The Adaptive Response to Dietary Zinc in Mice Involves the Differential Cellular Localization and Zinc Regulation of the Zinc Transporters ZIP4 and ZIP5
2004; Elsevier BV; Volume: 279; Issue: 47 Linguagem: Inglês
10.1074/jbc.m409962200
ISSN1083-351X
AutoresJodi Dufner‐Beattie, Yien–Ming Kuo, Jane Gitschier, Glen K. Andrews,
Tópico(s)Aluminum toxicity and tolerance in plants and animals
ResumoThe ZIP5 gene encodes a protein closely related to ZIP4, a zinc transporter mutated in the human genetic disorder acrodermatitis enteropathica. Herein, we demonstrate that mouse ZIP5 and ZIP4 genes are co-expressed in several tissues involved in zinc homeostasis (intestine, pancreas, embryonic yolk sac). However, unlike expression of the ZIP4 gene, which is induced during periods of zinc deficiency, ZIP5 gene expression is unaltered by dietary zinc. Immunohistochemistry localizes ZIP5 to the basolateral surfaces of enterocytes, acinar cells, and visceral endoderm cells in mice fed a zinc-adequate diet. However, this protein is removed from these cell surfaces and internalized during dietary zinc deficiency. In contrast, ZIP4 is induced and recruited to the apical surface of enterocytes and endoderm cells during zinc deficiency. In the pancreas, ZIP4 is expressed in β-cells, whereas ZIP5 is expressed in acinar cells. These results suggest that the function of ZIP5 is antagonistic to that of ZIP4 in the control of zinc homeostasis; rather than functioning in the acquisition of dietary zinc, as does ZIP4, ZIP5 may function in the removal of zinc from the body. Thus, during periods when dietary zinc is replete, ZIP5 may function to remove zinc from the blood via the pancreas and intestine, the major sites of zinc excretion in mammals, whereas the acquisition of dietary zinc by intestinal ZIP4 would be minimal. In contrast, during periods of dietary zinc deficiency when secretion of zinc by the pancreas and intestine is minimized, ZIP5 is removed from the cell surface, and the intestinal uptake of zinc is augmented by induction of ZIP4. The ZIP5 gene encodes a protein closely related to ZIP4, a zinc transporter mutated in the human genetic disorder acrodermatitis enteropathica. Herein, we demonstrate that mouse ZIP5 and ZIP4 genes are co-expressed in several tissues involved in zinc homeostasis (intestine, pancreas, embryonic yolk sac). However, unlike expression of the ZIP4 gene, which is induced during periods of zinc deficiency, ZIP5 gene expression is unaltered by dietary zinc. Immunohistochemistry localizes ZIP5 to the basolateral surfaces of enterocytes, acinar cells, and visceral endoderm cells in mice fed a zinc-adequate diet. However, this protein is removed from these cell surfaces and internalized during dietary zinc deficiency. In contrast, ZIP4 is induced and recruited to the apical surface of enterocytes and endoderm cells during zinc deficiency. In the pancreas, ZIP4 is expressed in β-cells, whereas ZIP5 is expressed in acinar cells. These results suggest that the function of ZIP5 is antagonistic to that of ZIP4 in the control of zinc homeostasis; rather than functioning in the acquisition of dietary zinc, as does ZIP4, ZIP5 may function in the removal of zinc from the body. Thus, during periods when dietary zinc is replete, ZIP5 may function to remove zinc from the blood via the pancreas and intestine, the major sites of zinc excretion in mammals, whereas the acquisition of dietary zinc by intestinal ZIP4 would be minimal. In contrast, during periods of dietary zinc deficiency when secretion of zinc by the pancreas and intestine is minimized, ZIP5 is removed from the cell surface, and the intestinal uptake of zinc is augmented by induction of ZIP4. Members