Metal-responsive Transcription Factor Research Articles

The metal-responsive transcription factor-1 protein is elevated in human tumors

We previously identified metal-responsive transcription factor-1 (MTF-1) as a positive contributor to mouse fibrosarcoma growth through effects on cell survival, proliferation, tumor angiogenesis, and extracellular matrix remodeling. In the present study, we investigated MTF-1 protein expression in human tissues by specific immunostaining of both normal and tumor tissue samples. Immunohistochemical (IHC) staining of a human tissue microarray (TMA), using a unique anti-human MTF-1 antibody, indicated constitutive MTF-1 expression in most normal tissues, with liver and testis displaying comparatively high levels of expression. Nevertheless, MTF-1 protein levels were found to be significantly elevated in diverse human tumor types, including breast, lung, and cervical carcinomas. IHC analysis of a separate panel of full-size tissue sections of human breast cancers, including tumor and normal adjacent, surrounding tissue, confirmed and extended the results of the TMA analysis. Taken with our previous findings, this new study suggests a role for MTF-1 in human tumor development, growth or spread. Moreover, the study suggests that MTF-1 could be a novel therapeutic target that offers the opportunity to manipulate metal or redox homeostasis in tumor cells.

A Drosophila model of Menkes disease reveals a role for DmATP7 in copper absorption and neurodevelopment

Human Menkes disease is a lethal neurodegenerative disorder of copper metabolism that is caused by mutations in the ATP7A copper-transporting gene. In the present study, we attempted to construct a Drosophila model of Menkes disease by RNA interference (RNAi)-induced silencing of DmATP7 , the Drosophila orthologue of mammalian ATP7A , in the digestive tract. Here, we show that a lowered level of DmATP7 mRNA in the digestive tract results in a reduced copper content in the head and the rest of the body of surviving adults, presumably owing to copper entrapment in the gut. Similar to Menkes patients, a majority of flies exhibit an impaired neurological development during metamorphosis and die before eclosion. In addition, we show that survival to the adult stage is highly dependent on the copper content of the food and that overexpression of the copper homeostasis gene, metal-responsive transcription factor-1 ( MTF-1 ), enhances survival to the adulthood stage. Taken together, these results highlight the role of DmATP7-mediated copper uptake in the neurodevelopment of Drosophila melanogaster and provide a framework for the analysis of potential gene interactions influencing Menkes disease.

The metal-responsive transcription factor-1 protein is elevated in human tumors

The metal-responsive transcription factor-1 protein is elevated in human tumors

A Drosophila model of Menkes disease reveals a role for DmATP7 in copper absorption and neurodevelopment

Human Menkes disease is a lethal neurodegenerative disorder of copper metabolism that is caused by mutations in the ATP7A copper-transporting gene. In the present study, we attempted to construct a Drosophila model of Menkes disease by RNA interference (RNAi)-induced silencing of DmATP7, the Drosophila orthologue of mammalian ATP7A, in the digestive tract. Here, we show that a lowered level of DmATP7 mRNA in the digestive tract results in a reduced copper content in the head and the rest of the body of surviving adults, presumably owing to copper entrapment in the gut. Similar to Menkes patients, a majority of flies exhibit an impaired neurological development during metamorphosis and die before eclosion. In addition, we show that survival to the adult stage is highly dependent on the copper content of the food and that overexpression of the copper homeostasis gene, metal-responsive transcription factor-1 (MTF-1), enhances survival to the adulthood stage. Taken together, these results highlight the role of DmATP7-mediated copper uptake in the neurodevelopment of Drosophila melanogaster and provide a framework for the analysis of potential gene interactions influencing Menkes disease.

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.

Loss of liver-intestine cadherin in human intrahepatic cholangiocarcinoma promotes angiogenesis by up-regulating metal-responsive transcription factor-1 and placental growth factor

