Artículo 1-Cadmio Metalotioneinas

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Glen K. AndrewsRudravajhala Ravindra, Huimin Jiang andIrina V. Smirnova, Douglas C. Bittel, Element-binding Transcription Factor-1Nuclear Translocation of Metal ResponseZinc and Cadmium Can Promote RapidREGULATION:GENES: STRUCTURE AND

doi: 10.1074/jbc.275.13.9377 2000, 275:9377-9384.J. Biol. Chem.

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

(Received for publication, October 29, 1999, and in revised form, December 28, 1999)

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

From the Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas 66160-7421

Metal response element-binding transcription fac-tor-1 (MTF-1) is a six-zinc finger protein that plays anessential role in activating metallothionein expressionin response to the heavy metals zinc and cadmium. Lowaffinity interactions between zinc and specific zinc fin-gers in MTF-1 reversibly regulate its binding to themetal response elements in the mouse metallothionein-Ipromoter. This study examined the subcellular distribu-tion and DNA binding activity of MTF-1 in cells treatedwith zinc or cadmium. Immunoblot analysis of cytosolic

and nuclear extracts demonstrated that in untreatedcells, about 83% of MTF-1 is found in the cytosolic ex-tracts and is not activated to bind to DNA. In sharpcontrast, within 30 min of zinc treatment (100 M),MTF-1 is detected only in nuclear extracts and is acti-vated to bind to DNA. The activation to bind to DNA and nuclear translocation of MTF-1 occurs in the ab-sence of increased MTF-1 content in the cell. Further-more, immunocytochemical localization and immuno-blotting assays demonstrated that zinc induces thenuclear translocation of MTF-1-FLAG, expressed fromthe cytomegalovirus promoter in transiently trans-fected dko7 (MTF-1 double knockout) cells. Immunoblotanalysis of cytosolic and nuclear extracts from cadmi-um-treated cells demonstrated that concentrations of

cadmium (10 M) that actively induce metallothioneingene expression cause only a small increase in theamount of nuclear MTF-1. In contrast, an overtly toxicconcentration of cadmium (50 M) rapidly induced thecomplete nuclear translocation and activation of DNA binding activity of MTF-1. These studies are consistentwith the hypothesis that MTF-1 serves as a zinc sensorthat responds to changes in cytosolic free zinc concen-trations. In addition, these data suggest that cadmiumactivation of metallothionein gene expression may beaccompanied by only small changes in nuclear MTF-1.

Metallothioneins (MT) 1 are small cysteine-rich proteins,

which play a role in zinc homeostasis, cadmium detoxication,and protection from reactive free radicals (16). The rapidinduction of MT-I and -II gene transcription by heavy metals(1) is mediated by metal response elements (MREs), present inmultiple copies in the proximal promoters of MT genes (7). A protein that binds directly and specifically to MREs (8) andtransactivates MT gene expression is referred to as MTF-1 (9).

MTF-1 is a six-zinc finger protein in the Cys 2 His 2 family of transcription factors. Human, mouse, and pufferfish MTF-1 havebeen cloned (1012), and this protein has been highly conserved,particularly in the zinc finger domain. The C terminus of mam-malian MTF-1contains three transactivation domains, whichareacidic, proline-rich, and serine/threonine-rich, respectively (13).The DNA binding activity of native and recombinant MTF-1 isreversibly modulated by zinc interactions with the finger domain(14). The zinc fingers are heterogeneous in function and at leasttwo exhibit low affinity binding of zinc (15, 16).

Treatment of cells with zinc in vivo results in a rapid, dra-matic increase in the DNA binding activity of MTF-1 measuredin vitro (10, 14) and the concomitant occupancy of MREs in theMT-I promoter in vivo (3). In contrast, the DNA binding activ-ity of MTF-1 is apparently not activated by transition metalsother than zinc (8, 17, 18), although cadmium is a particularlypotent inducer of MT gene expression. Homozygous deletion of the mouse MTF-1 gene revealed that MTF-1 is essential forzinc and cadmium induction, as well as for basal expression of the mouse MT-I and -II genes in embryonic stem cells (19).MTF-1 is also essential for induction of these genes by oxida-tive stress (3) and hypoxia (20). Thus, several signal transduc-tion pathways may impinge on the activities of MTF-1. Micehomozygous for targeted deletions of the MTF-1 gene die inutero due to failure of liver development, demonstrating thatthe MTF-1 gene is an essential gene (21), unlike the mouseMT-I and -II genes (22).