of the ZIP superfamily of metal ion uptake transporters (solute carrier family 39A) are found in all eukaryotes (1Eng B.H. Guerinot M.L. Eide D. Saier Jr., M.H. J. Membr. Biol. 1998; 166: 1-7Crossref PubMed Scopus (207) Google Scholar, 2Kambe T. Yamaguchi-Iwai Y. Sasaki R. Nagao M. Cell. Mol. Life Sci. 2004; 61: 49-68Crossref PubMed Scopus (334) Google Scholar, 3Harris E.D. Nutr. Rev. 2002; 60: 121-124Crossref PubMed Scopus (45) Google Scholar). In mice 14 members of the ZIP family have been identified based on sequence homology, and almost all of these genes are conserved in humans (1Eng B.H. Guerinot M.L. Eide D. Saier Jr., M.H. J. Membr. Biol. 1998; 166: 1-7Crossref PubMed Scopus (207) Google Scholar, 4Guerinot M.L. Biochim. Biophys. Acta. 2000; 1465: 190-198Crossref PubMed Scopus (837) Google Scholar, 5Taylor K.M. Nicholson R.I. Biochim. Biophys. Acta. 2003; 1611: 16-30Crossref PubMed Scopus (212) Google Scholar). ZIP proteins have eight predicted transmembrane domains, and transmembrane domain IV contains conserved histidyl, seryl, and glycyl residues in an amphipathic α-helix. These proteins often contain an intracellular loop between transmembrane domains III and IV and an extracellular amino terminus that is histidine-rich and may play roles in metal transport. Members of the ZIP superfamily have been shown to transport zinc, iron, or manganese into cells (6Eide D. Curr. Opin. Cell Biol. 1997; 9: 573-577Crossref PubMed Scopus (82) Google Scholar) and have been implicated in early development of the zebrafish embryo (7Yamashita S. Miyagi C. Fukada T. Kagara N. Che Y.S. Hirano T. Nature. 2004; 429: 298-302Crossref PubMed Scopus (310) Google Scholar) and zinc homeostasis in humans (8Wang K. Zhou B. Kuo Y.-M. Gitschier J. Am. J. Hum. Genet. 2002; 71: 66-73Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar, 9Kury S. Dreno B. Bezieau S. Giraudet S. Kharfi M. Kamoun R. Moisan J.P. Nat. Genet. 2002; 31: 239-240Crossref PubMed Scopus (410) Google Scholar). Despite this growing body of knowledge, little is known about the structure and function of most members of this diverse family of proteins.The ZIP family can be subdivided into four subfamilies, named Subfamilies I and II, gufA, and LIV-1 (5Taylor K.M. Nicholson R.I. Biochim. Biophys. Acta. 2003; 1611: 16-30Crossref PubMed Scopus (212) Google Scholar). The three members of subfamily II (ZIP1-3) have been well conserved in mammals, and each functions as zinc-specific transporter (10Gaither L.A. Eide D.J. J. Biol. Chem. 2001; 276: 22258-22264Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 11Gaither L.A. Eide D.J. J. Biol. Chem. 2000; 275: 5560-5564Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, 12Dufner-Beattie J. Langmade S.J. Wang F. Eide D. Andrews G.K. J. Biol. Chem. 2003; 278: 50142-50150Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). However, the physiological roles of these transporters remain to be determined. The LIV-1 subfamily in mice contains 9 members (5Taylor K.M. Nicholson R.I. Biochim. Biophys. Acta. 2003; 1611: 16-30Crossref PubMed Scopus (212) Google Scholar) of which only three (ZIP4, -6, and -7) have been characterized in any detail (13Taylor K.M. Morgan H.E. Johnson A. Hadley L.J. Nicholson R.I. Biochem. J. 2003; 375: 51-59Crossref PubMed Scopus (134) Google Scholar, 14Taylor K.M. Morgan H.E. Johnson A. Nicholson R.I. Biochem. J. 2004; 377: 131-139Crossref PubMed Scopus (113) Google Scholar, 15Dufner-Beattie J. Wang F. Kuo Y.M. Gitschier J. Eide D. Andrews G.K. J. Biol. Chem. 2003; 278: 33474-33481Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). The founding member of this family, LIV-1 (ZIP6; SLC39A6), was identified as a breast cancer-associated protein whose expression is induced by estrogen in breast cancer cells (13Taylor K.M. Morgan H.E. Johnson A. Hadley L.J. Nicholson R.I. Biochem. J. 2003; 375: 51-59Crossref PubMed Scopus (134) Google Scholar, 16Manning D.L. Daly R.J. Lord P.G. Kelly K.F. Green C.D. Mol. Cell. Endocrinol. 