Zinc and Cadmium Can Promote Rapid Nuclear Translocation of Metal Response Element-binding Transcription Factor-1

Metal response element-binding transcription factor-1 (MTF-1) is a six-zinc finger protein that plays an essential role in activating metallothionein expression in response to the heavy metals zinc and cadmium. Low affinity interactions between zinc and specific zinc fingers in MTF-1 reversibly regulate its binding to the metal response elements in the mouse metallothionein-I promoter. This study examined the subcellular distribution and DNA binding activity of MTF-1 in cells treated with zinc or cadmium. Immunoblot analysis of cytosolic and nuclear extracts demonstrated that in untreated cells, about 83% of MTF-1 is found in the cytosolic extracts and is not activated to bind to DNA. In sharp contrast, within 30 min of zinc treatment (100 μm), MTF-1 is detected only in nuclear extracts and is activated to bind to DNA. The activation to bind to DNA and nuclear translocation of MTF-1 occurs in the absence of increased MTF-1 content in the cell. Furthermore, immunocytochemical localization and immunoblotting assays demonstrated that zinc induces the nuclear translocation of MTF-1-FLAG, expressed from the cytomegalovirus promoter in transiently transfected dko7 (MTF-1 double knockout) cells. Immunoblot analysis of cytosolic and nuclear extracts from cadmium-treated cells demonstrated that concentrations of cadmium (10 μm) that actively induce metallothionein gene expression cause only a small increase in the amount of nuclear MTF-1. In contrast, an overtly toxic concentration of cadmium (50 μm) rapidly induced the complete nuclear translocation and activation of DNA binding activity of MTF-1. These studies are consistent with the hypothesis that MTF-1 serves as a zinc sensor that responds to changes in cytosolic free zinc concentrations. In addition, these data suggest that cadmium activation of metallothionein gene expression may be accompanied by only small changes in nuclear MTF-1. Metal response element-binding transcription factor-1 (MTF-1) is a six-zinc finger protein that plays an essential role in activating metallothionein expression in response to the heavy metals zinc and cadmium. Low affinity interactions between zinc and specific zinc fingers in MTF-1 reversibly regulate its binding to the metal response elements in the mouse metallothionein-I promoter. This study examined the subcellular distribution and DNA binding activity of MTF-1 in cells treated with zinc or cadmium. Immunoblot analysis of cytosolic and nuclear extracts demonstrated that in untreated cells, about 83% of MTF-1 is found in the cytosolic extracts and is not activated to bind to DNA. In sharp contrast, within 30 min of zinc treatment (100 μm), MTF-1 is detected only in nuclear extracts and is activated to bind to DNA. The activation to bind to DNA and nuclear translocation of MTF-1 occurs in the absence of increased MTF-1 content in the cell. Furthermore, immunocytochemical localization and immunoblotting assays demonstrated that zinc induces the nuclear translocation of MTF-1-FLAG, expressed from the cytomegalovirus promoter in transiently transfected dko7 (MTF-1 double knockout) cells. Immunoblot analysis of cytosolic and nuclear extracts from cadmium-treated cells demonstrated that concentrations of cadmium (10 μm) that actively induce metallothionein gene expression cause only a small increase in the amount of nuclear MTF-1. In contrast, an overtly toxic concentration of cadmium (50 μm) rapidly induced the complete nuclear translocation and activation of DNA binding activity of MTF-1. These studies are consistent with the hypothesis that MTF-1 serves as a zinc sensor that responds