Metallothionein Gene Transcription Research Articles

Type I Interferons Ameliorate Zinc Intoxication of Candida glabrata by Macrophages and Promote Fungal Immune Evasion.

SummaryHost and fungal pathogens compete for metal ion acquisition during infectious processes, but molecular mechanisms remain largely unknown. Here, we show that type I interferons (IFNs-I) dysregulate zinc homeostasis in macrophages, which employ metallothionein-mediated zinc intoxication of pathogens as fungicidal response. However, Candida glabrata can escape immune surveillance by sequestering zinc into vacuoles. Interestingly, zinc-loading is inhibited by IFNs-I, because a Janus kinase 1 (JAK1)-dependent suppression of zinc homeostasis affects zinc distribution in macrophages as well as generation of reactive oxygen species (ROS). In addition, systemic fungal infections elicit IFN-I responses that suppress splenic zinc homeostasis, thereby altering macrophage zinc pools that otherwise exert fungicidal actions. Thus, IFN-I signaling inadvertently increases fungal fitness both in vitro and in vivo during fungal infections. Our data reveal an as yet unrecognized role for zinc intoxication in antifungal immunity and suggest that interfering with host zinc homeostasis may offer therapeutic options to treat invasive fungal infections.

Metal-mediated Epigenetic Regulation of Gene Expression.

Epigenetics is the study of heritable changes in gene expression that occur without alterations in the DNA sequence. Several studies have shown that environmental chemicals can alter epigenetic modifications, including histone modifications and DNA methylation. Environmental chemicals may show toxic effects via epigenetic mechanism-regulated changes in gene expression. Previously, we reported that zinc treatment rapidly decreased Lys(4)-trimethylated and Lys(9)-acetylated histone H3 in the metallothionein (MT) promoter, and also decreased total histone H3. The chromatin structure in the MT promoter may be locally disrupted by zinc-induced nucleosome removal. We also showed that chromium (VI) inhibited MT gene transcription by modifying the transcription potential of the co-activator p300. MT is a small cysteine-rich protein that is active in zinc homeostasis, cadmium detoxification, and protection against reactive oxygen species. Epigenetic changes might influence the cytoprotective function of the MT gene. In this review, I briefly summarize the results of previous studies and discuss the mechanisms and toxicological significance of metal-mediated epigenetic modifications.

Digestive cell lysosomes as main targets for Ag accumulation and toxicity in marine mussels, Mytilus galloprovincialis, exposed to maltose-stabilised Ag nanoparticles of different sizes

Bioavailability and toxicity of maltose-stabilised AgNPs of different sizes (20, 40 and 100 nm) in mussels were compared with bulk and aqueous forms of the metal through a two-tier experimental approach. In the first tier, mussels were exposed for 3 d to a range of concentrations (0.75, 75, 750 μg Ag/l) in the form of Ag20-Mal, Ag40-Mal, Ag100-Mal, bulk Ag and aqueous Ag (as AgNO3), as well as to the concentrations of maltose used in the formulation of NPs. Mortality, bioaccumulation, tissue and cell distribution and lysosomal responses were investigated. In the second tier, mussels were exposed for 21 d to Ag20-Mal, Ag100-Mal, bulk Ag and aqueous Ag at the lowest effective concentration selected after Tier 1 (0.75 μg Ag/l), biomarkers and toxicopathic effects were investigated. Aqueous Ag was lethal within 3 d at 75 μg Ag/l; Ag NPs or bulk Ag did not produce significant mortality at 750 μg Ag/l. Ag accumulation was limited and metallothionein gene transcription was not regulated although metal accumulation occurred in digestive, brown and stomach epithelial cells and in gut lumen after exposure to AgNPs and aqueous Ag starting at low concentrations after 1 d. Electrondense particles (<10 nm) in lysosomes and residual bodies after exposure to AgNPs contained Ag and S (X-ray). Intralysosomal metal accumulation and lysosomal membrane destabilisation were enhanced after exposure to all the forms of Ag and more marked after exposure to Ag20-Mal than to larger NPs. 21 d exposure to AgNPs provoked digestive cell loss and loss of digestive gland integrity, resulting in atrophy-necrosis in digestive alveoli and oedema/hyperplasia in gills (Ag NP), vacuolisation in digestive cells (aqueous Ag) and haemocyte infiltration of connective tissue (all treatments). Intralysosomal metal accumulation, lysosomal responses and toxicopathic effects are enhanced at decreasing sizes and appear to be caused by Ag+ ions released from NPs, although the metal was not substantially accumulated.

