Cadmium-mediated Activation of the Metal Response Element in
Human Neuroblastoma Cells Lacking Functional Metal Response
Element-binding Transcription Factor-1*
Waihei A.
Chu
,
Jeffrey D.
Moehlenkamp
§,
Doug
Bittel¶
,
Glen K.
Andrews¶, and
Jeffrey A.
Johnson
**
From the
Departments of Pharmacology, Toxicology, and
Therapeutics and the ¶ Departments of Biochemistry and
Molecular Biology, University of Kansas Medical Center,
Kansas City, Kansas 66160-7417
 |
ABSTRACT |
Metal response element-binding transcription
factor-1 (MTF-1) binds specifically to metal response elements (MREs)
and transactivates metallothionein (MT) gene expression in response to
zinc and cadmium. This investigation contrasts the mechanism of mouse
MT gene (mMT-I) promoter activation by cadmium and zinc in IMR-32 human
neuroblastoma cells to determine whether MTF-1 binding to the MRE is
necessary for activation by these metals. Cadmium activated a mMT-1
promoter (
150 base pairs) luciferase reporter 20-25-fold through a
MRE-dependent mechanism. In contrast, zinc had little
effect on the mMT-1 luciferase reporter. IMR-32 cells lacked MRE
binding activity, and treatment with zinc in vitro or
in vivo did not generate a MTF-1·MRE complex, suggesting
that IMR-32 cells lack functional MTF-1. Overexpression of mMTF-1
regenerated a zinc-mediated induction of the MRE without affecting
cadmium activation. Because no other transition metals tested activated
the MRE, this effect appeared to be cadmium-specific. These data
demonstrate that in IMR-32 human neuroblastoma cells, zinc and cadmium
can use independent mechanisms for activation of the mMT-I promoter and
cadmium-mediated MRE activation is independent of MTF-1 and zinc.
 |
INTRODUCTION |
Metallothioneins (MTs)1
constitute a conserved family of cysteine-rich heavy metal binding
proteins (1). In the mouse, MT-I and MT-II display a wide tissue
distribution and have been demonstrated to participate in
detoxification of cadmium (2, 3), zinc homeostasis (4), and protection
against oxidative stress (5). MT-I and MT-II gene transcription is
dramatically increased by heavy metals (i.e. zinc and
cadmium) (6). Metal response elements (MREs) are essential for this
induction, and these elements are present in multiple copies in the
proximal promoters of MT genes. MREs were initially shown to mediate
transcriptional response of MT genes to zinc and cadmium (7), and more
recently to mediate, in part, the transcriptional response to oxidative
stress in mouse hepatoma cells (8, 9).
A protein that binds specifically to MREs and that transactivates MT
gene expression has been identified in mouse and human and is termed
MRE-binding transcription factor-1 (MTF-1) (10, 11). MTF-1 is a zinc
finger transcription factor in the Cys2His2 family. Targeted disruption of both MTF-1 alleles in mouse embryonic stem cells demonstrated its essential role for basal as well as heavy
metal-induced MT gene expression (12). The 5'-flanking region of the
mouse MT-I gene (mMT-I) also contains cis-acting elements that bind the
trans-acting factors Sp1 and upstream stimulatory factor (USF). An
antioxidant responsive element (ARE) overlaps the USF binding site, but
the protein(s) that bind to the ARE have not been identified (9). This
composite element was shown to contribute to the induction of mMT-I by
pro-oxidants in mouse hepatoma cells (Hepa) (9). More recently, our
laboratories have demonstrated that the USF/ARE participates in
cadmium-mediated induction of mMT-I in Hepa cells and that this
composite site does not contribute to mMT-1 induction by zinc (13).
