64, 67–76 (2001)
Copyright © 2001 by the Society of Toxicology
TOXICOLOGICAL SCIENCES
Zinc-Metallothionein Levels Are Correlated with Enhanced
Glucocorticoid Responsiveness in Mouse Cells Exposed
to ZnCl 2, HgCl 2, and Heat Shock
Janice M. DeMoor,* Wendy A. Kennette,† Olga M. Collins,* and James Koropatnick* ,† ,‡ ,§ ,¶ ,1
*London Regional Cancer Centre, 790 Commissioners Road East, London, Ontario, Canada N6A 4L6; Departments of †Microbiology and Immunology,
‡Oncology, §Pathology, and ¶Pharmacology and Toxicology, University of Western Ontario, London, Ontario, Canada
Received April 25, 2001; accepted August 16, 2001
Koropatnick and Leibbrandt, 1995; O’Halloran, 1993). Intracellular free zinc concentrations are extremely low (Jiang et al.,
1998; Simons, 1991). Rather, zinc is bound to a wide variety of
molecules, raising the possibility that regulation of zinc availability may modulate the activity of zinc-requiring proteins.
Multiple membrane-associated zinc transporters have been
identified (reviewed in McMahon and Cousins, 1998) and may
play a role in regulating zinc. In addition, metallothioneins
have been identified as major intracellular zinc-binding proteins that can act as a source of zinc for apoproteins (Vallee,
1995; Zeng and Kägi, 1995).
Metallothioneins (MTs) constitute a family of proteins that
bind both essential and toxic metals. Due to their binding
capacity (i.e., up to 7 zinc or 12 copper ions per molecule),
ubiquitous nature, and the capacity of some isoforms (MT-I,
MT-II) to be induced by metals, it has been suggested that MTs
may participate in essential metal trafficking to and from many
metal-dependent proteins (Koropatnick and Leibbrandt, 1995;
Vallee, 1995; Zeng and Kägi, 1995). Zinc-bound metallothionein has been shown to reactivate various zinc-requiring
apoenzymes in vitro (Jiang et al., 1998; Udom and Brady,
1980). Zinc transfer has been demonstrated from zinc-finger
transcription factors to apothionein (Sp1 [Zeng et al., 1991a];
TFIIIA [Zeng et al., 1991b]). More recently, reversible zinc
exchange between MT and the estrogen receptor (Cano-Gauci
and Sarkar, 1996), MT and the yeast transcription factor GAL4
(Maret et al., 1997), and MT and a 2-zinc-finger peptide of the
transcription factor Tramtrack (Roesijadi et al., 1998) have
been reported. The potential ability of MT to interact with
zinc-requiring proteins in vivo could lead to MT-based modulation of gene expression and signal transduction pathways. In
support of this, we have previously shown that enhanced MT
expression accompanies lipopolysaccharide (LPS)-induced activation of human monocytes/macrophages (Leibbrandt and
Koropatnick, 1994). Furthermore, transient antisense downregulation of metallothionein expression abolishes the capacity
of LPS to induce a respiratory burst in these cells (Leibbrandt
et al., 1994). Also, treatment of monocytes with low, nontoxic
levels of mercury, cadmium, or zinc to induce MT expression
Metallothioneins (MTs) are the major low molecular weight,
zinc-binding proteins in mammalian cells. It has been hypothesized that they play a role in the function of zinc-dependent signal
transduction proteins and transcription factors. We investigated
the capacity of zinc and other metal ions and conditions to increase both Zn-associated MT levels and the receptiveness of cells
to transcriptional activation mediated by the zinc-dependent glucocorticoid receptor (GR). We studied, in a GR-responsive mouse
mammary-tumor cell line, the ability of dexamethasone (DEX) to
stimulate transcription of a chloramphenicol acetyltransferase
(CAT) gene controlled by a mouse mammary-tumor virus promoter. In cells pretreated with 20 to 100 ␮M ZnCl 2, DEX-induced
CAT activity correlated with zinc-induced MT levels. However,
0.05 to 0.5 ␮M CdCl 2 had no effect on CAT activity, despite an
increase in Cd-associated MT. Copper-associated MT was detected in cells treated with 20 ␮M CuCl 2, but there was no change
in the level of Zn-MT, nor was CAT activity altered in cells
exposed to 5 to 20 ␮M CuCl 2. These results may reflect a functional difference between zinc-associated MT, and MT associated
with other metals. Significantly more CAT activity was observed
in both heat-shocked cells and in cells exposed to 40 or 50 nM
HgCl 2. Although absolute amounts of MT were unchanged by
these two treatments, a higher percentage of total cellular zinc was
associated with the MT protein fractions after treatment. Changes
in GR levels could not account for variations in CAT activity.
These data indicate that hormonal signalling can be altered by
exposure to metal salts and heat shock, and the effect is correlated
with the level of Zn-MT.
Key Words: metallothionein; metals; zinc; glucocorticoid receptor; heat shock.
Various metal ions (e.g., zinc, copper, iron) are essential for
both prokaryotic and eukaryotic cell function. More than 300
proteins, including signal transduction molecules, transcription
factors, and metalloenzymes, require zinc for their activity
and/or their structure (reviewed in Berg and Shi, 1996;
1
To whom correspondence should be addressed at London Regional Cancer
Centre, 790 Commissioners Road East, London, Ontario, Canada N6A 4L6.
Fax: (519) 685-8646. E-mail: [email protected].
67
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DEMOOR ET AL.
alters the ability of these cells to be activated by bacterial LPS
(Koropatnick and Zalups, 1997; Leibbrandt and Koropatnick,
1994) or phorbol myristate acetate (PMA; Koropatnick, 1999).