The 2013 William B. Coley Award for Distinguished Research In Basic and Tumor Immunology.

The 2013 William B. Coley Award for Distinguished Research In Basic and Tumor Immunology.

Hypothermia enhances induction of protective protein metallothionein under ischemia

BackgroundHypothermic protection against ischemic stroke has been reported by many studies. Hypothermia is supposed to mitigate the effects of deleterious genes and proteins and promote the activity of protective genes and proteins in the ischemic brain. Metallothionein (MT)-1/2 is thought to be a crucial factor for metal homeostasis, immune function, and apoptosis. This protein was found to exert protective effects in models of brain injury as well. In the present study, we investigated the effect of hypothermia on MT expression and the underlying mechanisms.MethodsCultured bEnd.3 brain endothelial cells were exposed to oxygen glucose deprivation and reperfusion (OGD+R). Reverse transcription PCR and western blot analyses were performed to measure the expression of MT, transcription factors, and methylation regulating factors. Transcription factor binding assays were also performed. Methylation profiles of the promoter area were obtained with pyrosequencing.ResultsHypothermia protected bEnd.3 cells from OGD+R. When the cells were exposed to OGD+R, MT expression was induced. Hypothermia augmented MT levels. While OGD+R-induced MT expression was mainly associated with metal regulatory transcription factor 1 (MTF-1), MT expression promoted by hypothermia was primarily mediated by the signal transducer and activator of transcription 3 (STAT3). Significantly increased STAT3 phosphorylation at Ser727 was observed with hypothermia, and JSI-124, a STAT-3 inhibitor, suppressed MT expression. The DNA demethylating drug 5-aza-2′-deoxycytidine (5-Aza) enhanced MT expression. Some of the CpG sites in the promoter MT=> it should be “the CpG sites in the MT promoter” showed different methylation profiles and some methylation regulating factors had different expressional profiles in the presence of OGD+R and hypothermia.ConclusionsWe demonstrated that hypothermia is a potent inducer of MT gene transcription in brain endothelial cells, and enhanced MT expression might contribute to protection against ischemia. MT gene expression is induced by hypothermia mainly through the STAT3 pathway. DNA methylation may contribute to MT gene regulation under ischemic or hypothermic conditions.

Bis(l-cysteinato)zincate(lI) as a coordination compound that induces metallothionein gene transcription without inducing cell-stress-related gene transcription

Zinc is an essential micronutrient, deficiency of which results in growth retardation, immunodeficiency, and neurological diseases such as dysgeusia. Several zinc coordination compounds are used for zinc supplementation; however, supplemented zinc ions have no specificity and interact with various groups of molecules. Here, we found that, from a library of 30 zinc coordination compounds, bis(L-cysteinato)zincate(II), designated Z01, functioned as a metallothionein (MT) inducer. Z01 induced MT expression mediated by the transcription factor MTF-1, without inducing cell-stress-related heme oxygenase-1 gene expression at specific concentration. The zinc ion was necessary for the MT induction. 65Zn incorporation following treatment with 65Zn-labeled Z01 suggested that Z01 did not act as zinc ionophore despite its hydrophilicity. Electrophoretic mobility shift assays revealed that Z01 facilitates MTF-1–MRE complex formation, and, by inference, transfer of zinc from Z01 to MTF-1. Phosphorylated ERK levels were increased by ZnSO4 treatment but not by Z01. Although our data do not definitely prove that Z01 is an MTF-1-specific activator, our observations suggest that zinc coordination compounds can regulate zinc distribution and act as zinc donors for specific molecules.