Transcriptional activation of mMT-I has been extensively studied in a
variety of cell types; however, there is little data on regulation of
the mMT-I promoter in cells of neural origin. Data generated in
vivo and in primary neuronal and glial cell cultures demonstrate
that mMT-I mRNA and protein are increased in response to metals
(14-20), immobilization stress (19), steroids (17, 18, 20, 21), and
lipopolysaccarides (21, 22). These data imply that neurons and glia
possess the components necessary for transcriptional activation of
mMT-I. The identity of transcription factors, response elements and
signal transduction cascades involved in mMT-I induction in neurons and
glial cells remain to be determined. Thus, the purpose of these
experiments was to contrast the mechanism of mMT-I promoter activation
by cadmium and zinc and determine whether MTF-1 binding to the MRE is
necessary for mMT-I activation by metals in IMR-32 human neuroblastoma cells.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Cadmium chloride, zinc chloride, copper sulfate,
sodium arsenite, sodium dichromate, cobalt chloride, nickel chloride,
lead acetate, mercuric chloride, manganese chloride, bismuth nitrate, ferric chloride, and phorbol 12-myristate 13-acetate were obtained from
Sigma. tert-Butylhydroquinone (tBHQ) was obtained from Acros Organics (Fairlawn, NJ). D-Luciferin was obtained from
Analytical Luminescence Laboratories, Inc. (Ann Arbor, MI). Reagents
for the Galacto-LightTM chemiluminescent reporter assay for
-galactosidase activity were purchased from Tropix, Inc. (Bedford,
MA). Tissue culture supplies were purchased from Life Technologies,
Inc. All other reagents were purchased from Fisher.
Reporter Plasmids--
The proximal mMT-I promoter fragments
150 to +66 promoter (numbers relative to the transcription start
point in the mMT-I gene),
150
USF/ARE to +66 promoter (deletion of
100 to
89), and the minimal
42 to +66 promoter (9) were subcloned
into a luciferase reporter construct, pGL-2 basic (Promega Biotech, Madison, WI). Five tandem copies of the MREd (MREd5) with
the mMT-I minimal promoter (
42 to +66) (9) were also subcloned into
pGL-2 basic. The TATA box and transcription start site were provided by
the mMT-I promoter in these fusion genes. A single forward oriented
copy of the USF/ARE
(5'-GATCCGCGGGGCGCGTGACTATGCGTGGGCTGGA-3') was
subcloned into the BglII site preceding the adenovirus major late minimal promoter-luciferase reporter construct, pTi-luciferase, provided by Dr. William Fahl (University of Wisconsin, Madison, WI)
(23). The bold portion of the sequence identifies the core ARE and USF
binding site. In this construct the TATA box and transcription start
site are provided by the adenovirus major late minimal promoter. The
mammalian expression vector CMV-mMTF-1 (24) was generated by inserting
the mouse MTF-1 cDNA into the NotI site of a CMV expression vector kindly provided by Dr. James Smith (Baylor College of
Medicine, Houston, TX).
Transient Transfections--
IMR-32 human neuroblastoma or HepG2
human hepatoma cells were plated at a density of 6.0 × 104 cells/well in 24-well plates and grown in 0.5 ml of
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum. Transient transfections using the calcium phosphate
method were done as described previously (25). IMR-32 and HepG2 cells
were cotransfected 72 and 48 h after plating, respectively, with
430 ng of promoter-driven reporter plasmids and 20 ng of
CMV-
-galactosidase to correct for transfection efficiency. Cells
were incubated overnight with the transfection mix and subsequently
treated for 18-24 h. For dose-response studies, cells were treated
with 0.1 mM HCl or increasing doses of cadmium or zinc in
0.1 mM HCl. Cells were also treated with 0.4% ethanol or
increasing doses of tBHQ in ethanol. To down-regulate protein kinase C
(PKC), cells were treated for 24 h prior to transfection with
0.4% ethanol or 1.0 µM phorbol 12-myristate 13-acetate
in ethanol. In the continued presence of phorbol 12-myristate
13-acetate, cells were transiently transfected, as described above, and
treated with zinc (100 µM) or cadmium (IMR-32, 1.0 µM; HepG2, 2.0 µM). In all experiments, the
medium was removed, and cells were lysed by freeze-thawing in 100 µl of extraction buffer (100 mM KPO4, pH 7.4, 4.0 mM ATP, 1.5 mM MgSO4, 1.0 mM dithiothreitol, and 0.1% Triton X-100). Cell lysates (25 µl) were added to 350 µl of luciferase assay buffer (100 mM KPO4, pH 7.4, 4.0 mM ATP, and
1.5 mM MgSO4), and then 100 µl of 1.0 mM D-Luciferin was injected into each culture
tube by a luminometer that integrated the luminescence over 10 s.
