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• W2105280572 abstract "Metal regulation of the mouse zinc transporter (ZnT)-1 gene was examined in cultured cells and in the developing conceptus. Zinc or cadmium treatment of cell lines rapidly (3 h) and dramatically (about 12-fold) induced ZnT1 mRNA levels. In cells incubated in medium supplemented with Chelex-treated fetal bovine serum, to remove metal ions, levels of ZnT1 mRNA were reduced, and induction of this message in response to zinc or cadmium was accentuated (up to 31-fold induction). Changes in ZnT1 gene expression in these experiments paralleled those of metallothionein I (MT-I). Inhibition of RNA synthesis blocked metal induction of ZnT1 and MT-I mRNAs, whereas inhibition of protein synthesis did not. Metal response element-binding transcription factor (MTF)-1 mediates metal regulation of the metallothionein I gene. In vitroDNA-binding assays demonstrated that mouse MTF-1 can bind avidly to the two metal-response element sequences found in the ZnT1 promoter. Using mouse embryo fibroblasts with homozygous deletions of the MTF-1 gene, it was shown that this transcription factor is essential for basal as well as metal (zinc and cadmium) regulation of the ZnT1 gene in these cells. In vivo, ZnT1 mRNA was abundant in the midgestation visceral yolk sac and placenta. Dietary zinc deficiency during pregnancy down-regulated ZnT1 and MT-I mRNA levels (4–5-fold and >20-fold, respectively) in the visceral yolk sac, but had little effect on these mRNAs in the placenta. Homozygous knockout of the MTF-1 gene in transgenic mice also led to a 4–6-fold reduction in ZnT1 mRNA levels and a loss of MT-I mRNA in the visceral yolk sac. These results suggest that MTF-1 mediates the response to metal ions of both the ZnT1 and the MT-I genes the visceral yolk sac. Overall, these studies suggest that MTF-1 directly coordinates the regulation of genes involved in zinc homeostasis and protection against metal toxicity. Metal regulation of the mouse zinc transporter (ZnT)-1 gene was examined in cultured cells and in the developing conceptus. Zinc or cadmium treatment of cell lines rapidly (3 h) and dramatically (about 12-fold) induced ZnT1 mRNA levels. In cells incubated in medium supplemented with Chelex-treated fetal bovine serum, to remove metal ions, levels of ZnT1 mRNA were reduced, and induction of this message in response to zinc or cadmium was accentuated (up to 31-fold induction). Changes in ZnT1 gene expression in these experiments paralleled those of metallothionein I (MT-I). Inhibition of RNA synthesis blocked metal induction of ZnT1 and MT-I mRNAs, whereas inhibition of protein synthesis did not. Metal response element-binding transcription factor (MTF)-1 mediates metal regulation of the metallothionein I gene. In vitroDNA-binding assays demonstrated that mouse MTF-1 can bind avidly to the two metal-response element sequences found in the ZnT1 promoter. Using mouse embryo fibroblasts with homozygous deletions of the MTF-1 gene, it was shown that this transcription factor is essential for basal as well as metal (zinc and cadmium) regulation of the ZnT1 gene in these cells. In vivo, ZnT1 mRNA was abundant in the midgestation visceral yolk sac and placenta. Dietary zinc deficiency during pregnancy down-regulated ZnT1 and MT-I mRNA levels (4–5-fold and >20-fold, respectively) in the visceral yolk sac, but had little effect on these mRNAs in the placenta. Homozygous knockout of the MTF-1 gene in transgenic mice also led to a 4–6-fold reduction in ZnT1 mRNA levels and a loss of MT-I mRNA in the visceral yolk sac. These results suggest that MTF-1 mediates the response to metal ions of both the ZnT1 and the MT-I genes the visceral yolk sac. Overall, these studies suggest that MTF-1 directly coordinates the regulation of genes involved in zinc homeostasis and protection against metal toxicity. zinc transporter bovine serum albumin Dulbecco's modified Eagle's medium electrophoretic mobility shift assay fetal bovine serum mouse embryo fibroblast metal response element metallothionein metal response element-binding transcription factor-1 zinc-adequate diet zinc-deficient diet base pair(s) reverse transcriptase polymerase chain reaction day Zinc metabolism is controlled by uptake and efflux, as well as by storage in peripheral tissues, but the mechanisms regulating homeostasis of this metal are poorly defined. Zinc absorption occurs in the intestinal mucosa (1Oestreicher P. Cousins R.J. J. Nutr. 1989; 119: 639-646Crossref PubMed Scopus (29) Google Scholar), and zinc is primarily lost in the bile-pancreatic secretions (2Walsh C.T. Sandstead H.H. Prasad A.S. Newberne P.M. Fraker P.J. Environ. Health Perspect. 1994; 102 Suppl. 