to changes in cytosolic free zinc concentrations. In addition, these data suggest that cadmium activation of metallothionein gene expression may be accompanied by only small changes in nuclear MTF-1. metallothionein cytosolic extract Dulbecco's modified Eagle's medium-high glucose electrophoretic mobility shift assay fetal bovine serum metal response elements metal response element-binding transcription factor-1 nuclear extract nuclear localization signal phosphate-buffered saline cytomegalovirus Metallothioneins (MT)1are small cysteine-rich proteins, which play a role in zinc homeostasis, cadmium detoxication, and protection from reactive free radicals (1.Andrews G.K. Prog. Food Nutr. Sci. 1990; 14: 193-258PubMed Google Scholar, 2.Dalton T.P. Palmiter R.D. Andrews G.K. Nucleic Acids Res. 1994; 22: 5016-5023Crossref PubMed Scopus (251) Google Scholar, 3.Dalton T.P. Li Q. Bittel D. Liang L.C. Andrews G.K. J. Biol. Chem. 1996; 271: 26233-26241Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 4.Stuart G.W. Searle P.F. Chen H.Y. Brinster R.L. Palmiter R.D. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 7318-7322Crossref PubMed Scopus (249) Google Scholar, 5.Dalton T.P. Fu K. Palmiter R.D. Andrews G.K. J. Nutr. 1996; 126: 825-833Crossref PubMed Scopus (107) Google Scholar, 6.Andrews G.K. Geiser J. J. Nutr. 1999; 129: 1643-1648Crossref PubMed Scopus (62) Google Scholar). The rapid induction of MT-I and -II gene transcription by heavy metals (1.Andrews G.K. Prog. Food Nutr. Sci. 1990; 14: 193-258PubMed Google Scholar) is mediated by metal response elements (MREs), present in multiple copies in the proximal promoters of MT genes (7.Stuart G.W. Searle P.F. Palmiter R.D. Nature. 1985; 317: 828-831Crossref PubMed Scopus (270) Google Scholar). A protein that binds directly and specifically to MREs (8.Koizumi S. Suzuki K. Ogra Y. Yamada H. Otsuka F. Eur. J. Biochem. 1999; 259: 635-642Crossref PubMed Scopus (102) Google Scholar) and transactivates MT gene expression is referred to as MTF-1 (9.Westin G. Schaffner W. EMBO J. 1988; 7: 3763-3770Crossref PubMed Scopus (234) Google Scholar). MTF-1 is a six-zinc finger protein in the Cys2His2 family of transcription factors. Human, mouse, and pufferfish MTF-1 have been cloned (10.Radtke F. Heuchel R. Georgiev O. Hergersberg M. Gariglio M. Dembic Z. Schaffner W. EMBO J. 1993; 12: 1355-1362Crossref PubMed Scopus (339) Google Scholar, 11.Brugnera E. Georgiev O. Radtke F. Heuchel R. Baker E. Sutherland G.R. Schaffner W. Nucleic Acids Res. 1994; 22: 3167-3173Crossref PubMed Scopus (168) Google Scholar, 12.Maur A.A.D. Belser T. Elgar G. Georgiev O. Schaffner W. Biol. Chem. Hoppe Seyler. 1999; 380: 175-185Google Scholar), and this protein has been highly conserved, particularly in the zinc finger domain. The C terminus of mammalian MTF-1 contains three transactivation domains, which are acidic, proline-rich, and serine/threonine-rich, respectively (13.Radtke F. Georgiev O. Müller H.-P. Brugnera E. Schaffner W. Nucleic Acids Res. 1995; 23: 2277-2286Crossref PubMed Scopus (138) Google Scholar). The DNA binding activity of native and recombinant MTF-1 is reversibly modulated by zinc interactions with the finger domain (14.Dalton T.D. Bittel D. Andrews G.K. Mol. Cell. Biol. 1997; 17: 2781-2789Crossref PubMed Scopus (105) Google Scholar). The zinc fingers are heterogeneous in function and at least two exhibit low affinity binding of zinc (15.Chen X.H. Agarwal A. Giedroc D.P. Biochemistry. 1998; 37: 11152-11161Crossref PubMed Scopus (67) Google Scholar, 16.Chen X. Chu M. Giedroc D.P. Biochemistry. 