Metal-responsive Transcription Factor Research Articles

Dumpy-30 family members as determinants of male fertility and interaction partners of metal-responsive transcription factor 1 (MTF-1) in Drosophila

BackgroundMetal-responsive transcription factor 1 (MTF-1), which binds to metal response elements (MREs), plays a central role in transition metal detoxification and homeostasis. A Drosophila interactome analysis revealed two candidate dMTF-1 interactors, both of which are related to the small regulatory protein Dumpy-30 (Dpy-30) of the worm C. elegans. Dpy-30 is the founding member of a protein family involved in chromatin modifications, notably histone methylation. Mutants affect mating type in yeast and male mating in C. elegans.ResultsConstitutive expression of the stronger interactor, Dpy-30L1 (CG6444), in transgenic flies inhibits MTF-1 activity and results in elevated sensitivity to Cd(II) and Zn(II), an effect that could be rescued by co-overexpression of dMTF-1. Electrophoretic mobility shift assays (EMSA) suggest that Dpy-30L1 interferes with the binding of MTF-1 to its cognate MRE binding site. Dpy-30L1 is expressed in the larval brain, gonads, imaginal discs, salivary glands and in the brain, testes, ovaries and salivary glands of adult flies. Expression of the second interactor, Dpy-30L2 (CG11591), is restricted to larval male gonads, and to the testes of adult males. Consistent with these findings, dpy-30-like transcripts are also prominently expressed in mouse testes. Targeted gene disruption by homologous recombination revealed that dpy-30L1 knockout flies are viable and show no overt disruption of metal homeostasis. In contrast, the knockout of the male-specific dpy-30L2 gene results in male sterility, as does the double knockout of dpy-30L1 and dpy-30L2. A closer inspection showed that Dpy-30L2 is expressed in elongated spermatids but not in early or mature sperm. Mutant sperm had impaired motility and failed to accumulate in sperm storage organs of females.ConclusionOur studies help to elucidate the physiological roles of the Dumpy-30 proteins, which are conserved from yeast to humans and typically act in concert with other nuclear proteins to modify chromatin structure and gene expression. The results from these studies reveal an inhibitory effect of Dpy-30L1 on MTF-1 and an essential role for Dpy-30L2 in male fertility.

Identification and characterization of two mtf-1 genes in common carp

The Metal-responsive Transcription Factor (MTF-1) serves as an essential regulator of Zn 2+ homeostasis via the activation of metallothionein gene expression. Only a single mtf-1 gene has been identified in any organism investigated previously. We report here the first evidence of the existence of two genes encoding MTF-1 proteins ( mtf-1.1 and mtf-1.2). The expression patterns were followed in the liver, kidney, muscle, brain and heart by means of Northern hybridization and reverse transcription coupled polymerase chain reactions (RT-PCR). mtf-1.1 mRNA was detected in all tissues examined, with the highest level in the brain, and the lowest in the kidney and the liver. mtf-1.2 expression was detected exclusively in the brain. Cold shock and Cd 2+ exposure influence the gene expression at the transcriptional level, in a stress-specific manner.

Copper sensing function of Drosophila metal-responsive transcription factor-1 is mediated by a tetranuclear Cu(I) cluster

Drosophila melanogaster MTF-1 (dMTF-1) is a copper-responsive transcriptional activator that mediates resistance to Cu, as well as Zn and Cd. Here, we characterize a novel cysteine-rich domain which is crucial for sensing excess intracellular copper by dMTF-1. Transgenic flies expressing mutant dMTF-1 containing alanine substitutions of two, four or six cysteine residues within the sequence 547CNCTNCKCDQTKSCHGGDC565 are significantly or completely impaired in their ability to protect flies from copper toxicity and fail to up-regulate MtnA (metallothionein) expression in response to excess Cu. In contrast, these flies exhibit wild-type survival in response to copper deprivation thus revealing that the cysteine cluster domain is required only for sensing Cu load by dMTF-1. Parallel studies show that the isolated cysteine cluster domain is required to protect a copper-sensitive S. cerevisiae ace1Δ strain from copper toxicity. Cu(I) ligation by a Cys-rich domain peptide fragment drives the cooperative assembly of a polydentate [Cu4-S6] cage structure, characterized by a core of trigonally S3 coordinated Cu(I) ions bound by bridging thiolate ligands. While reminiscent of Cu4-L6 (L = ligand) tetranuclear clusters in copper regulatory transcription factors of yeast, the absence of significant sequence homology is consistent with convergent evolution of a sensing strategy particularly well suited for Cu(I).