2: 5-46PubMed Google Scholar, 3McClain C.J. J. Lab. Clin. Med. 1990; 116: 275-276PubMed Google Scholar). Four mammalian genes involved in zinc transport have been identified (4McMahon R.J. Cousins R.J. J. Nutr. 1998; 128: 667-670Crossref PubMed Scopus (180) Google Scholar). Zinc transporters (ZnT)1 1–4 are proteins with six membrane-spanning domains; these four proteins function in the efflux or vesicular storage of zinc (5Palmiter R.D. Cole T.B. Findley S.D. EMBO J. 1996; 15: 1784-1791Crossref PubMed Scopus (393) Google Scholar, 6Palmiter R.D. Cole T.B. Quaife C.J. Findley S.D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14934-14939Crossref PubMed Scopus (595) Google Scholar). Mouse ZnT2 causes the vesicular accumulation of zinc in endosomal vesicles (5Palmiter R.D. Cole T.B. Findley S.D. EMBO J. 1996; 15: 1784-1791Crossref PubMed Scopus (393) Google Scholar) and is most similar in structure to ZnT3, which is responsible for the accumulation of zinc in synaptic vesicles in the brain (7Wenzel H.J. Cole T.B. Born D.E. Schwartzkroin P.A. Palmiter R.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12676-12681Crossref PubMed Scopus (287) Google Scholar, 8Cole T.B. Wenzel H.J. Kafer K.E. Schwartzkroin P.A. Palmiter R.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1716-1721Crossref PubMed Scopus (449) Google Scholar). Targeted deletion of ZnT3 is not lethal (8Cole T.B. Wenzel H.J. Kafer K.E. Schwartzkroin P.A. Palmiter R.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1716-1721Crossref PubMed Scopus (449) Google Scholar). ZnT4 was identified during a search for the Lethal Milk locus in the mouse (9Huang L.P. Gitschier J. Nat. Genet. 1997; 17: 292-297Crossref PubMed Scopus (307) Google Scholar). This zinc effluxer is highly expressed in the mammary gland, but may be involved in more general zinc homeostasis in the adult (9Huang L.P. Gitschier J. Nat. Genet. 1997; 17: 292-297Crossref PubMed Scopus (307) Google Scholar). ZnT1 functions to efflux zinc from cells, is localized to the plasma membrane, and is expressed ubiquitously (5Palmiter R.D. Cole T.B. Findley S.D. EMBO J. 1996; 15: 1784-1791Crossref PubMed Scopus (393) Google Scholar, 10Palmiter R.D. Findley S.D. EMBO J. 1995; 14: 639-649Crossref PubMed Scopus (636) Google Scholar). ZnT1 is an essential gene, and homozygous knockout of the ZnT1 gene is lethal to the embryo. 2R. D. Palmiter, personal communication.2R. D. Palmiter, personal communication. Zinc induction of ZnT1 mRNA had been documented in cultured neurons (11Tsuda M. Imaizumi K. Katayama T. Kitagawa K. Wanaka A. Tohyama M. Takagi T. J. Neurosci. 1997; 17: 6678-6684Crossref PubMed Google Scholar), and in the rat intestine after oral gavage with zinc (12McMahon R.J. Cousins R.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4841-4846Crossref PubMed Scopus (255) Google Scholar, 13Davis S.R. McMahon R.J. Cousins R.J. J. Nutr. 1998; 128: 825-831Crossref PubMed Scopus (81) Google Scholar). Furthermore, ZnT1 expression in enterocytes can be regulated by dietary zinc (12McMahon R.J. Cousins R.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4841-4846Crossref PubMed Scopus (255) Google Scholar). These preliminary studies suggested that zinc may regulate ZnT1 gene expression.In higher eukaryotes, the best understood metal-regulated genes are the metallothioneins (MT) (for review, see Ref. 14Andrews G.K. Biochem. Pharmacol. 2000; 59: 95-104Crossref PubMed Scopus (708) Google Scholar). Transcription of the mouse MT-I gene, for example, is regulated by zinc and cadmium, and this regulation is mediated by metal response element-binding transcription factor-1 (MTF-1) (15Heuchel R. Radtke F. Georgiev O. Stark G. Aguet M. Schaffner W. EMBO J. 1994; 13: 2870-2875Crossref PubMed Scopus (403) Google Scholar). MTF-1 is a six zinc-finger (Cys2His2) transcription factor, which functions as a sensor of intracellular zinc (for review, see Ref. 14Andrews G.K. Biochem. Pharmacol. 2000; 59: 95-104Crossref PubMed Scopus (708) Google Scholar). MTF-1 is activated by zinc to bind to metal response elements (MREs) in the MT-I promoter, resulting in an increased rate of transcription of this gene (15Heuchel R. Radtke F. Georgiev O. Stark G. Aguet M. Schaffner W. EMBO J. 1994; 13: 2870-2875Crossref PubMed Scopus (403) Google Scholar, 16Dalton T.P. Li Q.W. Bittel D. Liang L.C. Andrews G.K. J. Biol. Chem. 1996; 271: 26233-26241Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 17Koizumi S. Suzuki K. Ogra Y. Yamada H. Otsuka F. Eur. J. Biochem. 