Transition metal regulation of gene expression has beendocumented in species from every kingdom of organisms. Inmany instances, the transition metal itself directly interactswith a preexisting metalloregulatory protein, and this interac-tion leads to a change in conformation of the protein and analteration in the DNA or RNA binding activity of the protein(23). The available evidence suggests that MTF-1 is a metal-loregulatory protein that serves as an intracellular zinc sensorto activate gene expression. This model predicts that MTF-1would be located in the cytoplasm to facilitate direct interactionwith free zinc. Since previous studies have not addressed thesubcellular localization of MTF-1, it is not clear whether thiscellular response to metal ions is initiated in the cytosol ornucleus. Furthermore, it is unclear how MTF-1 senses the toxicmetal cadmium. Therefore, we examined the effects of zinc andcadmium treatment on the subcellular localization and DNA binding activity of mouse MTF-1.

* This work was supported by National Institutes of Health Grant ES05704 (to G. K. A.), and National Research Service Award F32 ES05753 (to D. C. B.). The costs of publication of this article were defrayedin part by the payment of page charges. This article must therefore behereby marked advertisement in accordance with 18 U.S.C. Section1734 solely to indicate this fact.

To whom correspondence should be addressed: G. K. Andrews,Department of Biochemistry and Molecular Biology, University of Kan-sas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160-7421.Tel.: 913-588-6935; Fax: 913-588-7035; E-mail: [email protected].

1 The abbreviations used are: MT, metallothionein; CE, cytosolic ex-tract; DMEM, Dulbeccos modified Eagles medium-high glucose;EMSA, electrophoretic mobility shift assay; FBS, fetal bovine serum;MRE, metal response elements; MTF-1, metal response element-bind-ing transcription factor-1; NE, nuclear extract; NLS, nuclear localiza-tion signal; PBS, phosphate-buffered saline; CMV, cytomegalovirus.

THE J OURNAL OF B IOLOGICAL CHEMISTRY Vol. 275, No. 13, Issue of March 31, pp. 93779384, 2000 2000 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

This paper is available on line at http://www.jbc.org 9377


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EXPERIMENTAL PROCEDURES

Materials The following reagents were used in this study: in vitroTnT coupled reticulocyte lysate transcription/translation system (Pro-mega Corporation, Madison, WI); Microcon 10 (Millipore Corp., Bed-ford, MA); nonfat dry milk, protein assay reagent (Bio-Rad); NE-PERnuclear and cytoplasmic extraction reagents and BCA protein assayreagent (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.); fourchamber glass slides (Nalge Nunc International, Naperville, IL); rabbitpolyclonal antibodies against Sp1 (PEP 2), USF1 (C-20) and FLAG-probe (D-8) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); goatanti-rabbit IgG conjugated to peroxidase (Jackson ImmunoResearchLaboratories, West Grove, PA); rat monoclonal antibody against Hsp90and 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 polyclonalantiserum against purified bacterial recombinant mouse MTF-1 fusedto glutathione S -transferase was raised in rabbits (Covance ResearchProducts, Inc., Denver, CO) and purified by protein A chromatographyfollowed by passage through glutathione S-transferase-agarose to re-move glutathione S -transferase antibodies (20).

Cell Culture Mouse Hepa cells were maintained in Dulbeccos mod-ified Eagles medium-high glucose (DMEM) supplemented with 2% fetalbovine serum (FBS). The mouse dko7 cell line is a simian virus 40 largeT-antigen-immortalized fibroblast derived from embryonic stem cellslacking MTF-1 (MTF-1 double knockout) and was a generous gift of Dr.Walter Schaffner, University of Zurich (Zurich, Switzerland) (13).These cells were maintained in DMEM supplemented with 10% FBS.For nuclear and cytosolic extract preparations, cells (2 10 6 ) wereplated in 15-cm Petri dishes and grown to 80% confluency. For trans-fection followed by extract preparation, cells (1.2 10 5 ) were plated insix-well plates (9.4 cm 2 ) and grown to 50% confluency. For transfectionand subsequent immunocytochemistry, cells (2.5 10 4 ) were plated infour-chamber glass slides (1.8 cm 2 ) and grown to 50% confluency. All FBSwas heat-inactivated prior to use; all media were supplemented with 50units/ml penicillin, 50 g/ml streptomycin, and 2 m M L-glutamine.

