S. Joshua Langmade

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 of MTF-1 in this response was ZnT1 gene was found to be to zinc and as well as to These rapidly induced the synthesis of ZnT1 and MT-I mRNAs in cultured cells. In assays demonstrated that mouse MTF-1 can bind to the sequences present in the mouse ZnT1 and studies of MTF-1 knockout mice and mouse fibroblast cells an essential for MTF-1 in metal of these studies that expression of the mouse ZnT1 gene is in by the zinc and cadmium, and suggest that MTF-1 is the transcription factor that mediates this MTF-1 coordinates the expression of genes that in zinc as well as in protection from metal toxicity. of cells to zinc results in the increased expression of which the intracellular zinc storage proteins PubMed Scopus Google Scholar), and the expression of which the metal from the cell R.D. Findley S.D. EMBO J. 1995; 14: 639-649Crossref PubMed Scopus (636) Google Scholar). of zinc are to a of zinc T.P. K. Palmiter R.D. Andrews G.K. J. Nutr. 1996; PubMed Scopus Google Scholar, G.K. J. J. Nutr. 1999; PubMed Scopus Google Scholar), and the efflux of zinc ZnT1 is (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) to of this metal in the MT-I and Quaife C.J. Palmiter R.D. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar), MTF-1 R. Georgiev O. P. H. S. A. Schaffner W. EMBO J. 1998; 17: PubMed Scopus Google Scholar) and are essential for of the This that metal efflux a more during of the embryo metal cadmium also the expression of MT-I and ZnT1 that ZnT1 may also a in from cadmium as Quaife C.J. Palmiter R.D. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar, Y. J. Andrews G.K. Palmiter R.D. Pharmacol. 1995; PubMed Scopus Google Scholar). with this are the that of ZnT1 cells from zinc R.D. Findley S.D. EMBO J. 1995; 14: 639-649Crossref PubMed Scopus (636) Google Scholar), and that cells as well as these cells also increased efflux of cadmium and increased to cadmium to be of the gene in which is a is also regulated by zinc and cadmium M. S. Biochem. Biol. 2000; MTF-1 directly or ZnT1 gene expression to be and the lines of are with the that MTF-1 directly ZnT1 gene expression in response to both zinc and cadmium the and synthesis of ZnT1 and MT-I mRNAs in cultured cells that but not in those MTF-1. both ZnT1 and MT-I mRNAs are in the visceral during of the both genes to dietary zinc and both are in mice MTF-1. MTF-1 can bind with to two found in the ZnT1 promoter, as it can with sequences from the mouse MT-I promoter. these studies the ZnT1 did not metal regulation R.D. Findley S.D. EMBO J. 1995; 14: 639-649Crossref PubMed Scopus (636) Google Scholar). for this are and also in the mechanisms of regulation of the ZnT1 and MT-I the mouse MT-I promoter, which in the promoter, the ZnT1 two MTF-1 an but in regulating the ZnT1 gene in visceral cells in the basal of expression of the ZnT1 gene is on transcription MTF-1. potential for the zinc-finger transcription factor is present of the in the MT-I promoter, whereas four are found in the ZnT1 promoter. studies of the structure and function of the ZnT1 are that the visceral yolk sac both the ZnT1 gene and the genes that this an in zinc and protection from zinc during studies rat ZnT1 by R. J. of in the visceral of the yolk These cells are also the of synthesis of G.K. Suzuki M. and Scholar). cells are the cell to from the of the cell and the of the visceral yolk sac, which the embryo in pregnancy These cells are responsible for the synthesis of serum and the visceral yolk sac is the of visceral a and for of the studies demonstrated that the mouse genes to metal ions the of the of MTF-1 in metal regulation of as well as ZnT1 these studies suggest that ZnT1 gene expression may also be activated and to this of studies are to this these studies that the mouse ZnT1 gene can be regulated by zinc as well as cadmium, and that this regulation is on the transcription factor MTF-1. was demonstrated that expression of the ZnT1 gene is highly in the visceral yolk sac of the developing and this expression is on MTF-1 and dietary MTF-1 was to regulate expression of the genes in but the genes are In the MTF-1 gene is essential for which suggested that this transcription factor also the expression of an essential 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 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 of MTF-1 in this response was ZnT1 gene was found to be to zinc and as well as to These rapidly induced the synthesis of ZnT1 and MT-I mRNAs in cultured cells. In assays demonstrated that mouse MTF-1 can bind