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Glen K.

Andrews
Rudravajhala Ravindra, Huimin Jiang and
Irina V. Smirnova, Douglas C. Bittel,

Element-binding Transcription Factor-1
Nuclear Translocation of Metal Response
Zinc and Cadmium Can Promote Rapid
REGULATION:
GENES: STRUCTURE AND
doi: 10.1074/jbc.275.13.9377
2000, 275:9377-9384. J. Biol. Chem.

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Zinc and Cadmium Can Promote Rapid Nuclear Translocation of
Metal Response Element-binding Transcription Factor-1*
(Received for publication, October 29, 1999, and in revised form, December 28, 1999)
Irina V. Smirnova, Douglas C. Bittel, Rudravajhala Ravindra, Huimin Jiang,
and Glen K. Andrews
From the Department of Biochemistry and Molecular Biology, University of Kansas Medical Center,
Kansas City, Kansas 66160-7421
Metal response element-binding transcription fac-
tor-1 (MTF-1) is a six-zinc finger protein that plays an
essential role in activating metallothionein expression
in response to the heavy metals zinc and cadmium. Low
affinity interactions between zinc and specific zinc fin-
gers in MTF-1 reversibly regulate its binding to the
metal response elements in the mouse metallothionein-I
promoter. This study examined the subcellular distribu-
tion and DNA binding activity of MTF-1 in cells treated
with zinc or cadmium. Immunoblot analysis of cytosolic
and nuclear extracts demonstrated that in untreated
cells, about 83% of MTF-1 is found in the cytosolic ex-
tracts and is not activated to bind to DNA. In sharp
contrast, within 30 min of zinc treatment (100 M),
MTF-1 is detected only in nuclear extracts and is acti-
vated to bind to DNA. The activation to bind to DNA
and nuclear translocation of MTF-1 occurs in the ab-
sence of increased MTF-1 content in the cell. Further-
more, immunocytochemical localization and immuno-
blotting assays demonstrated that zinc induces the
nuclear translocation of MTF-1-FLAG, expressed from
the cytomegalovirus promoter in transiently trans-
fected dko7 (MTF-1 double knockout) cells. Immunoblot
analysis of cytosolic and nuclear extracts from cadmi-
um-treated cells demonstrated that concentrations of
cadmium (10 M) that actively induce metallothionein
gene expression cause only a small increase in the
amount of nuclear MTF-1. In contrast, an overtly toxic
concentration of cadmium (50 M) rapidly induced the
complete nuclear translocation and activation of DNA
binding activity of MTF-1. These studies are consistent
with the hypothesis that MTF-1 serves as a zinc sensor
that responds to changes in cytosolic free zinc concen-
trations. In addition, these data suggest that cadmium
activation of metallothionein gene expression may be
accompanied by only small changes in nuclear MTF-1.
Metallothioneins (MT)
1
are small cysteine-rich proteins,
which play a role in zinc homeostasis, cadmium detoxication,
and protection from reactive free radicals (16). The rapid
induction of MT-I and -II gene transcription by heavy metals
(1) is mediated by metal response elements (MREs), present in
multiple copies in the proximal promoters of MT genes (7). A
protein that binds directly and specifically to MREs (8) and
transactivates MT gene expression is referred to as MTF-1 (9).
MTF-1 is a six-zinc finger protein in the Cys
2
His
2
family of
transcription factors. Human, mouse, and pufferfish MTF-1 have
been cloned (1012), and this protein has been highly conserved,
particularly in the zinc finger domain. The C terminus of mam-
malian MTF-1 contains three transactivation domains, which are
acidic, proline-rich, and serine/threonine-rich, respectively (13).
The DNA binding activity of native and recombinant MTF-1 is
reversibly modulated by zinc interactions with the finger domain
(14). The zinc fingers are heterogeneous in function and at least
two exhibit low affinity binding of zinc (15, 16).
Treatment of cells with zinc in vivo results in a rapid, dra-
matic increase in the DNA binding activity of MTF-1 measured
in vitro (10, 14) and the concomitant occupancy of MREs in the
MT-I promoter in vivo (3). In contrast, the DNA binding activ-
ity of MTF-1 is apparently not activated by transition metals
other than zinc (8, 17, 18), although cadmium is a particularly
potent inducer of MT gene expression. Homozygous deletion of
the mouse MTF-1 gene revealed that MTF-1 is essential for
zinc and cadmium induction, as well as for basal expression of
the mouse MT-I and -II genes in embryonic stem cells (19).
MTF-1 is also essential for induction of these genes by oxida-
tive stress (3) and hypoxia (20). Thus, several signal transduc-
tion pathways may impinge on the activities of MTF-1. Mice
homozygous for targeted deletions of the MTF-1 gene die in
utero due to failure of liver development, demonstrating that
the MTF-1 gene is an essential gene (21), unlike the mouse
MT-I and -II genes (22).