1999; 259: 635-642Crossref PubMed Scopus (101) Google Scholar). Cadmium activation of MT-I gene expression also requires MTF-1. In the present study, the hypothesis that zinc and cadmium regulate ZnT1 gene expression was tested and the potential role of MTF-1 in this response was examined. The ZnT1 gene was found to be responsive to zinc excess and deficiency, as well as to cadmium. These metals rapidly induced the coordinated synthesis of ZnT1 and MT-I mRNAs in cultured cells. In vitro DNA-binding assays demonstrated that recombinant mouse MTF-1 can bind to the MRE sequences present in the mouse ZnT1 promoter and studies of MTF-1 knockout mice and mouse embryonic fibroblast cells revealed an essential role for MTF-1 in metal responsiveness of these genes.DISCUSSIONThese studies demonstrate that expression of the mouse ZnT1 gene is regulated, in part, by the heavy metals zinc and cadmium, and suggest that MTF-1 is the transcription factor that mediates this response. Thus, MTF-1 coordinates the expression of genes that play roles in zinc homeostasis, as well as in protection from metal toxicity. Exposure of cells to excess zinc results in the increased expression of MT genes, which encode the major intracellular zinc storage proteins (40Kagi J.H.R. Methods Enzymol. 1991; 205: 613-626Crossref PubMed Scopus (737) Google Scholar), and the expression of ZnT1, which effluxes the metal from the cell (10Palmiter R.D. Findley S.D. EMBO J. 1995; 14: 639-649Crossref PubMed Scopus (636) Google Scholar). Reciprocally, under conditions of zinc deprivation, MTs are degraded to provide a biologically active labile pool of zinc (19Dalton T.P. Fu K. Palmiter R.D. Andrews G.K. J. Nutr. 1996; 126: 825-833Crossref PubMed Scopus (106) Google Scholar, 20Andrews G.K. Geiser J. J. Nutr. 1999; 129: 1643-1648Crossref PubMed Scopus (58) Google Scholar), and the efflux of zinc via ZnT1 is attenuated (4McMahon R.J. Cousins R.J. J. Nutr. 1998; 128: 667-670Crossref PubMed Scopus (180) Google Scholar, 10Palmiter R.D. Findley S.D. EMBO J. 1995; 14: 639-649Crossref PubMed Scopus (636) Google Scholar) leading to conservation of this metal in the cell. However, unlike MT-I and -II (41Masters B.A. Kelly E.J. Quaife C.J. Brinster R.L. Palmiter R.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 584-588Crossref PubMed Scopus (564) Google Scholar), MTF-1 (18Günes Ç. Heuchel R. Georgiev O. Müller K.H. Lichtlen P. Blüthmann H. Marino S. Aguzzi A. Schaffner W. EMBO J. 1998; 17: 2846-2854Crossref PubMed Scopus (220) Google Scholar) and ZnT12 are essential for embryonic development of the mouse. This suggests that metal efflux plays a more important role during development of the embryo than does metal storage. Remarkably, cadmium also coordinately regulates the expression of MT-I and ZnT1 genes, suggesting that ZnT1 may also play a role in protecting from cadmium toxicity, as does MT (41Masters B.A. Kelly E.J. Quaife C.J. Brinster R.L. Palmiter R.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 584-588Crossref PubMed Scopus (564) Google Scholar, 42Liu Y. Liu J. Iszard M.B. Andrews G.K. Palmiter R.D. Klaassen C.D. Toxicol. App. Pharmacol. 1995; 135: 222-228Crossref PubMed Scopus (133) Google Scholar). Consistent with this concept are the findings that overexpression of ZnT1 protects cells from zinc toxicity (10Palmiter R.D. Findley S.D. EMBO J. 1995; 14: 639-649Crossref PubMed Scopus (636) Google Scholar), and that zinc-resistant Hepa cells overexpress MT as well as ZnT1. 4R. Ravindra, unpublished results. Whether these cells also display increased efflux of cadmium and increased resistance to cadmium toxicity remains to be determined. A recent study of the ZnTA gene in Escherichia coli, which is a cadmium/zinc-exporting P1-type ATPase, is also regulated by zinc and cadmium (43Noll M. Lutsenko S. Biochem. Mol. Biol. Int. 2000; 49: 297-302Google Scholar).Whether MTF-1 directly or indirectly regulates ZnT1 gene expression remains to be determined, and the data presented herein cannot formally exclude either possibility. However, several lines of evidence are consistent with the concept that MTF-1 directly regulates ZnT1 gene expression in response to metals. First, both zinc and cadmium induce the rapid and coordinated synthesis of ZnT1 and MT-I mRNAs in cultured cells that contain MTF-1, but not in those lacking MTF-1. Second, both ZnT1 and MT-I mRNAs are specifically elevated in the visceral endoderm during early development of the embryo, both genes respond to dietary zinc deficiency, and both are reduced in mice lacking MTF-1. Third, MTF-1 can bind with avidity to two MREs found in the ZnT1 promoter, as it can with MRE sequences from the mouse MT-I promoter. Despite these findings, previous transfection studies using the ZnT1 promoter did not demonstrate metal regulation (10Palmiter R.D. Findley S.D. EMBO J. 1995; 14: 639-649Crossref PubMed Scopus (636) Google Scholar). The reason for this discrepancy