Molecular Mechanisms of Zinc-mediated Induction and Chromium(VI)-mediated Inhibition of Mouse Metallothionein-I Gene Transcription

The expression of metallothionein (MT), a heavy-metal-binding protein, is induced by heavy metals such as zinc, copper, cadmium, and mercury. This induction of MT maintains zinc homeostasis and defends against toxic heavy metals by sequestering these metals and lowering their concentrations at critical intracellular sites. However, MT cannot bind chromium(VI), a heavy metal that has been known for over 100 years to be a human carcinogen. Chromium(VI) enters cells via the sulfate anion transporter system and is reduced to intermediate oxidation states, such as chromium(V) and chromium(IV), in the process of forming stable chromium(III) forms. Chromium(VI) is known to inhibit MT gene transcription, and recently my colleagues and I reported that chromium(VI) inhibits zinc-induced MT gene transcription by modifying the transactivation potential of metal response element-binding transcription factor-1 (MTF-1), a zinc-finger transcription factor. Inhibition of MT gene transcription may therefore be involved in the carcinogenicity of chromium(VI). In this review, I briefly summarize the molecular mechanisms of heavy metal-induced MT gene transcription and discuss the current status of research on chromium(VI) toxicity and chromium(VI)-mediated inhibition of MT gene transcription.

Metal-Responsive Transcription Factor 1 (MTF-1) Activity Is Regulated by a Nonconventional Nuclear Localization Signal and a Metal-Responsive Transactivation Domain

Metal-responsive transcription factor 1 (MTF-1) mediates both basal and heavy metal-induced transcription of metallothionein genes and also regulates other genes involved in the cell stress response and in metal homeostasis. In resting cells, MTF-1 localizes to both the cytoplasm and the nucleus but quantitatively accumulates in the nucleus upon metal load and under other stress conditions. Here we show that within the DNA-binding domain, a region spanning zinc fingers 1 to 3 (amino acids [aa] 137 to 228 in human MTF-1) harbors a nonconventional nuclear localization signal. This protein segment confers constitutive nuclear localization to a cytoplasmic marker protein. The deletion of the three zinc fingers impairs nuclear localization. The export of MTF-1 to the cytoplasm is controlled by a classical nuclear export signal (NES) embedded in the acidic activation domain. We show that this activation domain confers metal inducibility in distinct cell types when fused to a heterologous DNA-binding domain. Furthermore, the cause of a previously described stronger inducibility of human versus mouse MTF-1 could be narrowed down to a 3-aa difference in the NES; "humanizing" mouse MTF-1 at these three positions enhanced its metal inducibility to the level of human MTF-1.

Dichotomous Effects of Lead Acetate on the Expression of Metallothionein in the Liver and Kidney of Mice

Metallothionein (MT) is a low-molecular-weight cysteine-rich protein which has a high affinity for metals and plays important roles in the protection against metal toxicity. As little information is available concerning the mechanism of MT induction by lead (Pb) compounds, we investigated the induction of MT by Pb acetate both at mRNA and protein levels in mice. Administration of Pb increased the levels of MT-I mRNA in the liver and kidney in six strains of mice. However, MT protein was detected only in the liver, and little or no increases in MT protein were detected in the kidney of any strains of mice. Speciation of metals in the liver cytosol showed that the major metal bound to MT was zinc but not Pb. The increases in plasma concentrations of interleukin-6 suggest that the production of interleukin-6 by Pb administration is involved in the induction of MT in the liver. Treatment of renal cells with Pb in vitro also resulted in the increase in MT mRNA but little increase in MT protein. These data suggest that Pb exerts a dual effect on MT expression; enhancement of MT gene transcription both in the liver and kidney and suppression of MT mRNA translation in the kidney.

Nuclear Factor-1 and Metal Transcription Factor-1 Synergistically Activate the Mouse Metallothionein-1 Gene in Response to Metal Ions