Preparation of Cell Extracts Whole cell extracts, nuclear extracts(NEs), and cytosolic extracts (CEs) were prepared essentially as de-scribed (14, 24). Briefly, for preparation of nuclear extracts, treatedcells were placed on ice, the medium was removed, and cells were

washed once with cold PBS. Cells were scraped off the dish and col-lected by centrifugation at 1,500 g for 5 min. The cell pellet wasresuspended in 5 ml of cell lysis buffer (10 m M HEPES (pH 7.9), 1.5 m MMgCl 2 , 10 m M KCl, 0.5 m M dithiothreitol, and 0.2 m M phenylmethyl-sulfonyl fluoride), and immediately centrifuged at 1,500 g for 5 min.Cells were resuspended in 2 times the original packed cell volume of celllysis buffer, allowed to swell on ice for 10 min, and homogenized with 10strokes of a Dounce homogenizer (B pestle). Nuclei were collected bycentrifugation at 3,300 g for 15 min at 4 C, and supernatant wassaved for cytosolic extracts. The nuclei were resuspended, using sixstrokes of a Teflon-glass homogenizer, in 3 volumes (about 750 l) of nuclear extraction buffer (20 m M HEPES, pH 7.9, 1.5 m M MgCl 2 , 400m M KCl, 0.5 m M dithiothreitol, 0.2 m M phenylmethylsulfonyl fluoride,and 25% glycerol). The nuclear suspension was stirred on ice for 30 minand then centrifuged at 89,000 g for 30 min. The supernatant wascollected and concentrated in a Microcon 10 concentrator by centrifu-gation at 14,000 g for 3 h at 4 C. For preparation of CE, the superna-tant obtained after removal of nuclei was mixed thoroughly with 0.11 volume of 10 cytoplasmic extraction buffer (1 cytoplasmic extractionbuffer: 30 m M HEPES (pH 7.9) at 4 C, 140 m M KCl, 3 m M MgCl 2 ) andthen centrifuged at 89,000 g for 1 h. The supernatant was collected andconcentrated in a Microcon 10 concentrator by centrifugation at 14,000 g for 1 h at 4 C. Protein concentration was determined using Bio-RadProtein Assay reagent with bovine serum albumin as the standard.

In transfection experiments, NE-PER nuclear and cytoplasmic ex-traction reagents was used to prepare extracts. However, because of thepresence of EDTA, the addition of exogenous zinc was required toactivate MTF-1 DNA binding activity in these extracts.

Preparation of Total Protein SDS Extracts SDS lysis of cells wasperformed on plates using 1 SDS sample buffer without reducing agent or bromphenol blue. Protein concentration was determined using BCA protein assay reagent and bovine serum albumin as the standard.

Electrophoretic Mobility Shift Assay (EMSA) EMSA was performed

as described previously (3). Extracts (1020 g of protein in 25 l)

were incubated in a total volume of 20 l for 15 min at 4 C in binding reaction buffer containing 12 m M HEPES (pH 7.9), 60 m M KCl, 0.5 m Mdithiothreitol, 12% glycerol, 5 m M MgCl 2 , 0.2 g of dI-dC/ g of proteinwith 24 fmol of end-labeled double-stranded oligonucleotide MRE-s orSp1 binding sequence (5,000 cpm/fmol) for MTF-1 or Sp1, respectively.Protein-DNA complexes were separated electrophoretically at 4 C in4% polyacrylamide gel (acrylamide/bisacrylamide, 80:1) at 15 V/cm. Thegel was polymerized in running buffer consisting of 0.19 M glycine (pH8.5), 25 m M Tris, and 0.5 m M EDTA. After electrophoresis, the gel wasdried, and labeled complexes were detected by autoradiography.

Immunoblotting Cell extracts (50100 g of protein) were sepa-rated by 10 or 12% SDS-polyacrylamide gel electrophoresis (25) underreduced conditions and transferred to nitrocellulose membranes. Themembranes were blocked overnight at 4 C in 10% nonfat dry milk inPBS, 0.1% Tween 20 and probed with primary antibody diluted in 3%nonfat dry milk in PBS, 0.1% Tween 20 for 1 h at room temperature.Membranes were then incubated with an appropriate secondary anti-body conjugated to horseradish peroxidase diluted in 3% nonfat drymilk in PBS, 0.1% Tween 20 for 30 min at room temperature, developedby chemiluminescence, and exposed to hyperfilm ECL. Relative bandintensities were quantitated using Biomax 1D image analysis software(Kodak Scientific Imaging Systems). Equal protein loading and transferwas verified visually by staining membranes with Ponceau solution.