The downstream metal‐regulatory element of Zip10 confers transcriptional gene repression

The mechanisms regulating zinc homeostasis are maintained under tight control, and operate under a wide range of zinc availability. Previously we identified hepatic Zip10 (Slc39a10) as a zinc-responsive gene. A recent study found that Zip10 is induced in mice lacking metal-response element binding transcription factor-1 (MTF-1) (Wimmer et al., Nucleic Acids Res., 2005, 33: 5715). Subsequently, we showed that zinc could cause repression of Zip10 through activation of MTF-1. Here we demonstrate through chromatin immunoprecipitation, that MTF-1 associates with the Zip10 promoter under supplemental zinc conditions, but not under deficient conditions. By creating Zip10-Luciferase constructs we show that the putative metal-response element located at +17 relative to the transcription start site of Zip10 is functional, allowing for zinc-responsiveness of the gene. Therefore, in this downstream promoter position, MTF-1 represses Zip10 expression. This is the first demonstration of the transcriptional activator MTF-1 negatively regulating a zinc transporter in response to zinc. Supported by NIH grant DK31127 and Boston Family Endowment funds (RJC) and CALS Alumni Fellowship (LAL).

Zinc protects endothelial cells from hydrogen peroxide via Nrf2-dependent stimulation of glutathione biosynthesis

Oxidative stress is one of the main causes of vascular disease. This study aims to investigate the antioxidant activity exerted by zinc in primary rat endothelial cells (EC). Using a 24-h treatment with hydrogen peroxide as a model for oxidative stress, we found that zinc supplementation protects from peroxide-induced cell death via increasing the transcription of the catalytic subunit (heavy chain) of glutamate-cysteine ligase (GCLC) and the concentrations of glutathione (GSH). Conversely, zinc depletion significantly decreased the expression of GCLC and the cellular GSH levels, resulting in an increased susceptibility of EC to oxidative stress. Using confocal microscopy and the RNA silencing technique, we found that zinc upregulates the expression of GCLC by activating the transcription factor Nrf2. Surprisingly, the intracellular zinc sensor, metal-responsive transcription factor-1, is not involved in the zinc-induced expression of GCLC. The present study shows that zinc controls the redox state of EC by regulating the de novo synthesis of GSH. This molecular mechanism may contribute to the elaboration of new nutritional and/or pharmaceutical approaches for protecting the endothelium against oxidative stress.

Metallothionein Induction by Hypoxia Involves Cooperative Interactions between Metal-Responsive Transcription Factor-1 and Hypoxia-Inducible Transcription Factor-1α

Mammalian metallothionein (MT) genes are transcriptionally activated by the essential metal zinc as well as by environmental stresses, including toxic metal overload and redox fluctuations. In addition to playing a key role in zinc homeostasis, MT proteins can protect against metal- and oxidant-induced cellular damage, and may participate in other fundamental physiologic and pathologic processes such as cell survival, proliferation, and neoplasia. Previously, our group reported a requirement for metal-responsive transcription factor-1 (MTF-1) in hypoxia-induced transcription of mouse MT-I and human MT-IIA genes. Here, we provide evidence that the protumorigenic hypoxia-inducible transcription factor-1alpha (HIF-1alpha) is essential for induction of MT-1 by hypoxia, but not zinc. Chromatin immunoprecipitation assays revealed that MTF-1 and HIF-1alpha are both recruited to the mouse MT-I promoter in response to hypoxia, but not zinc. In the absence of HIF-1alpha, MTF-1 is recruited to the MT-I promoter but fails to activate MT-I gene expression in response to hypoxia. Thus, HIF-1alpha seems to function as a coactivator of MT-I gene transcription by interacting with MTF-1 during hypoxia. Coimmunoprecipitation studies suggest interaction between MTF-1 and HIF-1alpha, either directly or as mediated by other factors. It is proposed that association of these important transcription factors in a multiprotein complex represents a common strategy to control unique sets of hypoxia-inducible genes in both normal and diseased tissue.