Metal activation of metallothionein (MT) gene transcription is dependent on the presence of metal regulatory elements (MREs), which are present in five non-identical copies (MREa through MREe) in the promoter of the mouse MT-1 gene and on the capacity of metal transcription factor-1 (MTF-1) to bind to the MREs in the presence of zinc. We detected a protein, distinct from MTF-1, specifically binding to the MREc region. DNA binding competition experiments using synthetic oligonucleotides and specific anti-NF1 antibodies showed that this protein binds to an NF1 site overlapping the MREc element as well as to a second site upstream of the Sp1a site and corresponds to NF1 or a related protein. Transfection experiments showed that loss of the two NF1 sites decreased metal-induced MT promoter activity by 55-70% in transiently transfected cells and almost completely abrogated metal and tert-butylhydroquinone (tBHQ) induction in stably transfected cells. Similarly, expression of an inactive NF1 protein strongly inhibited MT-1 promoter activity. Using synthetic promoters containing NF1 and MRE sites fused to a minimal MT promoter, we showed that these NF1 sites did not confer metal induction but enhanced metal-induced promoter activity. Chromatin immunoprecipitation assays confirmed that NF1 binds to the mouse MT-1 promoter in vivo and showed that NF1 binding is zinc-inducible. In addition, zinc-induced NF1 DNA binding was MTF-1-dependent. Taken together, these studies show that NF1 acts synergistically with MTF-1 to activate the mouse MT-1 promoter in response to metal ions and tert-butylhydroquinone and contributes to maximal activation of the gene.

Anti-fibrotic effects of tetrandrine on bile-duct ligated rats

Tetrandrine (Tet) (C38H42O8N2; molecular weight, 622), an alkaloid isolated from the Chinese medicinal herb Stephania tetrandra, has been shown to elicit anti-inflammatory and anti-fibrotic effects in pulmonary diseases, but the mechanism of action has yet to be investigated. In this study, we tested whether Tet exerts anti-fibrotic effects on rat hepatic fibrosis through anti-NFkappaB pathways. After bile-duct ligation, rats were given Tet (1 or 5 mg/kg) or silymarin (50 mg/kg, as a positive control) by gavage twice daily for 3 weeks. Liver sections were taken for Sirius red quantitative scoring, immunofluorescence double staining of alpha-smooth muscle actin (alpha-SMA) and NFkappaB, and for quantitative determinations of the mRNA expression levels of TGF-beta1, alpha-SMA, collagen 1alpha2, inducible nitric oxide synthase (iNOS), intercellular adhesion molecule 1 (ICAM-1), metallothionein, vascular endothelial growth factor (VEGF), and VEGF type II receptor (VEGFR2) genes. The results showed that both Tet and silymarin treatment significantly reduced the fibrosis scores and hepatic collagen content of BDL rats, compared with no treatment. Both Tet and silymarin treatments decreased the number of alpha-SMA- and NFkappaB-positive cells in fibrotic livers. Moreover, Tet and silymarin treatments attenuated the mRNA expression levels of TGF-beta1,alpha-SMA, collagen 1alpha2, iNOS, ICAM-1, VEGF, and VEGFR2 genes, and induced the mRNA expression of the metallothionein gene. This study suggests that the anti-fibrotic effects of Tet were related to the reduction of fibrosis-related gene transcription, the attenuation of NFkappaB-activated pathways, and the induction of metallothionein gene transcription in the livers of BDL rats.

Induction and activity of oxidative stress-related proteins during waterborne Cu-exposure in brown trout ( Salmo trutta)

Induction of gene transcription for proteins and enzymes involved in metal-mediated oxidative stress were studied in brown trout transferred to a Cu-contaminated river in the Røros region in Central Norway. In addition to metallothionein (MT-A), Cu/Zn-superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR) gene transcription, protein levels of MT and enzyme activities of SOD and CAT were analyzed in gill, liver and kidney. MT-A, SOD and GR transcription increased significantly along with uptake of Cu in gills, while only transcription of MT-A was found to respond in liver and kidney during the exposure. Already present MT proteins in gills seemed to be oxidized during the exposure, probably caused by Cu-mediated oxidative stress, and no increase in MT protein levels were observed in gills. SOD and CAT enzyme levels were affected in all tissues during the exposure. A negative correlation between SOD and CAT activities was observed in gills, and we suggest that the activities of these enzymes were influenced not only through transcription. GPx and GR transcription levels correlated positively with each other in gills and liver, indicating their shared function in GSH-turnover. Levels of MT and activity of SOD and CAT dealing with metal-induced oxidative stress appear to be regulated not only through gene transcription, but also through post-translational mechanisms.