Transition metal regulation of gene expression has been
documented in species from every kingdom of organisms. In
many instances, the transition metal itself directly interacts
with a preexisting metalloregulatory protein, and this interac-
tion leads to a change in conformation of the protein and an
alteration in the DNA or RNA binding activity of the protein
(23). The available evidence suggests that MTF-1 is a metal-
loregulatory protein that serves as an intracellular zinc sensor
to activate gene expression. This model predicts that MTF-1
would be located in the cytoplasmto facilitate direct interaction
with free zinc. Since previous studies have not addressed the
subcellular localization of MTF-1, it is not clear whether this
cellular response to metal ions is initiated in the cytosol or
nucleus. Furthermore, it is unclear how MTF-1 senses the toxic
metal cadmium. Therefore, we examined the effects of zinc and
cadmium treatment on the subcellular localization and DNA
binding activity of mouse MTF-1.
* This work was supported by National Institutes of Health Grant ES
05704 (to G. K. A.), and National Research Service Award F32 ES
05753 (to D. C. B.). The costs of publication of this article were defrayed
in part by the payment of page charges. This 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: G. K. Andrews,
Department of Biochemistry and Molecular Biology, University of Kan-
sas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160-7421.
Tel.: 913-588-6935; Fax: 913-588-7035; E-mail: gandrews@kumc.edu.
1
The abbreviations used are: MT, metallothionein; CE, cytosolic ex-
tract; DMEM, Dulbeccos modified Eagles medium-high glucose;
EMSA, electrophoretic mobility shift assay; FBS, fetal bovine serum;
MRE, metal response elements; MTF-1, metal response element-bind-
ing transcription factor-1; NE, nuclear extract; NLS, nuclear localiza-
tion signal; PBS, phosphate-buffered saline; CMV, cytomegalovirus.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 275, No. 13, Issue of March 31, pp. 93779384, 2000
2000 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
This paper is available on line at http://www.jbc.org 9377

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EXPERIMENTAL PROCEDURES
MaterialsThe following reagents were used in this study: in vitro
TnT coupled reticulocyte lysate transcription/translation system (Pro-
mega Corporation, Madison, WI); Microcon 10 (Millipore Corp., Bed-
ford, MA); nonfat dry milk, protein assay reagent (Bio-Rad); NE-PER
nuclear and cytoplasmic extraction reagents and BCA protein assay
reagent (Pierce); Protran nitrocellulose membrane (Schleicher &
Schuell); ECL Western blotting (immunoblotting) detection reagent,
Hyperfilm ECL (Amersham Life Science, Arlington Heights, IL); X-
Omat film for autoradiography (Eastman Kodak Co., Rochester, NY);
LipofectAMINE and LipofectAMINE Plus (Life Technologies, Inc.); four
chamber glass slides (Nalge Nunc International, Naperville, IL); rabbit
polyclonal antibodies against Sp1 (PEP 2), USF1 (C-20) and FLAG-
probe (D-8) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); goat
anti-rabbit IgG conjugated to peroxidase (Jackson ImmunoResearch
Laboratories, West Grove, PA); rat monoclonal antibody against Hsp90
and rabbit anti-rat IgG conjugated to peroxidase (Stressgen, Victoria,
Canada); and the DAB kit (Zymed Laboratories Inc., San Francisco,
CA). All other chemicals were purchased from Sigma. The polyclonal
antiserum against purified bacterial recombinant mouse MTF-1 fused
to glutathione S-transferase was raised in rabbits (Covance Research
Products, Inc., Denver, CO) and purified by protein A chromatography
followed by passage through glutathione S-transferase-agarose to re-
move glutathione S-transferase antibodies (20).
Cell CultureMouse Hepa cells were maintained in Dulbeccos mod-
ified Eagles medium-high glucose (DMEM) supplemented with 2% fetal
bovine serum (FBS). The mouse dko7 cell line is a simian virus 40 large
T-antigen-immortalized fibroblast derived from embryonic stem cells
lacking MTF-1 (MTF-1 double knockout) and was a generous gift of Dr.
Walter Schaffner, University of Zurich (Zurich, Switzerland) (13).
These cells were maintained in DMEM supplemented with 10% FBS.
For nuclear and cytosolic extract preparations, cells (2 10
6
) were
plated in 15-cm Petri dishes and grown to 80% confluency. For trans-
fection followed by extract preparation, cells (1.2 10
5
) were plated in
six-well plates (9.4 cm
2
) and grown to 50% confluency. For transfection
and subsequent immunocytochemistry, cells (2.5 10
4
) were plated in
four-chamber glass slides (1.8 cm
2
) and grown to 50% confluency. All FBS
was heat-inactivated prior to use; all media were supplemented with 50
units/ml penicillin, 50 g/ml streptomycin, and 2 mM L-glutamine.
Preparation of Cell ExtractsWhole cell extracts, nuclear extracts
(NEs), and cytosolic extracts (CEs) were prepared essentially as de-
scribed (14, 24). Briefly, for preparation of nuclear extracts, treated
cells were placed on ice, the medium was removed, and cells were
washed once with cold PBS. Cells were scraped off the dish and col-
lected by centrifugation at 1,500 g for 5 min. The cell pellet was
resuspended in 5 ml of cell lysis buffer (10 mM HEPES (pH 7.9), 1.5 mM
MgCl
2
, 10 mM KCl, 0.5 mM dithiothreitol, and 0.2 mM phenylmethyl-
sulfonyl fluoride), and immediately centrifuged at 1,500 g for 5 min.