An in vitro method to assess toxicity of waterborne metals to fish

The transcription of metal-responsive genes in the rainbow trout ( Oncorhynchus mykiss) gill tissue can be used to detect effects of bioreactive metals in natural waters. Here we take advantage of an in vitro gill epithelium, which can be directly exposed to test water samples. The in vitro gill epithelial model mimics the molecular response of in vivo gill epithelial cells to waterborne contaminants. The same culture system can detect trace metals and organic waterborne contaminants. Furthermore, combining this epithelial model with transcriptomic profiling yields an extremely discriminatory biomonitoring tool able to detect and differentiate waterborne metal contaminants. The bioreactive fraction of metal in the water sample is detected using the cells naturally occurring metal sensor, metal-responsive transcription factor 1 (MTF1), which acts upon Metal Response Elements (MRE's) in the enhancer region of metal regulated genes. Induction of the MTF1 responsive genes, metallothionein-A (MTA), metallothionein-B (MTB), and zinc transporter 1 (ZnT-1) in the cell culture was strongly dependent of the concentrations of bioreactive zinc and silver in the test water. Importantly, gene expression in cell culture reflected animal toxicity, measured as inhibition of Ca 2+ and Na + influx, in live rainbow trout exposed to the same waters. A cDNA microarray was deployed to determine the differential profiles of transcripts characteristic of exposure to silver, copper or cadmium within this in vitro system. These experiments illustrated the potential power of combining the in vitro gill model epithelium with genetic profiling for accurate characterisation and identification of bioreactive toxicants in waterborne samples.

Understanding the mechanisms of zinc-sensing by metal-response element binding transcription factor-1 (MTF-1)

The regulation of divalent zinc has been observed in a wide range of organisms. Since this metal is an essential nutrient, but also toxic in excess, zinc homeostasis is crucial for normal cellular functioning. The metal-responsive-element-binding transcription factor-1 (MTF-1) is a key regulator of zinc in higher eukaryotes ranging from insects to mammals. MTF-1 controls the expression of metallothioneins ( MTs) and a number of other genes directly involved in the intracellular sequestration and transport of zinc. Although the diverse functions of MTF-1 extend well beyond zinc homeostasis to include stress-responses to heavy metal toxicity, oxidative stress, and selected chemical agents, in this review we focus on the recent advances in understanding the mechanisms whereby MTF-1 regulates MT gene expression to protect the cell from fluctuations in environmental zinc. Particular emphasis is devoted to recent studies involving the Cys 2His 2 zinc finger DNA-binding domain of MTF-1, which is an important contributor to the zinc-sensing and metal-dependent transcriptional activation functions of this protein.

Copper homeostasis in Drosophila by complex interplay of import, storage and behavioral avoidance

Copper is an essential but potentially toxic trace element. In Drosophila, the metal-responsive transcription factor (MTF-1) plays a dual role in copper homeostasis: at limiting copper concentrations, it induces the Ctr1B copper importer gene, whereas at high copper concentrations, it mainly induces the metallothionein genes. Here we find that, despite the downregulation of the Ctr1B gene at high copper concentrations, the protein persists on the plasma membrane of intestinal cells for many hours and thereby fills the intracellular copper stores. Drosophila may risk excessive copper accumulation for the potential benefit of overcoming a period of copper scarcity. Indeed, we find that copper-enriched flies donate a vital supply to their offspring, allowing the following generation to thrive on low-copper food. We also describe two additional modes of copper handling: behavioral avoidance of food containing high (>or=0.5 mM) copper levels, as well as the ability of DmATP7, the Drosophila homolog of Wilson/Menkes disease copper exporters, to counteract copper toxicity. Regulated import, storage, export, and avoidance of high-copper food establish an adequate copper homeostasis under variable environmental conditions.