The
-galactosidase activity was measured using the
Galacto-LightTM chemiluminescent reporter assay. Cell
lysates (25 µl) were added to 100 µl of
-galactosidase reaction
buffer (100 mM sodium phosphate, pH 8.0, 1 mM
MgCl2, and GalactonTM) incubated for 35 min,
and then 100 µL of EmeraldTM luminescence amplifier was
injected by a luminometer, which integrated the luminescence over
5 s. The data are expressed as a ratio of luciferase to
-galactosidase activities or are presented as the fold activation
relative to the vehicle control. Statistics were performed using the
Student's t test (p < 0.05).
Electrophoretic Mobility Shift Assay--
EMSAs were performed
using whole cell extracts prepared as described previously (26) with
modifications (8, 24, 27). IMR-32 human neuroblastoma cells or Hepa
mouse hepatoma cells were plated at a density of 3.0 × 106 cells in 100-mm dishes. IMR-32 cells were grown in 10 ml of Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum, and mouse Hepa cells were grown in Dulbecco's modified
Eagle's medium supplemented with 2% fetal bovine serum. Protein
concentration was determined using the BCA protein kit (Pierce). Whole
cell extracts (20 µg of protein in 1 µl) or zinc-activated MTF-1
from an in vitro transcription/translation reaction (1 µl
of a 50-µl reaction) were incubated in buffer containing 12 mM HEPES (pH 7.9), 60 mM KCl, 0.5 mM dithiothreitol, 12% glycerol, 5 mM
MgCl2, 0.2 µg of poly(dI-dC)/µg protein, 8 fmol of
end-labeled double-stranded oligonucleotide (5000 cpm/fmol) in a total
volume of 20 µl for 20 min on ice (8, 24). EMSA was performed using
the oligonucleotide sequence MRE-s
(5'-GATCCAGGGAGCTCTGCACACGGCCCGAAAAGTA) (11). Protein-DNA complexes were separated at 4 °C using 4% polyacrylamide gel (acrylamide:bisacrylamide 80:1) electrophoresis at
15 V/cm. The gel was polymerized and run in buffer consisting of 0.19 M glycine, 25 mM Tris (pH 8.5), 0.5 mM EDTA. After electrophoresis, the gel was dried, and
labeled complexes were detected by autoradiography.
 |
RESULTS |
Transcriptional Activation of mMT-1 Promoter in IMR-32
Cells--
IMR-32 human neuroblastoma cells were transfected with a
150 mMT-1-luciferase reporter construct and treated with tBHQ,
cadmium, and zinc (Fig. 1). Cadmium
activated the
150 mMT-1-luciferase reporter 20-25-fold at doses of
0.5-2.0 µM (Fig. 1B) In contrast, tBHQ did
not change the luciferase expression (Fig. 1A), and zinc caused only a modest but statistically significant increase of 1.6-2.0-fold (Fig. 1C). Activation of the mMT-I promoter by
heavy metals and oxidative stress in Hepa cells has been attributed to
interaction between MTF-1 and MRE (7-9). Thus, the absence of
functional MTF-1 in IMR-32 cells could account for the lack of
responsiveness to zinc and/or tBHQ.

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Fig. 1.
Transcriptional Activation of mMT-1 promoter
in IMR-32 cells. 72 h after plating, IMR-32 human
neuroblastoma cells were transfected with a 150 mMT-1-luciferase
reporter construct and treated with increasing concentrations of tBHQ
(A, ), cadmium (B, ), and zinc
(C, ). After 24 h, the medium was removed from the
cells, the cells were lysed in extraction buffer, and cell extracts
were assayed for luciferase and -galactosidase activity. The data
are expressed as the luciferase/ -galactosidase ratio. Each value
represents the mean ± S.E. (n = 6). Error
bars not visible on the graph are covered by the
symbol. a, significantly different from the
corresponding control value (p < 0.05).
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The hypothesis that this human neuroblastoma cell line has no
functional MTF-1 was evaluated by EMSAs on whole cell extracts from
IMR-32 cells compared with mouse Hepa cells (Fig.