Although treatment with certain metals can impair glucocorticoid receptor-mediated function (e.g., Cd repression of GR
activity in rats [Dundjerski et al., 1996] and Pb repression of
GR function in hepatoma cells [Heiman and Tonner, 1995]),
metal-induced enhancement of GR function has not been reported. Because of the requirement of GR for zinc, and the
proposed role for MT in mediating zinc availability, we investigated the capacity of zinc and other metal ions and conditions
to increase both Zn-associated MT levels and the receptiveness
of cells to dexamethasone-induced GR signals.
To investigate the roles of MT and metals in mediating the
activity of a well-characterized zinc-requiring transcription
factor in vivo, we used a derivative of the C127 mouse mammary tumor cell line (denoted 2305) that harbors a stable
episomal bovine papilloma virus (BPV)-based vector containing a chloramphenicol acetyltransferase (CAT) reporter gene
under the control of the mouse mammary tumor virus (MMTV)
promoter (Mymryk et al., 1995). This promoter responds to
dexamethasone (DEX) signals transduced to the nucleus by the
glucocorticoid receptor (GR), which is a zinc-finger, DNAbinding protein. Nuclear GR interacts with hormone response
elements in the MMTV promoter to induce CAT mRNA,
protein and enzyme activity. The addition of zinc to 2305 cells
at concentrations that induced MT expression significantly
increased DEX-induced CAT activity. Following heat shock or
HgCl 2 treatment, a higher proportion of total cellular zinc was
associated with the MT protein fractions and correlated with
enhanced CAT activity. CAT activity was not increased following CdCl 2 or CuCl 2 exposure nor was an increase in Zn-MT
detected. These data indicate that, in addition to suppressing
response to glucocorticoids, certain metals (Zn and Hg) can
enhance glucocorticoid-induced gene expression, possibly perhaps through a Zn-MT-mediated pathway.
MATERIALS AND METHODS
Cell Culture
Mouse 2305 cells (kindly supplied by Dr. Trevor Archer, Laboratory of
Reproductive and Developmental Toxicology, NIEHS) were produced by
bovine papilloma virus (BPV) transformation of C127 mouse mammary tumor
cells (Mymryk et al., 1995). The 2305 cells contain stably incorporated
BPV-based episomes, carrying a glucocorticoid-inducible mouse mammary
tumor-virus (MMTV) promoter regulating the expression of the chloramphenicol-acetyltransferase (CAT) reporter gene. Cells were maintained in DMEM
supplemented with 10% fetal bovine serum, 2-mM glutamine, and 5-mM
HEPES.
Dexamethasone (DEX; Sigma-Aldrich Canada, Ltd., Oakville, ON, Canada),
or the equivalent volume of ethanol, was added 24 h later to a final concentration of 10 –7 M. Samples were harvested for CAT assay following an
additional 24 h. The cells were washed 3⫻ in PBS, scraped off the plate with
a rubber policeman, washed again in PBS, and then frozen at – 80°C until use.
Cells collected for MT, Western blot, or atomic absorption spectrophotometry
analyses were seeded at 1 ⫻ 10 6 per plate (48 h time point) or 2 ⫻ 10 6 per plate
(6 or 24 h time points) in 100-mm plates in a final volume of 15 ml, and treated
and harvested as above. No DEX was added to 6- or 24-h samples. For GR
localization studies, cells were harvested at 26 h (2-h DEX treatment).
Cat Reporter Gene Transcription Measurement
Run-on transcription in isolated nuclei. Approximately 1 ⫻ 10 7 mouse
2305 cells, treated with and without metal salts or heat shock, plus or minus
DEX induction, were prepared, pelleted, and frozen at – 80°C at the same time
cells were prepared for MT and Western blot analysis. Nuclei were isolated
from the pelleted cells and assessed for relative transcription of transfected
CAT reporter genes as described in detail previously (Zalups and Koropatnick,
2000). Briefly, frozen cell pellets were homogenized in 10 volumes of buffer
1 [0.32 M sucrose, 2 mM CaCl 2 2 mM Mg(OAc) 2, 0.1 mM EDTA, 0.1%
Triton X-100, 1 mM dithiothreitol (DTT), 10 mM Tris-HCl, pH 8.0], combined
with 2 volumes of buffer II [2 M sucrose, 5 mM Mg(OAc) 2, 0.1 mM EDTA,
10 mM Tris-HCl, pH 8.0], layered over a 10-ml cushion (2 M sucrose, 5 mM
magnesium acetate, 0.1 mM EDTA, 10 mM Tris-HCl, pH 8.0) and pelleted by
ultracentrifugation at 24,000 rpm for 60 min in an SW-28 rotor at 4°C. The
pelleted nuclei were suspended in nuclear buffer (40% glycerol, 5 mM MgCl 2,
50 mM Tris-HCl, pH 8.0, 0.1 mM EDTA), counted by hemacytometer, and
adjusted to 5 ⫻ 10 4 nuclei/ml. RNA elongation reactions were performed using
20 ␮l of nuclei in nuclear buffer plus 200 ␮l of sterile 2X reaction buffer (10
mM ATP, 1 mM CTP, 1 mM GTP, 5 mM DTT, 2 ␮l [␣- 32P]CTP (⬃3000
Ci/mmol, 10 Ci/ml; Amersham Pharmacia Biotech, Baie d’Urfé, Québèc).