Cells were resuspended in 2 times the original packed cell volume of cell
lysis buffer, allowed to swell on ice for 10 min, and homogenized with 10
strokes of a Dounce homogenizer (B pestle). Nuclei were collected by
centrifugation at 3,300 g for 15 min at 4 C, and supernatant was
saved for cytosolic extracts. The nuclei were resuspended, using six
strokes of a Teflon-glass homogenizer, in 3 volumes (about 750 l) of
nuclear extraction buffer (20 mM HEPES, pH 7.9, 1.5 mM MgCl
2
, 400
mM KCl, 0.5 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride,
and 25% glycerol). The nuclear suspension was stirred on ice for 30 min
and then centrifuged at 89,000 g for 30 min. The supernatant was
collected and concentrated in a Microcon 10 concentrator by centrifu-
gation at 14,000 g for 3 h at 4 C. For preparation of CE, the superna-
tant obtained after removal of nuclei was mixed thoroughly with 0.11
volume of 10 cytoplasmic extraction buffer (1 cytoplasmic extraction
buffer: 30 mM HEPES (pH 7.9) at 4 C, 140 mM KCl, 3 mM MgCl
2
) and
then centrifuged at 89,000 g for 1 h. The supernatant was collected and
concentrated in a Microcon 10 concentrator by centrifugation at 14,000
g for 1 h at 4 C. Protein concentration was determined using Bio-Rad
Protein Assay reagent with bovine serum albumin as the standard.
In transfection experiments, NE-PER nuclear and cytoplasmic ex-
traction reagents was used to prepare extracts. However, because of the
presence of EDTA, the addition of exogenous zinc was required to
activate MTF-1 DNA binding activity in these extracts.
Preparation of Total Protein SDS ExtractsSDS lysis of cells was
performed on plates using 1 SDS sample buffer without reducing
agent or bromphenol blue. Protein concentration was determined using
BCA protein assay reagent and bovine serum albumin as the standard.
Electrophoretic Mobility Shift Assay (EMSA)EMSA was performed
as described previously (3). Extracts (1020 g of protein in 25 l)
were incubated in a total volume of 20 l for 15 min at 4 C in binding
reaction buffer containing 12 mM HEPES (pH 7.9), 60 mM KCl, 0.5 mM
dithiothreitol, 12% glycerol, 5 mM MgCl
2
, 0.2 g of dI-dC/g of protein
with 24 fmol of end-labeled double-stranded oligonucleotide MRE-s or
Sp1 binding sequence (5,000 cpm/fmol) for MTF-1 or Sp1, respectively.
Protein-DNA complexes were separated electrophoretically at 4 C in
4% polyacrylamide gel (acrylamide/bisacrylamide, 80:1) at 15 V/cm. The
gel was polymerized in running buffer consisting of 0.19 M glycine (pH
8.5), 25 mM Tris, and 0.5 mM EDTA. After electrophoresis, the gel was
dried, and labeled complexes were detected by autoradiography.
ImmunoblottingCell extracts (50100 g of protein) were sepa-
rated by 10 or 12% SDS-polyacrylamide gel electrophoresis (25) under
reduced conditions and transferred to nitrocellulose membranes. The
membranes were blocked overnight at 4 C in 10% nonfat dry milk in
PBS, 0.1% Tween 20 and probed with primary antibody diluted in 3%
nonfat dry milk in PBS, 0.1% Tween 20 for 1 h at room temperature.
Membranes were then incubated with an appropriate secondary anti-
body conjugated to horseradish peroxidase diluted in 3% nonfat dry
milk in PBS, 0.1% Tween 20 for 30 min at room temperature, developed
by chemiluminescence, and exposed to hyperfilm ECL. Relative band
intensities were quantitated using Biomax 1D image analysis software
(Kodak Scientific Imaging Systems). Equal protein loading and transfer
was verified visually by staining membranes with Ponceau solution.
Expression Vector ConstructionThe CMV-MTF-1 expression vector
was described previously (14). The MTF-1-FLAG construct was created
by polymerase chain reaction amplification from this template using a
sense primer that encompassed the translation start codon and an
antisense primer against the carboxyl terminus that also incorporated
the FLAG coding sequence. The amplified product was cloned into the
CMV vector. Vectors were verified by DNA sequencing.
Transient Transfectiondko7 cells were transfected using Lipo-
fectAMINE according to the manufacturers instructions. Cells were
grown to 50% confluence. After washing the cells with serum-free
DMEM, DNA and LipofectAMINE mixture prepared in serum-free
DMEM was added. For immunocytochemistry, the mixture consisted of
2 l/well LipofectAMINE, 4 ng/well CMV-MTF-1-FLAG expression vec-
tor, and 300 ng/well SV--gal, as an internal control for transfection
efficiency, in 250 l of DMEM. In experiments in which preparation of
nuclear and cytosolic extracts was performed, cells were treated with 4
l/well LipofectAMINE, 100 ng/well CMV-MTF-1 expression vector,
and 1 g/well SV--gal in 1.2 ml of DMEM. After 5 h, an equal volume
of DMEM containing 2 FBS was added, and the incubation was
continued overnight. The following morning, the medium was removed
and replaced with fresh DMEM containing 1FBS. Zinc treatment was
initiated in the afternoon of day 2. After 1 h, cells were processed for
either immunocytochemistry, as described below, or for nuclear and
cytosolic protein isolation.
