-gcagtcggaatgtccGgaaacACTaCtGaTagaagtaaagcggtacc-3'. mMTI-Luc
was constructed by inserting a blunt-ended fragment encompassing the
promoter (
The NLS (133KRKEVKR139), NES
(336LCLSDLSLL344), and NES'
(417LSLPLVLQPGL427) sequences of human MTF-1
were cloned in the HindIII-KpnI sites of double
peGFP (peGFP2x), which is located in the C terminus. The
peGFP2x vector containing tandemly repeated eGFP coding
sequences was a kind gift from Dr. Ulrike Kutay (ETH,
Zürich, Switzerland).
Cell Cultures and Transfections--
Adenovirus-transformed
human embryonal kidney 293 cells, U2OS human osteosarcoma cells, and
SV40-transformed MTF-1 Indirect Immunofluorescence--
293 and U2OS cells were plated
onto coverslips in 6-well tissue culture dishes in DMEM
supplemented with 10% FBS 16 h before transfection. Cells were
transfected with 2 µg/well plasmid DNA using the calcium phosphate
method. Sixteen h after transfection, cells were washed and incubated
for another 24 h in the medium containing 10% FBS. The cells were
switched to the medium in which serum was replaced by 0.5% BSA
(DMEM-BSA). After 24 h in the serum-limited condition, the
treatments were initiated as indicated. To monitor redistribution of
MTF-1 from the nucleus to the cytoplasm after zinc or cadmium
withdrawal, cells were pretreated with 100 µM ZnCl2 or 60 µM CdCl2 for 1 h
and washed three times with phosphate-buffered saline and three times
with DMEM-BSA. Further incubations were performed in DMEM-BSA as
indicated. For treatment of cells with low pH, the pH of DMEM
supplemented with 0.5% BSA was adjusted by the addition of HCl from pH
~7.4 to pH 6.0. This pH remained stable throughout the experiment.
For fixation, cells were treated with 4% paraformaldehyde for 15 min
at room temperature and permeabilized with 0.5% Triton X-100 in
phosphate-buffered saline for 3 min. After blocking nonspecific binding
by incubation in 5% newborn calf serum in phosphate-buffered
saline for 10 min, the cells were incubated with anti-VSV antibody
(Sigma) diluted 1:250 in 5% newborn calf serum at room temperature for
30 min. After rinsing, cells were incubated with fluorescein-coupled
rabbit anti-mouse IgG (Molecular Probes) diluted 1:200 in 5% newborn
calf serum at room temperature for 1 h. After three washings,
nuclei were stained with 4',6-diamidino-2-phenylindole (0.001 mg/ml)
for 2 min, and cells were imaged by using a Reichert-Jung Polyvar
upright fluorescence microscope equipped with fluorescein filters
(excitation, 475-495 nm; emission, 520-560 nm),
4',6-diamidino-2-phenylindole filters (excitation, 330-380 nm;
emission, 420 nm/long pass), and Nomarski optics. Images were
recorded through a ×40 objective (oil immersion) and collected on an
8-bit charge-coupled device camera (Hamamatsu C 5405) online to an
Apple Macintosh computer, as described previously (26). Digital images
were processed using photoshop software (Adobe).
Determination of MTF-1 Localization--
Multiple fields were
examined to count at least 200 positive cells, and localizations of
MTF-1 were classsified as nuclear, cytoplasmic, and both nuclear and
cytoplasmic. Mean and S.D. values of the number of cells showing
nuclear, cytoplasmic, and both nuclear and cytoplasmic staining of
MTF-1 were derived from three independent experiments, each sampling
about 200 different cells.
293 cells plated on coverslips were transfected with 2 µg of the eGFP
plasmid constructs. 24 h after transfection, cells were fixed with
4% paraformaldehyde for 15 min and analyzed by fluorescence microscopy
as described above.