Nascent RNA transcripts were allowed to elongate for 30 min at 30°C on a
shaking platform, and this was followed by addition of 60 ␮l of RNase-free
DNase 1 [(0.04 U of RQ1 DNase 1, RNase-free; Promega, Madison WI), 0.5
M NaCl, 50 mM MgCl 2, 2 mM CaCl 2, 10 mM Tris-HCl, pH 7.4]. The
32
P-labeled RNA was isolated using Trizol (Life Technologies, Inc., Grand
Island, NY), and the final precipitated RNA was dissolved in Church hybridization buffer (1 mM EDTA, 0.5 M NaHPO 4, pH 7.2, 7% sodium lauryl
sulfate), to a final concentration of 4 ⫻ 10 6 cpm/ml.
Hybridization of radiolabeled RNA to immobilized, unlabeled probes.
Target DNA (immobilized on nylon filters in triplicate dots, 2 ␮g per dot)
consisted of unlabeled cDNA of 2 separate types: (1) a 1.68 kb Xba1/HindIII
cDNA full-length CAT cDNA excised from a pCAT-3 Basic Vector (Promega,
Madison, WI), and (2) a full-length glyceraldehyde phosphate dehydrogenase
(GAPDH) cDNA (Denhardt et al., 1988). Both cDNAs were denatured and
immobilized on the same nylon filters. Hybridization of radiolabeled RNA to
these dots assessed transcription of GAPDH genes as internal standards against
which to measure change in CAT gene transcription. Filters were prehybridized and hybridized to 2 ml of radiolabeled RNA resulting from 30 min of
run-on transcription for 48 h at 65°C, as described previously (Zalups and
Koropatnick, 2000), and hybridization visualized by phosphorimage analysis
and the ImageQuant data reduction program (Molecular Dynamics, Inc.,
Sunnyvale, CA). The ratio of CAT gene transcription to the transcription of the
housekeeping GAPDH gene was used as a measure of the relative rate of CAT
gene transcription.
CAT Activity Measurement
Metal and Heat Shock Treatments
For CAT assay samples, 60-mm tissue culture plates were seeded with
250,000 cells that were allowed to adhere for 1 h prior to treatment. Metals
were then added to the desired concentration in a final volume of 5 ml. Heat
shock consisted of a 1-h incubation at 42°C followed by a return to 37°C.
Pelleted cells were lysed in 100 ␮l 0.25 M Tris-HCl (pH 7.8) by 3 rounds
of freezing and thawing. CAT enzyme activity was assessed by a 2-phase fluor
diffusion assay (Neumann et al., 1987) and values were normalized to total
protein content (measured by the Bio-Rad Protein Assay, Bio-Rad Laboratories, Hercules, CA).
METALLOTHIONEIN AND GLUCOCORTICOID RESPONSIVENESS
MT Protein Measurement
Pelleted cells were lysed in 200 ␮l 1% Tween 20 in PBS by 3 cycles of
freezing and thawing, then centrifuged at 12,000 ⫻ g at 4°C for 5 min. MT
protein concentrations in supernatants were determined by an ELISA method
based on Chan et al. (1992) and Leibbrandt et al. (1991). Cell supernatants, or
known concentrations (0 to 200 ␮g/ml) of rabbit MT-I (Sigma-Aldrich Canada) diluted in 1% Tween 20 in PBS were each combined with an equal
volume (160 ␮l) of diluted (1:1000 in 1% Tween 20 in PBS) mouse monoclonal anti-MT antibody (Dako Corp., Carpinteria, CA) in polypropylene
microfuge tubes and incubated overnight at 4°C. A Nunc-ImmunoPlate (Polysorp Surface) was coated (100 ␮l/well) with 2 ␮g/ml rabbit MT-I in 0.1 M
NaHCO 3 (pH 9.6) overnight at 4°C in a humidified chamber. All subsequent
steps were carried out at room temperature. The MT-I-coated plate was washed
3⫻ with 0.05% Tween 20 in PBS (buffer was left in the wells for 5 min
between washes), then blocked for 30 min with 150 ␮l per well 0.3% gelatin,
0.05% Tween 20 in PBS. The plate was washed as before, then 100 ␮l from
each antibody/sample or antibody/MT-I standard mixture were transferred to
each of triplicate wells on the blocked plate and incubated for 1 h. The plate
was washed and 100 ␮l of biotinylated goat antimouse IgG (Jackson
ImmunoResearch Laboratories, Inc., West Grove, PA) were added per well
and allowed to incubate for 1 h. Subsequently, the plate was again washed,
then incubated with 100 ␮l per well of alkaline phosphatase-conjugated
streptavidin (Jackson ImmunoResearch Laboratories, Inc.) for 1 h. Following
a final wash, 100 ␮l of 1 mg/ml p-nitrophenyl phosphate in 100 mM Tris-HCl
(pH 9.5), 100 mM NaCl, and 5 mM MgCl 2 were added per well. Color
development was stopped when desired by the addition of 50 ␮l of 0.2 M
EDTA disodium salt. The color change was quantitated using a Bio-Rad
microplate reader at 405 nm. This method is capable of detecting both MT-I
and MT-II isoforms. Results were expressed as ng MT per ␮g total. In some
cases, a fluorescence-based modification of this assay was used (manuscript in
preparation). In the DELFIA (dissociation-enhanced lanthanide fluoroimmunoassay) method, the addition of biotinylated goat antimouse IgG, and all
subsequent ELISA steps, were omitted. Europium-labeled antimouse IgG
(1:200 in DELFIA assay buffer [Wallac Oy, Turku, Finland]) was added at 100
␮l per well, and the plate was incubated for 1 h on an orbital shaker. After
washing, 100 ␮l of DELFIA enhancement solution were added per well and
the plate was incubated for 5 min on an orbital shaker. Time-resolved fluorescence was measured using a Wallac 1420 Victor 2 multilabel counter
(PerkinElmer Life Sciences, Boston, MA).