1988; 59: 205-212Crossref PubMed Scopus (72) Google Scholar). In transfected cells, ZIP6 localizes to the plasma membrane and may function to increase zinc uptake (13Taylor K.M. Morgan H.E. Johnson A. Hadley L.J. Nicholson R.I. Biochem. J. 2003; 375: 51-59Crossref PubMed Scopus (134) Google Scholar). Very recently, ZIP7 was found to localize to intracellular membranes in transfected cells and to enhance zinc accumulation (14Taylor K.M. Morgan H.E. Johnson A. Nicholson R.I. Biochem. J. 2004; 377: 131-139Crossref PubMed Scopus (113) Google Scholar). However, ZIP6 expression appears to be at its lowest level in the human intestine, and ZIP7 appears to be fairly ubiquitously expressed at low levels (13Taylor K.M. Morgan H.E. Johnson A. Hadley L.J. Nicholson R.I. Biochem. J. 2003; 375: 51-59Crossref PubMed Scopus (134) Google Scholar, 14Taylor K.M. Morgan H.E. Johnson A. Nicholson R.I. Biochem. J. 2004; 377: 131-139Crossref PubMed Scopus (113) Google Scholar). This suggests that they do not play key roles in the acquisition of dietary zinc.In contrast, ZIP4 (SLC39A4) is mutated in the human genetic disorder of zinc metabolism acrodermatitis enteropathica (8Wang K. Zhou B. Kuo Y.-M. Gitschier J. Am. J. Hum. Genet. 2002; 71: 66-73Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar, 9Kury S. Dreno B. Bezieau S. Giraudet S. Kharfi M. Kamoun R. Moisan J.P. Nat. Genet. 2002; 31: 239-240Crossref PubMed Scopus (410) Google Scholar). ZIP4 is also well conserved during evolution, and the mouse ZIP4 gene is induced, and this protein is recruited to the apical surface of enterocytes and embryonic visceral endoderm cells during periods of zinc deficiency (15Dufner-Beattie J. Wang F. Kuo Y.M. Gitschier J. Eide D. Andrews G.K. J. Biol. Chem. 2003; 278: 33474-33481Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). The intracellular trafficking of ZIP4 is also regulated (e.g. zinc stimulates the endocytosis of ZIP4) (17Kim B.E. Wang F.D. Dufner-Beattie J. Andrews G.K. Eide D.J. Petris M.J. J. Biol. Chem. 2004; 279: 4523-4530Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Thus, ZIP4 plays a key role in zinc homeostasis by sensing dietary zinc levels.Herein, we studied ZIP5 (SLC39A5), a member of the mouse LIV-1 subfamily that is closely related to ZIP4. We demonstrate that the mouse ZIP5 gene is most actively expressed in tissues involved in zinc homeostasis (intestine, visceral endoderm, pancreas) but is not induced during zinc deficiency. Instead, ZIP5 is localized to the basolateral surface of these cells under zinc-replete conditions but is internalized during periods of dietary zinc deficiency. In related studies, it was recently demonstrated that mZIP5 functions as a bona fide zinc transporter that is specific for zinc as a substrate (18Wang F. Kim B.E. Petris M.J. Eide D.J. J. Biol. Chem. 2004; (August 20, 10.1074/jbc.M408361200)Google Scholar). Our studies suggest that ZIP5 plays a central role in mammalian zinc homeostasis by antagonizing the actions of ZIP4.MATERIALS AND METHODSAnimal Care and Use; Dietary Zinc ManipulationAll experiments involving mice were conducted in accordance with National Institutes of Health guidelines for the care and use of experimental animals and were approved by the Institutional Animal Care and Use Committee. CD-1 mice (48-60 days old) were purchased from Charles River Breeding Laboratories (criver.com). Mouse diets were purchased from Harlan