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J. Biol. Chem., Vol. 275, Issue 26, 19906-19912, June 30, 2000

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A Novel Mammalian Iron-regulated Protein Involved in Intracellular Iron Metabolism^*

Sherry Abboud¶ and David J. Haile^§¶

From the  Department of Pathology and the ^§ Department of Medicine, University of Texas Health Science Center, San Antonio, Texas 78229 and the Audie Murphy Veterans Affairs Hospital, South Texas Veterans Health System, San Antonio, Texas 78284

Received for publication, January 31, 2000, and in revised form, March 15, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We have isolated and characterized a novel iron-regulated gene that is homologous to the divalent metal transporter 1 family of metal transporters. This gene, termed metal transporter protein (mtp1), is expressed in tissues involved in body iron homeostasis including the developing and mature reticuloendothelial system, the duodenum, and the pregnant uterus. MTP1 is also expressed in muscle and central nervous system cells in the embryo. At the subcellular level, MTP1 is localized to the basolateral membrane of the duodenal epithelial cell and a cytoplasmic compartment of reticuloendothelial system cells. Overexpression of MTP1 in tissue culture cells results in intracellular iron depletion. In the adult mouse, MTP1 expression in the liver and duodenum are reciprocally regulated. Iron deficiency induces MTP1 expression in the duodenum but down-regulates expression in the liver. These data indicate that MTP1 is an iron-regulated membrane-spanning protein that is involved in intracellular iron metabolism.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The uptake of iron by the duodenum and by individual cells in the body is regulated by total body iron levels and intracellular iron levels, respectively. Individual cells take up iron bound to transferrin using the transferrin receptor. Iron not immediately utilized is stored as cytosolic ferritin. The iron-dependent regulation of ferritin and transferrin receptor is mediated by the post-transcriptional interaction of iron-responsive elements (IREs),¹ found in the untranslated regions (UTRs) of the mRNAs of these genes, with cytosolic RNA-binding proteins called iron-regulatory proteins (IRP1 and IRP2) (1). The IRE is a well conserved RNA stem-loop found in the 5'-UTR of iron-regulated genes such as ferritin and the 3'-UTR of transferrin receptor and dmt1 (2, 3).

Intestinal iron acquisition requires uptake of iron at the brush border of the duodenal epithelial cell and subsequent export of the iron across the basal border. DMT1 (NRAMP2, DCT1) transports iron into the cell at the apical brush border of the duodenal epithelial cell. A mouse mutant in DMT1 in unable to take up intestinal iron (3, 4). An alternative pathway for intestinal iron absorption that involves a cell surface ₃ integrin, a calreticulin-like molecule called mobilferrin, and a ferrireductase has been described (5). Iron export from the epithelial cell also requires a copper-dependent ferroxidase called hephaestin (6), but otherwise little is known about this process. Another major area of iron metabolism that is not well understood is that of the recycling of hemoglobin-derived iron by the RE system. Tissue macrophages ingest recycled iron from senescent erythrocytes. Export of heme-derived iron as ferritin and low molecular weight iron by macrophages has been described (7, 8), but details of the regulation of the process and molecular mechanisms are lacking.

This paper reports the cloning and characterization of a novel iron-regulated iron transporter called MTP1. MTP1 is related to the DMT1 class of divalent metal transporters and a yeast manganese transporter (SMF1) (9). The unique characteristics of this protein suggest that it functions as an iron-exporting molecule and that it is involved in iron acquisition from the environment and iron recycling in the body.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Animals-- C57/BL6 mice were used for all in vivo studies. Three- to six-month-old mice were made iron-deficient by feeding a low iron diet (Harlan Biosciences) for 4-6 weeks. Mice were made iron-replete by injection of 1 mg of iron dextran, intramuscularly. Mice were killed 1-2 weeks after injection, and the organs were harvested and fixed in formalin or frozen for immunohistochemistry.