Insertion of a yeast metallothionein gene into the model insect Drosophila melanogaster (Diptera: Drosophilidae) to assess the potential for its use in genetic improvement programs with natural enemies

The use of copper-containing fungicides, such as Kocide, negatively aVects nontarget beneWcial insects in Florida citrus. To determine if the insertion of the yeast metallothionein gene (CUP1) into the genome of beneWcial insects could produce populations resistant to copper, the model insect Drosophila melanogaster Meigen (Diptera: Drosophilidae) was transformed with the Drosophila metallothionein promoter Mtn fused to the CUP1 open reading frame. Two transformed lines, conWrmed by high-Wdelity polymerase chain reaction and Southern blot analysis, were assessed for tolerance to copper hydroxide (CuOH). Transgenic lines 8 and 14 exhibited 1.5- to 2-fold increases in tolerance when held at 24 °C, »75% RH, and 14L:10D. In the absence of copper, lines 8 and 14 exhibited Wtness costs as compared to the ww control line. In addition, each line (ww control, lines 8 and 14), when pretreated with 2.5 mM CuOH and held at »22– 24 °C, »50–75% RH, and 14L:10D, was more tolerant to copper, with line 14 exhibiting the greatest increase. The increased copper tolerance of the pretreated transgenic lines could be a result of induced transcription of both the CUP1 and native metallothionein (MT) genes whereas the increase in the pretreated ww control could be a due to induced transcription of native MT genes. Both transgenic lines exhibited reduced Wtness compared to the ww control line, which could be due to the eVects of inbreeding or to the insertion of the CUP1 construct into the genome. The data suggest that CUP1 may be useful for the genetic improvement of natural enemies where copper is applied.

Alkaline induces metallothionein gene expression and potentiates cell proliferation in Chinese hamster ovary cells

Metallothionein (MT) gene expression is increased in cadmium resistant Chinese hamster ovary cells (CHO Cd(R)) upon medium (regular or serum-free) change during culturing. Among the major components of the medium, NaHCO3 was found to be able to induce MT gene expression in a dose- and time-dependent manner. The same effect was observed with other alkaline solutions, such as HEPES and NaOH. Using MT promoter-luciferase reporter gene constructs, we found that the presence of metal response elements (MREs) in the promoter region is necessary for NaHCO3-induced MT gene transcription. This finding is further supported by the observation that the binding activity between the metal-responsive transcription factor 1 (MTF-1) and the MRE were increased after NaHCO3 treatment. Following NaHCO3 treatment, an increase in cell proliferation was observed in CdR cells but not in the parental CHO K1 cells that do not express MT transcripts due to MT gene methylation. Using synchronized cells, an increase in cell proliferation was observed 9 h after NaHCO3 addition. Notably, proliferation of CHO K1 cells was increased when transfected with an MT gene. The effect of MT on cell growth was affirmed by treating CHO K1 cells with 5-azacytidine (Aza) to demethylate the MT gene. Proliferation increased in Aza-treated CHO K1 cells after NaHCO3 treatment. These results demonstrate that NaHCO3 stimulates MT gene expression and causes an enhancement of cell proliferation in CHO cells.

Metal-responsive transcription factor-1 (MTF-1) is essential for embryonic liver development and heavy metal detoxification in the adult liver.

Metal-responsive transcription factor-1 (MTF-1) activates the transcription of metallothionein genes and other target genes in response to heavy metal load and other stresses such as hypoxia and oxidative stress. It also has an essential function during embryogenesis: targeted disruption of Mtf1 in the mouse results in lethal liver degeneration on day 14 of gestation. Here we studied Mtf1 knockout mice at embryonic and adult stages, the latter by means of conditional knockout. Hepatocytes from Mtf1 null mutant and wild-type embryos were taken into culture on day 12.5 of gestation. Both initially appeared normal, but mutant cells were lost within a few days. Furthermore, Mtf1 null hepatocytes were poorly, if at all, rescued by cocultivation with wild-type rat embryo hepatocytes, indicating a cell-autonomous defect. When the Mtf1 gene was excised by Cre recombinase after birth in liver and bone marrow and to a lesser extent in other organs, mice were viable under non-stress conditions but highly susceptible to cadmium toxicity, in support of a role of MTF-1 in coping with heavy metal stress. An additional MTF-1 function was revealed upon analysis of the hematopoietic system in conditional knockout mice where leukocytes, especially lymphocytes, were found to be severely underrepresented. Together, these findings point to a critical role of MTF-1 in embryonic liver formation, heavy metal toxicity, and hematopoiesis.