Liver-intestine cadherin (LI-cadherin) represents a novel type of cadherin within the cadherin superfamily that comprises seven cadherin repeats and a short cytoplasmic domain. In this study, we first examined LI-cadherin expression immunohistochemically in 34 specimens of human intrahepatic cholangiocarcinoma (ICC). LI-cadherin expression was positive (defined as positivity in > or = 10% of cells) in 18 of the ICCs (52.9%). LI-cadherin negativity was significantly correlated with tumor dedifferentiation (P=0.026) and vascular invasion (P=0.015). The cumulative survival rate of patients with LI-cadherin-negative ICC was significantly shorter than that of patients with LI-cadherin-positive ICC (P=0.021). Multivariate analysis identified the extent of LI-cadherin staining as an independent prognostic factor for ICC survival (P=0.027). Next, to elucidate the mechanism of loss of LI-cadherin-mediated aggressiveness in ICC, we knocked down LI-cadherin expression in an ICC cell line using small interfering RNA (siRNA) technology, and screened for genes that were expressed differentially between these cells and ICC cells transfected with scrambled siRNA using microarray analysis with real-time polymerase chain reaction confirmation. Among 21 identified genes, we focused on metal-responsive transcription factor-1 (MTF-1), whose target genes might contribute to tumor aggressiveness. Expression of placental growth factor (PlGF), one of the MTF-1 target genes, was up-regulated in the ICC cells transfected with LI-cadherin siRNA. Likewise, PlGF expression was up-regulated in LI-cadherin-negative ICC specimens. There was a significant inverse relationship between these expressions (P=0.033). Furthermore, the microvessel density of LI-cadherin-negative ICC specimens was higher than that of LI-cadherin-positive specimens. These findings suggest that loss of LI-cadherin in ICC is associated with tumor dedifferentiation and vascular invasion, and thus poor prognosis. Loss of LI-cadherin results in up-regulation of MTF-1 and PlGF, thereby regulating angiogenesis in ICC.

Metal transcription factor-1 regulation via MREs in the transcribed regions of selenoprotein H and other metal-responsive genes

Selenoprotein H is a redox-sensing DNA binding protein that upregulates genes involved in antioxidant responses. Given the known links between oxidative stress and heavy metals, we investigated the potential for regulation of selenoprotein H by metals. In silico analysis of the selenoprotein H genes from nine species reveals multiple predicted metal response elements (MREs). To validate MRE function, we investigated the effects of zinc or cadmium addition and metal-responsive transcription factor 1 (MTF-1) knockout on selenoprotein H mRNA levels. Chromatin immunoprecipitation was used to directly assess physical binding of the transcription factor to MREs in the human and mouse selenoprotein H genes. The results reported herein show that selenoprotein H is a newly identified target for MTF-1. Further, whereas nearly all prior studies of MREs focused on those located in promoters, we demonstrate binding of MTF-1 to MREs located downstream of the transcription start sites in the human and murine selenoprotein H genes. Finally, we identified MREs in downstream sequences in 15 additional MTF-1 regulated genes lacking promoter MREs, and demonstrated MTF-1 binding in three of these genes. This regulation via sequences downstream of promoters highlights a new direction for identifying previously unrecognized target genes for MTF-1.

Silver ions induce oxidative stress and intracellular zinc release in human skin fibroblasts

Silver compounds used as topical antimicrobial agents are known to exert toxic effects on skin cells. The aim of this study was to investigate whether the toxicity of silver ions, in analogy to other transition metal ions, depends on pro-oxidant effects. We treated human skin fibroblasts with concentrations of AgNO 3 not affecting cell proliferation, mitochondrial activity, or cell viability and found that Ag + strongly increases the production of reactive oxygen species, including superoxide anion radicals. These effects correspond to a strong decrease in intracellular reduced glutathione and to an increased susceptibility to H 2O 2-induced cell death. In addition, AgNO 3 down-regulates the expression of antioxidant genes such as the transcription factor Nrf2 and its target gene glutamate–cysteine ligase catalytic subunit. Furthermore Ag + induces a transient intracellular zinc release and increases the mRNA and protein expression of the zinc-binding protein metallothionein by activating the metal-responsive transcription factor 1, as verified by RNA interference. In conclusion, we show for the first time that Ag + induces oxidative stress and affects intracellular zinc homeostasis in human skin fibroblasts. The understanding of the mechanism involved in silver toxicity might contribute to new strategies for managing the therapy of skin infections.