2). Whole cell extracts were prepared
from vehicle-treated, 10 µM tBHQ-treated, 1.0 µM cadmium-treated, and 100 µM zinc-treated
cells (2 and 8 h). IMR-32 cell extracts did not contain detectable
MRE binding activity at either 2 h (Fig. 2, lanes 2-5)
or 8 h (Fig. 2, lanes 6-9). As has been demonstrated
previously (8, 24, 27), treating Hepa cells for 1 h with 100 µM zinc results in significant MRE binding activity due
to MTF-1 activation (Fig. 2, lane 1). We have previously
shown that addition of unlabeled MRE oligonucleotide competes for the
MRE·MTF-1 complex (8) and that the MRE·MTF-1 complex is
supershifted specifically by a polyclonal antibody raised against MTF-1
(28).

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Fig. 2.
IMR-32 cell extracts do not contain MRE
binding activity. Electrophoretic mobility shift assays were
performed with whole cell extracts from control, zinc-treated,
cadmium-treated, and tBHQ-treated IMR-32 cells. Mouse Hepa cells were
treated with 100 µM zinc for the indicated time (h).
IMR-32 cells were treated with 100 µM zinc, 1.0 µM cadmium, or 10 µM tBHQ for the indicated
time (h). Whole cell extracts were made, 20 µg of total protein was
added to the EMSA buffer containing labeled MRE oligonucleotide, and
the reactions were incubated on ice for 15 min. Reactions were
subjected to polyacrylamide gel electrophoresis, the gel was dried, and
the label was detected by autoradiography. The arrow
indicates the MRE·MTF-1 complex.
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MRE·MTF-1 binding activity can be increased in vitro by
addition of exogenous zinc to whole cell extracts from untreated cells (8, 24, 27). Incubation of IMR-32 extracts with zinc in vitro did not generate a MTF-1·MRE complex (Fig.
3, lane 6). In contrast,
treatment of Hepa control cell extract with zinc in vitro
resulted in a large increase in MRE binding activity (Fig. 3,
lanes 5 and 9). A possible explanation for these
results is that IMR-32 cells contain an inhibitor of MTF-1 activation
and subsequent MRE·MTF-1 complex formation. A combined whole cell extract was prepared from a mixture of IMR-32 and Hepa cells to test
this possibility (Fig. 3, lanes 4 and 8). The
IMR-32 cell extract had no effect on activation of MTF-1 in Hepa cell
extract, implying that IMR-32 cells do not possess a constitutively
expressed repressor of MTF-1 activation.

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Fig. 3.
MTF-1 binding activity is not activated
in vitro by addition of exogenous zinc in IMR-32 cell
extracts. EMSAs were performed with whole cell extracts from
control and zinc-treated IMR-32 and mouse Hepa cells. Mouse Hepa cells
were treated with 100 µM zinc for 1 h (lanes
5 and 9). IMR-32 cells were treated with vehicle
(lanes 2 and 6) or 100 µM zinc for
2 h (lanes 3 and 7). To test for the
presence of an inhibitor of MTF-1 binding in IMR-32 cells, Hepa and
IMR-32 cells treated with zinc were mixed prior to extract preparation,
and the combined extract was used in the EMSA (lanes 4 and
8). For reactions with zinc added in vitro,
control and treated extracts were brought to 30 µM zinc
and incubated at 37 °C for 15 min and returned to ice (lanes
6-9). Reactions with no exogenous zinc remained on ice
(lanes 2-5). Radiolabeled MRE oligonucleotide (2-4 fmol,
5,000 cpm/fmol) was added, and incubation on ice was continued for an
additional 15 min. Reactions were subjected to polyacrylamide gel
electrophoresis, the gels were dried, and the label was detected by
autoradiography. Zinc-activated MTF-1 from an in vitro
transcription/translation reaction was used as a positive control
(lane 1). The arrow indicates the MRE·MTF-1
complex.