Cloning of MTP1 and Plasmid Constructions-- In order to identify new genes that are important in the regulation of iron metabolism, a library of mRNA sequences enriched for IRP1 binding was constructed using SELEX technology (10). From this library, a 200-bp sequence corresponding to the 5'-UTR of MTP1 was isolated.²

The novel cDNA, containing a 5'-UTR IRE, was used to probe a day-15.5 mouse embryo cDNA library (Superscript cDNA library in pSPORT2 CMV vector, Life Technologies, Inc.). A plasmid clone containing a 2.1-kb insert, which included the entire protein coding region and most of the 5'-untranslated region of MTP1, was isolated (pSPORT2 MTP1, GenBank^TM accession number AF215637). An MTP1 plasmid clone lacking the IRE (IRE-MTP1) was also made by excision of an SmaI fragment from pSPORT2 MTP1. ESTs corresponding to the 5' and 3' ends of the human MTP1 cDNA were identified in the GenBank^TM. These sequences were used to amplify the entire human gene by PCR from commercially obtained human small intestinal cDNA (Marathon Human Small Intestine cDNA, CLONTECH). The first 1700 bases of the human cDNA were sequenced (GenBank^TM accession number AF215636). The remaining human MTP1 sequence was assembled by overlapping of multiple ESTs from the GenBank^TM. The rat sequence is available at GenBank^TM accession number U76714. Errors in the published rat nucleotide sequence that resulted in frameshifts in the cDNA reading frame were corrected by substituting the corresponding mouse cDNA bases in order to obtain a translatable reading frame. A 13-kb mouse genomic  phage clone was isolated from a 129SvJ mouse genomic library (FIX II, Stratagene) using a probe from the 5' end of the mouse MTP1 cDNA.

5'-Rapid amplification of cDNA ends (RACE) was done using the Marathon PCR-ready human small intestine and mouse embryo cDNAs (CLONTECH) per manufacturer's recommendation. Five clones of the race product from both the mouse and human cDNA RACE reactions were sequenced.

For iron-dependent regulation studies of the mouse MTP1 5'-UTR, a luciferase reporter containing the mouse MTP1 promoter and the MTP1 5'-UTR was constructed (IRE-LUC) as follows. A PCR-generated 5'-UTR of MTP1 was cloned into the NcoI/HindIII site of PGL3 control vector (Promega, Madison, WI). A 2.7-kb fragment (HindIII/SmaI) from the MTP1 genomic DNA (GenBank^TM accession number AF216834) was subcloned into the BglII/SmaI site of the MTP1 5'-UTR containing PGL3 vector to complete the IRE-LUC vector. This vector contains 3.4 kb of 5'-flanking sequence and the entire MTP1 5'-UTR. The IRE-LUC vector was constructed by deletion of the BamHI/SmaI segment of the MTP1 5'-UTR from the IRE-LUC vector.

Cell Transfections-- For studies of the iron-dependent regulation of the MTP1 5'-UTR, 50-60% confluent COS7 cells (24-well format) were transiently transfected with IRE-LUC or IRE-LUC plasmids along with a control Renilla luciferase plasmid (Dual Luciferase Assay Kit, Promega) using LipofectAMINE. Twenty four hours after transfection, the cells were treated overnight with either 50 µM hemin (Sigma) or 50 µM desferrioxamine (Sigma). Firefly and Renilla luciferase activities of lysates were assayed. All luciferase measurements were normalized to the Renilla luciferase expression in order to correct for differences in transfection efficiency. For MTP1 overexpression studies, 50-60% confluent HEK293T cells were transfected using IRE-MTP1 or pSPORT2 CMV vectors with Superfect reagent (Qiagen). Twenty four to 48 h after transfection, cells were harvested, washed in phosphate-buffered saline, and detergent extracts prepared using 1% Triton in 10 mM Tris- HCl, pH 7.4, 50 mM NaCl, 1 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride (Triton lysis buffer).

Western Blotting-- Five µg of protein from cell lysates were separated by SDS gel electrophoresis, transferred to nitrocellulose, and probed using a rabbit anti-horse spleen ferritin antiserum (Sigma) and rabbit anti-actin (Sigma) both at 1:1000 dilution, and a horseradish peroxidase-labeled secondary antibody. Signal was detected using the enhanced chemiluminescence kit.