Teklad (Teklad.com) and have been described in detail previously (12Dufner-Beattie J. Langmade S.J. Wang F. Eide D. Andrews G.K. J. Biol. Chem. 2003; 278: 50142-50150Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 15Dufner-Beattie J. Wang F. Kuo Y.M. Gitschier J. Eide D. Andrews G.K. J. Biol. Chem. 2003; 278: 33474-33481Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 19Dalton T.P. Fu K. Palmiter R.D. Andrews G.K. J. Nutr. 1996; 126: 825-833Crossref PubMed Scopus (106) Google Scholar). Zinc levels in the diets were as follows: zinc-deficient (ZnD), 1The abbreviations used are: ZnD, zinc-deficient; ZnA, zinc-adequate; HA, hemagglutinin; TRITC, tetramethylrhodamine isothiocyanate. 1 ppm Zn; zinc-adequate (ZnA), 50 ppm Zn.To examine the tissue-specific expression of ZIP5, CD-1 female or male mice (6 per group) maintained on ZnA feed were killed, and the indicated tissues were harvested and snap-frozen in liquid nitrogen for subsequent extraction of RNA and Northern blot analysis. Pancreas RNA was extracted from fresh tissue.To examine the effects of zinc on ZIP5 expression, female mice (six mice per group) were subjected to dietary zinc deficiency during pregnancy as described previously (15Dufner-Beattie J. Wang F. Kuo Y.M. Gitschier J. Eide D. Andrews G.K. J. Biol. Chem. 2003; 278: 33474-33481Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 20Andrews G.K. Geiser J. J. Nutr. 1999; 129: 1643-1648Crossref PubMed Scopus (58) Google Scholar). The visceral yolk sac and maternal small intestine were harvested and either fixed for immunohistochemistry, snap-frozen in liquid nitrogen for Northern blotting, or processed fresh for Western blotting. Dietary zinc deficiency was initiated on day 8 of pregnancy, and tissues were harvested on the days of pregnancy indicated in the figure legends (up to day 15). The maternal intestine was isolated as an intact tissue (not a mucosal scrape) but was subdivided as follows. The first six centimeters were considered the proximal (nearest the duodenum), and the next 6 cm were considered the distal small intestine. Previous studies have documented that little anorexia occurs under these experimental conditions, and results using pair-fed controls do not differ from those obtained using mice allowed free access to feed (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).Computer Analyses of Sequence DataMultiple sequence alignments were performed using the Vector NTI Suite Program (Invitrogen.com).RNA Extraction and Northern Blot HybridizationTissue RNAs were isolated as described in detail previously (21Andrews G.K. Lee D.K. Ravindra R. Lichtlen P. Sirito M. Sawadogo M. Schaffner W. EMBO J. 2001; 20: 1114-1122Crossref PubMed Scopus (84) Google Scholar, 22Langmade S.J. Ravindra R. Daniels P.J. Andrews G.K. J. Biol. Chem. 2000; 275: 34803-34809Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar). Total RNA (3 μg) was size-fractionated by agarose formaldehyde gel electrophoresis, transferred, and cross-linked to nylon membranes. Northern blot membranes were hybridized and washed under stringent conditions as described (19Dalton T.P. Fu K. Palmiter R.D. Andrews G.K. J. Nutr. 1996; 126: 825-833Crossref PubMed Scopus (106) Google Scholar, 22Langmade S.J. Ravindra R. Daniels P.J. Andrews G.K. J. Biol. Chem. 2000; 275: 34803-34809Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar, 23Dalton T.P. Palmiter R.D. Andrews G.K. Nucleic Acids Res. 1994; 22: 5016-5023Crossref PubMed Scopus (250) Google Scholar). Hybrids were detected by autoradiography with intensifying screens at -70 °C. Duplicate gels were stained with acridine orange, or the same membrane was rehybridized with a β-actin probe to monitor RNA loading