warrants further investigation. Clearly, there are similarities and also distinct differences in the mechanisms of regulation of the ZnT1 and MT-I genes. Unlike the mouse MT-I promoter, which contains five functional MREs in the proximal 200-bp promoter, the ZnT1 proximal promoter contains only two MRE sequences. MTF-1 plays an important, but nonessential, role in regulating the ZnT1 gene in visceral endoderm cells in vivo. Thus, the basal level of expression of the ZnT1 gene is clearly dependent on transcription factors other than MTF-1. One potential binding site for the zinc-finger transcription factor Sp1 is present upstream of the MREs in the proximal MT-I promoter, whereas at least four such sites are found in the ZnT1 promoter. Further studies of the structure and function of the ZnT1 promoter are required.The finding that the visceral yolk sac actively expresses both the ZnT1 gene and the MT-I/II genes suggests that this organ plays an important role in zinc homeostasis, and protection from excess zinc during pregnancy. Preliminary immunolocalization studies using rat ZnT1 antisera (provided by R. J. Cousins, University of Florida, Gainsville, FL) detected immunoreactivity specifically in the visceral endoderm layer of the yolk sac.4 These cells are also the site of synthesis of MT (36Andrews G.K. McMaster M.T. De S.K. Paria B.C. Dey S.K. Suzuki K.T. Imura N. Kimura M. Metallothionein III: Biological Roles and Medical Implications. Birkhauser Verlag, Basel, Switzerland1993: 351-362Google Scholar). Visceral endoderm cells are the second cell type to differentiate from the primitive endoderm of the inner cell mass and they form the secretory layer of the visceral yolk sac, which surrounds the embryo until late in pregnancy (d19). These cells are responsible for the synthesis of serum proteins, and the visceral yolk sac is the first site of hematopoiesis. The visceral endoderm plays a nutritive and supportive role for embryonic development of the mouse. Previous studies demonstrated that the mouse MT genes become responsive to metal ions first at the morula/blastocyst stage of development. Given the role of MTF-1 in metal regulation of MT as well as ZnT1 genes, these studies suggest that ZnT1 gene expression may also be activated and responsive to metals first at this stage of preimplantation development. Further studies are required to address this possibility.In summary, these studies demonstrate that the mouse ZnT1 gene can be regulated by zinc as well as cadmium, and that this regulation is dependent on the transcription factor MTF-1. It was further demonstrated that expression of the ZnT1 gene is highly active in the visceral yolk sac of the developing embryo, and this expression is partially dependent on MTF-1 and dietary zinc. MTF-1 was known to regulate expression of the MT-I/II genes in mice, but the MT genes are nonessential. In contrast, the MTF-1 gene is essential for development, which suggested that this transcription factor also regulates the expression of an essential gene(s). One such gene is the ZnT1 gene. Zinc metabolism is controlled by uptake and efflux, as well as by storage in peripheral tissues, but the mechanisms regulating homeostasis of this metal are poorly defined. Zinc absorption occurs in the intestinal mucosa (1Oestreicher P. Cousins R.J. J. Nutr. 1989; 119: 639-646Crossref PubMed Scopus (29) Google Scholar), and zinc is primarily lost in the bile-pancreatic secretions (2Walsh C.T. Sandstead H.H. Prasad A.S. Newberne P.M. Fraker P.J. Environ. Health Perspect. 1994; 102 Suppl. 2: 5-46PubMed Google Scholar, 3McClain C.J. J. Lab. Clin. Med. 1990; 116: 275-276PubMed Google Scholar). Four mammalian genes involved in zinc transport have been identified (4McMahon R.J. Cousins R.J. J. Nutr. 1998; 128: 667-670Crossref PubMed Scopus (180) Google Scholar). Zinc transporters (ZnT)1 1–4 are proteins with six membrane-spanning domains; these four proteins function in the efflux or vesicular storage of zinc (5Palmiter R.D. Cole T.B. Findley S.D. EMBO J. 1996; 15: 1784-1791Crossref PubMed Scopus (393) Google Scholar, 6Palmiter R.D. Cole T.B. Quaife C.J. Findley S.D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14934-14939Crossref PubMed Scopus (595) Google Scholar). Mouse ZnT2 causes the vesicular accumulation of zinc in endosomal vesicles (5Palmiter R.D. Cole T.B. Findley S.D. EMBO J. 1996; 15: 1784-1791Crossref PubMed Scopus (393) Google Scholar) and is most similar in structure to ZnT3, which is responsible for the accumulation of zinc in synaptic vesicles in the brain (7Wenzel H.J. Cole T.B. Born D.E. Schwartzkroin P.A. Palmiter R.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12676-12681Crossref PubMed Scopus (287) Google Scholar, 8Cole T.B. Wenzel H.J. Kafer K.E. Schwartzkroin P.A. Palmiter R.