ImmunocytochemistryTwenty-four hours after transfection, dko7
cells were washed twice with serum-free medium and then incubated
for 6 h in DMEM containing 1% (w/v) bovine serum albumin. Cells were
then treated for 30 min with 100 M ZnSO
4
in this medium, washed
with PBS, and fixed with 70% ethanol for 5 min. Slides were blocked for
1 h at room temperature with 10% goat serum in PBS-Triton X-100 and
incubated overnight at 4 C with rabbit polyclonal FLAG antibody or
Sp1 antibody diluted 1:500 or 1:100, respectively. Slides were then
incubated with anti-rabbit IgG conjugated to peroxidase and stained
with a DAB kit.
In Vitro Transcription/Translation of Mouse MTF-1Synthesis of
recombinant mouse MTF-1 was performed using the TnT coupled re-
ticulocyte lysate transcription/translation system (TnT lysate), as de-
scribed in detail previously (18).
RESULTS
Specificity of Mouse MTF-1 AntiseraPrevious studies dem-
onstrated that the rabbit polyclonal antisera against bacteri-
ally expressed glutathione S-transferase-MTF-1 was specific
for MTF-1 in supershift EMSA (20). The specificity of this
MTF-1 polyclonal antisera was examined by immunoblotting
(Fig. 1). Recombinant mouse MTF-1 synthesized in vitro in a
TnT lysate system was used as a positive control (Fig. 1, lane
1), and an extract from dko7 (MTF-1 double knockout) cells was
used as a negative control (Fig. 1, lane 2). Mouse MTF-1 mi-
grates with an apparent molecular mass of 100 kDa (Fig. 1,
lane 3), despite its predicted size of 72.5 kDa (10). This aber-
rant mobility may reflect the clustering of acidic, serine, and
proline residues in the structure of MTF-1 (10, 13) and is not
MTF-1 Translocates to the Nucleus 9378

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unique among transcription factors (26, 27). The MTF-1 band
was absent in extracts from dko7 cells (Fig. 1, lane 2) but was
detected in whole cell extracts from mouse Hepa cells (Fig. 1,
lane 3) and from dko7 cells transiently transfected with an
MTF-1 expression vector (see below). Two other bands with
apparent molecular masses of 200 and 57 kDa were de-
tected in cell extracts from both dko7 and Hepa cells (Fig. 1).
MTF-1 Is Localized in the Cytosol in Untreated Cells and in
the Nucleus in Zinc-treated CellsThe subcellular distribution
of MTF-1 in untreated and zinc-treated cells was investigated
by immunoblotting. Nuclear and cytosolic extracts were pre-
pared from mouse Hepa cells, and it was noted that approxi-
mately 4-fold more protein was extracted in the cytosolic versus
the nuclear extracts obtained from the same number of cells.
On average, these extraction procedures recovered 88 pg of
cytosolic protein and 21 pg of nuclear protein per cell. Immu-
noblotting of these extracts was performed using MTF-1 anti-
serum, and extracted proteins were normalized per cell for
analysis. To account for differences in the amount of protein
recovered in nuclear versus cytosolic extracts, 50 g of nuclear
and 200 g of cytosolic protein were loaded (Fig. 2A, right
panel). Quantitation of relative intensities of the MTF-1 bands
in these samples suggested that 17 9% of the immunoreac-
tive MTF-1 was extracted in the nuclear fraction whereas 83
9% of the MTF-1 was extracted in the cytosolic fraction from
control cells. In the remaining figures, equal quantities of pro-
tein per lane were applied to the gels. Some variability between
experiments in the amount of MTF-1 extracted from nuclei
versus cytosol was noted, as is demonstrated by the S.D. value
shown above. This variability was accentuated in the nuclear
FIG. 1. Characterization of a rabbit polyclonal antisera
against mouse MTF-1. Proteins were separated by 12% SDS-poly-
acrylamide gel electrophoresis followed by immunoblotting as described
under Experimental Procedures. Lane 1, recombinant mouse MTF-1
synthesized in vitro in a TnT coupled reticulocyte lysate system; lane 2,
dko7 (MTF-1 double knockout) whole cell extract; lane 3, Hepa whole
cell extract. The arrow shows the position of MTF-1. Relative mobilities
of molecular mass protein markers (kDa) are indicated to the left.
FIG. 2. Immunoblot detection of MTF-1 in nuclear and cytosolic extracts from Hepa cells treated with zinc. Hepa cells were treated
with 100 M ZnSO
4
for 1 h. NEs and CEs were prepared from treated and untreated cells and analyzed by immunoblotting using antisera against
the following: mouse MTF-1 (A); Sp1 (B); USF1 (C); or Hsp90 (D). A, the left panel represents a membrane where equal amounts of protein (100
g/lane) were loaded onto each lane; the right panel shows a membrane where 50 and 200 g of protein/lane was loaded for nuclear and cytosolic
extracts, respectively, to normalize per cell number (see text for explanation). Arrows show the positions of MTF-1, Sp1, USF1, and Hsp90. Relative
mobilities of molecular mass protein markers (kDa) are indicated to the left.
MTF-1 Translocates to the Nucleus 9379

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extracts relative to cytoplasmic extracts and may reflect subtle
differences in cell density, cell passage number, or culture
conditions. Each experiment contained an internal control of
untreated cells cultured in parallel under identical conditions.