and integrity.The mouse MT-I, β-actin, ZIP1, and ZIP4 probes were as described (12Dufner-Beattie J. Langmade S.J. Wang F. Eide D. Andrews G.K. J. Biol. Chem. 2003; 278: 50142-50150Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 15Dufner-Beattie J. Wang F. Kuo Y.M. Gitschier J. Eide D. Andrews G.K. J. Biol. Chem. 2003; 278: 33474-33481Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 19Dalton T.P. Fu K. Palmiter R.D. Andrews G.K. J. Nutr. 1996; 126: 825-833Crossref PubMed Scopus (106) Google Scholar). The protein-coding region of ZIP5 mRNA (GenBank™ accession numbers AK008448 and AK007473) was amplified by reverse transcription-PCR from mouse intestinal RNA using Improm-II reverse transcriptase (promega.com), Pfu DNA polymerase (stratagene.com), and primers mZIP5(S) (5′-CGGAATTCGTCTCAAAGGAAATAGAAGTCAGAAACAGAGC-3′) and mZIP5HA(AS) (5′-GGACTAGTGCCGTCAGGAACCGTGGGC-3′). The product was digested with EcoRI and SpeI, and the purified product was ligated into EcoRI-digested pcDNA3.1Puro(+) (24Thomas L.R. Stillman D.J. Thorburn A. J. Biol. Chem. 2002; 277: 34343-34348Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar) along with annealed oligonucleotides (5′-CTAGTGGCTATCCATATGATGTTCCAGATTATGCTTGAT-3′ and 5′-AATTCTCAAGCATAATCTGGAACATCATATGGATAGCCA-3′) that contain SpeI and EcoRI compatible termini and encode a carboxyl-terminal hemagglutinin (HA) tag. The product (pcDNA3.1Puro(+)-mZIP5HA) was confirmed by DNA sequencing. Probes were labeled using the Random Primers DNA Labeling System according to the manufacturer's instructions (invitrogen.com). Probes had specific activities of about 1 to 3 × 109 dpm/μg.Western BlottingZIP4 and -5 Antibodies—A rabbit polyclonal antiserum was generated against the following mZIP5 peptide: (C)ASEPEVQGQRENRQS. The peptide was coupled to keyhole limpet hemocyanin and used to immunize rabbits as described previously for ZIP4 (8Wang K. Zhou B. Kuo Y.-M. Gitschier J. Am. J. Hum. Genet. 2002; 71: 66-73Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar).Preparation of Membrane and Cytosolic Proteins—Membrane proteins were prepared using a modified protocol (25Rodriguez E. Pulido N. Romero R. Arrieta F. Panadero A. Rovira A. Endocrinology. 2004; 145: 679-685Crossref PubMed Scopus (22) Google Scholar, 26Sagawa N. Fujita H. Banno Y. Nozawa Y. Katoh H. Kuzumaki N. Br. J. Cancer. 2003; 88: 606-612Crossref PubMed Scopus (49) Google Scholar, 27Krotova K.Y. Zharikov S.I. Block E.R. Am. J. Physiol. Lung Cell. Mol. Physiol. 2003; 284: 1037-1044Crossref PubMed Scopus (50) Google Scholar). Maternal proximal small intestine or embryonic visceral yolk sac (∼0.1 g) was collected and homogenized using a Polytron homogenizer in 1 ml of ice-cold lysis buffer (20 mm Tris-HCl (pH 7.4), 1 mm EDTA, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 2 mm NaF, 5 mm Na3VO4) containing a Protease Inhibitor Mixture (roche-applied-science.com). The homogenate was centrifuged at 500 × g for 10 min at 4 °C to pellet insoluble debris. The supernatant was collected and centrifuged at 100,000 × g for 30 min at 4 °C to pellet membranes. The supernatant (cytosolic fraction) was recovered, and the membranes were rinsed with wash buffer (150 mm NaCl, 5 mm phosphate buffer (pH 7.0), 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 2 mm NaF, 5 mm Na3VO4, and Protease Inhibitor Mixture) and collected by centrifugation at 20,000 × g for 10 min at 4 °C. The membrane pellet was then resuspended in 200 μl of radioimmune precipitation assay buffer mix (150 mm NaCl, 50 mm Tris-HCl (pH 7.4), 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mm EDTA, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 2 mm NaF, 5 mm Na3VO4, and Protease Inhibitor