Western Blot Protocol
Cell pellets were boiled 5 min in 500 ␮l 2% sodium dodecyl sulfate (SDS),
100 mM dithiothreitol (DTT), and 60 mM Tris-HCl (pH 6.8), then sonicated
for 30 s (50% pulse, output 3) using a VibraCell sonicator (Sonics and
Materials Inc., Danbury, CT). Debris was removed by centrifugation at
10,000 ⫻ g for 10 min at 4°C. Nuclear and cytoplasmic extracts were prepared
according to the method of Scheinman et al. (1993). Protein concentrations
were determined using the Bio-Rad Protein Assay. Glycerol (to 10%) and
bromophenol blue (to 0.01%) were added prior to loading. Proteins were
separated on a 7.5% polyacrylamide gel using a Mini-Protean II Dual Slab Cell
(Bio-Rad Laboratories) according to manufacturer’s instructions. Each gel was
loaded with equivalent amounts (50 to 100 ␮g) of protein per lane. Proteins
were electroblotted at 25 V overnight onto a Hybond ECL nitrocellulose
membrane (Amersham Canada, Ltd.) in transfer buffer (173 mM glycine, 22.5
mM Tris base, 1.15 mM SDS, 12.5% methanol [Towbin et al., 1979]). Proteins
were detected using enhanced chemiluminescence (ECL, Amersham Canada,
Ltd.) according to manufacturer’s instructions. All antibodies were diluted in
1% skim milk powder (Carnation, Inc., Toronto, ON, Canada) in TBS-T (20
mM Tris-HCl, pH 7.6, 137 mM NaCl, 0.05% Tween-20). Five percent skim
milk powder in TBS-T was used as a blocking agent. A mouse monoclonal
anti-GR antibody (BuGR) was a generous gift from Dr. B. Gametchu, University of Texas Medical Branch, Galveston, TX (Gametchu and Harrison,
1984). ␤-Tubulin was detected by a mouse monoclonal antibody (Chemicon
69
International, Inc., Temecula, CA). Horseradish peroxidase-linked sheep antimouse IgG (Amersham Canada) was used as the secondary antibody. Chemiluminescence autoradiographs were densitometry-scanned and -quantitated
using the ImageQuant program (Molecular Dynamics, Sunnyvale, CA).
MT Metal Analysis
Cells were homogenized in 0.5 M glycine (pH 8.5), sonicated as for Western
blots, and centrifuged at 10,000 ⫻ g for 20 min. The resulting supernatants
were fractionated on Sephadex G-75 columns. One-ml fractions were digested
in an equal volume of concentrated nitric acid and analyzed for zinc, cadmium,
or copper using a Varian Spectra-30 (Varian Canada, Georgetown, ON, Canada) atomic absorption spectrophotometer with an air-acetylene flame. Mercury is not quantifiable by this method.
RESULTS
The glucocorticoid response is enhanced by zinc at concentrations that induce MT. The 2305 cells were treated with 20,
40, 80, or 100 ␮M ZnCl 2 for 48 h. DEX-induced CAT activity
(Fig. 1A) was significantly enhanced in those cells that also
responded to zinc treatment (80 or 100 ␮M) by MT induction
(Fig. 1B, 3A). No enhancement of CAT activity was seen in
cells exposed to 40 ␮M ZnCl 2, a zinc concentration that is
substantially higher than the 4 ␮M zinc found in normal 2305
culture medium (measured by atomic absorption spectrophotometry, results not shown; Palmiter, 1995) but too low to
induce MT expression in these cells (Fig. 1B). The correlation
(r 2 ⫽ 0.94) between CAT activity and MT protein levels is
shown in Fig. 1C. Logarithmic increases in MT level are
reflected by linear increases in CAT activity. Zinc treatment
(100 ␮M) in the absence of DEX did not induce CAT activity
(Fig. 1A).
Enhanced DEX-induced CAT activity is observed in cells
that respond to metal or heat-shock treatment by an increase in
zinc-associated MT. The concentrations of each metal used to
treat 2305 cells did not reach cytotoxic levels, as assessed by a
cell-growth assay, in comparison with control cells at time of
harvest (48 h). Viability was also unaffected by heat shock
(results not shown).
Pretreatment with CdCl 2 did not result in enhancement of
CAT activity in 2305 cells, despite an increase in MT protein
at 0.5 ␮M CdCl 2 (Fig. 2). However, cadmium, rather than zinc,
was the predominant metal ion associated with MT in CdCl 2treated cells (Fig. 3A, Table 1). Exposure to 40 or 50 nM
HgCl 2 resulted in a significant increase in CAT activity (Fig.
2B), but unlike ZnCl 2 treatment, did not induce MT protein.
However, a higher percentage of total cellular zinc was detected in the MT-containing protein fractions, when compared
to control cells (Fig. 3A, Table 1). In CuCl 2-treated cells, CAT
activity was not altered (Fig. 2A), nor was MT protein induced
(Fig. 2B). Despite detection of Cu-associated MT, the level of
Zn-MT was unchanged in 20-␮M CuCl 2-treated cells, compared to untreated control cells (Fig 3A, Table 1).
A significantly higher level of DEX-induced CAT enzyme
activity was also detected in cells exposed to 42°C for 1 h as
70
DEMOOR ET AL.
increase in the proportion of total zinc associated with MT was
observed in heat-treated 2305 cells over control cells (Fig. 3A,
Table 1).