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1716-1721Crossref PubMed Scopus (449) Google Scholar). Targeted deletion of ZnT3 is not lethal (8Cole T.B. Wenzel H.J. Kafer K.E. Schwartzkroin P.A. Palmiter R.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1716-1721Crossref PubMed Scopus (449) Google Scholar). ZnT4 was identified during a search for the Lethal Milk locus in the mouse (9Huang L.P. Gitschier J. Nat. Genet. 1997; 17: 292-297Crossref PubMed Scopus (307) Google Scholar). This zinc effluxer is highly expressed in the mammary gland, but may be involved in more general zinc homeostasis in the adult (9Huang L.P. Gitschier J. Nat. Genet. 1997; 17: 292-297Crossref PubMed Scopus (307) Google Scholar). ZnT1 functions to efflux zinc from cells, is localized to the plasma membrane, and is expressed ubiquitously (5Palmiter R.D. Cole T.B. Findley S.D. EMBO J. 1996; 15: 1784-1791Crossref PubMed Scopus (393) Google Scholar, 10Palmiter R.D. Findley S.D. EMBO J. 1995; 14: 639-649Crossref PubMed Scopus (636) Google Scholar). ZnT1 is an essential gene, and homozygous knockout of the ZnT1 gene is lethal to the embryo. 2R. D. Palmiter, personal communication.2R. D. Palmiter, personal communication. Zinc induction of ZnT1 mRNA had been documented in cultured neurons (11Tsuda M. Imaizumi K. Katayama T. Kitagawa K. Wanaka A. Tohyama M. Takagi T. J. Neurosci. 1997; 17: 6678-6684Crossref PubMed Google Scholar), and in the rat intestine after oral gavage with zinc (12McMahon R.J. Cousins R.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4841-4846Crossref PubMed Scopus (255) Google Scholar, 13Davis S.R. McMahon R.J. Cousins R.J. J. Nutr. 1998; 128: 825-831Crossref PubMed Scopus (81) Google Scholar). Furthermore, ZnT1 expression in enterocytes can be regulated by dietary zinc (12McMahon R.J. Cousins R.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4841-4846Crossref PubMed Scopus (255) Google Scholar). These preliminary studies suggested that zinc may regulate ZnT1 gene expression. In higher eukaryotes, the best understood metal-regulated genes are the metallothioneins (MT) (for review, see Ref. 14Andrews G.K. Biochem. Pharmacol. 2000; 59: 95-104Crossref PubMed Scopus (708) Google Scholar). Transcription of the mouse MT-I gene, for example, is regulated by zinc and cadmium, and this regulation is mediated by metal response element-binding transcription factor-1 (MTF-1) (15Heuchel R. Radtke F. Georgiev O. Stark G. Aguet M. Schaffner W. EMBO J. 1994; 13: 2870-2875Crossref PubMed Scopus (403) Google Scholar). MTF-1 is a six zinc-finger (Cys2His2) transcription factor, which functions as a sensor of intracellular zinc (for review, see Ref. 14Andrews G.K. Biochem. Pharmacol. 2000; 59: 95-104Crossref PubMed Scopus (708) Google Scholar). MTF-1 is activated by zinc to bind to metal response elements (MREs) in the MT-I promoter, resulting in an increased rate of transcription of this gene (15Heuchel R. Radtke F. Georgiev O. Stark G. Aguet M. Schaffner W. EMBO J. 1994; 13: 2870-2875Crossref PubMed Scopus (403) Google Scholar, 16Dalton T.P. Li Q.W. Bittel D. Liang L.C. Andrews G.K. J. Biol. Chem. 1996; 271: 26233-26241Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 17Koizumi S. Suzuki K. Ogra Y. Yamada H. Otsuka F. Eur. J. Biochem. 1999; 259: 635-642Crossref PubMed Scopus (101) Google Scholar). Cadmium activation of MT-I gene expression also requires MTF-1. In the present study, the hypothesis that zinc and cadmium regulate ZnT1 gene expression was tested and the potential role of MTF-1 in this response was examined. The ZnT1 gene was found to be responsive to zinc excess and deficiency, as well as to cadmium. These metals rapidly induced the coordinated synthesis of ZnT1 and MT-I mRNAs in cultured cells. In vitro DNA-binding assays demonstrated that recombinant mouse MTF-1 can bind to the MRE sequences present in the mouse ZnT1 promoter and studies of MTF-1 knockout mice and mouse embryonic fibroblast cells revealed an essential role for MTF-1 in metal responsiveness of these genes. DISCUSSIONThese studies demonstrate that expression of the mouse ZnT1 gene is regulated, in part, by the heavy metals zinc and cadmium, and suggest that MTF-1 is the transcription factor that mediates this response. Thus, MTF-1 coordinates the expression of genes that play roles in zinc homeostasis, as well as in protection from metal toxicity. Exposure of cells to excess zinc results in the increased expression of MT genes, which encode the major intracellular zinc storage proteins (40Kagi J.H.R. Methods Enzymol. 