Gene expression analysis of gallium-resistant and gallium-sensitive lymphoma cells reveals a role for metal-responsive transcription factor-1, metallothionein-2A, and zinc transporter-1 in modulating the antineoplastic activity of gallium nitrate

Several clinical trials have shown gallium nitrate to be an active agent in the treatment of lymphoma. Whereas gallium is known to target cellular iron homeostasis, the basis for lymphoma cell resistance to gallium is not known. Understanding mechanisms of resistance may suggest strategies to enhance the clinical efficacy of gallium. In the present study, we used a focused DNA microarray to compare the expression of genes related to metal metabolism in gallium-resistant and gallium-sensitive lymphoma cell lines developed by us. Gallium-resistant cells were found to display a marked increase in gene expression for metallothionein-2A and the zinc transporter ZnT-1. Cells exposed to gallium nitrate displayed an increase in the binding of metal-responsive transcription factor-1 to metal response element sequences involved in the transcriptional regulation of metallothionein and ZnT-1 genes. Gallium nitrate induced metallothionein-2A and ZnT-1 expression in cells. A role for metallothionein in modulating the antineoplastic activity of gallium was confirmed by showing that the induction of metallothionein expression by zinc provided partial protection against the cytotoxicity of gallium and by showing that the level of endogenous metallothionein in lymphoma cell lines correlated with their sensitivity to gallium nitrate. Immunohistochemical staining of lymphomatous tissues revealed metallothionein protein to be variably expressed in different lymphomas. Our studies show for the first time that gallium acts on pathways related to zinc metabolism and that metal-responsive transcription factor-1 activity and metallothionein expression contribute to the development of gallium drug resistance. Furthermore, the endogenous level of metallothionein in lymphoma may be an important determinant of clinical response to gallium nitrate.

Mtf-1 lymphoma-susceptibility locus affects retention of large thymocytes with high ROS levels in mice after γ-irradiation

Mouse strains exhibit different susceptibilities to γ-ray-induced thymic lymphomas. Our previous study identified Mtf-1 (metal responsive transcription factor-1) as a candidate susceptibility gene, which is involved in the radiation-induced signaling pathway that regulates the cellular reactive oxygen species (ROS). To reveal the mechanism for the increased susceptibility conferred by Mtf-1 locus, we examined early effects of γ-ray on ROS levels in vivo and its difference between Mtf-1 susceptible and resistant congenic mice. Here, we show the detection of clonally growing thymocytes at 4 weeks after irradiation, indicating the start of clonal expansion at a very early stage. We also show that large thymocytes with higher ROS levels and a proliferation capacity were more numerous in the Mtf-1 susceptible mice than the resistant mice when examined at 7 days after irradiation, although such tendency was not found in mice lacking one allele of Bcl11b tumor suppressor gene. This high retention of the large thymocytes, at a high risk for ROS-induced mutation, is a compensatory proliferation and regeneration response to depletion of the thymocytes after irradiation and the response is likely to augment the development of prelymphoma cells leading to thymic lymphomas.

A Cadmium-transporting P1B-type ATPase in Yeast Saccharomyces cerevisiae

Detoxification and homeostatic acquisition of metal ions are vital for all living organisms. We have identified PCA1 in yeast Saccharomyces cerevisiae as an overexpression suppressor of copper toxicity. PCA1 possesses signatures of a P1B-type heavy metal-transporting ATPase that is widely distributed from bacteria to humans. Copper resistance conferred by PCA1 is not dependent on catalytic activity, but it appears that a cysteine-rich region located in the N terminus sequesters copper. Unexpectedly, when compared with two independent natural isolates and an industrial S. cerevisiae strain, the PCA1 allele of the common laboratory strains we have examined possesses a missense mutation in a predicted ATP-binding residue conserved in P1B-type ATPases. Consistent with a previous report that identifies an equivalent mutation in a copper-transporting P1B-type ATPase of a Wilson disease patient, the PCA1 allele found in laboratory yeast strains is nonfunctional. Overexpression or deletion of the functional allele in yeast demonstrates that PCA1 is a cadmium efflux pump. Cadmium as well as copper and silver, but not other metals examined, dramatically increase PCA1 protein expression through post-transcriptional regulation and promote subcellular localization to the plasma membrane. Our study has revealed a novel metal detoxification mechanism in yeast mediated by a P1B-type ATPase that is unique in structure, substrate specificity, and mode of regulation.