Enhanced CAT activity was observed in cells that responded
to treatment by an increase in Zn-associated MT, but not
necessarily by an increase in absolute levels of MT. The
correlation between the percentage of enhancement of CAT
activity and the percentage of total cellular zinc associated with
MT in shown in Fig. 3B (r 2 ⫽ 0.65).
It was formally possible that variation in CAT mRNA or
FIG. 1. ZnCl 2 treatment enhances glucocorticoid-induced gene expression
in 2305 cells. (A) Cells were incubated with increasing concentrations of
ZnCl 2 for 48 h. Ten –7 M dexamethasone (hatched bars) or ethanol vehicle
(solid bars) was added to the cultures for the last 24 h. Bars represent CAT
activity (4-h time point) of triplicate samples ⫾ SE. Asterisks indicate values
significantly different from controls (Student’s t-test, p ⬍ 0.05, n ⫽ 3). (B)
Metallothionein induction after 48-h ZnCl 2 treatment, as measured by the
ELISA assay. Bars represent a single measurement, consistent with results of
at least 2 independent repeat experiments (results not shown). (C) Correlation
of CAT activity with MT levels in ZnCl 2-treated cells.
compared to control (Fig. 2A). Heat shock in the absence of
DEX did not induce CAT activity (Fig. 2A). Like HgCl 2, heat
shock did not induce MT protein (Fig. 2B), but a substantial
FIG. 2. Effect of metal or heat-shock pretreatment on CAT activity and
MT levels in 2305 cells. Cells were incubated at 37°C with the indicated metal
concentrations for 48 h. Alternatively, cells were incubated at 42°C for 1 h,
then returned to 37°C for the remaining 47 h (heat shock; HS), or maintained
at 37°C for the entire experiment (control) (cont). (A) Bars represent CAT
activity (4-h time point, or 3-h time point for HS samples) of triplicate
samples ⫾ SE. Ten –7 M dexamethasone (hatched bars) or ethanol vehicle
(solid bars) was added to the cultures for the last 24 h. Asterisks indicate values
significantly different from controls (Student’s t-test, p ⬍ 0.05, n ⫽ 3). (B)
Metallothionein induction 24 h after metal or HS treatment (no dexamethasone), as measured by the DELFIA assay. Bars represent a single measurement, consistent with results of at least 2 independent repeat experiments
(results not shown).
METALLOTHIONEIN AND GLUCOCORTICOID RESPONSIVENESS
71
porter genes in cells untreated with metal salts or heat shock, or
treated with zinc chloride, cadmium chloride, or mercuric
chloride, followed with and without induction by DEX. The
results were enhancement of DEX-induced CAT gene transcription after treatment with zinc and mercury salts and heat
shock, but not with cadmium or copper salts (Table 2).
Metal-induced enhancement of GR activity is not mediated
by increased overall or nuclear GR levels. The effect of
metal or heat treatment on GR levels is shown in Figure 4A.
␤-tubulin content was used as a loading control. There was an
increase (ranging from 34% to 79%) in the GR:tubulin ratio
seen in all treated groups at 6 hr and 24 hr as compared to
controls. Although CdCl 2 treatment did not increase CAT
activity, the enhancement of GR level observed was similar to
that seen after treatments that did enhance CAT activity
(ZnCl 2, heat shock). The increase in GR levels, under conditions where no increase in GR-dependent CAT activity was
evident (i.e., CdCl 2 induction), suggested that the effects of
metal or heat treatment on CAT activity in 2305 cells could not
be accounted for by a corresponding change in GR levels at 6
or 24 h (time of DEX addition). The addition of DEX resulted
in substantially less GR in all samples harvested at 48 h (time
of CAT measurement) in accordance with the glucocorticoidmediated downregulation of GR gene expression (DuBois et
al., 1995; Silva et al., 1994).
Upon DEX administration, GR shifted from a predominantly
cytoplasmic to a predominantly nuclear location in the cell.
This shift was observed in all groups regardless of treatment
(Fig. 4B). There was no consistent observable difference in a
total of 3 experiments (including the one shown in Fig. 4B) in
the degree of GR translocation into the nucleus under conditions where GR activity was enhanced (ZnCl 2, heat).
DISCUSSION
FIG. 3. Metal content of MT from treated 2305 cells. Cells were grown in
the absence (Control), or presence of the indicated metal for 24 h, or incubated
for 1 h at 42°C, followed by 23 h at 37°C (heat shock). (A) Cell extracts were
separated on a Sephadex G-75 column, then the zinc (Zn), and cadmium (Cd)
or copper (Cu) contents of 1-ml fractions were determined by air-acetylene
flame atomic absorption spectrophotometry. (B) The correlation of MT-associated zinc and enhancement of CAT activity following metal or heat treatment.
protein stability could be altered by metal salts or heat. To
avoid this potential problem, CAT gene transcription was
assessed directly in run-on transcription assays of CAT re-
The multiple circumstances under which MTs are expressed
(Andrews, 1990; Cherian and Chan, 1993), and the ability of
MTs to bind metals, has led to the suggestion that they may
participate in essential metal trafficking to and from many
metal-dependent proteins. To investigate the role of MT in
mediating the activity of a zinc-requiring transcription factor in
vivo, we monitored CAT activity after metal or heat treatment
in a mouse mammary tumor cell line (2305) harboring a
DEX-inducible, GR-responsive MMTV-CAT gene construct.