Luciferase Reporter Gene Assay--
293 and DKO7 cells were
cultured in DMEM supplemented with 10% FBS. Transient transfections
were carried out in 100-mm tissue culture plates with 5 µg of
reporter plasmid, 3 µg of LacZ expression vector, and 5 µg of each
MTF-1 expression vector by the calcium phosphate method. 16 h
after transfection, the medium was removed, and cells were fed with
medium containing 10% FBS. After 24 h, media were changed to
0.5% BSA-supplemented medium, and cells were serum-starved in that
medium by incubation for another 24 h. Cells were then harvested
and assayed for luciferase activity as described previously (27). The
results were normalized to the LacZ expression from the cotransfected
reference gene.
Electrophoretic Mobility Shift Assay--
Electrophoretic
mobility shift assay was performed as described previously (6). Binding
reactions were performed by incubating 2-5 fmol of an end-labeled,
31-base pair-long oligonucleotide containing a strong MRE
consensus sequence (MRE-s), with nuclear extracts obtained according to
the protocol of Schreiber et al. (28). For competition
experiments, 2 pmol of unlabeled oligonucleotides were added to the
reaction mixture before the addition of the extracts.
MTF-1 Shows Stress-inducible Nuclear Import--
To determine the
effects of zinc and cadmium on the intracellular localization of MTF-1,
293 (adenovirus-transformed human embryonal kidney) cells and U2OS
(human osteosarcoma) cells were transiently transfected with an
expression vector encoding a VSV-tagged MTF-1. This allowed for easy
identification of functional MTF-1 (data not shown). 24 h after
serum deprivation, subcellular localization of the MTF-1-VSV fusion
protein was determined by indirect immunofluorescence. Under normal
growth conditions in serum-containing cell culture medium, a
considerable fraction of MTF-1 localizes to the nucleus (Ref. 24; data
not shown). Here, we have addressed this point and find that MTF-1
stays almost exclusively in the cytoplasm in serum-starved 293 cells
(Fig. 1A). In U2OS cells,
however, the fluorescence signal of tagged MTF-1 was detected in both
the cytoplasm and the nucleus (Fig. 1B). Exposure of the
cells to 200 µM zinc chloride (ZnCl2) (Fig.
1, C and D) or 60 µM cadmium chloride (CdCl2) (Fig. 1, E and F)
for 1 h resulted in a striking nuclear accumulation of MTF-1 in
both 293 and U2OS cells. In addition, the treatment of 293 cells with
heat shock (43 °C, 1 h), H2O2 (800 µM, 3 h), low pH (pH 6.0, 1 h), cycloheximide
(10 µg/ml, 3 h), and serum factors (10% FBS, 3 h) caused
nuclear translocation of MTF-1 (Fig.
2A). The calculated
percentages of nuclear accumulation upon each different treatment are
shown in Fig. 2B. The cells were also treated with
substances that induce nuclear translocation of other transcription
factors: tumor necrosis factor Heavy Metal-induced Nuclear Translocation of MTF-1 Is
Concentration- and Time-dependent--
293 cells
transfected with a plasmid encoding VSV-tagged human MTF-1 were treated
with different concentrations of zinc chloride or cadmium chloride, and
MTF-1 localization was analyzed at several time points by indirect
immunofluorescence staining. Rapid translocation of MTF-1-VSV was
obtained at 100 µM zinc chloride, with a half-maximal nuclear transport time (t1/2) of 23 min (Fig.
3A). Nuclear translocation was
nearly complete after 30 min of exposure. A higher zinc concentration (200 µM) had little accelerating effect (Fig.
3A). Similarly, increasing concentrations of cadmium
decreased t1/2 from 25 min (40 µM)
to 21 min (120 µM cadmium chloride) (Fig.
3B).
Analysis of the Nuclear Translocation of MTF-1--
Human MTF-1
contains near the first zinc finger the motif
(133KRKEVKR139) that fits the consensus of a
classical NLS sequence. To evaluate the contribution to nuclear
accumulation of this motif, we performed site-directed mutagenesis to
replace the three basic amino acids Lys133-Arg134-Lys135 with
Leu-Leu-Ile (Fig. 4). The mutated protein
(LLI(133-135)MTF-1-VSV) was localized exclusively in the
cytoplasm of resting cells. Nevertheless, when the
LLI(133-135)MTF-1-VSV-expressing cells were treated with
either 200 µM zinc chloride or 60 µM
cadmium chloride for 1 h, 72% of the transfected cells had
fluorescent nuclei (Fig. 5A).