Mixture), sonicated, and allowed to sit on ice for 30-60 min. This solution was then centrifuged at 20,000 × g for 10 min at 4 °C to pellet insoluble debris. The supernatant was collected, and its protein concentration was determined using a BCA protein assay kit (piercenet.com).Immunoblotting—Membrane proteins (25 μg) were resolved on a 10% SDS-polyacrylamide gel and transferred to polyvinylidene difluoride membranes. The membranes were blocked overnight at 4 °C in blocking solution (150 mm NaCl, 10 mm Tris-HCl (pH 7.4), 0.1% Tween 20, 5% (w/v) nonfat dry milk) and then incubated with primary antibody diluted in blocking solution (1:500 for ZIP5 and 1:1000 for ZIP4) for 1 h at room temperature. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibody diluted in blocking solution for 30 min at room temperature. Immunoreactive bands were visualized using ECL Plus Western blotting detection system with Hyperfilm ECL (amershambiosciences.com). As a positive control for ZIP5, the plasmid pcDNA3.1Puro(+)-mZIP5HA was transiently transfected into HEK293 cells and grown for 48 h, and a total protein lysate was prepared in 1× SDS sample buffer. As a negative control, the antisera were each neutralized by preincubation for 2 h at room temperature with 6 × 10-5m peptide before incubation with the membranes.ImmunohistochemistryImmunohistochemistry was performed using the Zymed Laboratories Inc. Histostain-SP kit (zymed.com) for rabbit primary antibody and diaminobenzidine substrate staining. Tissues were fixed overnight in Bouin's fixative at 4 °C, embedded in paraffin, and sectioned. Sections were deparaffinized, and subjected to antigen retrieval in 10 mm citrate (pH 6.0) at 95 °C for 6-8 min (28Shi S.R. Chaiwun B. Young L. Cote R.J. Taylor C.R. J. Histochem. Cytochem. 1993; 41: 1599-1604Crossref PubMed Scopus (262) Google Scholar, 29Shi S.R. Key M.E. Kalra K.L. J. Histochem. Cytochem. 1991; 39: 741-748Crossref PubMed Scopus (2420) Google Scholar). Sections were then treated with 1% peroxide for 10 min, blocked with 10% normal goat serum for 10 min, and incubated for 1 h at room temperature with the primary antiserum (ZIP5, 1:300 dilution; ZIP4, 1:600 dilution). Controls included antisera neutralized by incubation for 2 h at room temperature with 6 × 10-5m peptide before application to the tissue sections, use of preimmune serum, and omission of primary antiserum (results not shown). All of these controls confirmed the specificity of these antisera.ImmunofluoresenceTissues were fixed overnight in 4% paraformaldehyde, and 5-μm cryosections were prepared as described (30Barthel L.K. Raymond P.A. J. Histochem. Cytochem. 1990; 38: 1383-1388Crossref PubMed Scopus (292) Google Scholar). Sections were incubated in phosphate-buffered saline (150 mm NaCl, 5 mm phosphate buffer (pH 7.0)) containing 50 mm lysine for 20 min, permeabilized by incubation for 20 min in phosphate-buffered saline containing 0.1% Tween 20 and 0.1% Triton X-100, and then blocked using 10% normal goat serum or 10% normal donkey serum during insulin detection. Co-expression of insulin and mZIP4 in the pancreas was examined by immunofluoresence using guinea pig anti-porcine insulin (DakoCytomation.com) detected with donkey fluorescein isothiocyanate-labeled anti-guinea pig serum (JacksonImmuno.com) and rabbit anti-mouse ZIP4 incubated with biotin-labeled anti rabbit anti-serum (JacksonImmuno.com) and detected using Qdot-605-streptavidin conjugate (Qdots.com) with a TRITC filter set. Nuclei were stained with 4,6-diamidino-2-phenylindole.Accession NumbersGenBank™ accession numbers for mouse the ZIP5 cDNA are AK008448 and AK007473 and those for the mouse ZIP5 gene are AC124670 and AC122159.RESULTSIdentification