亜鉛応答性転写因子MTF-1とCr（VI）毒性</br>—6価クロムの発がん性と遺伝子発現抑制作用との関係—

Chromium exists in many different oxidation states in the environment, Cr(VI) and Cr(III) being the most stable forms. Chromium has been known for over 100 years to be a human carcinogen. The greatest risk of cancer from chromium exposure is associated with Cr(VI). Cr(VI) enters cells via the sulfate anion transporter system and is reduced to intermediate oxidation states, such as Cr(V) and Cr(IV), in the process of forming stable Cr(III) forms. It is known that Cr(VI) affects expression of various genes. Metal responsive element-binding transcription factor-1 (MTF-1) is involved in sensing heavy metal load and the induced transcription of several protective genes, including metallothionein (MT)-I, MT-II, zinc transporter-1, and gamma-glutamylcysteine synthetase. Cr(VI) inhibits zinc-induced MT transcription via modifying transactivation potential of MTF-1. However, the molecular mechanism for the Cr(VI)-mediated inhibition of MTF-1 has not been fully elucidated. In this review, I briefly summarize the previous studies and discuss the current status of research on Cr(VI) toxicity and Cr(VI)-mediated inhibition against transcription.

Expression pattern of metallothionein, MTF‐1 nuclear translocation, and its dna‐binding activity in zebrafish (Danio rerio) induced by zinc and cadmium

Metallothionein is a small (6-kDa), cysteine-rich protein expressed by a six-zinc finger protein called metal-responsive element-binding transcription factor-1 (MTF-1) in response to Zn and Cd. Our previous reports have shown the basal expression of metallothionein (mt) and MTF-I (mtf-1) genes in embryo and early larval stages of zebrafish (Danio rerio). In the present study, we investigated the mt expression in zebrafish early larvae induced by exposure to Cd and Zn (48, 72, 96, and 120 h postfertilization). Whole-mount in situ hybridization showed that Zn induced mt expression in the olfactory pit, cerebellum, ceratobranchials, liver, chloride cells, and neuromasts of the lateral line. Cadmium also induced mt expression in all the above regions except the cerebellum. Using fluorescence techniques, we have shown that Zn and Cd mediate cytoplasmic and nuclear translocation of MTF- 1-enhanced green fluorescent protein fusion protein in zebrafish liver cell line. The MTF-1 protein was produced recombinantly by inserting zebrafish mtf-1 cDNA (1.8 kb) into pET-20b(+) expression vector and expressing in Escherichia coli BL21 (DE3) pLysS host strain competent cell on induction with isopropyl-beta-D-thiogalactopyranoside. The protein was then purified by affinity chromatography on a nickel-nitrilotriacetic acid column. Electrophoretic mobility shift assay revealed binding of the recombinant MTF-1 in response to Zn and Cd at the putative metal-responsive elements (MREs) in the promoter region of the mt gene. Taken together, these results suggest that Zn and Cd are efficiently involved with mt expression induced in zebrafish embryos and with MTF-1 nuclear translocation and that this induction is achieved through the activation of MTF-1 binding at the MREs.