to the sequences present in the mouse ZnT1 and studies of MTF-1 knockout mice and mouse fibroblast cells an essential for MTF-1 in metal of these studies that expression of the mouse ZnT1 gene is in by the zinc and cadmium, and suggest that MTF-1 is the transcription factor that mediates this MTF-1 coordinates the expression of genes that in zinc as well as in protection from metal toxicity. of cells to zinc results in the increased expression of which the intracellular zinc storage proteins PubMed Scopus Google Scholar), and the expression of which the metal from the cell R.D. Findley S.D. EMBO J. 1995; 14: 639-649Crossref PubMed Scopus (636) Google Scholar). of zinc are to a of zinc T.P. K. Palmiter R.D. Andrews G.K. J. Nutr. 1996; PubMed Scopus Google Scholar, G.K. J. J. Nutr. 1999; PubMed Scopus Google Scholar), and the efflux of zinc ZnT1 is (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) to of this metal in the MT-I and Quaife C.J. Palmiter R.D. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar), MTF-1 R. Georgiev O. P. H. S. A. Schaffner W. EMBO J. 1998; 17: PubMed Scopus Google Scholar) and are essential for of the This that metal efflux a more during of the embryo metal cadmium also the expression of MT-I and ZnT1 that ZnT1 may also a in from cadmium as Quaife C.J. Palmiter R.D. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar, Y. J. Andrews G.K. Palmiter R.D. Pharmacol. 1995; PubMed Scopus Google Scholar). with this are the that of ZnT1 cells from zinc R.D. Findley S.D. EMBO J. 1995; 14: 639-649Crossref PubMed Scopus (636) Google Scholar), and that cells as well as these cells also increased efflux of cadmium and increased to cadmium to be of the gene in which is a is also regulated by zinc and cadmium M. S. Biochem. Biol. 2000; MTF-1 directly or ZnT1 gene expression to be and the lines of are with the that MTF-1 directly ZnT1 gene expression in response to both zinc and cadmium the and synthesis of ZnT1 and MT-I mRNAs in cultured cells that but not in those MTF-1. both ZnT1 and MT-I mRNAs are in the visceral during of the both genes to dietary zinc and both are in mice MTF-1. MTF-1 can bind with to two found in the ZnT1 promoter, as it can with sequences from the mouse MT-I promoter. these studies the ZnT1 did not metal regulation R.D. Findley S.D. EMBO J. 1995; 14: 639-649Crossref PubMed Scopus (636) Google Scholar). for this are and also in the mechanisms of regulation of the ZnT1 and MT-I the mouse MT-I promoter, which in the promoter, the ZnT1 two MTF-1 an but in regulating the ZnT1 gene in visceral cells in the basal of expression of the ZnT1 gene is on transcription MTF-1. potential for the zinc-finger transcription factor is present of the in the MT-I promoter, whereas four are found in the ZnT1 promoter. studies of the structure and function of the ZnT1 are that the visceral yolk sac both the ZnT1 gene and the genes that this an in zinc and protection from zinc during studies rat ZnT1 by R. J. of in the visceral of the yolk These cells are also the of synthesis of G.K. Suzuki M. and Scholar). cells are the cell to from the of the cell and the of the visceral yolk sac, which the embryo in pregnancy These cells are responsible for the synthesis of serum and the visceral yolk sac is the of visceral a and for of the studies demonstrated that the mouse genes to metal ions the of the of MTF-1 in metal regulation of as well as ZnT1 these studies suggest that ZnT1 gene expression may also be activated and to this of studies are to this these studies that the mouse ZnT1 gene can be regulated by zinc as well as cadmium, and that this regulation is on the transcription factor MTF-1. was demonstrated that expression of the ZnT1 gene is highly in the visceral yolk sac of the developing and this expression is on MTF-1 and dietary MTF-1 was to regulate expression of the genes in but the genes are In the MTF-1 gene is essential for which suggested that this transcription factor also the expression of an essential gene is the ZnT1 gene. These studies that expression of the mouse ZnT1 gene is in by the zinc and cadmium, and suggest that MTF-1 is the transcription factor that mediates this MTF-1 coordinates the expression of genes that in zinc as well as in protection from metal toxicity. of cells to zinc results in the increased expression of which the intracellular zinc storage proteins PubMed Scopus Google Scholar), and the expression of which the metal from the cell R.D. Findley S.D. EMBO J. 1995; 14: 639-649Crossref PubMed Scopus (636) Google Scholar). of zinc are to a of zinc T.P. K. Palmiter R.D. Andrews G.K. J. Nutr. 1996; PubMed Scopus Google Scholar, G.K. J. J. Nutr. 1999; PubMed Scopus Google Scholar), and the efflux of zinc ZnT1 is (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) to of this metal in the MT-I and Quaife C.J. Palmiter R.D. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar), MTF-1 R. Georgiev O. P. H. S. A. Schaffner W. EMBO J. 1998; 17: PubMed Scopus Google Scholar) and are essential for of the This that metal efflux a more during of the embryo metal cadmium also the expression of MT-I and ZnT1 that ZnT1 may also a in from cadmium as Quaife C.J. Palmiter R.D. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar, Y. J. Andrews G.K. Palmiter R.D. Pharmacol. 1995; PubMed Scopus Google Scholar). with this are the that of ZnT1 cells from zinc R.D. Findley S.D. EMBO J. 1995; 14: 639-649Crossref PubMed Scopus (636) Google Scholar), and that cells as well as these cells also increased efflux of cadmium and increased to cadmium to be of the gene in which is a is also regulated by zinc and cadmium M. S. Biochem. Biol. 2000; Scholar). MTF-1 directly or ZnT1 gene expression to be and the lines of are with the that MTF-1 directly ZnT1 gene expression in response to both zinc and cadmium the and synthesis of ZnT1 and MT-I mRNAs in cultured cells that but not in those MTF-1. both ZnT1 and MT-I mRNAs are in the visceral during of the both genes to dietary zinc and both are in mice MTF-1. MTF-1 can bind with to two found in the ZnT1 promoter, as it can with sequences from the mouse MT-I promoter. these studies the ZnT1 did not metal regulation R.D. Findley S.D. EMBO J. 1995; 14: 639-649Crossref PubMed Scopus (636) Google Scholar). for this are and also in the mechanisms of regulation of the ZnT1 and MT-I the mouse MT-I promoter, which in the promoter, the ZnT1 two MTF-1 an but in regulating the ZnT1 gene in visceral cells in the basal of expression of the ZnT1 gene is on transcription MTF-1. potential for the zinc-finger transcription factor is present of the in the MT-I promoter, whereas four are found in the ZnT1 promoter. studies of the structure and function of the ZnT1 are that the visceral yolk sac both the ZnT1 gene and the genes that this an in zinc and protection from zinc during studies rat ZnT1 by R. J. of in the visceral of the yolk These cells are also the of synthesis of G.K. Suzuki M. and Scholar). cells are the cell to from the of the cell and the of the visceral yolk sac, which the embryo in pregnancy These cells are responsible for the synthesis of serum and the visceral yolk sac is the of visceral a and for of the studies demonstrated that the mouse genes to metal ions the of the of MTF-1 in metal regulation of as well as ZnT1 these studies suggest that ZnT1 gene expression may also be activated and to this of studies are to this In these studies that the mouse ZnT1 gene can be regulated by zinc as well as cadmium, and that this regulation is on the transcription factor MTF-1. was demonstrated that expression of the ZnT1 gene is highly in the visceral yolk sac of the developing and this expression is on MTF-1 and dietary MTF-1 was to regulate expression of the genes in but the genes are In the MTF-1 gene is essential for which suggested that this transcription factor also the expression of an essential gene is the ZnT1 gene. are to and for

Niemann-Pick type C1 (NPC1) disease is a rare progressive neurodegenerative disorder characterized by accumulation of cholesterol in the endolysosomes. Previous studies implicating oxidative stress in NPC1 disease pathogenesis raised the possibility that nonenzymatic formation of cholesterol oxidation products could serve as disease biomarkers. We measured these metabolites in the plasma and tissues of the Npc1(-/-) mouse model and found several cholesterol oxidation products that were elevated in Npc1(-/-) mice, were detectable before the onset of symptoms, and were associated with disease progression. Nonenzymatically formed cholesterol oxidation products were similarly increased in the plasma of all human NPC1 subjects studied and delineated an oxysterol profile specific for NPC1 disease. This oxysterol profile also correlated with the age of disease onset and disease severity. We further show that the plasma oxysterol markers decreased in response to an established therapeutic intervention in the NPC1 feline model. These cholesterol oxidation products are robust blood-based biochemical markers for NPC1 disease that may prove transformative for diagnosis and treatment of this disorder, and as outcome measures to monitor response to therapy.