Ultraviolet irradiation increases the sensitivity of cultured human skin cells to cadmium probably through the inhibition of metallothionein gene expression

We previously developed an apparatus that can irradiate cultured cells with monochromatic ultraviolet (UV) rays to exactly assess the biological effects of UV components on mammalian cells. Using this device, we studied the effects of UV in and near the UVB region on the general as well as specific protein synthesis of the human skin-derived NB1RGB cells. We found that Cd-induced synthesis of metallothioneins (MTs), which are the proteins involved in the protection against heavy metals and oxidative stress, is inhibited by UV at 280 nm more extensively than total protein synthesis. Such an inhibition was observed when MTs were induced by different inducers such as Cd, Zn, and dexamethasone in three human cell lines, indicating that it is not an event specific to a certain inducer or a certain cell type. By contrast, UV at 300 or 320 nm showed only a marginal effect. UV at 280 nm was likely to block MT gene transcription because Cd-induced increase of MT mRNA was strongly inhibited by irradiation. Cd induction of 70-kDa heat shock protein mRNA was also inhibited by UV irradiation, suggesting that the expression of inducible genes are commonly sensitive to UV. Furthermore, we observed that the irradiation of UV at 280 nm renders NB1RGB cells extremely susceptible to Cd, probably due to the reduced MT synthesis. These observations strongly suggest that UV at 280 nm severely damages cellular inducible protective functions, warning us of a new risk of UV exposure.

Metal ion affinities of the zinc finger domains of the metal responsive element-binding transcription factor-1 (MTF1).

Metal response element (MRE) binding transcription factor-1 (MTF1) is a six Cys(2)His(2) zinc finger-containing transcription factor required for basal and zinc-induced transcription of metallothionein genes. The cobalt(II) and zinc(II) affinities of a protein fragment comprising the six zinc finger domains have been examined to reveal apparent dissociation constants (for the six domains collectively) of 0.5 +/- 0.2 microM for cobalt(II) and 31 +/- 14 pM for zinc(II). Two approaches have been used to determine the metal ion affinities of the individual domains. First, the six domains have been examined as single domain peptides revealing dissociation constants ranging from 0.3 to 1.7 microM for cobalt(II). The domains fall into two sets with peptides corresponding to domains 2, 3, and 4 showing relatively high affinity (K(d)(Co(II)) 0.3-0.5 microM) and peptides corresponding to domains 1, 5, and 6 showing lower affinity (K(d)(Co(II)) 1.6-1.7 microM). Second, we examined the affinity of each domain in the context of the six zinc finger domain protein by individually mutating one metal-binding His residue to Cys to allow independent monitoring of the cobalt(II) occupancy of each site. The affinity of each domain was higher in this context than as a single domain peptide with affinities (corrected for the effect of the mutation) ranging from 0.02 to 0.5 microM. The increase in affinity for the individual domains ranged from factors of 1.1 to 20. The order of affinities (from higher to lowest) was observed to be 4 > 2 approximately 5 > 6 approximately 3 approximately 1. These results reveal that none of the Cys(2)His(2) zinc finger domains of MTF1 have dramatically low metal ion affinities, certainly none low enough to respond to changes in free zinc ion concentrations in the micromolar range. Nonetheless, the metal ion affinities of some domains do differ by a factor of 25 with domains at both the amino- and carboxyl-termini showing lower intrinsic affinities for metal ions than the central domains.

Activity of metal-responsive transcription factor 1 by toxic heavy metals and H2O2 in vitro is modulated by metallothionein.