However, the kinetics of nuclear localization upon zinc treatment was
delayed, with a pronounced extension of the initial lag phase (Fig.
5B). The most pronounced difference was observed in a
short-term incubation (30 min) with 200 µM zinc chloride
in which 75% of the cells transfected with wild type MTF-1 showed
nuclear staining, but only 6.5% of the cells contained nuclear
NLS-mutated MTF-1. At 60 min of zinc treatment, this
difference had almost leveled out in that wild type and mutant MTF-1
were localized in the nucleus in 90.5% and 72% of the cells,
respectively. Thus, it was not unexpected to find only marginal
differences in metallothionein promoter activation by wild type
compared with NLS mutant MTF-1, as measured during a 3-h time period.
In these experiments, the first time point with clearly elevated
reporter gene transcripts was 2.5 h after stress induction for
both wild type and NLS mutant (Fig. 5C). However, a strong
effect of the NLS mutant was observed in other stress conditions.
Translocation of NLS mutant MTF-1 to the nucleus by administration of
hydrogen peroxide, low pH, cycloheximide, and serum factors was
severely impaired, reaching only about 10% of wild type MTF-1 levels,
even after extended incubation times of up to 4 h (data not
shown).
LMB Induces Nuclear Accumulation of MTF-1 without Affecting Nuclear
Import--
To define the mechanism(s) responsible for the
steady-state cytoplasmic retention of MTF-1, we decided to use LMB, a
specific inhibitor of the nuclear export protein CRM1 (19, 32). Upon LMB treatment of MTF-1 transfected cells for 3 h, 63 ± 6%
of the transfected cells showed nuclear accumulation (Fig.
6). The shift from the cytoplasm to the
nucleus was detectable within 30 min of LMB administration and was
nearly complete after 6 h (data not shown). Treatment with LMB at
4 °C did not cause any nuclear MTF-1 accumulation (data not shown).
To test the effect of LMB on the metal-induced nuclear accumulation of
MTF-1, 293 cells were pretreated with 10 ng/ml LMB for 15 min and
incubated at 37 °C for 60 min in the presence of 200 µM zinc chloride or 60 µM cadmium chloride.
As expected for an export blocker, simultaneous treatments with LMB and
heavy metals did not impair the nuclear import of MTF-1 (data not
shown).
MTF-1 Has a Functional Nuclear Export Signal
Sequence--
Examination of the sequence of human MTF-1 revealed two
candidate NES sequences (tentatively designated NES and NES'),
336LCLSDLSLL344 (NES) and
417LSLPLVLQPGL427 (NES'), respectively (Fig.
4). The putative MTF-1 NES motifs are similar in sequence to the
well-established leucine-rich NES sequences in other proteins such as
human immunodeficiency virus Rev (18), protein kinase A inhibitor (16),
and mitogen-activated protein kinase kinase (17) (Fig.
7). This figure also shows a comparison
to MTF-1 NES candidate motifs from three different species (human,
mouse, and Japanese puffer fish (Fugu rubripes)). To
determine whether the putative NES sequences are functional, we
generated three human MTF-1 mutants in which the four most critical
leucine residues in either NES, NES', or both were replaced by alanines
(MTF-1NESmut, MTF-1NES'mut, and
MTF-1NESNES'mut, respectively) (Fig. 4). VSV-tagged
versions of these mutants were transfected into 293 cells in parallel
to wild type MTF-1. The subcellular distribution of each protein was
determined by indirect immunofluorescence staining. The wild type MTF-1
and the MTF-1NES'mut localized exclusively to the cytoplasm
of serum-starved cells. In contrast, MTF-1NESmut and
MTF-1NESNES'mut localized to the nucleus, with 92 ± 3% nuclear staining (the remaining cells showed both cytoplasmic and
nuclear fluorescence) (Fig. 8). These
data indicate that NES but not NES' is required for efficient
cytoplasmic localization of MTF-1.