Differential spatial expression of metal responsive transcription factor MTF-1 is related to functional organization of pituitary cells in Cyprinus carpio

Differential spatial expression of metal responsive transcription factor MTF-1 is related to functional organization of pituitary cells in Cyprinus carpio

Metallothionein expression in HaCaT and C6 cell lines exposed to cadmium

Metallothioneins (MT) are low-molecular weight, cysteine-rich metal-binding proteins. MT play a role in the homeostasis of essential metals such as zinc (Zn) and copper (Cu), detoxification of toxic metals such as cadmium (Cd) and protection against oxidative stress. In this study, we examined the expression of MT in HaCaT and C6 cells as a strategy to enhance protection against Cd-mediated toxicity. At basal level, HaCaT cells showed higher MT level than C6 cells which could explain the resistance of HaCaT cells. Western blot showed that C6 cells treated with 20 μmol/L Cd for 24 h did not express any MT. MT were initially expressed in the cytoplasmic or periplasmic compartment and were then translocated in the nucleus after 24 h treatment by Cd both in HaCaT and C6 cells. In addition, the cell treatment with Cd was followed by an increase in the cellular zinc level but the electrophoretic mobility shift assay (EMSA) experiment did not show any translocation of metal-responsive transcription factor-1 (MTF-1) to the nucleus of HaCaT cells. These absence of translocation could be due to the presence of MT in these cells at the basal state. The translocation study in HaCaT cells suggested that the MT translocation in the nucleus was greater than observed in C6 cells. The latter observation could explain HaCaT cells resistance to Cd concentrations up to 50 μmol/L. Our results suggested that the C6 cell sensitivity was correlated with the decrease in MT level at 20 μmol/L Cd occurring after the transcription of MT gene.

Identification of a splice variant of the metal-responsive transcription factor MTF-1 in common carp

Transactivation of the expression of metallothionein genes involves the Metal-responsive Transcription Factor (MTF-1). We report here the identification of mtf-1.1a, the first known splice variant of mtf-1.1 mRNA, in common carp ( Cyprinus carpio). The lack of a 103 nt internal segment results in a frame shift, causing the early termination of translation. mtf-1.1a mRNA encodes a protein consisting of the first 349 amino acids of MTF-1.1 plus an additional 64 amino acids, with no significant similarity to any of the proteins in the databases. The predicted MTF-1.1a protein carries the Zn-finger domain and the nuclear exporting and nuclear localization signals, and lacks the transcription activation domains. mtf-1.1a was detected in all tissues examined but the liver, with the highest level in the brain. Arsenic alters the levels of both mtf- 1.1 and mtf- 1.1a transcripts, in an isoform- and tissue-specific manner.

The “metallo-specific” response of proteins: A perspective based on the Escherichia coli transcriptional regulator NikR

Transition metal ions are required by all cells but an excess of metal ions beyond physiological requirements has toxic consequences. Optimal cellular concentrations of transition metals are commonly maintained by metal-responsive transcription factors that regulate genes encoding the proteins responsible for transport, sequestration and/or use of the metals. These metalloregulators must discriminate between the bioavailable metals to properly effect metal homeostasis, but how this metal selectivity is achieved is poorly understood. This perspective examines the metal-selective response of the Escherichia coli Ni(II)-responsive metalloregulator NikR. Biochemical and structural studies of E. coli NikR reveal that the mechanism of metal-selective regulation is more complex than that defined by simple metal-binding thermodynamics. Here we examine the metal-dependent allosteric changes on NikR structure that affect DNA binding and discuss the correspondence with other metalloregulators. Given what we have learned of how metal selectivity is achieved by E. coli NikR, we propose a complete scheme for the regulatory function of NikR in E. coli nickel homeostasis.

Metal Response Element-binding Transcription Factor-1 Is Activated by Degradation of Metallothionein

Cytosolic zinc-binding protein, metallothionein (MT), is normally saturated with Zn. It is thought that Zn-saturated MT (Zn-MT) acts as a major intracellular Zn pool. Metal-response element-binding transcription factor-1 (MTF-1) plays an important role in Zn-mediated MT transcription. Here, we showed that degradation of Zn-MT activates MTF-1. We measured activated MTF-1 using an electrophoretic mobility shift assay. Interleukin-6 induced MT expression and increased MTF-1 activity. MTF-1 activation was not observed in MT-overexpressing cells. MT-dependent MTF-1 activation was observed only after treating MT-overexpressing cells with cycloheximide (CHX), a protein synthesis inhibitor. CHX-treatment increased the degradation/synthesis ratio of protein. An increase in the degradation/synthesis ratio for the MT protein is expected to increase the level of labile Zn and activate MTF-1. Recombinant MTF-1 was activated by H2O2 only in the presence of Zn-MT. Oxidative stress activated MTF-1 DNA-binding activity in primary cultured hepatocytes but not in MT-deficient hepatocytes. These findings suggest that degradation of Zn-MT activates MTF-1, and that MT plays an important role in zinc-mediated signal transduction.