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The MRE Not USF/ARE Mediates Induction of the mMT-I Promoter by
Cadmium--
We have recently demonstrated that the USF/ARE from the
mMT-I promoter participates in cadmium-mediated induction of mMT-I in
Hepa cells and that this composite site does not contribute to
induction of mMT-1 by zinc (13). IMR-32 cells were transfected with
150 mMT-1-luciferase,
150
USF/ARE-luciferase, and
USF/ARE-luciferase reporter constructs to determine the role of USF/ARE
in cadmium-mediated activation of mMT-1 promoter (Table
I). Deletion of the USF/ARE from the
intact
150 mMT-I promoter sequence had no effect on induction of
luciferase by cadmium in IMR-32 cells. The
150 mMT-I-luciferase reporter and
150
USF/ARE-luciferase were activated 21.5- and 20.3-fold, respectively (Table I). In addition, the isolated USF/ARE
element in a heterologous promoter construct was not activated by
cadmium (Table I). Because the IMR-32 cells apparently do not have
functional MTF-1, the MREd5-luciferase was included to serve as a negative control. Contrary to this assumption, the data
showed that MREd5-luciferase was activated 30.2-fold by
treatment with 1.0 µM cadmium (Table I). Thus,
cadmium-mediated transcriptional activation of mMT-1 promoter is
MRE-dependent, yet MTF-1-independent in IMR-32
neuroblastoma cells.
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Table I
Transcriptional activation of the MT promoter by cadmium is dependent
on the MRE and independent of the USF/ARE
IMR-32 cells were plated at a density of 6.0 × 104
cells/well in 24-well plates and grown in 0.5 ml of Dulbecco's
modified Eagle's medium supplemented with 10% fetal bovine serum. The
cells were transiently transfected and subsequently treated for 24 h. The medium was removed, and cells were lysed in extraction buffer.
Cell lysates were then assayed for luciferase and -galactosidase
activity. Each value represents the mean ± S.E.
(n = 6-12). NS, not significant.
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Overexpression of mMTF-1 Restores Zinc-mediated Induction of the
MRE--
IMR-32 cells were cotransfected with increasing
concentrations of a mammalian expression vector for mMTF-1 (CMV-mMTF-1)
and the MREd5-luciferase reporter construct (Fig.
4). Basal level expression of the
MRE-driven fusion construct was significantly increased in a
dose-dependent manner with increasing concentrations of
CMV-mMTF-1 (Fig. 4A). At a concentration of 20 ng of
CMV-mMTF-1/well there was a 9-fold increase in basal level
transcription of the MRE. In addition, overexpression of MTF-1
regenerated a zinc-mediated induction of the
MREd5-luciferase reporter gene (Fig. 4B). The magnitude of zinc induction was 9-fold and maximal at 1 ng of CMV-mMTF-1. Notably, overexpression of mMTF-1 had no effect on activation of MREd5-luciferase by cadmium and actually
significantly decreased the extent of activation with increasing doses
of CMV-mMTF-1 (Fig. 4B).

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Fig. 4.
Overexpression of mMTF-1 increases basal
level expression and restores zinc-mediated activation of
MREd5-luciferase. 72 h after plating, IMR-32
human neuroblastoma cells were transiently transfected with a
MREd5-luciferase reporter construct and increasing amounts
of CMV-mMTF-1 mammalian expression vector. The effect of mMTF-1 on
basal level expression (A, ) and vehicle-treated ( ),
10 µM tBHQ-treated ( ), 1.0 µM
cadmium-treated ( ) and 100 µM zinc-treated ( )
IMR-32 cells (B) was determined. After 24 h, medium was
removed from the cells, the cells were lysed in extraction buffer, and
cell extracts were assayed for luciferase and -galactosidase
activity. The data are expressed as the ratio of luciferase to
-galactosidase activities (A) and fold activation
versus the corresponding control value (B). Each
value represents the mean ± S.E. (n = 6).
Error bars not visible on the graph are covered by the
symbol. a, significantly different from the
corresponding value in the absence of CMV-mMTF-1 (p < 0.05); b, significantly different from the corresponding
vehicle-treated value (p < 0.05); c,
significantly different from zinc-treated value in the presence of 1 ng
of CMV-mMTF-1/well; d, significantly different from
cadmium-treated value in the absence of CMV-mMTF-1.