GR-dependent, DEX-induced CAT activity was significantly
enhanced by the addition of exogenous ZnCl 2, but only at those
concentrations that induced MT expression (80, 100 ␮M
ZnCl 2; Fig. 1). Increasing zinc concentrations 5- to 10-fold (20
and 40 ␮M, respectively) without increasing MT protein levels
was not sufficient to enhance GR-mediated transcription, suggesting that MT (and not zinc unassociated with MT), modulated DEX-inducible gene expression. Zinc transfer in vitro
between MT and metalloenzymes (Jiang et al., 1998; Udom
and Brady, 1980; Suzuki and Kuroda, 1995) or MT and zinc-
72
DEMOOR ET AL.
TABLE 1
Changes in Zn-Associated MT Levels in Mouse 2305 Cells Induced by Treatment with Metal Salts or Heat Shock
% of control
Sample
(ZnMT)/(Total Zn) a
Trial 1
Trial 2
Trial 3
Mean ⫾ SE
Significance
Control
ZnCl 2 (100 ␮M)
HgCl 2 (40 nM)
Heat shock
CdCl 2 (0.25 ␮M)
CuCl 2 (20 ␮M)
.273
.520
.345
.550
.163
.265
100
191
126
202
60
97
100
165
122
155
81
100
100
214
141
183
88
91
NA
190 ⫾ 14
130 ⫾ 6
180 ⫾ 14
76 ⫾ 9
96 ⫾ 3
NA
Yes (p ⫽ 0.003)
Yes (p ⫽ 0.007)
Yes (p ⫽ 0.004)
Yes (p ⫽ 0.048)
No (p ⫽ 0.288)
Note. Data from 3 independent experiments is shown. Cells were separately treated with Zn, Hg, Cd, Cu, and heat shock, and independently and separately
analyzed for total zinc and zinc associated with MT. Values for Trial 1 are derived from the data shown in Figure 3A. The percentage of total cellular zinc
associated with MT in treated cells was determined by comparing the area under the curve in the MT peak (approximately fractions 20 to 30, confirmed by the
DELFIA assay and SDS-polyacrylamide gel electrophoresis [results not shown]) to the total area under the curve for zinc). NA, not applicable. Values are given
as mean ⫾ SE.
a
Only [ZnMT:Total Zn] ratios for Trial 1 are shown.
b
Significantly different from control cells (p ⱕ 0.05).
requiring transcription factors (Zeng et al., 1991a,b; CanoGauci and Sarkar, 1996; Maret et al., 1997; Roesijadi et al.,
1998) has been reported. In vivo, constitutive expression of
MT-I in stably transfected BHK cells increased the expression
TABLE 2
The Influence of Metal Salt or Heat Shock Pretreatment on
DEX-Induced Transcription of CAT Genes in 2305 Cells a
Relative CAT gene
transcription a
Treatment
– DEX
⫹ DEX
Fold
increase b
Control
ZnCl 2 (100 ␮M)
Control
HgCl 2 (40 nM)
Control
Heat shock
Control
CdCl 2 (0.25 ␮M)
Control
CuCl 2 (20 ␮M)
1.2 ⫾ 0.4
1.3 ⫾ 0.3
3.6 ⫾ 1.3
5.0 ⫾ 2.1
2.6 ⫾ 0.7
2.9 ⫾ 0.6
3.2 ⫾ 1.5
4.3 ⫾ 0.9
5.7 ⫾ 1.1
9.4 ⫾ 2.7
14.5 ⫾ 1.9
27.8 ⫾ 3.5*
88.7 ⫾ 3.1
138.6 ⫾ 11.6*
31.2 ⫾ 4.1
53.9 ⫾ 3.7*
84.6 ⫾ 2.9
80.3 ⫾ 5.1
129.5 ⫾ 10.5
118.4 ⫾ 5.8
1.00
1.92*
1.00
1.56*
1.00
1.73*
1.00
0.95
1.00
0.91
Significance
Yes (p ⫽ 0.004)
Yes (p ⫽ 0.002)
Yes (p ⫽ 0.002)
No (p ⫽ 0.273)
No (p ⫽ 0.184)
Note. 24 h after metal salt or control vehicle addition, or heat shock
treatment, DEX was added to a concentration of 10 –7 M as described in
Materials and Methods. Control cells were treated with vehicle with metal
salts, or were incubated at 37°C, as described in Materials and Methods.
Values are given as mean ⫾ SE.
a
Run-on transcription of CAT and GAPDH genes was measured as described in Materials and Methods and the ratio (CAT transcription/GAPDH
transcription) is presented as “relative CAT gene transcription”.
b
Fold-enhancement of CAT transcription in DEX-induced cells pre-treated
with metals or heat shock, relative to control cells treated with DEX alone.
CAT transcription was assigned an arbitrary value of 1.00 in every case.
*Significantly different from control cells (p ⱕ 0.05), assessed as described
in Materials and Methods.
of a zinc-sensitive reporter gene at low zinc concentrations
(Palmiter, 1995), and a mouse fibroblast cell line adapted to
extreme zinc deprivation constitutively expresses high levels of
MT mRNA (Suhy et al., 1999). In addition, transgenic mice
that overexpress MT-I are resistant to dietary zinc deficiency
(Dalton et al., 1996), and MT-I and II knockout mice are more
sensitive to zinc restriction than control mice (Kelly et al.,
1996). Taken together, these data suggest that MT-I can influence the amount of biologically labile zinc under low zinc
conditions in living cells and mammals.