Mercury and cadmium trigger expression of the copper importer Ctr1B, which enables Drosophila to thrive on heavy metal-loaded food

Organisms from insects to mammals respond to heavy metal load (copper, zinc, cadmium, and mercury) by activating the metal-responsive transcription factor 1 (MTF-1). MTF-1 binds to short DNA sequence motifs, termed metal response elements, and boosts transcription of a number of genes, notably those for metallothioneins. In Drosophila, MTF-1 somewhat counter-intuitively also activates transcription of a copper importer gene (Ctr1B) in response to copper starvation. Here, we report that mutant flies lacking Ctr1B are extremely sensitive to cadmium and mercury treatment, but can be rescued by excess copper in the food. We thus propose that copper, by competing for binding sites on cellular proteins, alleviates the toxic effects of mercury and cadmium. Such a scenario also explains a seemingly fortuitous metal response, namely, that cadmium and mercury strongly induce the expression of a Ctr1B reporter gene. Thus, the transcription enhancer/promoter region of the Ctr1B copper importer gene is subject to three modes of regulation. All of them depend on MTF-1 and all make biological sense, namely, (i) induction by copper starvation, (ii) repression by copper abundance, and (iii), as shown here, induction by cadmium or mercury at normal copper supply.

Metal Transcription Factor-1 Is Involved in Hypoxia-Dependent Regulation of Placenta Growth Factor in Trophoblast-Derived Cells

Placenta growth factor (PlGF) is a placental angiogenic factor. Metal-responsive transcription factor (MTF)-1 was reported to take part in the hypoxic induction of PlGF in RAS-transformed mouse fibroblasts. We contrarily showed that PlGF mRNA and protein levels decreased under hypoxia in a choriocarcinoma BeWo cell line derived from trophoblast. In this report, we examined whether hypoxia-dependent regulation of the PlGF gene in these cells also depends on MTF-1. We analyzed the effect of hypoxia on MTF-1 expression, and it was revealed to be decreased. Moreover, MTF-1 small interfering RNA treatment decreased PlGF mRNA level. To investigate the transcription of PlGF under hypoxia, we cloned promoter region of the human PlGF. Promoter deletion analysis suggested that triple repeats of metal-responsive element located between -511 and -468 bp in the promoter are important for the hypoxic regulation of PlGF. Treatment with MTF-1 small interfering RNA resulted in the significant decreased luciferase activity in PlGF reporter constructs. Chromatin immunoprecipitation showed the binding of the MTF-1 protein to the promoter region. We examined MTF-1 immunoreactivity in trophoblasts of term placental tissue from patients with normal pregnancies and preeclampsia, which represents a condition of placental hypoxia. Immunoreactivity of the MTF-1 protein was decreased in placentas from pregnant women with preeclampsia when compared with those from normal pregnant women. Taken together, these findings suggest that MTF-1 is involved in hypoxia-dependent regulation of PlGF in trophoblast-derived cells.

Mechanisms of mammalian zinc-regulated gene expression

Mechanisms through which gene expression is regulated by zinc are central to cellular zinc homoeostasis. In this context, evidence for the involvement of zinc dyshomoeostasis in the aetiology of diseases, including Type 2 diabetes, Alzheimer's disease and cancer, highlights the importance of zinc-regulated gene expression. Mechanisms elucidated in bacteria and yeast provide examples of different possible modes of zinc-sensitive gene regulation, involving the zinc-regulated binding of transcriptional activators and repressors to gene promoter regions. A mammalian transcriptional regulatory mechanism that mediates zinc-induced transcriptional up-regulation, involving the transcription factor MTF1 (metal-response element-binding transcription factor 1), has been studied extensively. Gene responses in the opposite direction (reduced mRNA levels in response to increased zinc availability) have been observed in mammalian cells, but a specific transcriptional regulatory process responsible for such a response has yet to be identified. Examples of single zinc-sensitive transcription factors regulating gene expression in opposite directions are emerging. Although zinc-induced transcriptional repression by MTF1 is a possible explanation in some specific instances, such a mechanism cannot account for repression by zinc of all mammalian genes that show this mode of regulation, indicating the existence of as yet uncharacterized mechanisms of zinc-regulated transcription in mammalian cells. In addition, recent findings reveal a role for effects of zinc on mRNA stability in the regulation of specific zinc transporters. Our studies on the regulation of the human gene SLC30A5 (solute carrier 30A5), which codes for the zinc transporter ZnT5, have revealed that this gene provides a model system by which to study both zinc-induced transcriptional down-regulation and zinc-regulated mRNA stabilization.