In contrast to the results obtained using extracts from un-
treated Hepa cells, immunoblotting revealed that all MTF-1 im-
munoreactivity was present in nuclear extracts fromcells treated
with 100 M ZnSO
4
for 1 h. The cytosolic extract was devoid of
detectable MTF-1 (Fig. 2A). Fig. 2A (left panel) is an immunoblot
where equal amounts of nuclear and cytosolic proteins were
applied to the gel. The amount of immunoreactive MTF-1 de-
tected in nuclear extracts increased about 4-fold after zinc treat-
FIG. 3. EMSA detection of MTF-1 in
nuclear and cytosolic extracts from
Hepa cells treated with zinc. Hepa
cells were treated with 100 M ZnSO
4
for
1 h. NEs and CEs were prepared from
treated and untreated cells and analyzed
for DNA binding activity using a labeled
MRE-s oligonucleotide (A), or an Sp1 fam-
ily specific oligonucleotide (B). The arrows
point to specific complexes of MTF-1 or Sp1
and their respective oligonucleotides.
FIG. 4. Immunoblot and EMSA detection of MTF-1 in nuclear extracts fromHepa cells at different times after zinc treatment. Hepa
cells were treated with 100 M ZnSO
4
for 5 or 30 min. A, nuclear extracts were prepared from treated and untreated cells and analyzed by
immunoblotting. Upper panel, recombinant mouse MTF-1 synthesized in vitro in a TnT lysate was used as a positive control. Lower panel, the
extracts were immunoblotted with USF1 antibody. The arrows show the positions of MTF-1 and USF1. B, upper panel, nuclear extracts were
analyzed for DNA binding activity using a labeled MTF-1-specific MRE-s oligonucleotide, as described under Experimental Procedures. Lower
panel, the same extracts were assayed using an Sp1-specific oligonucleotide. The arrows point to specific MTF-1 and Sp1 complexes with their
respective oligonucleotides.
MTF-1 Translocates to the Nucleus 9380

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ment of the cells. This is consistent with the data suggesting that
about 83% of MTF-1 is initially found in the cytosolic fraction
from untreated cells. Immunoblot analysis of proteins remaining
in the nuclear pellet fraction after extraction revealed no MTF-1,
Sp1, and USF1. Thus, MTF-1 is not preferentially extracted from
nuclei of zinc-treated cells (data not shown). In contrast to
MTF-1, two other transcription factors, known for their consti-
tutive nuclear localization, Sp1 (Fig. 2B) and USF1 (Fig. 2C),
were detected only in nuclear extracts, and the amount of immu-
noreactive protein was unaffected by zinc treatment. Thus, the
cytosolic extracts were not significantly contaminated with nu-
clear transcription factors. Furthermore, immunoblotting with
antisera against the predominantly cytosolic heat shock protein
90 (Hsp90) (28, 29) revealed that the majority of Hsp90 was
detected in cytosolic extracts (Fig. 2D). Thus, nuclear extracts
were not significantly contaminated with cytosolic proteins.
Nuclear Localization of MTF-1 Is Accompanied by Activation
of DNA Binding ActivityEMSA was used to detect the MRE
binding activity of MTF-1 (3, 10, 14) in extracts from untreated
and zinc-treated Hepa cells. Nuclear and cytosolic extracts
from untreated cells contained little MTF-1 that was active to
bind to DNA (Fig. 3A). Previous studies demonstrated that
MTF-1 in whole cell extracts from control cells can be activated
in vitro by the addition of zinc (530 M) followed by incubation
at 37 C (14). After zinc treatment, however, the DNA binding
activity of MTF-1 increased 812-fold in nuclear extracts,
while cytosolic extracts exhibited no detectable MTF-1 DNA
binding activity (Fig. 3A). The identity of the MTF-1MRE-s
complex was confirmed by supershift EMSA using the MTF-1
antisera (data not shown). The rapid and dramatic increase in
MTF-1 DNA binding activity in nuclear extracts from cells
treated with zinc correlates with the immunoblotting data and
suggest that zinc induces the nuclear translocation and activa-
tion of MTF-1. Sp1 was detected only in nuclear extracts, and its
DNAbinding activity was unaffected by exogenous zinc (Fig. 3B).
Zinc-induced Nuclear Accumulation of MTF-1 Is RapidA
time course for zinc-dependent nuclear accumulation of MTF-1
protein and of MRE binding activity of MTF-1 was determined
using Hepa cells treated with 100 M ZnSO
4
(Fig. 4). The amount
of immunoreactive MTF-1 in the nucleus increased about 2-fold
by 5 min after the addition of zinc and 4-fold by 30 min (Fig. 4A).
As a control for potential differences in protein loading, MTF-1
was compared with immunoreactive USF1 in these same ex-
tracts (Fig. 4A). Nuclear USF1 levels remained constant during
zinc treatment. MREbinding activity of MTF-1 was monitored by
EMSA (Fig. 4B). A 2.5-fold increase in MRE binding activity was
detected by 5 min after zinc treatment and a 7-fold increase was
detected by 30 min, consistent with previous observations (18).
Sp1 DNA binding activity remained constant (Fig. 4B).
