Zinc-Metallothionein Levels Are Correlated with Enhanced Glucocorticoid Responsiveness in Mouse Cells Exposed to ZnCl2, HgCl2, and Heat Shock

Janice M. DeMoor*, Wendy A. Kennette{dagger}, Olga M. Collins* and James Koropatnick*,{dagger},{ddagger},§,1

* London Regional Cancer Centre, 790 Commissioners Road East, London, Ontario, Canada N6A 4L6; Departments of {dagger} Microbiology and Immunology, {ddagger} Oncology, § Pathology, and Pharmacology and Toxicology, University of Western Ontario, London, Ontario, Canada

Received April 25, 2001; accepted August 16, 2001


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 µM ZnCl2, DEX-induced CAT activity correlated with zinc-induced MT levels. However, 0.05 to 0.5 µM CdCl2 had no effect on CAT activity, despite an increase in Cd-associated MT. Copper-associated MT was detected in cells treated with 20 µM CuCl2, but there was no change in the level of Zn-MT, nor was CAT activity altered in cells exposed to 5 to 20 µM CuCl2. 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 HgCl2. 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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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, 1996Go; Koropatnick and Leibbrandt, 1995Go; O'Halloran, 1993Go). Intracellular free zinc concentrations are extremely low (Jiang et al., 1998Go; Simons, 1991Go). 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, 1998Go) 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, 1995Go; Zeng and Kägi, 1995Go).

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, 1995Go; Vallee, 1995Go; Zeng and Kägi, 1995Go). Zinc-bound metallothionein has been shown to reactivate various zinc-requiring apoenzymes in vitro (Jiang et al., 1998Go; Udom and Brady, 1980Go). Zinc transfer has been demonstrated from zinc-finger transcription factors to apothionein (Sp1 [Zeng et al., 1991aGo]; TFIIIA [Zeng et al., 1991bGo]). More recently, reversible zinc exchange between MT and the estrogen receptor (Cano-Gauci and Sarkar, 1996Go), MT and the yeast transcription factor GAL4 (Maret et al., 1997Go), and MT and a 2-zinc-finger peptide of the transcription factor Tramtrack (Roesijadi et al., 1998Go) 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, 1994Go). Furthermore, transient antisense downregulation of metallothionein expression abolishes the capacity of LPS to induce a respiratory burst in these cells (Leibbrandt et al., 1994Go). Also, treatment of monocytes with low, nontoxic levels of mercury, cadmium, or zinc to induce MT expression alters the ability of these cells to be activated by bacterial LPS (Koropatnick and Zalups, 1997Go; Leibbrandt and Koropatnick, 1994Go) or phorbol myristate acetate (PMA; Koropatnick, 1999Go).

Although treatment with certain metals can impair glucocorticoid receptor-mediated function (e.g., Cd repression of GR activity in rats [Dundjerski et al., 1996Go] and Pb repression of GR function in hepatoma cells [Heiman and Tonner, 1995Go]), 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., 1995Go). This promoter responds to dexamethasone (DEX) signals transduced to the nucleus by the glucocorticoid receptor (GR), which is a zinc-finger, DNA-binding 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 HgCl2 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 CdCl2 or CuCl2 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
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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., 1995Go). 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.

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. 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 3x 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 x 106 per plate (48 h time point) or 2 x 106 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 x 107 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, 2000Go). Briefly, frozen cell pellets were homogenized in 10 volumes of buffer 1 [0.32 M sucrose, 2 mM CaCl2 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 MgCl2, 50 mM Tris-HCl, pH 8.0, 0.1 mM EDTA), counted by hemacytometer, and adjusted to 5 x 104 nuclei/ml. RNA elongation reactions were performed using 20 µl of nuclei in nuclear buffer plus 200 µl of sterile 2X reaction buffer (10 mM ATP, 1 mM CTP, 1 mM GTP, 5 mM DTT, 2 µl [{alpha}-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 µl of RNase-free DNase 1 [(0.04 U of RQ1 DNase 1, RNase-free; Promega, Madison WI), 0.5 M NaCl, 50 mM MgCl2, 2 mM CaCl2, 10 mM Tris-HCl, pH 7.4]. The 32P-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 NaHPO4, pH 7.2, 7% sodium lauryl sulfate), to a final concentration of 4 x 106 cpm/ml.

Hybridization of radiolabeled RNA to immobilized, unlabeled probes.
Target DNA (immobilized on nylon filters in triplicate dots, 2 µg 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., 1988Go). 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, 2000Go), 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
Pelleted cells were lysed in 100 µl 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., 1987Go) and values were normalized to total protein content (measured by the Bio-Rad Protein Assay, Bio-Rad Laboratories, Hercules, CA).