DEX, used to induce CAT activity, can itself induce MT
synthesis via binding of DEX-GR to two GREs in the 5⬘
flanking sequences of both MT-I and MT-II (Kelly et al.,
1997). MT levels increase 2- to 3-fold in HeLa cells in the
presence of 10 –7 M DEX (Karin et al., 1981), the same concentration used for CAT induction. It is possible that DEX
induction of MT indicates a necessary role for Zn-MT in
DEX-induced signal transduction.
We report here that zinc exposure prior to DEX treatment
induces MT and enhances DEX-induced transcription of CAT
reporter genes and CAT enzyme activity. However, increasing
MT with regard to the specific metal ions associated with it did
not lead to elevated CAT activity under all circumstances. At
0.5 ␮M, CdCl 2 induced MT but did not alter CAT activity in
response to DEX (Figs. 2A and 2B). Cadmium-induced MT
was associated mainly with cadmium, unlike the predominant
Zn-MT form detected after zinc treatment (Fig. 3A, Table 1).
Similarly, in CuCl 2-treated cells, Cu-MT was detected but
CAT activity was unchanged. Therefore, enhancement of CAT
activity occurred in the presence of Zn-MT but not Cd-MT or
Cu-MT. This is consistent with the hypothesis that a capacity
of MT to regulate zinc (and not increased MT level, regardless
of the associated metal ion) is a key part of the process of
metal-induced enhancement of GR responsiveness. It is impor-
METALLOTHIONEIN AND GLUCOCORTICOID RESPONSIVENESS
FIG. 4. Treatment effects on GR levels (A) and GR localization (B) in
2305 cells. Mouse 2305 cells were incubated with 100 ␮M ZnCl 2 (Zn), or
1 ␮M CdCl 2 (or 0.25 ␮M CdCl 2 in panel B (Cd), or heat-shocked (42°C)
(HS) for the first hour of the culture period. Control cells (cont) were
untreated. In A, cells were harvested after 6, 24, or 48 h and protein
analyzed by the Western blot assay. DEX (10 –7 M) (ⴙ) or the equivalent
volume of ethanol (⫺) was added at 24 h to cells harvested at 48 h. For GR
localization studies (B), cells were treated with DEX for only 2 h before the
preparation of nuclear and cytoplasmic extracts. Blots were sequentially
probed with antibodies recognizing the glucocorticoid receptor (GR) and
␤-tubulin [Tubulin]). The ratios of the chemiluminescence signals for GR
to tubulin are presented.
73
tant to note, however, that cadmium and copper are toxic
metals that may exert effects on signal transduction through
events not associated with MT. In fact, exposure of 2305 cells
to 1 ␮M CdCl 2 resulted in significantly decreased CAT activity
(results not shown). However, unlike the lower CdCl 2 concentrations used in this study, 1 ␮M CdCl 2 significantly decreased
cell growth. Although low levels of cadmium can restore
DNA-binding activity of zinc-depleted GR in vitro (Freedman
et al., 1988), administration of cadmium to rats reduces the
ability of GR to bind both DEX and DNA and also reduces the
activity of liver tyrosine aminotransferase, a protein whose
gene is transcriptionally regulated by GR (Dundjerski et al.,
1996). In vitro reconstitution of a zinc-finger peptide of the
transcription factor Tramtrack by cadmium results in an altered
peptide secondary structure and reduced DNA-binding ability,
which can be restored by metal exchange with equimolar
Zn-MT (Roesijadi et al., 1998). In our experiments, it is clear
that Zn-MT, and not Cd-MT, is associated with increased
GR-mediated transcription in vivo.
Our results indicate that Zn-MT enhances GR-mediated
transcription from the MMTV promoter. Others have shown an
increase in CAT activity encoded by an MMTV-CAT construct
in response to heat shock in the presence of DEX (Sanchez et
al., 1994), and we confirmed these observations (Fig. 2). Although heat shock does not induce MT protein production (Fig.
2B, Bauman et al., 1993), twice as much of the total cellular
zinc is associated with the MT protein fractions (Fig. 3A, Table
1) after heat shock. A similar, but less substantial shift in
association of zinc with MT is seen in HgCl 2-treated cells
(Figs. 3A and 3B). The increase in Zn-MT, without a change in
MT protein levels, suggests several possible scenarios. First,
zinc may displace non-zinc metal ions in MT molecules. MT
can exist in association with a mixed complement of metals.
That is, with less than 7 zinc ions in combination with non-zinc
metal ions (Zn, CuMT, for example; Bofill et al., 2001), or a
mixed population of MT molecules associated exclusively with
zinc, or with non-zinc metal ions (ZnMT plus CuMT, for
example; Ebara et al., 2000). Treatment with zinc, mercury, or
heat could result in cellular rearrangement of metals to increase
the amount of zinc associated with MT. Considering that
isolated MT has a higher affinity for zinc than for copper
(Hamer, 1986) a simple process of direct displacement of Cu
by Zn to achieve this appears unlikely; more complex processes would be required. For example, new production of
Zn-MT may be induced but is balanced by degradation of
existing MT wholly or partly associated with non-zinc metals.
This possibility remains to be investigated in future studies of
MT gene transcription and mRNA translation in response to
Hg or heat shock. Alternatively, MT, prior to treatment with
exogenous metal salts, may exist without a full complement of
metal ions (i.e., partially unsaturated, or apo-MT). This hypothesis requires that zinc become available from a non-MT
source, either by transport from medium into cells or mobilization from intracellular sources, and be taken up by apo-MT
74
DEMOOR ET AL.
to generate MT with a full complement of seven zinc ions per
MT molecule. The presence of apo-MT in tumors has been
previously reported by Pattanaik et al. (1994), but the role of
apo-MT in glucocorticoid responsiveness remains to be explored.