1999; 38: 12915-12925Crossref PubMed Scopus (98) Google Scholar). Treatment of cells with zinc in vivo results in a rapid, dramatic increase in the DNA binding activity of MTF-1 measuredin vitro (10.Radtke F. Heuchel R. Georgiev O. Hergersberg M. Gariglio M. Dembic Z. Schaffner W. EMBO J. 1993; 12: 1355-1362Crossref PubMed Scopus (339) Google Scholar, 14.Dalton T.D. Bittel D. Andrews G.K. Mol. Cell. Biol. 1997; 17: 2781-2789Crossref PubMed Scopus (105) Google Scholar) and the concomitant occupancy of MREs in the MT-I promoter in vivo (3.Dalton T.P. Li Q. Bittel D. Liang L.C. Andrews G.K. J. Biol. Chem. 1996; 271: 26233-26241Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). In contrast, the DNA binding activity of MTF-1 is apparently not activated by transition metals other than zinc (8.Koizumi S. Suzuki K. Ogra Y. Yamada H. Otsuka F. Eur. J. Biochem. 1999; 259: 635-642Crossref PubMed Scopus (102) Google Scholar, 17.Koizumi S. Yamada H. Suzuki K. Otsuka F. Eur. J. Biochem. 1992; 210: 555-560Crossref PubMed Scopus (40) Google Scholar, 18.Bittel D. Dalton T. Samson S. Gedamu L. Andrews G.K. J. Biol. Chem. 1998; 273: 7127-7133Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar), although cadmium is a particularly potent inducer of MT gene expression. Homozygous deletion of the mouse MTF-1 gene revealed that MTF-1 is essential for zinc and cadmium induction, as well as for basal expression of the mouse MT-I and -II genes in embryonic stem cells (19.Heuchel R. Radtke F. Georgiev O. Stark G. Aguet M. Schaffner W. EMBO J. 1994; 13: 2870-2875Crossref PubMed Scopus (408) Google Scholar). MTF-1 is also essential for induction of these genes by oxidative stress (3.Dalton T.P. Li Q. Bittel D. Liang L.C. Andrews G.K. J. Biol. Chem. 1996; 271: 26233-26241Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar) and hypoxia (20.Murphy B.J. Andrews G.K. Bittel D. Discher D.J. McCue J. Green C.J. Yanovsky M. Giaccia A. Sutherland R.M. Laderoute K.R. Webster K.A. Cancer Res. 1999; 59: 1315-1322PubMed Google Scholar). Thus, several signal transduction pathways may impinge on the activities of MTF-1. Mice homozygous for targeted deletions of the MTF-1 gene die in utero due to failure of liver development, demonstrating that the MTF-1 gene is an essential gene (21.Günes Ç. Heuchel R. Georgiev O. Müller K.H. Lichtlen P. Blüthmann H. Marino S. Aguzzi A. Schaffner W. EMBO J. 1998; 17: 2846-2854Crossref PubMed Scopus (224) Google Scholar), unlike the mouse MT-I and -II genes (22.Masters B.A. Kelly E.J. Quaife C.J. Brinster R.L. Palmiter R.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 584-588Crossref PubMed Scopus (571) Google Scholar). Transition metal regulation of gene expression has been documented in species from every kingdom of organisms. In many instances, the transition metal itself directly interacts with a preexisting metalloregulatory protein, and this interaction leads to a change in conformation of the protein and an alteration in the DNA or RNA binding activity of the protein (23.O'Halloran T.V. Science. 1993; 261: 715-725Crossref PubMed Scopus (558) Google Scholar). The available evidence suggests that MTF-1 is a metalloregulatory protein that serves as an intracellular zinc sensor to activate gene expression. This model predicts that MTF-1 would be located in the cytoplasm to facilitate direct interaction with free zinc. Since previous studies have not addressed the subcellular localization of MTF-1, it is not clear whether this cellular response to metal ions is initiated in the cytosol or nucleus. Furthermore, it is unclear how MTF-1 senses the toxic metal cadmium. Therefore, we examined the effects of zinc and cadmium treatment on the subcellular localization and DNA binding activity of mouse MTF-1. The following reagents were used in this study:in vitro TnT coupled reticulocyte lysate transcription/translation system (Promega Corporation, Madison, WI); Microcon 10 (Millipore Corp., Bedford, MA); nonfat dry milk, protein assay reagent (Bio-Rad); NE-PER nuclear and cytoplasmic extraction reagents and BCA protein assay reagent (Pierce); Protran nitrocellulose membrane (Schleicher & Schuell); ECL Western blotting (immunoblotting) detection reagent, Hyperfilm ECL (Amersham Life Science, Arlington Heights, IL); X-Omat film for autoradiography (Eastman Kodak Co., Rochester, NY); LipofectAMINE and LipofectAMINE Plus (Life Technologies, Inc.); four chamber glass slides (Nalge Nunc International, Naperville, IL); rabbit polyclonal antibodies against Sp1 (PEP 2), USF1 (C-20) and FLAG-probe (D-8) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); goat anti-rabbit IgG conjugated to peroxidase (Jackson ImmunoResearch Laboratories, West Grove, PA); rat monoclonal antibody against Hsp90 and rabbit anti-rat IgG conjugated to peroxidase (Stressgen, Victoria, Canada); and the DAB kit (Zymed Laboratories Inc., San Francisco, CA). All other chemicals were purchased from Sigma. The polyclonal antiserum against purified bacterial recombinant mouse MTF-1 fused to glutathioneS-transferase was raised in rabbits (Covance Research Products, Inc., Denver, CO) and purified by protein A chromatography followed by passage through glutathioneS-transferase-agarose to remove glutathioneS-transferase antibodies (20.Murphy B.J. Andrews G.K. Bittel D. Discher D.J. McCue J. Green C.J. Yanovsky M. Giaccia A. Sutherland R.M. Laderoute K.R. Webster K.A. Cancer Res. 1999; 59: 1315-1322PubMed Google Scholar). Mouse Hepa cells were maintained in Dulbecco's modified Eagle's medium-high glucose (DMEM) supplemented with 2% fetal bovine serum (FBS). The mouse dko7 cell line is a simian virus 40 large T-antigen-immortalized fibroblast derived from embryonic stem cells lacking MTF-1 (MTF-1 double knockout) and was a generous gift of Dr. Walter Schaffner, University of Zurich (Zurich, Switzerland) (13.Radtke F. Georgiev O. Müller H.-P. Brugnera E. Schaffner W. Nucleic Acids Res. 1995; 23: 2277-2286Crossref PubMed Scopus (138) Google Scholar). These cells were maintained in DMEM supplemented with 10% FBS. For nuclear and cytosolic extract preparations, cells (2 × 106) were plated in 15-cm Petri dishes and grown to 80% confluency. For transfection followed by extract preparation, cells (1.2 × 105) were plated in six-well plates (9.4 cm2) and grown to 50% confluency. For transfection and subsequent immunocytochemistry, cells (2.5 × 104) were plated in four-chamber glass slides (1.8 cm2) and grown to 50% confluency. All FBS was heat-inactivated prior to use; all media were supplemented with 50 units/ml penicillin, 50 μg/ml streptomycin, and 2 mml-glutamine. Whole cell extracts, nuclear extracts (NEs), and cytosolic extracts (CEs) were prepared essentially as described (14.Dalton T.D. Bittel D. Andrews G.K. Mol. Cell. Biol. 1997; 17: 2781-2789Crossref PubMed Scopus (105) Google Scholar, 24.Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9164) Google Scholar). Briefly, for preparation of nuclear extracts, treated cells were placed on ice, the medium was removed, and cells were washed once with cold PBS. Cells were scraped off the dish and collected by centrifugation at 1,500 × g for 5 min. The cell pellet was resuspended in 5 ml of cell lysis buffer (10 mm HEPES (pH 7.9), 1.5 mm MgCl2, 10 mm KCl, 0.5 mm dithiothreitol, and 0.2 mm phenylmethylsulfonyl fluoride), and immediately centrifuged at 1,500 × g for 5 min. Cells were resuspended in 2 times the original packed cell volume of cell lysis buffer, allowed to swell on ice for 10 min, and homogenized with 10 strokes of a Dounce homogenizer (B pestle). Nuclei were collected by centrifugation