An MTF-1-derived NES Directs Nuclear Export of a Heterologous
Protein, Most Likely via Interaction with the Nuclear Export Protein
CRM1--
To test whether the activities of the NLS and NES sequences
of MTF-1 are transferable to another protein, we transfected the NLS-
or NES-eGFP2x constructs into 293 cells and observed their localizations 24 h after transfection. The tandem duplication of
GFP protein is suitable for such studies because it apparently distributes almost equally to cytoplasm and nucleus (Fig.
9A). In agreement with a role
in nuclear export for the NES motif, NES-eGFP2x was
detected exclusively in the cytoplasm (Fig. 9C). By
contrast, the NLS and NES' sequences of MTF-1 did not change the
cellular localization of the double GFP protein (Fig. 9, B and E versus A). To test for a
possible involvement of CRM1 in the MTF-1 nuclear export, we analyzed
the effect of leptomycin B on the subcellular localization of
NES-eGFP2x fusion proteins in 293 cells. Treatment with
leptomycin B abolished nuclear export of the fusion protein containing
the NES sequence (Fig. 9D). These results show that the NES
of MTF-1 is a functional nuclear export signal that may be recognized
by CRM1.
Nuclear Accumulation of MTF-1 per se Is Not Sufficient for
Transcriptional Activation of the Mouse Metallothionein Gene
Promoter--
To determine whether nuclear accumulation of MTF-1 is
sufficient for transcriptional activation, we performed transcription assays on native 293 cells treated with heavy metals,
H2O2, low pH (pH 6.0), heat shock,
cycloheximide, serum, or LMB. The effect of MTF-1 on gene expression
was measured with a luciferase reporter gene driven by the
metallothionein I promoter. As expected, zinc treatment resulted in a
strong, 5-fold induction of luciferase activity, but all other
conditions tested showed markedly lower luciferase activities (Fig.
10A). The export blocker
LMB, which also induces nuclear accumulation of MTF-1, had no effect on
transcription, confirming the notion that nuclear localization of MTF-1
per se is not sufficient for transcriptional activity. In
addition, nuclear localization is not sufficient for DNA binding
activity. Nuclear extracts of MTF-1 null mutant cells (DKO7) that had
been transfected with wild type MTF-1 and exposed to non-metal stress
including LMB or H2O2, heat shock, serum, low
extracellular pH, and cycloheximide did not show any significant DNA
binding activity as measured by electrophoretic mobility shift assay
(data not shown).
Next, we determined the transcriptional activity of the NES mutants of
MTF-1. NES mutant MTF-1 did not activate transcription from the mMTI
promoter, even after zinc treatment. However, cells transfected with
NES' mutant MTF-1 vector activated transcription in response to zinc,
similar to wild type MTF-1 transfected cells. As expected, the double
mutant (NESNES') MTF-1 behaved in a manner similar to that of the NES
mutant MTF-1 (Fig. 10B). NES, NES', and NESNES' mutants did
not show any DNA binding activity in the absence of zinc in
vitro, but a relatively strong DNA binding activity was observed
upon zinc treatment of cells (Fig. 10C). Together, our
results demonstrate that neither nuclear localization of MTF-1 nor an
ability to bind DNA in vitro is sufficient to ensure
transcriptional activation of target genes.
We have analyzed the subcellular localization of MTF-1 in cultured
cells under resting conditions and also under various stress conditions. We demonstrate that this transcription factor localizes to
the cytoplasm of resting cells but rapidly translocates to the nucleus
under a variety of stress conditions, including exposure to heavy
metals, heat shock, hydrogen peroxide, low pH, inhibition of protein
synthesis by cycloheximide, and serum factors. The dual localization of
MTF-1 is conferred by an N-terminal classical NLS that facilitates
nuclear localization and a NES that promotes nuclear export of MTF-1
and is also functional on a reporter protein. Whereas nuclear
localization of MTF-1 is necessary for transcriptional activation of
metal-responsive genes, it is not sufficient. This was indicated by DNA
binding experiments and transcription assays of cells exposed to stress
conditions that did not directly involve heavy metals.