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MREd5-luciferase Is Activated by Zinc in Human Hepatoma
Cells Not Human Neuroblastoma Cells--
To validate that the observed
effects in IMR-32 cells were not due to differences between rodent and
human MTF-1, we transfected HepG2 human hepatoma cells with
MREd5-luciferase and treated them with zinc and cadmium.
Zinc and cadmium transcriptionally activated the
MREd5-driven luciferase expression by 17- and 30-fold,
respectively, in HepG2 human hepatoma cells (Fig.
5). Thus, HepG2 cells express functional
human MTF-1 that is activated by zinc, whereas this transcription
factor is apparently not functional or not expressed in IMR-32 cells.
Based on these data, we wanted to determine whether activation of the
MRE in IMR-32 cells was cadmium-specific. In addition to cadmium, we
tested copper sulfate (5-200 µM), sodium arsenite
(5-200 µM), sodium dichromate (0.1-20
µM), cobalt chloride (5-200 µM), nickel
chloride (5-200 µM), lead acetate (0.1-50
µM), mercuric chloride (0.5-20 µM),
manganese chloride (0.5-25 µM), bismuth nitrate (1-200
µM), and ferric chloride (5-200 µM).
IMR-32 cells transfected with
150 mMT-1-luciferase or
MREd5-luciferase were treated with the above compounds, but
only cadmium caused a significant increase in luciferase expression
(data not shown).

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Fig. 5.
MREd5-luciferase is activated by
zinc in human hepatoma cells. HepG2 cells were transfected 48 h after plating with MREd5-luciferase reporter. Cells were
treated with vehicle (open bar) and 100 µM
zinc (hatched bar) (A) or vehicle (open
bar), 1.0 µM cadmium (hatched bar), 2.0 µM cadmium (stippled bar), and 4.0 µM cadmium (filled/hatched bar)
(B). After 24 h, medium was removed from the cells, the
cells were lysed in extraction buffer, and cell extracts were assayed
for luciferase and -galactosidase activity. The data are expressed
as fold activation versus the corresponding control value.
Each value represents the mean ± S.E. (n = 6).
Error bars not visible on the graph are covered by the
bar. a, significantly different from the
corresponding control value (p < 0.05).
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MREd5-Luciferase Activation in IMR-32 Cells Is
PKC-independent--
Recently, Yu and co-workers (29) showed that
metal-induced MT gene expression can be inactivated by PKC inhibitors.
IMR-32 cells and HepG2 cells were PKC down-regulated by chronic
treatment with phorbol 12-myristate 13-acetate, transfected with
MREd5-luciferase reporter, and treated with zinc and
cadmium (Table II). In HepG2 cells,
cadmium-mediated activation of the MRE was completely blocked by PKC
down-regulation, and zinc-mediated activation was significantly reduced
from 20.5-fold in control cells to 3.12-fold in PKC down-regulated cells. In contrast, PKC down-regulation in the IMR-32 cells had no
effect on activation of the MRE by cadmium (Table II). Similarly, pretreatment of IMR-32 cells with the PKC inhibitors H7 and GF109203X had no effect on cadmium-mediated activation of the MRE (data not
shown).
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Table II
Effect of PKC down-regulation on zinc- and cadmium-mediated activation
of MREd5-luciferase
IMR-32 or HepG2 cells were plated at a density of 6.0 × 104 cells/well in 24-well plates and grown in 0.5 ml of
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum. 48 h after plating, cells were treated with ethanol
or 1 µM PMA and transiently transfected 24 h
later. Cells were treated for 24 h with vehicle or 1.0 µM (IMR-32) or 2.0 µM (HepG2) cadmium. The
medium was removed, and cells were lysed in extraction buffer. Cell
lysates were then assayed for luciferase and -galactosidase
activity. Each value represents the mean ± S.E.
(n = 6-12). ND, not determined. NS, not significant.
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DISCUSSION |
These data demonstrate that zinc and cadmium have independent
mechanisms for activation of the mMT-I promoter in IMR-32 cells. Because cotransfection of the mMT-I reporter constructs with a mammalian expression vector containing mMTF-1 restored zinc
responsiveness to the IMR-32 cells, the lack of a zinc effect can be
attributed to absence of functional MTF-1 in this cell type.