1991; 205: 613-626Crossref PubMed Scopus (737) Google Scholar), and the expression of ZnT1, which effluxes the metal from the cell (10Palmiter R.D. Findley S.D. EMBO J. 1995; 14: 639-649Crossref PubMed Scopus (636) Google Scholar). Reciprocally, under conditions of zinc deprivation, MTs are degraded to provide a biologically active labile pool of zinc (19Dalton T.P. Fu K. Palmiter R.D. Andrews G.K. J. Nutr. 1996; 126: 825-833Crossref PubMed Scopus (106) Google Scholar, 20Andrews G.K. Geiser J. J. Nutr. 1999; 129: 1643-1648Crossref PubMed Scopus (58) Google Scholar), and the efflux of zinc via ZnT1 is attenuated (4McMahon R.J. Cousins R.J. J. Nutr. 1998; 128: 667-670Crossref PubMed Scopus (180) Google Scholar, 10Palmiter R.D. Findley S.D. EMBO J. 1995; 14: 639-649Crossref PubMed Scopus (636) Google Scholar) leading to conservation of this metal in the cell. However, unlike MT-I and -II (41Masters B.A. Kelly E.J. Quaife C.J. Brinster R.L. Palmiter R.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 584-588Crossref PubMed Scopus (564) Google Scholar), MTF-1 (18Günes Ç. Heuchel R. Georgiev O. Müller K.H. Lichtlen P. Blüthmann H. Marino S. Aguzzi A. Schaffner W. EMBO J. 1998; 17: 2846-2854Crossref PubMed Scopus (220) Google Scholar) and ZnT12 are essential for embryonic development of the mouse. This suggests that metal efflux plays a more important role during development of the embryo than does metal storage. Remarkably, cadmium also coordinately regulates the expression of MT-I and ZnT1 genes, suggesting that ZnT1 may also play a role in protecting from cadmium toxicity, as does MT (41Masters B.A. Kelly E.J. Quaife C.J. Brinster R.L. Palmiter R.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 584-588Crossref PubMed Scopus (564) Google Scholar, 42Liu Y. Liu J. Iszard M.B. Andrews G.K. Palmiter R.D. Klaassen C.D. Toxicol. App. Pharmacol. 1995; 135: 222-228Crossref PubMed Scopus (133) Google Scholar). Consistent with this concept are the findings that overexpression of ZnT1 protects cells from zinc toxicity (10Palmiter R.D. Findley S.D. EMBO J. 1995; 14: 639-649Crossref PubMed Scopus (636) Google Scholar), and that zinc-resistant Hepa cells overexpress MT as well as ZnT1. 4R. Ravindra, unpublished results. Whether these cells also display increased efflux of cadmium and increased resistance to cadmium toxicity remains to be determined. A recent study of the ZnTA gene in Escherichia coli, which is a cadmium/zinc-exporting P1-type ATPase, is also regulated by zinc and cadmium (43Noll M. Lutsenko S. Biochem. Mol. Biol. Int. 2000; 49: 297-302Google Scholar).Whether MTF-1 directly or indirectly regulates ZnT1 gene expression remains to be determined, and the data presented herein cannot formally exclude either possibility. However, several lines of evidence are consistent with the concept that MTF-1 directly regulates ZnT1 gene expression in response to metals. First, both zinc and cadmium induce the rapid and coordinated synthesis of ZnT1 and MT-I mRNAs in cultured cells that contain MTF-1, but not in those lacking MTF-1. Second, both ZnT1 and MT-I mRNAs are specifically elevated in the visceral endoderm during early development of the embryo, both genes respond to dietary zinc deficiency, and both are reduced in mice lacking MTF-1. Third, MTF-1 can bind with avidity to two MREs found in the ZnT1 promoter, as it can with MRE sequences from the mouse MT-I promoter. Despite these findings, previous transfection studies using the ZnT1 promoter did not demonstrate metal regulation (10Palmiter R.D. Findley S.D. EMBO J. 1995; 14: 639-649Crossref PubMed Scopus (636) Google Scholar). The reason for this discrepancy warrants further investigation. Clearly, there are similarities and also distinct differences in the mechanisms of regulation of the ZnT1 and MT-I genes. Unlike the mouse MT-I promoter, which contains five functional MREs in the proximal 200-bp promoter, the ZnT1 proximal promoter contains only two MRE sequences. MTF-1 plays an important, but nonessential, role in regulating the ZnT1 gene in visceral endoderm cells in vivo. Thus, the basal level of expression of the ZnT1 gene is clearly dependent on transcription factors other than MTF-1. One potential binding site for the zinc-finger transcription factor Sp1 is present upstream of the MREs in the proximal MT-I promoter, whereas at least four such sites are found in the ZnT1 promoter. Further studies of the structure and function of the ZnT1 promoter are required.The finding that the visceral yolk sac actively expresses both the ZnT1 gene and the MT-I/II genes suggests that this organ plays an important role in zinc homeostasis, and protection from excess zinc during pregnancy. Preliminary immunolocalization studies using rat ZnT1 antisera (provided by R. J. Cousins, University of Florida, Gainsville, FL) detected