A major question concerns the mechanism(s) of MTF-1 induction in
response to stress. The zinc fingers of MTF-1 are required for DNA
binding, and it was noted early on that this DNA binding required
higher zinc concentrations than other zinc finger transcription factors, such as Sp1. In a recent study, Bittel et al. (33) have further characterized this zinc induction and found zinc finger 1 to play a crucial role, whereas other zinc fingers behaved "conventionally" as constitutive DNA binding domains. Whereas this
property of MTF-1 nicely explained the induction by zinc, it fell short
of explaining induction by other heavy metals, let alone by other
stress conditions. Indeed, attempts to induce DNA binding with other
heavy metals failed (34), suggesting that they could not substitute for
zinc. To make things worse, any stress condition other than zinc load
led to a loss of MTF-1 DNA binding
(34).2 Recently, we were able
to develop in our laboratory a cell-free transcription system that
depends on MTF-1 and yields DNA binding and transcriptional activation
by stress conditions such as cadmium and copper, provided that these
conditions could release zinc from zinc storage
proteins.3 These findings
suggest a unifying mechanism of MTF-1 activation via elevated
intracellular zinc concentration. The data presented here now indicate
an additional level of complexity, namely, a regulation of MTF-1
activity by subcellular compartmentalization. We found that various
stress conditions such as exposure to heavy metals (zinc and cadmium),
heat shock, H2O2, low pH, and inhibition of
protein synthesis by cycloheximide cause translocation of MTF-1 from
the cytoplasm to the nucleus. Thus MTF-1 joins a growing list of
transcription factors that are regulated at the level of nuclear import
in response to a variety of signals, including stress conditions
(35).
MTF-1 harbors, immediately N-terminal to the first zinc finger, a
putative SV40 T antigen-like NLS. Mutational analysis showed that the
candidate NLS facilitates nuclear accumulation of MTF-1 but that it is
not essential for import per se. However, the NLS failed to
induce nuclear translocation when fused to a reporter protein
(eGFP2x). One possible explanation could be that the basic sequence motif studied here is incomplete and part of a larger, perhaps
bipartite, NLS. MTF-1 may also cooperate with a cofactor that contains
an NLS of its own. However, such a cofactor would only be operative
under heavy metal stress because nuclear translocation of a candidate
NLS-mutated MTF-1 under the non-metal conditions was very poor.
Alternatively, MTF-1 might be specifically modified by heavy metal
stress, possibly exposing additional nuclear targeting signals. These
open questions seem to warrant further experiments on the mechanism of
MTF-1 nuclear import.
The situation with regard to the NES appears to be more
straightforward. We have characterized two candidate nuclear export signal sequences (NES and NES') that resemble leucine-rich NES sequences of other proteins. NES but not NES' turned out to be functional in nuclear export. Inhibition of the export activity by LMB
or amino acid mutations in NES changed MTF-1 localization from the
cytoplasm to the nucleus in 293 cells, indicating that the cytoplasmic
accumulation of MTF-1 under basal conditions is accomplished by the NES
element. Furthermore, the transfer of the NES sequences from MTF-1 to a
eGFP2x protein revealed that one copy of NES (but not of
NES') is sufficient to export the reporter protein from the nucleus to
the cytoplasm. Like export of MTF-1, export of eGFP2x-NES
was sensitive to LMB, suggesting that NES is responsible for the
cytoplasmic accumulation of MTF-1 under resting conditions and that
export is carried out through an interaction between NES and the export
protein CRM1. In transcription assays, mutation of NES but not of NES'
abolished the heavy metal-dependent activity. It is
possible that loss of activity of NESmut MTF-1 was due to
an impaired function of the strong acidic MTF-1 activation domain in
which NES is embedded. Alternatively, nucleo-cytoplasmic shuttling of
MTF-1 might be required to ensure correct inducibility, perhaps by
allowing for essential posttranslational modification(s) in the
cytoplasm. Furthermore, shuttling of MTF-1 between two compartments
may, on one hand, ensure removal of MTF-1 from its target genes and, on
the other hand, still allow for a rapid transcriptional response.