Expression Vector Construction The CMV-MTF-1 expression vectorwas described previously (14). The MTF-1-FLAG construct was createdby polymerase chain reaction amplification from this template using asense primer that encompassed the translation start codon and an

antisense primer against the carboxyl terminus that also incorporatedthe FLAG coding sequence. The amplified product was cloned into theCMV vector. Vectors were verified by DNA sequencing.

Transient Transfection dko7 cells were transfected using Lipo-fectAMINE according to the manufacturers instructions. Cells weregrown to 50% confluence. After washing the cells with serum-freeDMEM, DNA and LipofectAMINE mixture prepared in serum-freeDMEM was added. For immunocytochemistry, the mixture consisted of 2 l/well LipofectAMINE, 4 ng/well CMV-MTF-1-FLAG expression vec-tor, and 300 ng/well SV- -gal, as an internal control for transfectionefficiency, in 250 l of DMEM. In experiments in which preparation of nuclear and cytosolic extracts was performed, cells were treated with 4

l/well LipofectAMINE, 100 ng/well CMV-MTF-1 expression vector,and 1 g/well SV- -gal in 1.2 ml of DMEM. After 5 h, an equal volumeof DMEM containing 2 FBS was added, and the incubation wascontinued overnight. The following morning, the medium was removedand replaced with fresh DMEM containing 1 FBS. Zinc treatment wasinitiated in the afternoon of day 2. After 1 h, cells were processed foreither immunocytochemistry, as described below, or for nuclear andcytosolic protein isolation.

Immunocytochemistry Twenty-four hours after transfection, dko7cells were washed twice with serum-free medium and then incubatedfor 6 h in DMEM containing 1% (w/v) bovine serum albumin. Cells werethen treated for 30 min with 100 M ZnSO 4 in this medium, washedwith PBS, and fixed with 70% ethanol for 5 min. Slides were blocked for1 h at room temperature with 10% goat serum in PBS-Triton X-100 andincubated overnight at 4 C with rabbit polyclonal FLAG antibody orSp1 antibody diluted 1:500 or 1:100, respectively. Slides were thenincubated with anti-rabbit IgG conjugated to peroxidase and stainedwith a DAB kit.

In Vitro Transcription/Translation of Mouse MTF-1 Synthesis of recombinant mouse MTF-1 was performed using the TnT coupled re-ticulocyte lysate transcription/translation system (TnT lysate), as de-scribed in detail previously (18).

RESULTS

Specificity of Mouse MTF-1 Antisera Previous studies dem-onstrated that the rabbit polyclonal antisera against bacteri-ally expressed glutathione S-transferase-MTF-1 was specificfor MTF-1 in supershift EMSA (20). The specificity of thisMTF-1 polyclonal antisera was examined by immunoblotting (Fig. 1). Recombinant mouse MTF-1 synthesized in vitro in aTnT lysate system was used as a positive control (Fig. 1, lane1), and an extract from dko7 (MTF-1 double knockout) cells wasused as a negative control (Fig. 1, lane 2 ). Mouse MTF-1 mi-grates with an apparent molecular mass of 100 kDa (Fig. 1,lane 3 ), despite its predicted size of 72.5 kDa (10). This aber-rant mobility may reflect the clustering of acidic, serine, and

proline residues in the structure of MTF-1 (10, 13) and is not

MTF-1 Translocates to the Nucleus9378


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unique among transcription factors (26, 27). The MTF-1 bandwas absent in extracts from dko7 cells (Fig. 1, lane 2 ) but wasdetected in whole cell extracts from mouse Hepa cells (Fig. 1,lane 3 ) and from dko7 cells transiently transfected with anMTF-1 expression vector (see below). Two other bands withapparent molecular masses of 200 and 57 kDa were de-tected in cell extracts from both dko7 and Hepa cells (Fig. 1).