of the Mouse ZIP5 Gene and mRNA and Evolutionary Conservation of the Predicted ZIP5 Peptide—While searching the mouse-translated non-redundant data base for the ortholog of the human acrodermatitis enteropathica gene ZIP4, a cDNA was identified that encoded a protein with significant amino acid similarity to the carboxyl-terminal half of ZIP4. When aligned with mouse ZIP4, this protein was found to be 36% similar to ZIP4 across the entire length of the protein, but when only the carboxyl-terminal halves of the proteins were aligned, the similarity increased to 49%. Like ZIP4, this protein is also predicted to contain a peptide motif specifically found in transmembrane domain 5 of the other members of the LIV-1 subfamily (HEXPHEXGDFAXLLXXG) (5Taylor K.M. Nicholson R.I. Biochim. Biophys. Acta. 2003; 1611: 16-30Crossref PubMed Scopus (212) Google Scholar). In addition, this protein was predicted to have eight transmembrane domains, as expected for a ZIP family member (Fig. 1A). Based on these results, the gene encoding this protein was referred to as ZIP5 (SLC39A5), which was found later to be consistent with the nomenclature used on the NCBI website. Alignment of mouse and human ZIP5 revealed 87% amino acid similarity, with amino acid differences scattered throughout the protein (Fig. 1A).There were two entries in the mouse non-redundant data base that encoded the ZIP5 protein, AK008448 and AK007473. These sequences were used to search the mouse expressed sequence tag (EST) data base, identifying an additional 49 base pairs of sequence at the 5′ end of the cDNA, although it has not yet been experimentally determined whether this represents the actual 5′ end of the transcript. There were several notable differences between AK008448 and AK007473. First, there were three single nucleotide differences within the coding region. Two of these altered the encoded amino acid, whereas the third was silent (Fig. 1A). A survey of the entire EST data base revealed that each of these differences was present in several ESTs, suggesting that they likely represented polymorphisms rather that mutations or sequencing errors. The second difference between AK008448 and AK007473 was the deletion of 175 base pairs in AK08448 relative to AK007473, presumably due to the splicing in of a noncoding exon. Several other sequences demonstrated identical deletions, and an additional set of ESTs contained an extended deletion, again suggesting that these changes were not mutations or sequencing errors. These differences probably reflect alternate splicing that occurs within exon 2 but that does not alter the protein-coding sequence.The mZIP5 gene was identified by using the 5′ end-extended cDNA sequence to search the mouse high-throughput genomic sequences data base. The intron-exon structure of the gene was determined by aligning the gene with the cDNA and identifying splice donor and acceptor consensus sequences. From this alignment, it was determined that the two deletions noted previously in the cDNA sequences were not due to the splicing out of exons but, rather, due to the use of cryptic splicing signals (Fig. 1B). In addition, as was seen with the mZIP4 gene (15Dufner-Beattie J. Wang F. Kuo Y.M. Gitschier J. Eide D. Andrews G.K. J. Biol. Chem. 2003; 278: 33474-33481Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar), the mZIP5 gene contained 12 exons and was relatively short, spanning ∼5300 base pairs (Fig. 1B). Using the Map-Viewer application on the NCBI server, the mZIP5 gene was localized to chromosome 10D3.The Mouse ZIP5 Gene Is Expressed in Tissues Involved in Zinc Homeostasis—Northern blotting was used to examine the expression of the mZIP5 gene in total RNA extracted