Advances in understanding corticotrophin-releasing hormone gene expression

Glucocorticoids inhibit corticotrophin-releasing hormone (CRH) gene expression in the hypothalamic paraventricular nucleus (PVN), but stimulate expression in the placenta. In AtT20 cells (a model of PVN CRH production) cAMP produces a high level of promoter activity. Cyclic AMP stimulation occurs through the cAMP response element (CRE) and the caudal type homeobox protein response element (CDXARE). The CRE acts as part of a cAMP response unit that includes the hybrid steroid response element (HRE), ecdysone response element (EcRE), metal-responsive transcription factor-1 response element (MTFRE), ying yang 1 response element (YY1RE) and negative glucocorticoid response element (nGRE). Cyclic AMP acts on the HRE, EcRE and MTFRE to block YY1RE mediated inhibition of the CRE. Glucocorticoids acting at the nGRE inhibit cAMP activation of the CRE. In placental cells the CRH promoter has low intrinsic basal activity and cAMP causes a modest increase in activity. Stimulation by glucocorticoids and cAMP and inhibition by estrogen and estrogen receptor alpha occurs through the CRE. In AtT20 cells multiple response elements coordinate a response to cAMP and glucocorticoids while in placental cells the CRE acts in isolation. These differences in promoter function lead to responses that meet specific physiological needs.

Transcriptome response to heavy metal stress in Drosophila reveals a new zinc transporter that confers resistance to zinc

All organisms are confronted with external variations in trace element abundance. To elucidate the mechanisms that maintain metal homeostasis and protect against heavy metal stress, we have determined the transcriptome responses in Drosophila to sublethal doses of cadmium, zinc, copper, as well as to copper depletion. Furthermore, we analyzed the transcriptome of a metal-responsive transcription factor (MTF-1) null mutant. The gene family encoding metallothioneins, and the ABC transporter CG10505 that encodes a homolog of ‘yeast cadmium factor’ were induced by all three metals. Zinc and cadmium responses have similar features: genes upregulated by both metals include those for glutathione S-transferases GstD2 and GstD5, and for zinc transporter-like proteins designated ZnT35C and ZnT63C. Several of the metal-induced genes that emerged in our study are regulated by the transcription factor MTF-1. mRNA studies in MTF-1 overexpressing or null mutant flies and in silico search for metal response elements (binding sites for MTF-1) confirmed novel MTF-1 regulated genes such as ferritins, the ABC transporter CG10505 and the zinc transporter ZnT35C. The latter was analyzed in most detail; biochemical and genetic approaches, including targeted mutation, indicate that ZnT35C is involved in cellular and organismal zinc efflux and plays a major role in zinc detoxification.

The four members of the Drosophila metallothionein family exhibit distinct yet overlapping roles in heavy metal homeostasis and detoxification

Four metallothionein genes are present in the Drosophila melanogaster genome, designated MtnA, MtnB, MtnC, MtnD, all of which are transcriptionally induced by heavy metals through the same metal-responsive transcription factor, MTF-1. Here we show, by targeted mutagenesis, that the four metallothionein genes exhibit distinct, yet overlapping, roles in heavy metal homeostasis and toxicity prevention. Among the individual metallothionein mutants, the most prominent distinction between them was that MtnA-defective flies were the most sensitive to copper load, while MtnB-defective flies were the most sensitive to cadmium. Using various reporter gene constructs and mRNA quantification, we show that the MtnA promoter is preferentially induced by copper, while the MtnB promoter is preferentially induced by cadmium. Such a metal preference is also observed at the protein level as the stoichiometric, spectrometric and spectroscopic features of the copper and cadmium complexes with MtnA and MtnB correlate well with a greater stability of copper-MtnA and cadmium-MtnB. Finally, MtnC and MtnD, both of which are very similar to MtnB, display lower copper and cadmium binding capabilities compared to either MtnA or MtnB. In accordance with these binding studies, Drosophila mutants of MtnC or MtnD have a near wild type level of resistance against copper or cadmium load. Furthermore, eye-specific over-expression of MtnA and MtnB, but not of MtnC or MtnD, can rescue a "rough eye" phenotype caused by copper load in the eye. Taken together, while the exact roles of MtnC and MtnD remain to be determined, the preferential protection against copper and cadmium toxicity by MtnA and MtnB, respectively, are the result of a combination of promoter preference and metal binding.