MTF-1 Is Localized in the Cytosol in Untreated Cells and inthe Nucleus in Zinc-treated Cells The subcellular distributionof MTF-1 in untreated and zinc-treated cells was investigatedby immunoblotting. Nuclear and cytosolic extracts were pre-pared from mouse Hepa cells, and it was noted that approxi-mately 4-fold more protein was extracted in the cytosolic versusthe nuclear extracts obtained from the same number of cells.On average, these extraction procedures recovered 88 pg of cytosolic protein and 21 pg of nuclear protein per cell. Immu-noblotting of these extracts was performed using MTF-1 anti-serum, and extracted proteins were normalized per cell foranalysis. To account for differences in the amount of proteinrecovered in nuclear versus cytosolic extracts, 50 g of nuclearand 200 g of cytosolic protein were loaded (Fig. 2 A, right panel ). Quantitation of relative intensities of the MTF-1 bandsin these samples suggested that 17 9% of the immunoreac-tive MTF-1 was extracted in the nuclear fraction whereas 839% of the MTF-1 was extracted in the cytosolic fraction fromcontrol cells. In the remaining figures, equal quantities of pro-tein per lane were applied to the gels. Some variability betweenexperiments in the amount of MTF-1 extracted from nucleiversus cytosol was noted, as is demonstrated by the S.D. valueshown above. This variability was accentuated in the nuclear

F IG . 1. Characterization of a rabbit polyclonal antiseraagainst mouse MTF-1. Proteins were separated by 12% SDS-poly-acrylamide gel electrophoresis followed by immunoblotting as describedunder Experimental Procedures. Lane 1 , recombinant mouse MTF-1synthesized in vitro in a TnT coupled reticulocyte lysate system; lane 2 ,dko7 (MTF-1 double knockout) whole cell extract; lane 3 , Hepa wholecell extract. The arrow shows the position of MTF-1. Relative mobilitiesof molecular mass protein markers (kDa) are indicated to the left .

F IG . 2. Immunoblot detection of MTF-1 in nuclear and cytosolic extracts from Hepa cells treated with zinc. Hepa cells were treatedwith 100 M ZnSO 4 for 1 h. NEs and CEs were prepared from treated and untreated cells and analyzed by immunoblotting using antisera againstthe following: mouse MTF-1 ( A); Sp1 ( B); USF1 ( C); or Hsp90 ( D). A, the left panel represents a membrane where equal amounts of protein (100

g/lane) were loaded onto each lane; the right panel shows a membrane where 50 and 200 g of protein/lane was loaded for nuclear and cytosolicextracts, respectively, to normalize per cell number (see text for explanation). Arrows show the positions of MTF-1, Sp1, USF1, and Hsp90. Relative

mobilities of molecular mass protein markers (kDa) are indicated to the left .

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extracts relative to cytoplasmic extracts and may reflect subtledifferences in cell density, cell passage number, or cultureconditions. Each experiment contained an internal control of untreated cells cultured in parallel under identical conditions.

In contrast to the results obtained using extracts from un-

treated Hepa cells, immunoblotting revealed that all MTF-1 im-

munoreactivity waspresent in nuclear extracts from cells treatedwith 100 M ZnSO 4 for 1 h. The cytosolic extract was devoid of detectable MTF-1 (Fig. 2 A). Fig. 2 A (left panel ) is an immunoblotwhere equal amounts of nuclear and cytosolic proteins wereapplied to the gel. The amount of immunoreactive MTF-1 de-

tected in nuclear extracts increased about 4-fold after zinc treat-

F IG . 3. EMSA detection of MTF-1 innuclear and cytosolic extracts fromHepa cells treated with zinc. Hepacells were treated with 100 M ZnSO 4 for

1 h. NEs and CEs were prepared fromtreated and untreated cells and analyzedfor DNA binding activity using a labeledMRE-s oligonucleotide ( A), or an Sp1 fam-ily specific oligonucleotide ( B). The arrowspointto specific complexes of MTF-1 or Sp1and their respective oligonucleotides.

F IG . 4. Immunoblot andEMSA detection of MTF-1 in nuclear extracts from Hepa cells at different times after zinc treatment. Hepacells were treated with 100 M ZnSO 4 for 5 or 30 min. A, nuclear extracts were prepared from treated and untreated cells and analyzed byimmunoblotting. Upper panel , recombinant mouse MTF-1 synthesized in vitro in a TnT lysate was used as a positive control. Lower panel , theextracts were immunoblotted with USF1 antibody. The arrows show the positions of MTF-1 and USF1. B, upper panel , nuclear extracts wereanalyzed for DNA binding activity using a labeled MTF-1-specific MRE-s oligonucleotide, as described under Experimental Procedures. Lower panel , the same extracts were assayed using an Sp1-specific oligonucleotide. The arrows point to specific MTF-1 and Sp1 complexes with theirrespective oligonucleotides.