Interestingly, cadmium-driven activation of the MRE was not dependent
upon the presence of functional MTF-1 and was actually attenuated by
increased overexpression of MTF-1. Finally, this MTF-1-independent
mechanism was specific for cadmium and not mediated through activation
of PKC in IMR-32 cells.
In sharp contrast to our findings in IMR-32 cells, MTF-1, a zinc finger
transcription factor, has been shown to be essential in mediating
transcriptional activation of MT genes by zinc and cadmium in embryonic
stem cells and BHK cells (12, 30). These transition metals are the most
potent activators of mouse MT expression in many cell types and are
proposed to converge on the MRE through MTF-1 activation. Furthermore,
expression of MT-I and MT-II genes in MTF-1 null embryonic stem cells
could not be increased by treatment with zinc, cadmium, copper, nickel,
or lead, and there was no detectable basal level expression of either
MT gene (12). These data suggest that MTF-1·MRE complex formation is
essential for basal expression and induction of MT genes.
Zinc can reversibly and directly activate the DNA binding activity of
MTF-1 (24), and the extent of MTF-1 binding activity is rapidly
increased, as is occupancy of MRE in the mMT-I promoter, after
treatment of cells with zinc (8). In contrast to zinc (8, 24), cadmium
has little effect on the amount of MTF-1 binding activity in Hepa or
HeLa cell extracts (27). Similarly, the DNA binding activity of
recombinant human and mouse MTF-1 to MRE in vitro is also
dependent on zinc and not facilitated by cadmium (27). These data
support the hypothesis that cadmium may be activating MT genes by a
mechanism distinct from that of zinc.
Fortuitously, we found that zinc did not transcriptionally activate
mMT-1-luciferase reporter constructs in IMR-32 human neuroblastoma. But
as was seen in the MTF-1 null embryonic stem cells (12), transfection
of recombinant mMTF-1 into IMR-32 cells restored the zinc-mediated
activation of mMT-1 reporters and increased their basal level
expression. Indeed, it would appear that increased expression of MTF-1
actually attenuated transcriptional activation of the MRE by cadmium
and zinc (Fig. 4B). Because both zinc and cadmium are
affected and there is a corresponding increase in basal level
expression of the luciferase reporter (Fig. 4A), we hypothesize that the greater the basal level expression (increased occupancy of the MRE by MTF-1) the lesser the induction of the MRE-luciferase reporter. This can certainly explain the effect of MTF-1
overexpression on zinc activation but also implies that activation of
the MRE by cadmium can be competed for by MTF-1, suggesting that MTF-1
can effectively block the interaction of cadmium-responsive
transcription factor(s) with the MRE. The mechanism(s) by which cadmium
activates the MRE in IMR-32 cells remains to be determined.
The MRE may also bind the basal transcription factor Sp1 and
zinc-regulated factor (31). We examined Sp1 by transfecting IMR-32
cells with a heterologous promoter construct containing the Sp1
consensus DNA binding site. This Sp1-luciferase reporter construct was
not activated by cadmium, zinc, or tBHQ in the IMR-32 cells (data not
shown), suggesting that the binding of Sp1 is not involved in
activation of the MRE by cadmium in IMR-32 cells. The zinc-regulated
factor is distinct from MTF-1 and was isolated from the mouse for its
ability to activate MRE-driven reporter gene expression in yeast (31).
A human homolog of the zinc-regulated factor has not been described,
and the effects of cadmium versus zinc on its MRE binding
activity has not been examined.