at 3,300 × g for 15 min at 4 °C, and supernatant was saved for cytosolic extracts. The nuclei were resuspended, using six strokes of a Teflon-glass homogenizer, in 3 volumes (about 750 μl) of nuclear extraction buffer (20 mm HEPES, pH 7.9, 1.5 mm MgCl2, 400 mm KCl, 0.5 mm dithiothreitol, 0.2 mm phenylmethylsulfonyl fluoride, and 25% glycerol). The nuclear suspension was stirred on ice for 30 min and then centrifuged at 89,000 × g for 30 min. The supernatant was collected and concentrated in a Microcon 10 concentrator by centrifugation at 14,000 × g for 3 h at 4 °C. For preparation of CE, the supernatant obtained after removal of nuclei was mixed thoroughly with 0.11 volume of 10× cytoplasmic extraction buffer (1× cytoplasmic extraction buffer: 30 mmHEPES (pH 7.9) at 4 °C, 140 mm KCl, 3 mmMgCl2) and then centrifuged at 89,000 × gfor 1 h. The supernatant was collected and concentrated in a Microcon 10 concentrator by centrifugation at 14,000 ×g for 1 h at 4 °C. Protein concentration was determined using Bio-Rad Protein Assay reagent with bovine serum albumin as the standard. In transfection experiments, NE-PER nuclear and cytoplasmic extraction reagents was used to prepare extracts. However, because of the presence of EDTA, the addition of exogenous zinc was required to activate MTF-1 DNA binding activity in these extracts. SDS lysis of cells was performed on plates using 1× SDS sample buffer without reducing agent or bromphenol blue. Protein concentration was determined using BCA protein assay reagent and bovine serum albumin as the standard. EMSA was performed as described previously (3.Dalton T.P. Li Q. Bittel D. Liang L.C. Andrews G.K. J. Biol. Chem. 1996; 271: 26233-26241Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). Extracts (10–20 μg of protein in 2–5 μl) were incubated in a total volume of 20 μl for 15 min at 4 °C in binding reaction buffer containing 12 mm HEPES (pH 7.9), 60 mm KCl, 0.5 mm dithiothreitol, 12% glycerol, 5 mm MgCl2, 0.2 μg of dI-dC/μg of protein with 2–4 fmol of end-labeled double-stranded oligonucleotide MRE-s or Sp1 binding sequence (5,000 cpm/fmol) for MTF-1 or Sp1, respectively. Protein-DNA complexes were separated electrophoretically at 4 °C in 4% polyacrylamide gel (acrylamide/bisacrylamide, 80:1) at 15 V/cm. The gel was polymerized in running buffer consisting of 0.19 m glycine (pH 8.5), 25 mm Tris, and 0.5 mm EDTA. After electrophoresis, the gel was dried, and labeled complexes were detected by autoradiography. Cell extracts (50–100 μg of protein) were separated by 10 or 12% SDS-polyacrylamide gel electrophoresis (25.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar) under reduced conditions and transferred to nitrocellulose membranes. The membranes were blocked overnight at 4 °C in 10% nonfat dry milk in PBS, 0.1% Tween 20 and probed with primary antibody diluted in 3% nonfat dry milk in PBS, 0.1% Tween 20 for 1 h at room temperature. Membranes were then incubated with an appropriate secondary antibody conjugated to horseradish peroxidase diluted in 3% nonfat dry milk in PBS, 0.1% Tween 20 for 30 min at room temperature, developed by chemiluminescence, and exposed to hyperfilm ECL. Relative band intensities were quantitated using Biomax 1D image analysis software (Kodak Scientific Imaging Systems). Equal protein loading and transfer was verified visually by staining membranes with Ponceau solution. The CMV-MTF-1 expression vector was described previously (14.Dalton T.D. Bittel D. Andrews G.K. Mol. Cell. Biol. 1997; 17: 2781-2789Crossref PubMed Scopus (105) Google Scholar). The MTF-1-FLAG construct was created by polymerase chain reaction amplification from this template using a sense primer that encompassed the translation start