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ment of the cells. This is consistent with the data suggesting thatabout 83% of MTF-1 is initially found in the cytosolic fractionfrom 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 fromnuclei of zinc-treated cells (data not shown). In contrast toMTF-1, two other transcription factors, known for their consti-tutive nuclear localization, Sp1 (Fig. 2 B) and USF1 (Fig. 2 C),were detected only in nuclear extracts, and the amount of immu-noreactive protein was unaffected by zinc treatment. Thus, thecytosolic extracts were not significantly contaminated with nu-

clear transcription factors. Furthermore, immunoblotting withantisera against the predominantly cytosolic heat shock protein90 (Hsp90) (28, 29) revealed that the majority of Hsp90 wasdetected in cytosolic extracts (Fig. 2 D). Thus, nuclear extractswere not significantly contaminated with cytosolic proteins.

Nuclear Localization of MTF-1 Is Accompanied by Activationof DNA Binding Activity EMSA was used to detect the MREbinding activity of MTF-1 (3, 10, 14) in extracts from untreatedand zinc-treated Hepa cells. Nuclear and cytosolic extractsfrom untreated cells contained little MTF-1 that was active tobind to DNA (Fig. 3 A). Previous studies demonstrated thatMTF-1 in whole cell extracts from control cells can be activatedin vitro by the addition of zinc (530 M) followed by incubationat 37 C (14). After zinc treatment, however, the DNA binding activity of MTF-1 increased 812-fold in nuclear extracts,while cytosolic extracts exhibited no detectable MTF-1 DNA binding activity (Fig. 3 A). The identity of the MTF-1 MRE-scomplex was confirmed by supershift EMSA using the MTF-1antisera (data not shown). The rapid and dramatic increase inMTF-1 DNA binding activity in nuclear extracts from cellstreated with zinc correlates with the immunoblotting data andsuggest that zinc induces the nuclear translocation and activa-tion of MTF-1. Sp1 was detected only in nuclear extracts, and itsDNAbinding activity was unaffected by exogenous zinc (Fig. 3 B).

Zinc-induced Nuclear Accumulation of MTF-1 Is Rapid A time course for zinc-dependent nuclear accumulation of MTF-1protein and of MRE binding activity of MTF-1 was determinedusing Hepa cells treated with 100 M ZnSO 4 (Fig. 4). The amountof immunoreactive MTF-1 in the nucleus increased about 2-foldby 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-1was compared with immunoreactive USF1 in these same ex-tracts (Fig. 4 A). Nuclear USF1 levels remained constant during zinc treatment. MRE binding activity of MTF-1 wasmonitoredbyEMSA (Fig. 4 B). A 2.5-fold increase in MRE binding activity wasdetected by 5 min after zinc treatment and a 7-fold increase wasdetected by 30 min, consistent with previous observations (18).

Sp1 DNA binding activity remained constant (Fig. 4 B). Zinc Treatment Does Not Alter the Amount of MTF-1 Pro-

tein Immunoblotting of total cell SDS extracts was used todetermine whether zinc causes a rapid change in the steadystate levels of MTF-1 in Hepa cells. Hepa cells were treatedwith 100 M ZnSO 4 for 1 h, and the cells were lysed in situ inSDS sample buffer. Equal amounts of SDS-extracted proteinswere then examined by immunoblotting. There was no detect-able change in the amount of immunoreactive MTF-1 after this

F IG . 5. Immunoblot detection of MTF-1 in SDS lysates of Hepacells after zinc treatment. Hepa cells were treated with 100 MZnSO 4 for 1 h and lysed in situ in 1 SDS-sample buffer, and proteinsfrom untreated ( lane 1 ) and treated ( lane 2 ) cells were analyzed byimmunoblotting using MTF-1-, Sp1-, or USF1-specific antiserum. Thearrows show the MTF-1-, Sp1-, and USF1-immunoreactive bands.

F IG . 6. Immunoblot and EMSA de-tection of MTF-1 in nuclear and cyto-solic extracts from dko7 cells trans-fected with a CMV-MTF-1 expressionvector. dko7 cells were transfected withCMV-MTF-1 expression vector and treatedwith 100 M ZnSO 4 for 1 h. NEs and CEswere prepared using NE-PER extractionreagents and analyzed by immunoblotting ( A) and EMSA ( B). A, upper panel , recom-binant mouse MTF-1 synthesized in a TnTlysate was used as a positive control. Lower panel , the extracts were immunoblottedwith USF1 antibody. The arrows show thepositions of MTF-1 and USF1. B, upper panel , the extracts, which containedEDTA, were analyzed for DNA binding ac-tivity using a labeled MRE-s oligonucleo-tide andafter theaddition of 100 M ZnSO 4to activate MTF-1. Lower panel , the sameextracts were assayed using an Sp1-spe-cific oligonucleotide. The arrows point tospecific MTF-1 and Sp1 complexes with

their respective oligonucleotides.