from several adult organs as well as from the embryonic visceral yolk sac (Fig. 2). The ZIP5 and ZIP4 transcripts were readily detectable in the proximal and distal small intestine as well as in the embryonic visceral yolk sac. However, ZIP5 mRNA was also detected in the pancreas and kidney, whereas ZIP4 mRNA was not.Fig. 2Northern blot detection of mZIP5 transcripts in mouse organs. The indicated mouse organs were collected from adult mice, whereas the visceral yolk sac was collected from embryos on day 14 of pregnancy. Total RNA was extracted and assayed by Northern blotting using cDNA probes for mZIP5, mZIP4 (15Dufner-Beattie J. Wang F. Kuo Y.M. Gitschier J. Eide D. Andrews G.K. J. Biol. Chem. 2003; 278: 33474-33481Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar), and mZIP1 (12Dufner-Beattie J. Langmade S.J. Wang F. Eide D. Andrews G.K. J. Biol. Chem. 2003; 278: 50142-50150Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Hybrids were detected by autoradiography (upper panel). Integrity and loading of RNA was confirmed by acridine orange staining of a duplicate gel (lower panel). ZIP1 is expressed in most of these organs and also served as a loading control. Sm Int, small intestine.View Large Image Figure ViewerDownload (PPT)Mouse ZIP5 Gene Expression Is Not Altered during Zinc Deficiency—To determine whether ZIP5 gene expression is responsive to dietary zinc, as we have shown previously for the ZIP4 gene (15Dufner-Beattie J. Wang F. Kuo Y.M. Gitschier J. Eide D. Andrews G.K. J. Biol. Chem. 2003; 278: 33474-33481Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar), we examined the effects of zinc deficiency during pregnancy on the abundance of ZIP5 and ZIP4 transcripts in the maternal intestine (Fig. 3A) and embryonic visceral yolk sac (Fig. 3B). Pregnant mice were fed a ZnA or ZnD diet beginning on day 8 of pregnancy, and the maternal small intestine was harvested every 24 h thereafter (days 9-15). The relative abundance of MT-I mRNA was also examined because the transcription of this gene is diminished during dietary zinc deficiency (21Andrews G.K. Lee D.K. Ravindra R. Lichtlen P. Sirito M. Sawadogo M. Schaffner W. EMBO J. 2001; 20: 1114-1122Crossref PubMed Scopus (84) Google Scholar). In mice fed the ZnD diet, ZIP5 mRNA abundance was unaffected, whereas ZIP4 mRNA levels were detectably increased by 24 h of zinc deficiency and continued to increase dramatically for 5 days (Fig. 3A). MT-I mRNA abundance during this period was severely reduced. Similar overall effects of zinc deficiency on these mRNAs were noted in the visceral yolk sac, and by 5 days of a zinc-deficient diet, ZIP5 mRNA levels remained unchanged, whereas ZIP4 mRNA was dramatically induced, and MT-I mRNA was repressed (Fig. 3B).Fig. 3Effects of dietary zinc deficiency on mZIP5 mRNA abundance in the maternal intestine and embryonic visceral yolk sac. Pregnant CD-1 mice were fed the ZnA (dietary zinc +) or ZnD (dietary zinc -) diet beginning on day (d) 8 of pregnancy. Six mice per group were sacrificed on days 9-15 of pregnancy (from 1 to 7 days of ZnD diet), and the relative abundance of mZIP5 mRNA in the maternal small intestine (A) and embryonic visceral yolk sac (B) was determined by Northern blotting. Membranes were hybridized with mZIP5, mZIP4, mZIP1, metallothionein-I (MT-I) and β-actin probes. MT-I mRNA abundance is down-regulated by dietary zinc deficiency in these tissues (15Dufner-Beattie J. Wang F. Kuo Y.M. Gitschier J. Eide D. Andrews G.K. J. Biol. Chem. 2003; 278: 33474-33481Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar).View Large Image Figure ViewerDownload (PPT)ZIP5 Is Localized on the Basolat
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