Metal responsive transcription factor MTF-1 spatial expression related to functional organization of pituitary cells in Cyprinus carpio

Metal responsive transcription factor MTF-1 spatial expression related to functional organization of pituitary cells in Cyprinus carpio

Overexpression of metal-responsive transcription factor (MTF-1) in Drosophila melanogaster ameliorates life-span reductions associated with oxidative stress and metal toxicity

Heavy metals are essential components of many biological processes but are toxic at high concentrations. Our results illustrate that when metal homeostasis is compromised by a mutation in the metal-responsive transcription factor (MTF-1), the life-span is shortened. In contrast, MTF-1 overexpression results in resistant flies with prolonged longevity on iron or cadmium-supplemented media but shortened life-span on zinc-supplemented medium. This effect was mediated by the overexpression of MTF-1 in specific tissues, such as the gut, hemocytes and in particular in neurons, indicating that these tissues are particularly sensitive to the perturbance of metal homeostasis. Further, MTF-1 overexpression in a neuron-specific manner protects flies against hyperoxia and prolongs the life-span of Cu/Zn superoxide dismutase-deficient flies, suggesting the presence of a common mechanism for protection against both oxidative stress and metal toxicity. Finally, normal life-span is extended up to 40% upon MTF-1 overexpression in either the peripheral nervous system or motorneurons. These results document the tissue-specific import of heavy metal toxicity and oxidative damage in aging and life-span determination.

Characterization of Metal‐Responsive Transcription Factor (MTF‐1) from the Giant Rodent Capybara Reveals Features in Common with Human as Well as with Small Rodents (Mouse, Rat). Short Communication

From mammals to insects, metal-responsive transcription factor 1 (MTF-1) is essential for the activation of metallothionein genes upon heavy-metal load. We have previously found that human MTF-1 induces a stronger metal response than mouse MTF-1. The latter differs from the human one in a number of amino acid positions and is also shorter by 78 aa at its C-terminus. We reasoned that the weaker metal inducibility might be associated with a lesser demand for tight metal homeostasis in a low-weight, short-lived animal, and thus set out to determine the sequence of MTF-1 from the largest living rodent, the Brazilian capybara that can reach 65 kg and also has a considerably longer life span than smaller rodents. An expression clone for capybara MTF-1 was then tested for its activity in both mouse and human cells. Our analysis revealed three unexpected features: i) capybara MTF-1 in terms of amino acid sequence is much more closely related to human than to mouse MTF-1, suggesting an accelerated evolution of MTF-1 in the evolutionary branch leading to small rodents; ii) capybara MTF-1 is even 32 aa shorter at its C-terminus than mouse MTF-1, and iii) in an activity test, it is not more active than mouse MTF-1. The latter two findings might indicate that capybara has evolved in an environment with low heavy-metal load.

Synthesis and Anticancer Properties of Water-Soluble Zinc Ionophores

Several water-solubilized versions of the zinc ionophore 1-hydroxypyridine-2-thione (ZnHPT), synthesized as part of the present study, have been found both to increase the intracellular concentrations of free zinc and to produce an antiproliferative activity in exponential phase A549 human lung cancer cultures. Gene expression profiles of A549 cultures treated with one of these water-soluble zinc ionophores, PCI-5002, reveal the activation of stress response pathways under the control of metal-responsive transcription factor 1 (MTF-1), hypoxia-inducible transcription factor 1 (HIF-1), and heat shock transcription factors. Additional oxidative stress response and apoptotic pathways were activated in cultures grown in zinc-supplemented media. We also show that these water-soluble zinc ionophores can be given to mice at 100 micromol/kg (300 micromol/m(2)) with no observable toxicity and inhibit the growth of A549 lung and PC3 prostate cancer cells grown in xenograft models. Gene expression profiles of tumor specimens harvested from mice 4 h after treatment confirmed the in vivo activation of MTF-1-responsive genes. Overall, we propose that water-solubilized zinc ionophores represent a potential new class of anticancer agents.