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addition of the cadmium (Fig. 8 B). We previously reported thattreatment of cells with 10 M cadmium only modestly activatesMTF-1 DNA binding activity ( 2-fold) (14, 18). In contrast, 50

M cadmium caused a 7.5-fold increase in immunoreactivenuclear MTF-1 within 1 h of treatment, and increased MTF-1was evident by 15 min (Fig. 8 B). The DNA binding activity of MTF-1 was examined using the nuclear extracts from cellstreated with 50 M cadmium for 15 min and 1 h (Fig. 9). Underthese conditions, 50 M cadmium caused about a 5-fold induc-tion of DNA binding. Sp1 DNA binding activity was not alteredby cadmium treatment (Fig. 9, lower panel ).

DISCUSSION

Previous studies have documented that MTF-1 is essentialfor metal ion regulation of MT gene expression (19) and thatthis metalloregulatory protein is activated by zinc to bind toMREs in the MT promoter (3). Treatment of cells with zincresults in a rapid increase in the amount of DNA binding activity of MTF-1 detected in nuclear extracts. However, it hasbeen shown that zinc-induced MT gene expression is not de-pendent on de novo protein synthesis (32), that MTF-1 mRNA is not induced by zinc (10, 19, 33), and that whole cell extractsfrom untreated cells contain latent MTF-1 that can be activated to bind to DNA by exogenous zinc (14). Taken together,these observations demonstrate that MTF-1 is a preexisting cellular protein that is activated to bind to DNA by metal ions.The data reported herein reveal that a significant portion of

MTF-1 protein is present in the cytoplasm of unstressed cells and

that exposure of the cells to metal ions results in the rapidtranslocation of MTF-1 protein to the nucleus and the activationof its DNA binding activity. These results are consistent with theconcept that MTF-1 functions, in part, as a zinc sensor.

Cadmium is a potent inducer of MT gene expression, andMTF-1 is also an essential component of that signaling mech-anism. However, MTF-1 is activated to bind to DNA by revers-ible interactions of zinc with specific zinc fingers in the DNA-binding domain and not by interactions with cadmium or othertransition metals (810, 14, 1619, 34). We previously reported

that 6 M CdCl 2 has little effect on the amount of MTF-1 DNA binding activity in the nuclei of cultured cells (18). Cadmium(515 M) rapidly induces MT-I gene expression in these cells 2

and in other cell types (33). Herein, it was further shown thatcadmium (10 M) exerts only a small effect (15% increase) onthe amount of MTF-1 protein in the nucleus. However, higherconcentrations of cadmium caused the complete activation of DNA binding and translocation of MTF-1 to the nucleus. Thissuggests that cadmium may cause the redistribution of zinc inthe culture, which, in turn, may activate MTF-1 to bind to DNA and move to the nucleus. Other transition metals have beensuggested to act in this manner (33). However, this mechanism

2 I. V. Smirnova, D. C. Bittel, R. Ravindra, H. Jiang, and G. K.

Andrews, unpublished data.

F IG . 8. Immunoblot detection of MTF-1 in nuclear and cytoso-lic extracts from Hepa cells exposed to cadmium. A, Hepa cellswere treated with 10, 20, or 50 M CdCl 2 or with 100 M ZnSO 4 for 1 h.NEs and CEs were prepared from treated and untreated cells andanalyzed by immunoblotting using antisera against mouse MTF-1. B ,Hepa cells were treated with 10 or 50 M CdCl 2 for 15 min or 1 h. NEsand CEs were prepared from treated and untreated cells and analyzedby immunoblotting as in A. The arrow shows the position of immuno-reactive MTF-1.

F IG . 9. EMSA detection of MTF-1 DNA binding activity in nu-clear extracts from Hepa cells treated with cadmium. Hepa cellswere treated with 50 M CdCl 2 for 15 min or 1 h or with 100 M ZnSO 4for 1 h. Upper panel , nuclear extracts were prepared from treated anduntreated cells and analyzed by EMSA using a labeled MRE-s oligonu-cleotide. Lower panel , the same extracts were assayed using a labeled

Sp1-specific oligonucleotide. The arrows point to the specific MTF-1 andSp1 complexes with their respective oligonucleotides. Lane 1 , untreatedcells; lane 2 , cells treated with 100 M ZnSO 4 for 1 h; lane 3 , cells treatedwith 50 M CdCl 2 for 15 min; lane 4 , cells treated with 50 M CdCl 2 for 1 h.