codon and an antisense primer against the carboxyl terminus that also incorporated the FLAG coding sequence. The amplified product was cloned into the CMV vector. Vectors were verified by DNA sequencing. dko7 cells were transfected using LipofectAMINE according to the manufacturer's instructions. Cells were grown to ∼50% confluence. After washing the cells with serum-free DMEM, DNA and LipofectAMINE mixture prepared in serum-free DMEM was added. For immunocytochemistry, the mixture consisted of 2 μl/well LipofectAMINE, 4 ng/well CMV-MTF-1-FLAG expression vector, and 300 ng/well SV-β-gal, as an internal control for transfection efficiency, in 250 μl of DMEM. In experiments in which preparation of nuclear and cytosolic extracts was performed, cells were treated with 4 μl/well LipofectAMINE, 100 ng/well CMV-MTF-1 expression vector, and 1 μg/well SV-β-gal in 1.2 ml of DMEM. After 5 h, an equal volume of DMEM containing 2× FBS was added, and the incubation was continued overnight. The following morning, the medium was removed and replaced with fresh DMEM containing 1× FBS. Zinc treatment was initiated in the afternoon of day 2. After 1 h, cells were processed for either immunocytochemistry, as described below, or for nuclear and cytosolic protein isolation. Twenty-four hours after transfection, dko7 cells were washed twice with serum-free medium and then incubated for 6 h in DMEM containing 1% (w/v) bovine serum albumin. Cells were then treated for 30 min with 100 μmZnSO4 in this medium, washed with PBS, and fixed with 70% ethanol for 5 min. Slides were blocked for 1 h at room temperature with 10% goat serum in PBS-Triton X-100 and incubated overnight at 4 °C with rabbit polyclonal FLAG antibody or Sp1 antibody diluted 1:500 or 1:100, respectively. Slides were then incubated with anti-rabbit IgG conjugated to peroxidase and stained with a DAB kit. Synthesis of recombinant mouse MTF-1 was performed using the TnT coupled reticulocyte lysate transcription/translation system (TnT lysate), as described in detail previously (18.Bittel D. Dalton T. Samson S. Gedamu L. Andrews G.K. J. Biol. Chem. 1998; 273: 7127-7133Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Previous studies demonstrated that the rabbit polyclonal antisera against bacterially expressed glutathione S-transferase-MTF-1 was specific for MTF-1 in supershift EMSA (20.Murphy B.J. Andrews G.K. Bittel D. Discher D.J. McCue J. Green C.J. Yanovsky M. Giaccia A. Sutherland R.M. Laderoute K.R. Webster K.A. Cancer Res. 1999; 59: 1315-1322PubMed Google Scholar). The specificity of this MTF-1 polyclonal antisera was examined by immunoblotting (Fig.1). Recombinant mouse MTF-1 synthesizedin vitro in a TnT lysate system was used as a positive control (Fig. 1, lane 1), and an extract from dko7 (MTF-1 double knockout) cells was used as a negative control (Fig.1, lane 2). Mouse MTF-1 migrates with an apparent molecular mass of ∼100 kDa (Fig. 1, lane 3), despite its predicted size of 72.5 kDa (10.Radtke F. Heuchel R. Georgiev O. Hergersberg M. Gariglio M. Dembic Z. Schaffner W. EMBO J. 1993; 12: 1355-1362Crossref PubMed Scopus (339) Google Scholar). This aberrant mobility may reflect the clustering of acidic, serine, and proline residues in the structure of MTF-1 (10.Radtke F. Heuchel R. Georgiev O. Hergersberg M. Gariglio M. Dembic Z. Schaffner W. EMBO J. 1993; 12: 1355-1362Crossref PubMed Scopus (339) Google Scholar, 13.Radtke F. Georgiev O. Müller H.-P. Brugnera E. Schaffner W. Nucleic Acids Res. 1995; 23: 2277-2286Crossref PubMed Scopus (138) Google Scholar) and is not unique among transcription factors (26.Van Beveren C. van Straaten F. Curran T. Muller R. Verma I.M. Cell. 1983; 32: 1241-1255Abstract Full Text PDF PubMed Scopus (368) Google Scholar, 27.Chan J.Y. Han X.L. Kan Y.