Although the activity of a number of transcription factors is regulated
by cytoplasmic-nuclear transport, it is also clear that the mere
presence of a factor within the nucleus is not necessarily sufficient
for transcriptional activation. Rather, the factor has to be further
activated for optimal DNA binding and/or transactivation by protein
modifications, such as phosphorylation or acetylation (36,
37), and/or released from an inhibitory protein (38-40).
Consistent with a need for posttranslational modification, our
preliminary data suggest that phosphorylation is involved in
signal-dependent activation of
MTF-1.4 The characterization
of posttranslational modifications of MTF-1 will be necessary for a
full understanding of stress-mediated gene regulation.
Thus far, the metallothionein genes are the best characterized target
genes of MTF-1 in heavy metal signaling. Further target genes with MRE
motifs in their promoters were recently identified (9). Why MTF-1 is
imported into the nucleus under non-metal stress (heat shock,
H2O2, low pH, cycloheximide, and serum
factors), conditions that failed to activate MRE-containing promoters
in our assays, remains an open question. It is interesting to note that, at least in some species, heat induces metallothionein
transcription, and, conversely, heavy metals can induce heat shock gene
transcription. It is possible that novel target genes for non-metal
stress response are activated (or repressed) through MTF-1 via sequence
motifs different from the classical MREs, for example, in cooperation with other transcription factors. Within the framework of such a model,
we have begun to screen a random collection of oligonucleotides for
sequences that specifically bind MTF-1 in absence of zinc load.5 Besides activation of
novel target genes under non-metal stress, one can also envisage other
scenarios. One straightforward reason for nuclear import under many
conditions may be to remove MTF-1 from a cytoplasmic "danger zone"
into the better protected nuclear environment. Whereas this seems
reason enough to transport MTF-1 to the nucleus, preliminary findings
also point in another direction and suggest a synergistic activation of
metallothionein promoters by a combination of metal and nonmetal
stress.4
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B, and
nuclear factor of activated T cells, localize differentially between the cytoplasm and the nucleus in response to DNA damage, growth
signals, or environmental stimuli (12, 13). The import of proteins
destined for the nucleus requires a nuclear localization signal; the
export of large macromolecules from the nucleus is also an active
transport process mediated by nuclear export signals (NESs) (12-15).
NES sequences are short, leucine-rich motifs that were first identified
in the cellular proteins, protein kinase A inhibitor, mitogen-activated
protein kinase kinase, and the human immunodeficiency virus Rev protein
(16-18). Recognition of NES motifs by the nuclear export receptor CRM1
is specifically suppressed by the antifungal drug leptomycin B (LMB)
(19). Here we have addressed the molecular mechanisms of MTF-1
regulation by a variety of stress inducers to explore possible
regulatory mechanisms. We demonstrate that cytoplasmic MTF-1 is
imported into the nucleus of serum-starved cells by stress inducers,
including heavy metals. While this work was in progress, another group
also found a cytoplasmic-nuclear translocation of MTF-1 upon zinc and cadmium treatment of cells (20). Here we characterize a nuclear localization signal (NLS) of MTF-1 and show that MTF-1 enters the
nucleus not only upon metal load but also upon oxidative stress, heat
shock, low pH, inhibition of protein synthesis by cycloheximide, and
serum factors. Additionally, we find that MTF-1 has a functional NES
that is sensitive to LMB and mediates subcellular localization and
nuclear-cytoplasmic shuttling. Most importantly, we demonstrate that
the nuclear accumulation of MTF-1 per se does not result in
transcriptional activation via MRE sequences, indicating that nuclear
translocation and transcriptional activation functions can be separated.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
727 to
13) region of the mMTI gene (23) into the
SmaI site of the pGL3 basic vector (Promega).
/
embryonic
stem cells designated as DKO7 cells (24) were maintained in Dulbecco's
modified Eagle's medium (DMEM; Life Technologies, Inc.)
supplemented with 10% fetal bovine serum (ICN). Cells were transfected
by the calcium phosphate method (25).