MT Protein Measurement
Pelleted cells were lysed in 200 µl 1% Tween 20 in PBS by 3 cycles of freezing and thawing, then centrifuged at 12,000 x 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 µg/ml) of rabbit MT-I (Sigma-Aldrich Canada) diluted in 1% Tween 20 in PBS were each combined with an equal volume (160 µl) 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 µl/well) with 2 µg/ml rabbit MT-I in 0.1 M NaHCO3 (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 3x 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 µl per well 0.3% gelatin, 0.05% Tween 20 in PBS. The plate was washed as before, then 100 µl 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 µl 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 µl per well of alkaline phosphatase-conjugated streptavidin (Jackson ImmunoResearch Laboratories, Inc.) for 1 h. Following a final wash, 100 µl of 1 mg/ml p-nitrophenyl phosphate in 100 mM Tris-HCl (pH 9.5), 100 mM NaCl, and 5 mM MgCl2 were added per well. Color development was stopped when desired by the addition of 50 µl 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 µg 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 µl per well, and the plate was incubated for 1 h on an orbital shaker. After washing, 100 µl 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 Victor2 multilabel counter (PerkinElmer Life Sciences, Boston, MA).

Western Blot Protocol
Cell pellets were boiled 5 min in 500 µl 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 x 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 µg) 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., 1979Go]). 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, 1984Go). ß-Tubulin was detected by a mouse monoclonal antibody (Chemicon 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 x 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
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The glucocorticoid response is enhanced by zinc at concentrations that induce MT.
The 2305 cells were treated with 20, 40, 80, or 100 µM ZnCl2 for 48 h. DEX-induced CAT activity (Fig. 1AGo) was significantly enhanced in those cells that also responded to zinc treatment (80 or 100 µM) by MT induction (Fig. 1B, 3AGoGo). No enhancement of CAT activity was seen in cells exposed to 40 µM ZnCl2, a zinc concentration that is substantially higher than the 4 µM zinc found in normal 2305 culture medium (measured by atomic absorption spectrophotometry, results not shown; Palmiter, 1995Go) but too low to induce MT expression in these cells (Fig. 1BGo). The correlation (r2 = 0.94) between CAT activity and MT protein levels is shown in Fig. 1CGo. Logarithmic increases in MT level are reflected by linear increases in CAT activity. Zinc treatment (100 µM) in the absence of DEX did not induce CAT activity (Fig. 1AGo).



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FIG. 1. ZnCl2 treatment enhances glucocorticoid-induced gene expression in 2305 cells. (A) Cells were incubated with increasing concentrations of ZnCl2 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 ZnCl2 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 ZnCl2-treated cells.

 


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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.

 
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 CdCl2 did not result in enhancement of CAT activity in 2305 cells, despite an increase in MT protein at 0.5 µM CdCl2 (Fig. 2Go). However, cadmium, rather than zinc, was the predominant metal ion associated with MT in CdCl2-treated cells (Fig. 3AGo, Table 1Go). Exposure to 40 or 50 nM HgCl2 resulted in a significant increase in CAT activity (Fig. 2BGo), but unlike ZnCl2 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. 3AGo, Table 1Go). In CuCl2-treated cells, CAT activity was not altered (Fig. 2AGo), nor was MT protein induced (Fig. 2BGo). Despite detection of Cu-associated MT, the level of Zn-MT was unchanged in 20-µM CuCl2-treated cells, compared to untreated control cells (Fig 3AGo, Table 1Go).



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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).

 

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TABLE 1 Changes in Zn-Associated MT Levels in Mouse 2305 Cells Induced by Treatment with Metal Salts or Heat Shock CdCl
 
A significantly higher level of DEX-induced CAT enzyme activity was also detected in cells exposed to 42°C for 1 h as compared to control (Fig. 2AGo). Heat shock in the absence of DEX did not induce CAT activity (Fig. 2AGo). Like HgCl2, heat shock did not induce MT protein (Fig. 2BGo), but a substantial increase in the proportion of total zinc associated with MT was observed in heat-treated 2305 cells over control cells (Fig. 3AGo, Table 1Go).

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. 3BGo (r2 = 0.65).

It was formally possible that variation in CAT mRNA or 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 reporter 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 2Go).


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TABLE 2 The Influence of Metal Salt or Heat Shock Pretreatment on DEX-Induced Transcription of CAT Genes in 2305 Cellsa
 
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 4AGo. ß-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 CdCl2 treatment did not increase CAT activity, the enhancement of GR level observed was similar to that seen after treatments that did enhance CAT activity (ZnCl2, heat shock). The increase in GR levels, under conditions where no increase in GR-dependent CAT activity was evident (i.e., CdCl2 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 glucocorticoid-mediated downregulation of GR gene expression (DuBois et al., 1995Go; Silva et al., 1994Go).