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11371-11375Crossref PubMed Scopus (296) Google Scholar). The MTF-1 band was absent in extracts from dko7 cells (Fig. 1, lane 2) but was detected in whole cell extracts from mouse Hepa cells (Fig. 1, lane 3) and from dko7 cells transiently transfected with an MTF-1 expression vector (see below). Two other bands with apparent molecular masses of ∼200 and ∼57 kDa were detected in cell extracts from both dko7 and Hepa cells (Fig. 1). The subcellular distribution of MTF-1 in untreated and zinc-treated cells was investigated by immunoblotting. Nuclear and cytosolic extracts were prepared from mouse Hepa cells, and it was noted that approximately 4-fold more protein was extracted in the cytosolic versus the nuclear extracts obtained from the same number of cells. On average, these extraction procedures recovered 88 pg of cytosolic protein and 21 pg of nuclear protein per cell. Immunoblotting of these extracts was performed using MTF-1 antiserum, and extracted proteins were normalized per cell for analysis. To account for differences in the amount of protein recovered in nuclear versus cytosolic extracts, 50 μg of nuclear and 200 μg of cytosolic protein were loaded (Fig.2 A, right panel). Quantitation of relative intensities of the MTF-1 bands in these samples suggested that 17 ± 9% of the immunoreactive MTF-1 was extracted in the nuclear fraction whereas 83 ± 9% of the MTF-1 was extracted in the cytosolic fraction from control cells. In the remaining figures, equal quantities of protein per lane were applied to the gels. Some variability between experiments in the amount of MTF-1 extracted from nuclei versus cytosol was noted, as is demonstrated by the S.D. value shown above. This variability was accentuated in the nuclear extracts relative to cytoplasmic extracts and may reflect subtle differences in cell density, cell passage number, or culture conditions. Each experiment contained an internal control of untreated cells cultured in parallel under identical conditions. In contrast to the results obtained using extracts from untreated Hepa cells, immunoblotting revealed that all MTF-1 immunoreactivity was present in nuclear extracts from cells treated with 100 μm ZnSO4 for 1 h. The cytosolic extract was devoid of detectable MTF-1 (Fig. 2 A). Fig. 2 A(left panel) is an immunoblot where equal amounts of nuclear and cytosolic proteins were applied to the gel. The amount of immunoreactive MTF-1 detected in nuclear extracts increased about 4-fold after zinc treatment of the cells. This is consistent with the data suggesting that about 83% of MTF-1 is initially found in the cytosolic fraction from untreated cells. Immunoblot analysis of proteins remaining in the nuclear pellet fraction after extraction revealed no MTF-1, Sp1, and USF1. Thus, MTF-1 is not preferentially extracted from nuclei of zinc-treated cells (data not shown). In contrast to MTF-1, two other transcription factors, known for their constitutive nuclear localization, Sp1 (Fig. 2 B) and USF1 (Fig. 2 C), were detected only in nuclear extracts, and the amount of immunoreactive protein was unaffected by zinc treatment. Thus, the cytosolic extracts were not significantly contaminated with nuclear transcription factors. Furthermore, immunoblotting with antisera against the predominantly cytosolic heat shock protein 90 (Hsp90) (28.Ali A. Bharadwaj S. O'Carroll R. Ovsenek N. Mol. Cell. Biol. 1998; 18: 4949-4960Crossref PubMed Scopus (244) Google Scholar, 29.Kang K.I. Devin J. Cadepond F. Jibard N. Guiochon-Mantel A. Baulieu E.E. Catelli M.