Zinc Treatment Does Not Alter the Amount of MTF-1 Pro-
teinImmunoblotting of total cell SDS extracts was used to
determine whether zinc causes a rapid change in the steady
state levels of MTF-1 in Hepa cells. Hepa cells were treated
with 100 M ZnSO
4
for 1 h, and the cells were lysed in situ in
SDS sample buffer. Equal amounts of SDS-extracted proteins
were then examined by immunoblotting. There was no detect-
able change in the amount of immunoreactive MTF-1 after this
FIG. 5. Immunoblot detection of MTF-1 in SDS lysates of Hepa
cells after zinc treatment. Hepa cells were treated with 100 M
ZnSO
4
for 1 h and lysed in situ in 1 SDS-sample buffer, and proteins
from untreated (lane 1) and treated (lane 2) cells were analyzed by
immunoblotting using MTF-1-, Sp1-, or USF1-specific antiserum. The
arrows show the MTF-1-, Sp1-, and USF1-immunoreactive bands.
FIG. 6. Immunoblot and EMSA de-
tection of MTF-1 in nuclear and cyto-
solic extracts from dko7 cells trans-
fected with a CMV-MTF-1 expression
vector. dko7 cells were transfected with
CMV-MTF-1 expression vector and treated
with 100 M ZnSO
4
for 1 h. NEs and CEs
were prepared using NE-PER extraction
reagents and analyzed by immunoblotting
(A) and EMSA (B). A, upper panel, recom-
binant mouse MTF-1 synthesized in a TnT
lysate was used as a positive control. Lower
panel, the extracts were immunoblotted
with USF1 antibody. The arrows show the
positions of MTF-1 and USF1. B, upper
panel, the extracts, which contained
EDTA, were analyzed for DNA binding ac-
tivity using a labeled MRE-s oligonucleo-
tide and after the addition of 100 MZnSO
4
to activate MTF-1. Lower panel, the same
extracts were assayed using an Sp1-spe-
cific oligonucleotide. The arrows point to
specific MTF-1 and Sp1 complexes with
their respective oligonucleotides.
MTF-1 Translocates to the Nucleus 9381

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zinc treatment (Fig. 5). Likewise, there were no detectable
changes in the relative amount of immunoreactive Sp1 or
USF1. In addition, levels of immunoreactive MTF-1 in whole
cell extracts from control and zinc-treated Hepa cells were
unaffected by pretreatment of the cells with cycloheximide (10
nM to 100 M) for 1 h (data not shown). Taken together, these
data indicate that zinc does not cause a detectable increase in
the steady state levels of MTF-1 protein within 1 h in these
cells, nor is MTF-1 protein rapidly degraded.
MTF-1 Expressed in Transiently Transfected dko7 Cells Also
Translocates to the Nucleus after Zinc TreatmentTo further
address the effects of zinc on the subcellular distribution of
MTF-1, dko7 cells, which have no endogenous MTF-1, were
transiently transfected with a CMV-MTF-1 expression vector
and then treated with 100 M ZnSO
4
for 1 h. Nuclear and
cytosolic extracts were prepared using the NE-PER reagent
and analyzed by immunoblotting (Fig. 6). In untreated cells,
MTF-1 was detected in both the nuclear and cytosolic extracts.
In contrast, after zinc treatment, about 3-fold more immuno-
reactive MTF-1 was detected in nuclear extracts, and cytosolic
extracts were devoid of detectable MTF-1. These extraction
buffers contained EDTA, which inactivates the DNA binding
activity of MTF-1. This process is reversible by the readdition
of zinc. The addition of zinc also serves to activate cytosolic
MTF-1 previously not detected by EMSA. MTF-1 DNA binding
was activated by incubating the extracts with 100 M ZnSO
4
for
15 min at 37 C prior to EMSA. Cytosolic extracts from un-
treated cells contained MTF-1 that was activated to bind to
DNA in vitro, whereas cytosolic extracts from zinc-treated cells
were devoid of MTF-1 binding activity. The amount of MTF-1
binding activity in nuclear extracts increased 3.5-fold in cells
treated with zinc compared with untreated cells (Fig. 6B).
Immunocytochemistry was used to localize MTF-1-FLAG,
expressed in transiently transfected dko7 cells. This approach
was taken because the polyclonal antisera against MTF-1 pro-
duced too much background to allow unambiguous detection of
endogenous MTF-1. Therefore, cells were transiently transfected
with a CMV-MTF-1-FLAGvector, treated with 100 M ZnSO
4
for
1 h, and processed for immunocytochemistry with commercially
available FLAG probe antisera. Untreated and zinc-treated,
mock-transfected cells served as a control for specificity of the
antisera (Fig. 7, A and B, respectively). Slight background im-
munostaining was detected in mock-transfected cells. However,
strong cytoplasmic FLAG immunostaining was detected in
510%of the cells after transfection with the CMV-MTF-1-FLAG
vector (Fig. 7C). This percentage of cells approximated the trans-
fection efficiency based on -galactosidase staining of cells in situ
(data not shown). After treatment of transfected cells with zinc,
this immunostaining was localized in nuclei (Fig. 7D). By com-
parison, intense nuclear staining of Sp1 was detected in un-
treated, as well as zinc-treated transfected cells (Fig. 7, E and F,
respectively). These results provide convergent evidence that
MTF-1 translocates to the nucleus in response to zinc.