immunoreactivity specifically in the visceral endoderm layer of the yolk sac.4 These cells are also the site of synthesis of MT (36Andrews G.K. McMaster M.T. De S.K. Paria B.C. Dey S.K. Suzuki K.T. Imura N. Kimura M. Metallothionein III: Biological Roles and Medical Implications. Birkhauser Verlag, Basel, Switzerland1993: 351-362Google Scholar). Visceral endoderm cells are the second cell type to differentiate from the primitive endoderm of the inner cell mass and they form the secretory layer of the visceral yolk sac, which surrounds the embryo until late in pregnancy (d19). These cells are responsible for the synthesis of serum proteins, and the visceral yolk sac is the first site of hematopoiesis. The visceral endoderm plays a nutritive and supportive role for embryonic development of the mouse. Previous studies demonstrated that the mouse MT genes become responsive to metal ions first at the morula/blastocyst stage of development. Given the role of MTF-1 in metal regulation of MT as well as ZnT1 genes, these studies suggest that ZnT1 gene expression may also be activated and responsive to metals first at this stage of preimplantation development. Further studies are required to address this possibility.In summary, these studies demonstrate that the mouse ZnT1 gene can be regulated by zinc as well as cadmium, and that this regulation is dependent on the transcription factor MTF-1. It was further demonstrated that expression of the ZnT1 gene is highly active in the visceral yolk sac of the developing embryo, and this expression is partially dependent on MTF-1 and dietary zinc. MTF-1 was known to regulate expression of the MT-I/II genes in mice, but the MT genes are nonessential. In contrast, the MTF-1 gene is essential for development, which suggested that this transcription factor also regulates the expression of an essential gene(s). One such gene is the ZnT1 gene. These studies demonstrate that expression of the mouse ZnT1 gene is regulated, in part, by the heavy metals zinc and cadmium, and suggest that MTF-1 is the transcription factor that mediates this response. Thus, MTF-1 coordinates the expression of genes that play roles in zinc homeostasis, as well as in protection from metal toxicity. Exposure of cells to excess zinc results in the increased expression of MT genes, which encode the major intracellular zinc storage proteins (40Kagi J.H.R. Methods Enzymol. 1991; 205: 613-626Crossref PubMed Scopus (737) Google Scholar), and the expression of ZnT1, which effluxes the metal from the cell (10Palmiter R.D. Findley S.D. EMBO J. 1995; 14: 639-649Crossref PubMed Scopus (636) Google Scholar). Reciprocally, under conditions of zinc deprivation, MTs are degraded to provide a biologically active labile pool of zinc (19Dalton T.P. Fu K. Palmiter R.D. Andrews G.K. J. Nutr. 1996; 126: 825-833Crossref PubMed Scopus (106) Google Scholar, 20Andrews G.K. Geiser J. J. Nutr. 1999; 129: 1643-1648Crossref PubMed Scopus (58) Google Scholar), and the efflux of zinc via ZnT1 is attenuated (4McMahon R.J. Cousins R.J. J. Nutr. 1998; 128: 667-670Crossref PubMed Scopus (180) Google Scholar, 10Palmiter R.D. Findley S.D. EMBO J. 1995; 14: 639-649Crossref PubMed Scopus (636) Google Scholar) leading to conservation of this metal in the cell. However, unlike MT-I and -II (41Masters B.A. Kelly E.J. Quaife C.J. Brinster R.L. Palmiter R.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 584-588Crossref PubMed Scopus (564) Google Scholar), MTF-1 (18Günes Ç. Heuchel R. Georgiev O. Müller K.H. Lichtlen P. Blüthmann H. Marino S. Aguzzi A. Schaffner W. EMBO J. 1998; 17: 2846-2854Crossref PubMed Scopus (220) Google Scholar) and ZnT12 are essential for embryonic development of the mouse. This suggests that metal efflux plays a more important role during development of the embryo than does metal storage. Remarkably, cadmium also coordinately regulates the expression of MT-I and ZnT1 genes, suggesting that ZnT1 may also play a role in protecting from cadmium toxicity, as does MT (41Masters B.A. Kelly E.J. Quaife C.J. Brinster R.L. Palmiter R.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 584-588Crossref PubMed Scopus (564) Google Scholar, 42Liu Y. Liu J. Iszard M.B. Andrews G.K. Palmiter R.D. Klaassen C.D. Toxicol. App. Pharmacol. 1995; 135: 222-228Crossref PubMed Scopus (133) Google Scholar). Consistent with this concept are the findings that overexpression of ZnT1 protects cells from zinc toxicity (10Palmiter R.D. Findley S.D. EMBO J. 1995; 14: 639-649Crossref PubMed Scopus (636) Google Scholar), and that zinc-resistant Hepa cells overexpress MT as well as ZnT1. 