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 340-344Crossref PubMed Scopus (121) Google Scholar) revealed that the majority of Hsp90 was detected in cytosolic extracts (Fig. 2 D). Thus, nuclear extracts were not significantly contaminated with cytosolic proteins. EMSA was used to detect the MRE binding activity of MTF-1 (3.Dalton T.P. Li Q. Bittel D. Liang L.C. Andrews G.K. J. Biol. Chem. 1996; 271: 26233-26241Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 10.Radtke F. Heuchel R. Georgiev O. Hergersberg M. Gariglio M. Dembic Z. Schaffner W. EMBO J. 1993; 12: 1355-1362Crossref PubMed Scopus (339) Google Scholar, 14.Dalton T.D. Bittel D. Andrews G.K. Mol. Cell. Biol. 1997; 17: 2781-2789Crossref PubMed Scopus (105) Google Scholar) in extracts from untreated and zinc-treated Hepa cells. Nuclear and cytosolic extracts from untreated cells contained little MTF-1 that was active to bind to DNA (Fig.3 A). Previous studies demonstrated that MTF-1 in whole cell extracts from control cells can be activated in vitro by the addition of zinc (5–30 μm) followed by incubation at 37 °C (14.Dalton T.D. Bittel D. Andrews G.K. Mol. Cell. Biol. 1997; 17: 2781-2789Crossref PubMed Scopus (105) Google Scholar). After zinc treatment, however, the DNA binding activity of MTF-1 increased 8–12-fold in nuclear extracts, while cytosolic extracts exhibited no detectable MTF-1 DNA binding activity (Fig. 3 A). The identity of the MTF-1·MRE-s complex was confirmed by supershift EMSA using the MTF-1 antisera (data not shown). The rapid and dramatic increase in MTF-1 DNA binding activity in nuclear extracts from cells treated with zinc correlates with the immunoblotting data and suggest that zinc induces the nuclear translocation and activation of MTF-1. Sp1 was detected only in nuclear extracts, and its DNA binding activity was unaffected by exogenous zinc (Fig. 3 B). A time course for zinc-dependent nuclear accumulation of MTF-1 protein and of MRE binding activity of MTF-1 was determined using Hepa cells treated with 100 μm ZnSO4 (Fig.4). The amount of immunoreactive MTF-1 in the nucleus increased about 2-fold by 5 min after the addition of zinc and 4-fold by 30 min (Fig. 4 A). As a control for potential differences in protein loading, MTF-1 was compared with immunoreactive USF1 in these same extracts (Fig. 4 A). Nuclear USF1 levels remained constant during zinc treatment. MRE binding activity of MTF-1 was monitored by EMSA (Fig. 4 B). A 2.5-fold increase in MRE binding activity was detected by 5 min after zinc treatment and a 7-fold increase was detected by 30 min, consistent with previous observations (18.Bittel D. Dalton T. Samson S. Gedamu L. Andrews G.K. J. Biol. Chem. 1998; 273: 7127-7133Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Sp1 DNA binding activity remained constant (Fig.4 B). Immunoblotting of total cell SDS extracts was used to determine whether zinc causes a rapid change in the steady state levels of MTF-1 in Hepa cells. Hepa cells were treated with 100 μm ZnSO4 for 1 h, and the cells were lysed in situ in SDS sample buffer. Equal amounts of SDS-extracted proteins were then examined by immunoblotting. There was no detectable change in the amount of immunoreactive MTF-1 after this zinc treatment (Fig. 5). Likewise, there were no detectable changes in the relative amount of immunoreactive Sp1 or USF1. In addition, levels of immunoreactive MTF-1 in whole cell extracts from control and zinc-treated Hepa cells were unaffected by pretreatment of the cells with cycloheximide (10 nm to 100 μm) for 1 h (data not shown). Taken together, these data indicate that zinc does not cause a detectable increase in the steady state levels of MTF-1 protein within 1 h in these cells, nor is MTF-1 protein rapidly degraded. To further address the effects of zinc on the subcellular distri