Yu and co-workers (29) demonstrated that metal-induced MT gene
expression was blocked by inhibitors of PKC. The relatively nonspecific
inhibitor of PKC, H7, blocked induction of MT by cadmium and zinc. The
effect on zinc-mediated induction was due to a lack of uptake. H7
blocked the cellular accumulation of zinc. In contrast, the cellular
accumulation of cadmium was unaffected by H7 pretreatment, suggesting
that PKC could play an important role in regulating induction of MT by
cadmium. We clearly show that PKC is not involved in the
cadmium-mediated activation of MT reporters in IMR-32 cells. However,
in HepG2 cells, the transcriptional activation of MRE reporter by
cadmium is completely blocked by PKC down-regulation. Therefore, PKC is
important in HepG2 cells but not in IMR-32 cells with respect to
cadmium-mediated induction of MT gene expression. Alternatively,
cadmium can stimulate myosin light chain kinase (32), affect calmodulin
activity in the brain (33), and evoke inositol polyphosphate formation
(34) and superoxide anion production by macrophages (35). The possible
role for these pathways in mediating cadmium-driven MRE activation in
IMR-32 cells has not been evaluated.
Others have reported differential gene activation in response to
treatment with zinc versus cadmium (36). These investigators demonstrate that in HeLa-derived cadmium-resistant cells, cadmium but
not zinc increases Hsp70 and GRP78 expression. In addition, a
cis-acting element responsible for cadmium-mediated induction of human
heme oxygenase in HeLa cells has been identified (cadmium response
element) (37, 38). The core sequence is different from the MRE. EMSAs
show that a protein constitutively associates with the cadmium response
element, the DNA binding activity is not altered by cadmium, and the
cadmium response element binding protein does not bind to the MRE.
Furthermore, cadmium response element-luciferase reporters are
activated by cadmium but not zinc, whereas MRE-luciferase reporters are
activated by both cadmium and zinc in HeLa cells (38). Thus, it is
obvious that cadmium and zinc can activate different genes through
specific pathways/response elements. The clear separation of cadmium
and zinc pathways for MT induction in IMR-32 neuroblastoma cells
indicates that MRE activation by cadmium is not only independent of
MTF-1 but also of zinc.
Targeted disruption of the MTF-1 locus is lethal to embryos at
embryonic day 14 (39). The MTF-1 null embryos are devoid of MT-I and
MT-II expression and have significantly reduced basal levels of
-glutamylcysteine synthetase but normal expression of Sp1. Primary
cultures of mouse embryo fibroblasts and established mouse embryo
fibroblast cells lines from the MTF-1 null embryos were shown to have
increased susceptibility to cadmium-induced cytotoxicity (39). The
effect of cadmium on expression of MT genes in these MTF-1 null mouse
embryo fibroblasts was not evaluated. Based on our data, there is an
alternative mechanism by which to activate the MRE and MT gene
expression independent of MTF-1 expression. MTF-1-independent
activation of the MRE may be cell type-specific, and thus it is very
important to determine whether cadmium increases expression of MT genes
in MTF-1 null mouse embryo fibroblasts. Comparison of these data with
the data generated in IMR-32 cells should help us begin to sort out the
mechanism of activation by cadmium versus zinc. In
conclusion, the data presented herein reveal a novel pathway for the
induction of MT gene expression and MRE activation by cadmium. The
relationship of these data to the regulation of MTs and other
MRE-driven genes in vivo remains open to speculation.
 |
FOOTNOTES |
*
This work was supported in part by Grant ES08089 from the
NIEHS, National Institutes of Health (to J. A. J.), by a
Burroughs Wellcome New Investigator in Toxicology Award (to J. A. J.), and by Grants ES05704 and CA61262 from the National
Institutes of Health (to G. K. A.).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.
§
Supported by NIEHS, National Institutes of Health Toxicology
Program Predoctoral Fellowship T32 ES07079.
Supported by National Research Service Award Fellowship F32 ES05753.
**
To whom correspondence should be addressed: Dept. of Pharmacology,
Toxicology and Therapeutics, University of Kansas Medical Center, G017B
Breidenthal, 3901 Rainbow Blvd., Kansas City, KS 66160-7417. Tel.:
913-588-7517; Fax: 913-588-7501; E-mail: jjohnso2{at}kumc.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
MT, metallothionein;
mMT-I, mouse MT gene;
MRE, metal response element;
MTF-1, MRE-binding
transcription factor-1;
USF, upstream stimulatory factor;
ARE, antioxidant responsive element;
tBHQ, tert-butylhydroquinone;
PKC, protein kinase C;
EMSA, electrophoretic mobility shift assay;
CMV, cytomegalovirus.
 |
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