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FIG. 4. Treatment effects on GR levels (A) and GR localization (B) in 2305 cells. Mouse 2305 cells were incubated with 100 µM ZnCl2 (Zn), or 1 µM CdCl2 (or 0.25 µM CdCl2 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) (+) 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 ß-tubulin [Tubulin]). The ratios of the chemiluminescence signals for GR to tubulin are presented.

 
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. 4BGo). There was no consistent observable difference in a total of 3 experiments (including the one shown in Fig. 4BGo) in the degree of GR translocation into the nucleus under conditions where GR activity was enhanced (ZnCl2, heat).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The multiple circumstances under which MTs are expressed (Andrews, 1990Go; Cherian and Chan, 1993Go), 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 ZnCl2, but only at those concentrations that induced MT expression (80, 100 µM ZnCl2; Fig. 1Go). Increasing zinc concentrations 5- to 10-fold (20 and 40 µM, 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., 1998Go; Udom and Brady, 1980Go; Suzuki and Kuroda, 1995Go) or MT and zinc-requiring transcription factors (Zeng et al., 1991aGo,1991bGo; Cano-Gauci and Sarkar, 1996Go; Maret et al., 1997Go; Roesijadi et al., 1998Go) has been reported. In vivo, constitutive expression of MT-I in stably transfected BHK cells increased the expression of a zinc-sensitive reporter gene at low zinc concentrations (Palmiter, 1995Go), and a mouse fibroblast cell line adapted to extreme zinc deprivation constitutively expresses high levels of MT mRNA (Suhy et al., 1999Go). In addition, transgenic mice that overexpress MT-I are resistant to dietary zinc deficiency (Dalton et al., 1996Go), and MT-I and II knockout mice are more sensitive to zinc restriction than control mice (Kelly et al., 1996Go). 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., 1997Go). MT levels increase 2- to 3-fold in HeLa cells in the presence of 10–7 M DEX (Karin et al., 1981Go), 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 µM, CdCl2 induced MT but did not alter CAT activity in response to DEX (Figs. 2A and 2BGoGo). Cadmium-induced MT was associated mainly with cadmium, unlike the predominant Zn-MT form detected after zinc treatment (Fig. 3AGo, Table 1Go). Similarly, in CuCl2-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 important 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 µM CdCl2 resulted in significantly decreased CAT activity (results not shown). However, unlike the lower CdCl2 concentrations used in this study, 1 µM CdCl2 significantly decreased cell growth. Although low levels of cadmium can restore DNA-binding activity of zinc-depleted GR in vitro (Freedman et al., 1988Go), 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., 1996Go). 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., 1998Go). 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., 1994Go), and we confirmed these observations (Fig. 2Go). Although heat shock does not induce MT protein production (Fig. 2BGo, Bauman et al., 1993Go), twice as much of the total cellular zinc is associated with the MT protein fractions (Fig. 3AGo, Table 1Go) after heat shock. A similar, but less substantial shift in association of zinc with MT is seen in HgCl2-treated cells (Figs. 3A and 3BGoGo). 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., 2001Go), 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., 2000Go). 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, 1986Go) 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 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, 1996Go). It is conceivable that a portion of the zinc detected in the low molecular weight, MT-containing fractions in heat-shocked 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, 2000Go; Pratt, 1993Go). Although high concentrations of metals can induce HSPs, the maximum zinc and mercury concentrations used in this study (100 µM zinc and 50 nM mercury) do not induce HSP 70 or HSP 90 in rat hepatocytes (Bauman et al., 1993Go) or HSP 70 in HeLa cells (Hatayama et al., 1992Go). 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, 1995Go) 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. 4Go). 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., 1984Go). 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., 1995Go; Silva et al., 1994Go; Vedeckis et al., 1987Go). DEX induction resulted in a rapid translocation of GR from the cytoplasm to the nucleus (Fig. 4BGo). 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., 1994Go). 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, 1995Go]) 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., 1994Go; Michalska and Choo, 1993Go). Cells expressing antisense MT RNA from transfected expression vectors contain lower levels of MT mRNA but unaltered levels of GAPDH mRNA (Koropatnick et al., 1999Go; Leibbrandt et al., 1994Go). 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 µM ZnCl2, 40 or 50 nM HgCl2, or heat shock). However, CdCl2 or CuCl2 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 immunodetection of GR. This study was supported by a grant to J.K. from the Canadian Institutes for Health Research.


    NOTES
 
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: jkoropat{at}uwo.ca. Back


    REFERENCES
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 RESULTS
 DISCUSSION
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