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(50 ng/ml, 1 h) and
12-O-tetradecanoylphorbol-13-acetate (100 ng/ml, 1 h), which activate nuclear factor
B by inducing the nuclear
translocation of RelA (29), and sorbitol (400 mM, 1 h)
which is known to stimulate the nuclear translocation of signal
transducer- activators of transcription in mammalian cells (30) and
HOG1 mitogen-activated protein kinase in yeast (31); however, these
treatments did not induce any obvious change in the subcellular
localization of MTF-1 (data not shown). This indicates that the nuclear
translocation of MTF-1 in response to stress conditions is a specific
property of this transcription factor.
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Fig. 1.
Subcellular distribution of MTF-1 in 293 and
U2OS cells. An expression vector coding for VSV
epitope-tagged MTF-1 was used for transfection of human 293 and
U2OS cells. To reduce serum content, 24 h after transfection the
cells were incubated for another 24 h in a medium containing 0.5%
BSA replacing FBS. The cells were either left untreated (A
and B) or treated with 200 µM
ZnCl2 (C and D) or 60 µM CdCl2 (E and F), and
the cells were then fixed and stained with anti-VSV antibody, followed
by fluorescein-labeled secondary antibody (left panels) and
DNA staining with 4',6-diamidino-2-phenylindole (right
panels). Scale bar, 20 µm.
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Fig. 2.
Heat shock and other stress conditions also
induce nuclear import of MTF-1. Subcellular localization of
MTF-1-VSV exposed to various stress conditions was examined. 293 cells
transiently transfected with MTF-1-VSV were treated with heat
(43 °C, 1 h), hydrogen peroxide (800 µM, 3 h), low pH (pH 6.0, 1 h), serum (10% dialyzed FBS, 3 h), and
cycloheximide (10 µg/ml, 3 h) (A). Control cells were
processed in parallel as described in the Fig. 1 legend, and MTF-1 was
visualized by fluorescence microscopy. Scale bar, 20 µm.
B, about 200 MTF-1-positive cells were counted to quantify
nuclear versus cytoplasmic distribution of MTF-1, and the
results were classified as exclusively cytoplasmic (C),
exclusively nuclear (N), and both cytoplasmic and nuclear
(C+N). The mean values of cells with exclusively nuclear
MTF-1 were calculated from three different experiments using ~200
cells, with the corresponding S.D. (B).
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Fig. 3.
Time course of nuclear import of MTF-1 in the
presence of zinc or cadmium. Serum-starved 293 cells were
incubated with different concentrations of zinc chloride (A)
or cadmium chloride (B) for the indicated times at 37 °C
and analyzed for MTF-1 localization by indirect immunofluorescence. The
fraction of cells with exclusively nuclear staining was plotted for
each time point. t1/2 indicates the time of
half-maximal nuclear transport of each condition. The values show the
average of three independent experiments.
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Fig. 4.
Mutagenized amino acids in the putative NLS
and NES motifs of human MTF-1. Schematic representation of the
primary structure of human MTF-1 showing a putative NLS (residues
133-139) and two putative NES sequences (NES, residues
336-344; NES', residues 417-427) with the mutation sites
indicated.
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Fig. 5.
Nuclear accumulation of MTF-1 in response to
zinc or cadmium is delayed by mutations in the NLS-like basic amino
acid cluster. 293 and MTF-1-negative DKO7 cells were transfected
with either the expression plasmids coding for MTF-1-VSV or the
LLI(133-135)MTF-1-VSV. 24 h after transfection, cells
were serum-starved for another 24 h and treated with zinc for the
indicated times. LLI(133-135)MTF-1-VSV-expressing 293 cells were either untreated or treated with 200 µM
ZnCl2 for 1 h and visualized by fluorescence
microscopy (A). Scale bar, 20 µm. The kinetics
of zinc-induced nuclear import of the wild type or the NLS mutant MTF-1
was determined in 293 cells (B). After treatment with 200 µM ZnCl2 for 5, 15, 30, 60, and 180 min,
cells were fixed and processed for indirect immunofluorescence. The
percentages of the cells showing exclusively nuclear staining from
three independent experiments are presented graphically with the S.D.