An Overtly Toxic Concentration of Cadmium Can Induce
Nuclear Translocation and MRE Binding Activity of MTF-1 in
Hepa CellsSeveral studies have reported that cadmium does
not activate the DNA binding activity of MTF-1 either in vivo or
in vitro (8, 17, 18). However, MTF-1 is essential for cadmium
activation of MT gene expression (19). On a molar basis, cad-
mium is at least 10 times more toxic and more potent than zinc
as an inducer of MT-I gene expression (30, 31). To further
investigate the effects of cadmium on MTF-1, Hepa cells were
treated with 10 M CdCl
2
, which results in nearly maximal
induction of MT gene expression, or treated with 20 or 50 M
CdCl
2
. The latter concentration is very toxic to Hepa cells
under these culture conditions and will kill them within 24 h.
Cadmium caused a dose- and time-dependent increase in the
amount of immunoreactive MTF-1 in the nucleus (Fig. 8).
Based on comparisons of relative intensities of the MTF-1
bands, 10 M cadmium caused less than a 2-fold increase in
immunoreactive MTF-1 in the nuclear extract (Fig. 8A). In-
creased nuclear MTF-1 was apparent within 15 min of the
FIG. 7. Immunocytochemical local-
ization of MTF-1-FLAGin transfected
dko7 cells before and after zinc treat-
ment. dko7 cells were transfected with a
CMV-MTF-1-FLAG expression vector.
Cells were then treated with 100 M
ZnSO
4
for 1 h and analyzed by immuno-
cytochemistry using FLAG-probe (AD)
or Sp1 (EF) antiserum. A, mock-trans-
fected cells; B, mock-transfected cells
treated with zinc; C and E, CMV-MTF-1-
FLAG vector-transfected cells; D and F,
CMV-MTF-1-FLAG vector-transfected
cells treated with zinc. The arrows point
to immunostaining of MTF-1-FLAG in
transfected cells.
MTF-1 Translocates to the Nucleus 9382

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addition of the cadmium (Fig. 8B). We previously reported that
treatment of cells with 10 M cadmium only modestly activates
MTF-1 DNA binding activity (2-fold) (14, 18). In contrast, 50
M cadmium caused a 7.5-fold increase in immunoreactive
nuclear MTF-1 within 1 h of treatment, and increased MTF-1
was evident by 15 min (Fig. 8B). The DNA binding activity of
MTF-1 was examined using the nuclear extracts from cells
treated with 50 M cadmium for 15 min and 1 h (Fig. 9). Under
these conditions, 50 M cadmium caused about a 5-fold induc-
tion of DNA binding. Sp1 DNA binding activity was not altered
by cadmium treatment (Fig. 9, lower panel).
DISCUSSION
Previous studies have documented that MTF-1 is essential
for metal ion regulation of MT gene expression (19) and that
this metalloregulatory protein is activated by zinc to bind to
MREs in the MT promoter (3). Treatment of cells with zinc
results in a rapid increase in the amount of DNA binding
activity of MTF-1 detected in nuclear extracts. However, it has
been shown that zinc-induced MT gene expression is not de-
pendent on de novo protein synthesis (32), that MTF-1 mRNA
is not induced by zinc (10, 19, 33), and that whole cell extracts
from untreated cells contain latent MTF-1 that can be acti-
vated to bind to DNA by exogenous zinc (14). Taken together,
these observations demonstrate that MTF-1 is a preexisting
cellular protein that is activated to bind to DNA by metal ions.
The data reported herein reveal that a significant portion of
MTF-1 protein is present in the cytoplasmof unstressed cells and
that exposure of the cells to metal ions results in the rapid
translocation of MTF-1 protein to the nucleus and the activation
of its DNA binding activity. These results are consistent with the
concept that MTF-1 functions, in part, as a zinc sensor.
Cadmium is a potent inducer of MT gene expression, and
MTF-1 is also an essential component of that signaling mech-
anism. However, MTF-1 is activated to bind to DNA by revers-
ible interactions of zinc with specific zinc fingers in the DNA-
binding domain and not by interactions with cadmium or other
transition metals (810, 14, 1619, 34). We previously reported
that 6 M CdCl
2
has little effect on the amount of MTF-1 DNA
binding activity in the nuclei of cultured cells (18). Cadmium
(515 M) rapidly induces MT-I gene expression in these cells
2
and in other cell types (33). Herein, it was further shown that
cadmium (10 M) exerts only a small effect (15% increase) on
the amount of MTF-1 protein in the nucleus. However, higher
concentrations of cadmium caused the complete activation of
DNA binding and translocation of MTF-1 to the nucleus. This
suggests that cadmium may cause the redistribution of zinc in
the culture, which, in turn, may activate MTF-1 to bind to DNA
and move to the nucleus. Other transition metals have been
suggested to act in this manner (33). However, this mechanism
2
I. V. Smirnova, D. C. Bittel, R. Ravindra, H. Jiang, and G. K.
Andrews, unpublished data.
FIG. 8. Immunoblot detection of MTF-1 in nuclear and cytoso-
lic extracts from Hepa cells exposed to cadmium. A, Hepa cells
were treated with 10, 20, or 50 M CdCl
2
or with 100 M ZnSO
4
for 1 h.