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may only account in part for the effects of cadmium on activa-tion of the MT promoter by MTF-1.

The disassociation between the concentration of cadmiumrequired for activation of MT-I gene expression and that re-quired to cause most MTF-1 to be activated to bind to DNA andtranslocated to the nucleus suggests several possibilities in-cluding the following: 1) only small changes in MTF-1 levels inthe nucleus are sufficient for maximal activation of gene ex-pression; 2) cadmium causes a modification of MTF-1; 3) cad-mium and zinc cause the formation of distinct MTF-1 promotercomplexes; or 4) cadmium-responsive transcription factorsother than MTF-1 may also activate MREs. The concentration-response curves for zinc activation of MT gene expression andincreased MTF-1 DNA binding activity do not support the firstpossibility. However, the possibility of effects of cadmium onthe transactivation capacity of MTF-1 cannot be excluded. Thetransactivation domains present in the carboxyl-terminal half of the protein are also important for transduction of the metalsignal. Thus, the full biological functions of MTF-1 are depend-ent on a complex interplay of different functional domains (13).Little is known about those interactions or the interactions of MTF-1 with other proteins; thus, the second and third possibili-ties remain to be addressed. With regard to the fourth possibility,we recently found that MRE activity can be increased in theabsence of detectable MTF-1 in IMR cells (35). Cadmium-respon-sive factor(s) that can interact with MREs in vitro have beenreported previously (8, 3638), but the functional significance, if any, of those factors has not been determined. Therefore, otherMRE-binding proteins may play a role in regulating MT geneexpression in response to cadmium, at least in certain cell types.

The nuclear localization of many transcription factors is akey controlling point in regulating gene expression and accom-panies differentiation or changes in the metabolic state of eu-karyotic cells (39). Although the majority of transcription fac-tors are localized to the nucleus (40, 41), others predominantlyreside in the cytoplasm and are translocated to the nucleus inresponse to stimulus (4244). One mechanism associated with

transcription factor transport to the nucleus is the nuclearlocalization signal (NLS) (45). MTF-1 has a putative NLS(KRKEVVKR) that immediately precedes the zinc finger do-main (10). For a large protein to translocate to the nucleus, theNLS has to be exposed on the protein surface (45). Interactionsbetween zinc and MTF-1 probably cause conformationalchanges leading to uncovering of the NLS. Another mechanismthat exposes the NLS is phosphorylation of adjacent sites (39).MTF-1 has several potential phosphorylation sites in the vicin-ity of the NLS such as Thr 131 for protein kinase C, Tyr 139 fortyrosine kinase, and Thr 142 for casein kinase. Protein kinase Chas been suggested to play a role in metal induction of MT geneexpression (46). Finally, it is possible that the zinc fingersthemselves are involved in metal-induced nuclear transloca-

tion of MTF-1. Several mutations of zinc fingers in MTF-1 werefound to cause the cytoplasmic localization of MTF-1 expressedin transiently transfected cells (13). Three Cys 2 His 2 -type zincfingers within the DNA-binding domain of nerve growth factor-induced transcription factor 1-A (also known as Erg1 orKrox24) are necessary for nuclear localization (47), and the vitamin D receptors NLS signal is located between the two zincfingers (41). The entire DNA-binding domain of the glucocorti-coid receptor, as a functional unit, may be required for nucleartransfer and optimal retention in the nucleus (48).

In conclusion, the present study indicates that the majority of MTF-1 protein is located in the cytoplasm in cells cultured inmedium replete with zinc. However, increases in zinc in theculture medium promote the rapid transport into the nucleus and

the activation of DNA binding activity of MTF-1. These results

are consistent with the concept that MTF-1 serves as a sensor of cytoplasmic metal ions and suggest that zinc and cadmium mayutilize MTF-1 differently in the activation of gene expression.

Acknowledgments We are indebted to Jim Geiser and Steve Eklundfor excellent technical support. We thank Dr. Walter Schaffner (Uni- versity of Zurich, Zurich, Switzerland) for a generous gift of the dko7cell line.

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Artículo 1-Cadmio Metalotioneinas