4R. Ravindra, unpublished results. Whether these cells also display increased efflux of cadmium and increased resistance to cadmium toxicity remains to be determined. A recent study of the ZnTA gene in Escherichia coli, which is a cadmium/zinc-exporting P1-type ATPase, is also regulated by zinc and cadmium (43Noll M. Lutsenko S. Biochem. Mol. Biol. Int. 2000; 49: 297-302Google Scholar). Whether MTF-1 directly or indirectly regulates ZnT1 gene expression remains to be determined, and the data presented herein cannot formally exclude either possibility. However, several lines of evidence are consistent with the concept that MTF-1 directly regulates ZnT1 gene expression in response to metals. First, both zinc and cadmium induce the rapid and coordinated synthesis of ZnT1 and MT-I mRNAs in cultured cells that contain MTF-1, but not in those lacking MTF-1. Second, both ZnT1 and MT-I mRNAs are specifically elevated in the visceral endoderm during early development of the embryo, both genes respond to dietary zinc deficiency, and both are reduced in mice lacking MTF-1. Third, MTF-1 can bind with avidity to two MREs found in the ZnT1 promoter, as it can with MRE sequences from the mouse MT-I promoter. Despite these findings, previous transfection studies using the ZnT1 promoter did not demonstrate metal regulation (10Palmiter R.D. Findley S.D. EMBO J. 1995; 14: 639-649Crossref PubMed Scopus (636) Google Scholar). The reason for this discrepancy warrants further investigation. Clearly, there are similarities and also distinct differences in the mechanisms of regulation of the ZnT1 and MT-I genes. Unlike the mouse MT-I promoter, which contains five functional MREs in the proximal 200-bp promoter, the ZnT1 proximal promoter contains only two MRE sequences. MTF-1 plays an important, but nonessential, role in regulating the ZnT1 gene in visceral endoderm cells in vivo. Thus, the basal level of expression of the ZnT1 gene is clearly dependent on transcription factors other than MTF-1. One potential binding site for the zinc-finger transcription factor Sp1 is present upstream of the MREs in the proximal MT-I promoter, whereas at least four such sites are found in the ZnT1 promoter. Further studies of the structure and function of the ZnT1 promoter are required. The finding that the visceral yolk sac actively expresses both the ZnT1 gene and the MT-I/II genes suggests that this organ plays an important role in zinc homeostasis, and protection from excess zinc during pregnancy. Preliminary immunolocalization studies using rat ZnT1 antisera (provided by R. J. Cousins, University of Florida, Gainsville, FL) detected immunoreactivity specifically in the visceral endoderm layer of the yolk sac.4 These cells are also the site of synthesis of MT (36Andrews G.K. McMaster M.T. De S.K. Paria B.C. Dey S.K. Suzuki K.T. Imura N. Kimura M. Metallothionein III: Biological Roles and Medical Implications. Birkhauser Verlag, Basel, Switzerland1993: 351-362Google Scholar). Visceral endoderm cells are the second cell type to differentiate from the primitive endoderm of the inner cell mass and they form the secretory layer of the visceral yolk sac, which surrounds the embryo until late in pregnancy (d19). These cells are responsible for the synthesis of serum proteins, and the visceral yolk sac is the first site of hematopoiesis. The visceral endoderm plays a nutritive and supportive role for embryonic development of the mouse. Previous studies demonstrated that the mouse MT genes become responsive to metal ions first at the morula/blastocyst stage of development. Given the role of MTF-1 in metal regulation of MT as well as ZnT1 genes, these studies suggest that ZnT1 gene expression may also be activated and responsive to metals first at this stage of preimplantation development. Further studies are required to address this possibility. In summary, these studies demonstrate that the mouse ZnT1 gene can be regulated by zinc as well as cadmium, and that this regulation is dependent on the transcription factor MTF-1. It was further demonstrated that expression of the ZnT1 gene is highly active in the visceral yolk sac of the developing embryo, and this expression is partially dependent on MTF-1 and dietary zinc. MTF-1 was known to regulate expression of the MT-I/II genes in mice, but the MT genes are nonessential. In contrast, the MTF-1 gene is essential for development, which suggested that this transcription factor also regulates the expression of an essential gene(s). One such gene is the ZnT1 gene. We are indebted to Jim Geiser and Steve Eklund for excellent technical support." @default.
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• W2105280572 title "The Transcription Factor MTF-1 Mediates Metal Regulation of the Mouse ZnT1 Gene" @default.
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