NEs and CEs were prepared from treated and untreated cells and
analyzed by immunoblotting using antisera against mouse MTF-1. B,
Hepa cells were treated with 10 or 50 M CdCl
2
for 15 min or 1 h. NEs
and CEs were prepared from treated and untreated cells and analyzed
by immunoblotting as in A. The arrow shows the position of immuno-
reactive MTF-1.
FIG. 9. EMSA detection of MTF-1 DNA binding activity in nu-
clear extracts from Hepa cells treated with cadmium. Hepa cells
were treated with 50 M CdCl
2
for 15 min or 1 h or with 100 M ZnSO
4
for 1 h. Upper panel, nuclear extracts were prepared from treated and
untreated cells and analyzed by EMSA using a labeled MRE-s oligonu-
cleotide. Lower panel, the same extracts were assayed using a labeled
Sp1-specific oligonucleotide. The arrows point to the specific MTF-1 and
Sp1 complexes with their respective oligonucleotides. Lane 1, untreated
cells; lane 2, cells treated with 100 M ZnSO
4
for 1 h; lane 3, cells treated
with 50 MCdCl
2
for 15 min; lane 4, cells treated with 50 MCdCl
2
for 1 h.
MTF-1 Translocates to the Nucleus 9383

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may only account in part for the effects of cadmium on activa-
tion of the MT promoter by MTF-1.
The disassociation between the concentration of cadmium
required for activation of MT-I gene expression and that re-
quired to cause most MTF-1 to be activated to bind to DNA and
translocated to the nucleus suggests several possibilities in-
cluding the following: 1) only small changes in MTF-1 levels in
the nucleus are sufficient for maximal activation of gene ex-
pression; 2) cadmium causes a modification of MTF-1; 3) cad-
mium and zinc cause the formation of distinct MTF-1 promoter
complexes; or 4) cadmium-responsive transcription factors
other than MTF-1 may also activate MREs. The concentration-
response curves for zinc activation of MT gene expression and
increased MTF-1 DNA binding activity do not support the first
possibility. However, the possibility of effects of cadmium on
the transactivation capacity of MTF-1 cannot be excluded. The
transactivation domains present in the carboxyl-terminal half
of the protein are also important for transduction of the metal
signal. Thus, the full biological functions of MTF-1 are depend-
ent on a complex interplay of different functional domains (13).
Little is known about those interactions or the interactions of
MTF-1 with other proteins; thus, the second and third possibili-
ties remain to be addressed. With regard to the fourth possibility,
we recently found that MRE activity can be increased in the
absence of detectable MTF-1 in IMR cells (35). Cadmium-respon-
sive factor(s) that can interact with MREs in vitro have been
reported previously (8, 3638), but the functional significance, if
any, of those factors has not been determined. Therefore, other
MRE-binding proteins may play a role in regulating MT gene
expression in response to cadmium, at least in certain cell types.
The nuclear localization of many transcription factors is a
key controlling point in regulating gene expression and accom-
panies differentiation or changes in the metabolic state of eu-
karyotic cells (39). Although the majority of transcription fac-
tors are localized to the nucleus (40, 41), others predominantly
reside in the cytoplasm and are translocated to the nucleus in
response to stimulus (4244). One mechanism associated with
transcription factor transport to the nucleus is the nuclear
localization signal (NLS) (45). MTF-1 has a putative NLS
(KRKEVVKR) that immediately precedes the zinc finger do-
main (10). For a large protein to translocate to the nucleus, the
NLS has to be exposed on the protein surface (45). Interactions
between zinc and MTF-1 probably cause conformational
changes leading to uncovering of the NLS. Another mechanism
that exposes the NLS is phosphorylation of adjacent sites (39).
MTF-1 has several potential phosphorylation sites in the vicin-
ity of the NLS such as Thr
131
for protein kinase C, Tyr
139
for
tyrosine kinase, and Thr
142
for casein kinase. Protein kinase C
has been suggested to play a role in metal induction of MT gene
expression (46). Finally, it is possible that the zinc fingers
themselves are involved in metal-induced nuclear transloca-
tion of MTF-1. Several mutations of zinc fingers in MTF-1 were
found to cause the cytoplasmic localization of MTF-1 expressed
in transiently transfected cells (13). Three Cys
2
His
2
-type zinc
fingers within the DNA-binding domain of nerve growth factor-
induced transcription factor 1-A (also known as Erg1 or
Krox24) are necessary for nuclear localization (47), and the
vitamin D receptors NLS signal is located between the two zinc
fingers (41). The entire DNA-binding domain of the glucocorti-
coid receptor, as a functional unit, may be required for nuclear
transfer and optimal retention in the nucleus (48).
In conclusion, the present study indicates that the majority of
MTF-1 protein is located in the cytoplasm in cells cultured in
medium replete with zinc. However, increases in zinc in the
culture mediumpromote the rapid transport into the nucleus and
the activation of DNA binding activity of MTF-1. These results
are consistent with the concept that MTF-1 serves as a sensor of
cytoplasmic metal ions and suggest that zinc and cadmium may
utilize MTF-1 differently in the activation of gene expression.
AcknowledgmentsWe are indebted to Jim Geiser and Steve Eklund
for excellent technical support. We thank Dr. Walter Schaffner (Uni-
versity of Zurich, Zurich, Switzerland) for a generous gift of the dko7
cell line.
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