The heat-shock response is characterized by the production
of heat-shock proteins. They are believed to play a role in
protecting the integrity of proteins essential in mediating response to stress, and to promote degradation of certain proteins
not required under stress conditions (Sherman and Goldberg,
1996). It is conceivable that a portion of the zinc detected in the
low molecular weight, MT-containing fractions in heatshocked cells is bound to fragments of partially degraded
high-molecular-weight proteins. However, SDS-polyacrylamide gel electrophoresis of Sephadex-fractionated proteins
provided no evidence to support this (results not shown).
Heat shock and metal effects on CAT activity could act
through a common non-MT-mediated path. Heat shock protein
(HSP) 90 interacts with GR, FKBP-52 and HSP 70 to generate
and maintain a hormone-responsive aporeceptor complex (reviewed in DeFranco, 2000; Pratt, 1993). Although high concentrations of metals can induce HSPs, the maximum zinc and
mercury concentrations used in this study (100 ␮M zinc and 50
nM mercury) do not induce HSP 70 or HSP 90 in rat hepatocytes (Bauman et al., 1993) or HSP 70 in HeLa cells
(Hatayama et al., 1992). Therefore, the enhanced CAT activity
seen in zinc- or mercury-pretreated cells is not obviously due
to altered HSP effects on GR stability.
It is also possible that, although exposure to zinc, mercury,
or heat shock are all stressful events that lead to increased
Zn-MT and affect GR-mediated signal transduction, they may
act through different pathways. Li et al. (1999) postulated that
the potentiation of CAT enzyme expression in heat-shocked
cells may be mediated by a heat shock factor-induced gene
product, and others (Mitsiou and Alexis, 1995) have suggested
that CAT mRNA stabilization may contribute to elevated CAT
activity in heat-stressed cells. It remains to be determined
whether increases in Zn-MT participate in the multiple cellular
events mediating increased GR signaling in response to heat
stress.
Changes in total GR content could not account for the
overall pattern in changes in CAT activity after metal or heat
treatments (Fig. 4). Although increases in GR:tubulin ratios
were seen in cells treated with zinc, cadmium, or heat shock,
cadmium actually inhibited GR responsiveness, while zinc and
heat shock enhanced it. Metal-induced increases in GR levels
have not been previously reported nor is the underlying reason
for the increase known. However, increases in GR levels in
response to stress have been reported (reviewed in Munck et
al., 1984). DEX treatment led to a dramatic drop in GR protein
levels 24 h later under all conditions, in accord with the
observation by others of glucocorticoid-induced suppression of
GR gene expression. (DuBois et al., 1995; Silva et al., 1994;
Vedeckis et al., 1987). DEX induction resulted in a rapid
translocation of GR from the cytoplasm to the nucleus (Fig.
4B). There was no consistent increase in nuclear translocation
under conditions where GR-induced CAT activity was enhanced. Heat potentiation of GR-mediated gene transcription
in mouse L929 cells has previously been shown to be due to
events other than increased GR nuclear translocation or retention (Sanchez et al., 1994). Overall, metal- or heat-induced
enhancement of glucocorticoid responsiveness does not appear
to be mediated by increases in GR levels or translocation.
Rather, other events (perhaps MT-mediated zinc donation to
GR and associated factors, or to other components of transcription complexes or chromatin, [Koropatnick and Leibbrandt,
1995]) may be involved.
It is not reasonable to suggest that Zn-MT could mediate the
activity of all transcription factors, as evidenced by the viability and normal development of MT-I and II null mice under
normal laboratory conditions (Masters et al., 1994; Michalska
and Choo, 1993). Cells expressing antisense MT RNA from
transfected expression vectors contain lower levels of MT
mRNA but unaltered levels of GAPDH mRNA (Koropatnick et
al., 1999; Leibbrandt et al., 1994). However, Zn-MT does
appear to be associated with enhanced GR activity, and the
possibility exists that other transcription factors that can donate
or sequester zinc ions to and from MT in vitro (including Sp1,
TFIIIA, and the estrogen receptor) may be regulated by MT in
vivo.
In summary, responsiveness to the synthetic glucocorticoid
DEX is enhanced under conditions that favor the formation of
Zn-MT (exposure to 80 or 100 ␮M ZnCl 2, 40 or 50 nM HgCl 2,
or heat shock). However, CdCl 2 or CuCl 2 treatment, which
leads predominantly to the association of MT with metals other
than zinc, has no effect on DEX-induced CAT activity. Novel
metal-induced upregulation of responsiveness to GR (rather
than the suppression of hormone signaling that has, to date,
been considered to be the consequence of exposure to toxic
metals) has implications for cellular exposure to metal ions,
and changes in MT expression and association with specific
metals in response to those exposures. This is particularly
important when one considers the broad range of functions
played by glucocorticoids in modulating cellular and humoral
immune activity, neuroendocrine development and function,
and a host of other physiological events. The correlation between ZnMT and enhancement of GR responsiveness suggests
that MT may play a physiological role in mediating appropriate
GR function. Investigation of that role (particularly in animals
with genetically ablated MT genes, under conditions of low
and high zinc availability), and the consequence of disruption
of ZnMT for hormone responses, is warranted.
ACKNOWLEDGMENTS
We thank Dr. M. George Cherian for his assistance in the analysis of
metal-associated MT and Dr. Trevor Archer for his assistance in immunode-
METALLOTHIONEIN AND GLUCOCORTICOID RESPONSIVENESS
tection of GR. This study was supported by a grant to J.K. from the Canadian
Institutes for Health Research.
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