Zinc is an essential metal for all eukaryotes, and cells have evolved a complex system of proteins to maintain the precise balance of zinc uptake, intracellular storage, and efflux. In mammals, zinc uptake appears to be mediated by members of the Zrt/Irt-like protein (ZIP) superfamily of metal ion transporters. Herein, we have studied a subfamily of zip genes (zip1, zip2, and zip3) that is conserved in mice and humans. These eight-transmembrane domain proteins contain a conserved 12-amino acid signature sequence within the fourth transmembrane domain. All three of these mouse ZIP proteins function to specifically increase the uptake of zinc in transfected cultured cells, similar to the previously demonstrated functions of human ZIP1 and ZIP2 (Gaither, L. A., and Eide, D. J. (2000) J. Biol. Chem. 275, 5560-5564; Gaither, L. A., and Eide, D. J. (2001) J. Biol. Chem. 276, 22258-22264). No ZIP3 orthologs have been previously studied. Furthermore, this first systematic comparative study of the in vivo expression and dietary zinc regulation of this subfamily of zip genes revealed that 1) zip1 mRNA is abundant in many mouse tissues, whereas zip2 and zip3 mRNAs are very rare or moderately rare, respectively, and tissue-restricted in their accumulation; and 2) unlike mouse metallothionein I and zip4 mRNAs (Dufner-Beattie, J., Wang, F., Kuo, Y.-M., Gitschier, J., Eide, D., and Andrews, G. K. (2003) J. Biol. Chem. 278, 33474-33481), the abundance of zip1, zip2, and zip3 mRNAs is not regulated by dietary zinc in the intestine and visceral endoderm, tissues involved in nutrient absorption. These studies suggest that all three of these ZIP proteins may play cell-specific roles in zinc homeostasis rather than primary roles in the acquisition of dietary zinc.

Over 200 disease-causing mutations have been identified in the NPC1 gene. The most prevalent mutation, NPC1(I1061T), is predicted to lie within the cysteine-rich luminal domain and is associated with the classic juvenile-onset phenotype of Niemann-Pick type C disease. To gain insight into the molecular mechanism by which the NPC1(I1061T) mutation causes disease, we examined expression of the mutant protein in human fibroblasts homozygous for the NPC1(I1061T) mutation. Despite similar NPC1 mRNA levels between wild type and NPC1(I1061T) fibroblasts, NPC1 protein levels are decreased by 85% in NPC1(I1061T) cells. Metabolic labeling studies demonstrate that unlike wild type protein, which undergoes a glycosylation pattern shift from Endo H-sensitive to Endo H-resistant species, NPC1(I1061T) protein remains almost exclusively Endo H-sensitive and exhibits a reduced half-life (t((1/2)) 6.5 h) versus wild type Endo H-resistant species (t((1/2)) 42 h). Treatment with chemical chaperones, growth at permissive temperature, or inhibition of proteasomal degradation increases NPC1(I1061T) protein levels, indicating that the mutant protein is likely targeted for endoplasmic reticulum-associated degradation (ERAD) due to protein misfolding. Overexpression of NPC1(I1061T) in NPC1-deficient cells results in late endosomal localization of the mutant protein and complementation of the NPC mutant phenotype, likely due to a small proportion of the nascent NPC1(I1061T) protein that is able to fold correctly and escape the endoplasmic reticulum quality control checkpoints. Our findings provide the first description of an endoplasmic reticulum trafficking defect as a mechanism for human NPC disease, shedding light on the mechanism by which the NPC1(I1061T) mutation causes disease and suggesting novel approaches to treat NPC disease caused by the NPC1(I1061T) mutation.

Niemann-Pick type C1 (NPC1) disease is a fatal neurodegenerative disease characterized by neuronal lipid storage and progressive Purkinje cell loss in the cerebellum. We investigated whether therapeutic approaches to bypass the cholesterol trafficking defect in NPC1 disease might delay disease progression in the npc1(-/-) mouse model. We show that the neurosteroid allopregnanolone (ALLO) and T0901317, a synthetic oxysterol ligand, act in concert to delay onset of neurological symptoms and prolong the lifespan of npc1(-/-) mice. ALLO and T0901317 therapy preserved Purkinje cells, suppressed cerebellar expression of microglial-associated genes and inflammatory mediators, and reduced infiltration of activated microglia in the cerebellar tissue. To establish whether the mechanism of neuroprotection in npc1(-/-) mice involves GABA(A) receptor activation, we compared treatment of natural ALLO and ent-ALLO, a stereoisomer that has identical physical properties of natural ALLO but is not a GABA(A) receptor agonist. ent-ALLO provided identical functional and survival benefits as natural ALLO in npc1(-/-) mice, strongly supporting a GABA(A) receptor-independent mechanism for ALLO action. On the other hand, the efficacy of ALLO, ent-ALLO, and T0901317 therapy correlated with the ability of these compounds to activate pregnane X receptor-dependent pathways in vivo. These findings suggest that treatment with pregnane X receptor ligands may be useful clinically in delaying the progressive neurodegeneration in human NPC disease.