Water-Soluble Genistin Glycoside Isoflavones Up-Regulate Antioxidant Metallothionein Expression and Scavenge Free Radicals

Genistin has antioxidant activities; however, its insolubility in water often limits its biological availability in vivo. Using a novel transglycosylation process, the solubility of genistin glycosides was increased 1000 to 10000-fold, but it was not known whether these modified genistin glycosides maintained antioxidant activity. We found that both genistin and its glycosides similarly up-regulated the transcription of several metallothionein (MT) antioxidant genes (MT1A, MT2A, MT1E, and MT1X), as well as the glucose 6-phosphate dehydrogenase (G6PD) gene in HepG2 cells. This gene induction was mediated by the sequestration of zinc in the cytosol, which up-regulated the metal-responsive transcription factor-1 (MTF-1) that induced MT gene expression. Although not as effective as ascorbic acid, genistin glycosides possessed slightly greater reducing power than genistin. We concluded that genistin and genistin glycosides have a direct antioxidant effect and an indirect antioxidant effect, perhaps via induction of MT by activity of MTF-1.

MTF‐1 Contributes to the Activation of MMP‐2

We previously reported a unique role for the redox-sensitive, metal-responsive transcription factor-1 (MTF-1) in tumor development: loss of Mtf1 delays fibrosarcoma tumor growth, increases tumor collagen deposition, and increases expression and activation of tumor cell and whole tumor transforming growth factor and tissue transglutaminase, two pivotal profibrotic proteins. This ongoing study demonstrates another level of MTF-1-dependent control over extracellular matrix (ECM) remodeling. Using ras-transformed Mtf1 null mouse embryonic fibroblasts (MEFs) and retroviral rescue of Mtf1, we show that MTF-1 is essential for optimal activation of matrix metalloproteinase (MMP)-2. Loss of Mtf1 markedly reduced activated MMP-2, while Mtf1 rescue partly restored the wild type phenotype. Conversely, levels of tissue inhibitor of metalloproteinase (TIMP)-2 dramatically increased in Mtf1 null MEFs compared with wild type cells. Retroviral-directed rescue of Mtf1 significantly reduced these levels. Since the membrane type 1-metalloproteinase (MT1-MMP; MMP-14) forms a complex with TIMP-2 and MMP-2 in regulating activation of MMP-2, we also assessed its levels as a function of MTF-1 expression. Activity levels of MT1-MMP were significantly higher in wild type fibroblasts than in the Mtf1 knockout MEFs. The precise underlying mechanisms of this MTF-1-dependent system are under investigation. Together, our data indicate a role for MTF-1 in ECM remodeling and thus in important physiological and pathophysiological processes. This work was supported by NIH Grant CA 057692.

A Family Knockout of All Four Drosophila Metallothioneins Reveals a Central Role in Copper Homeostasis and Detoxification

Metallothioneins are ubiquitous, small, cysteine-rich proteins with the ability to bind heavy metals. In spite of their biochemical characterization, their in vivo function remains elusive. Here, we report the generation of a metallothionein gene family knockout in Drosophila melanogaster by targeted disruption of all four genes (MtnA to -D). These flies are viable if raised in standard laboratory food. During development, however, they are highly sensitive to copper, cadmium, and (to a lesser extent) zinc load. Metallothionein expression is particularly important for male viability; while copper load during development affects males and females equally, adult males lacking metallothioneins display a severely reduced life span, possibly due to copper-mediated oxidative stress. Using various reporter gene constructs, we find that different metallothioneins are expressed with virtually the same tissue specificity in larvae, notably in the intestinal tract at sites of metal accumulation, including the midgut's "copper cells." The same expression pattern is observed with a synthetic minipromoter consisting only of four tandem metal response elements. From these and other experiments, we conclude that tissue specificity of metallothionein expression is a consequence, rather than a cause, of metal distribution in the organism. The bright orange luminescence of copper accumulated in copper cells of the midgut is severely reduced in the metallothionein gene family knockout, as well as in mutants of metal-responsive transcription factor 1 (MTF-1), the main regulator of metallothionein expression. This indicates that an in vivo metallothionein-copper complex forms the basis of this luminescence. Strikingly, metallothionein mutants show an increased, MTF-1-dependent induction of metallothionein promoters in response to copper, cadmium, silver, zinc, and mercury. We conclude that free metal, but not metallothionein-bound metal, triggers the activation of MTF-1 and that metallothioneins regulate their own expression by a negative feedback loop.