Metallothioneins are small, cysteine-rich proteins that avidly bind heavy metals such as zinc, copper, and cadmium to reduce their concentration to a physiological or nontoxic level. Metallothionein gene transcription is induced by several stimuli, notably heavy metal load and oxidative stress. Transcriptional induction of metallothionein genes is mediated by the metal-responsive transcription factor 1 (MTF-1), an essential zinc finger protein that binds to specific DNA motifs termed metal-response elements. In cell-free DNA binding reactions with nuclear extracts, MTF-1 requires elevated zinc concentrations for efficient DNA binding but paradoxically is inactivated by other in vivo inducers such as cadmium, copper, and hydrogen peroxide. Here we have developed a cell-free, MTF-1-dependent transcription system which accurately reproduces the activation of metallothionein gene promoters not only by zinc but also by these other inducers. We found that while transcriptional induction by zinc can be achieved by elevated zinc concentration alone, induction by cadmium, copper, or H2O2 additionally requires the presence of zinc-saturated metallothionein. This is explained by the preferential binding of cadmium or copper to metallothionein or its oxidation by H2O2; the concomitant release of zinc in turn leads to the activation of transcription factor MTF-1. Conversely, thionein, the metal-free form of metallothionein, inhibits activation of MTF-1. The release of zinc from cellular components, including metallothioneins, and the sequestration of zinc by newly produced apometallothionein might be a basic mechanism to regulate MTF-1 activity upon cellular stress.

Chromium(VI) Down-regulates Heavy Metal-induced Metallothionein Gene Transcription by Modifying Transactivation Potential of the Key Transcription Factor, Metal-responsive Transcription Factor 1

The robust induction of metallothionein-I and II (MT-I and MT-II) genes by several heavy metals such as zinc and cadmium requires the specific transcription factor metal-responsive transcription factor 1 (MTF1). Chromium (VI), a major environmental carcinogen, not only failed to activate these genes but also inhibited their induction by Zn2+ or Cd2+. The heavy metal-induced expression of another MTF1 target gene, zinc transporter 1 (ZnT-1), was also down-regulated by Cr6+. By contrast, the expression of two MTF1-independent Cd2+-inducible genes, heme oxygenase 1 (HO-1) and HSP-70, was not sensitive to Cr6+. Cr6+ did not also affect the expression of housekeeping genes such as GAPDH or beta-actin. Stable cell lines overexpressing variable levels of MTF1, the key transactivator of the MT genes, demonstrated differential resistance toward the inhibitory effect of Cr6+, indicating MTF1 as a target of chromium toxicity. The basal and inducible binding of MTF1 to metal response elements was not affected by treatment of cells with Cr6+. Transient transfection studies showed that the ability of MTF1 to transactivate the MT-I promoter was significantly compromised by Cr6+. The fusion protein consisting of a Gal-4 DNA binding domain and one or more of the three transactivation domains of MTF1, namely the acidic domain, proline-rich domain, and serine-threonine rich domain, activated the GAL-4-driven luciferase gene to different degrees, but all were sensitive to Cr6+. MTF1 null cells were prone to apoptosis after exposure to Zn2+ or Cd2+ that was augmented in presence Cr6+, whereas the onset of apoptosis was significantly delayed in cells overexpressing MTF1.

Knockout of 'metal-responsive transcription factor' MTF-1 in Drosophila by homologous recombination reveals its central role in heavy metal homeostasis.

'Metal-responsive transcription factor-1' (MTF-1), a zinc finger protein, is conserved from mammals to insects. In the mouse, it activates metallothionein genes and other target genes in response to several cell stress conditions, notably heavy metal load. The knockout of MTF-1 in the mouse has an embryonic lethal phenotype accompanied by liver degeneration. Here we describe the targeted disruption of the MTF-1 gene in Drosophila by homologous recombination. Unlike the situation in the mouse, knockout of MTF-1 in Drosophila is not lethal. Flies survive well under laboratory conditions but are sensitive to elevated concentrations of copper, cadmium and zinc. Basal and metal-induced expression of Drosophila metallothionein genes MtnA (Mtn) and MtnB (Mto), and of two new metallothionein genes described here, MtnC and MtnD, is abolished in MTF-1 mutants. Unexpectedly, MTF-1 mutant larvae are sensitive not only to copper load but also to copper depletion. In MTF-1 mutants, copper depletion prevents metamorphosis and dramatically extends larval development/lifespan from normally 4-5 days to as many as 32 days, possibly reflecting the effects of impaired oxygen metabolism. These findings expand the roles of MTF-1 in the control of heavy metal homeostasis.