The kinetics of zinc-induced transcriptional activity of the wild type
and the NLS mutant MTF-1 was analyzed by luciferase assay using a
natural mouse metallothionein I promoter as a reporter in 293 cells
(C). After treatment with 200 µM
ZnCl2 for 15 min, 30 min, 1 h, 2 h, and 3 h,
cells were harvested and processed for luciferase assays. Results were
normalized to cotransfected -galactosidase activity. The data from
the three independent experiments are shown with the S.D.
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Fig. 6.
Nuclear export of MTF-1 is sensitive to
LMB. The transfected and serum-starved 293 cells were treated with
either LMB in ethanol (final concentration, 10 ng/ml and 0.1% v/v
ethanol) or ethanol alone (0.1% v/v) for 3 h at 37 °C. Cells
were then fixed and stained with anti-VSV antibody. Quantification of
this experiment revealed that MTF-1 accumulated in the nucleus in
63% of LMB-treated cells. Scale bar, 20 µm.
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Fig. 7.
MTF-1 contains two putative conserved
NESs. A comparison of the two putative NES sequences (NES and
NES') of human, mouse, and F. rubripes MTF-1 with the NES
consensus sequence of human immunodeficiency virus Rev, protein kinase
A inhibitor, and mitogen-activated protein kinase kinase
(MAPKK) is shown. Important hydrophobic residues (leucine
and isoleucine) in the sequences are boxed.
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Fig. 8.
Cytoplasmic localization of MTF-1 requires
nuclear export mediated by NES but not by NES'. 293 cells were
transfected with the expression vectors encoding MTF-1 wild
type, NESmut, NES'mut, or
NESNES'mut proteins. After 24 h of serum starvation,
the cells were treated with 200 µM ZnCl2 for
1 h and processed for indirect immunofluorescence. Scale
bar, 20 µm.
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Fig. 9.
MTF-1 NES induces nuclear export of a
heterologous protein. 293 cells were transfected with the GFP
vectors encoding the double GFP (eGFP2x) alone
(A), NLS-eGFP2x (B),
NES-eGFP2x (C and D), and
NES'-eGFP2x (E and F). After 24 h, the indicated samples were treated with 10 ng/ml LMB for 6 h
(D and F), and cells were fixed and analyzed by
fluorescence microscopy. Scale bar, 20 µm.
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Fig. 10.
Transcriptional activation and DNA binding
activities of NES mutants. 293 cells transfected with mMTI-Luc and
CMV-LacZ were starved for 24 h and then treated with
ZnCl2 (200 µM), leptomycin B (10 ng/ml), cycloheximide (10 µg/ml), low pH (pH 6.0), serum
(10% dialyzed FBS), H2O2 (800 µM) for 4 h, and heat shock (43 °C) for 1 h.
The cells were harvested and processed for luciferase assays
(A). The data presented are the mean values from three
independent experiments with the S.D. Wild type or NES mutant MTF-1
expression vectors were transfected into mouse DKO7 cells lacking
MTF-1. Serum-starved cells were either left untreated or treated with
200 µM ZnCl2 for 4 h before luciferase
assays (B) or electrophoretic mobility shift assays
(C).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Dr. Minoru Yoshida (University of Tokyo, Tokyo, Japan) and Dr. Ulrike Kutay for the generous gifts of leptomycin B and eGFP2x construct, respectively.
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FOOTNOTES |
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* This work was supported by the Swiss National Science Foundation and the Kanton Zürich.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 41-1-635-3150/51; Fax: 41-1-635-6811; E-mail: walter.schaffner@molbio.unizh.ch.
Published, JBC Papers in Press, April 16, 2001, DOI 10.1074/jbc.M009154200
2 B. Zhang, N. Saydam, O. Georgiev, and W. Schaffner, unpublished data.
3 B. Zhang, O. Georgiev, and W. Schaffner, unpublished data.
4 N. Saydam and W. Schaffner, unpublished data.
5 Y. Wang and W. Schaffner, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: MT, metallothionein; MRE, metal-responsive element; NES, nuclear export signal; MTF-1, metal-regulatory transcription factor 1; LMB, leptomycin B; NLS, nuclear localization signal; GFP, green fluorescent protein; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; BSA, bovine serum albumin.
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