Gel Retardation Assay-- The gel retardation assay was done using radiolabeled ferritin H-chain IRE probe by previously published protocols with the following modifications (11). 10 µg of protein from the total cell detergent extract in 18 µl of Triton lysis buffer (with 5% glycerol and Tris-HCl, pH 7.4, raised to 50 mM) was used for each assay point. 50,000 cpm of IRE probe (2 ng) was added followed by 1 unit of T1 nuclease and heparin to a final concentration of 5 mg/ml prior to electrophoresis.

Antibody Production and Immunostaining-- Recombinant peptide produced in Escherichia coli (amino acids 223-303 of mouse MTP1) was used to immunize a rabbit for production of anti-MTP1 antiserum. The antiserum was immunoaffinity purified using an immobilized 28-amino acid peptide (amino acids 236-263 of MTP1). Immunohistochemical staining was done using a commercially available kit (Envision Plus, Dako) with Vector VIP (Vector Laboratories) or diaminobenzidine substrate. Tissue indirect immunofluorescent studies were done on frozen sections fixed at 20 °C in acetone containing 1% paraformaldehyde at an antibody concentration of 12.5 µg/ml. Blocking for these studies was done using 10% milk and 10% horse or goat serum. Secondary antibody was Alexa 488-conjugated goat anti-rabbit IgG antibody (Molecular Probes). Fluorescent images were viewed and photographed using an Olympus BX-40 fluorescent microscope.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

All mtp1 Genes Contain IREs and Are Homologous to Known Metal Transporters-- Mouse, rat, and human MTP1 cDNAs contain IREs in the 5'-UTR (Fig. 1a). The mouse transcription start site was determined by 5'-RACE using cDNA derived from mouse embryo, and the human start site was determined using cDNA derived from the small intestine (Fig. 1a). For the mouse embryo, the start site was determined to be 53 bases proximal to the IRE stem. The transcription start site of the human small intestinal MTP1 cDNA is placed at a distance of 73 bases from the IRE stem (Fig. 1a). The IRE sequence from the mouse MTP1 5'-UTR folds into an appropriate IRE-like stem loop with a calculated free energy of 15.2 kcal/mol (Fig. 1b) and competes with human H-chain ferritin IRE for binding to IRP1 as demonstrated by the gel retardation assay (Fig. 1c).

View larger version (69K): [in this window] [in a new window]   Fig. 1.   mtp1 genes have 5'-UTR IRE sequences. a, alignment of IRE-containing 5'-UTR regions of mouse, human, and rat MTP1 cDNAs. IRE loop sequences are underlined. b, predicted mouse MTP1 IRE RNA secondary structure. c, the MTP1 IRE sequence competes with ferritin H-chain IRE for binding to IRP1. Gel retardation assay was performed as indicated above using 10 µg of HEK293T detergent-extracted cell lysate as source of IRP1 and radiolabeled ferritin H-chain IRE as probe, 1st lane. Indicated are competitions with 50-fold molar excess of tRNA (t) or 50-fold molar excess of MTP1 IRE RNA (m). Last lane indicates probe alone without any cell lysate added. d, multalin alignment of amino acid sequences of the mouse, human, and rat MTP1, human NRAMP2, SMF1 and two uncharacterized gene products AAC28758 (Arabidopsis thaliana) and AAB94213 (Caenorhabditis elegans).

The deduced amino acid sequence of the mouse, human, and rat mtp1 genes is illustrated below (Fig. 1d). The human, mouse, and rat MTP1 proteins are greater than 90% similar. These proteins are 62 kilodaltons and are predicted to contain at least 10 transmembrane regions using the TMHMM algorithm.

Homology of MTP1 to the DMT1 family, the SMF1 family, and two uncharacterized gene products (AAC28758 and AAB94213) has been identified using PROPSEARCH and Blast algorithms using the TMHMM algorithm.

Alignment of these amino acid sequences with the MTP1s demonstrates similarity (identical and conserved amino acids) throughout the amino acid sequence (Fig. 1d). Amino acid analysis using PROSITE found no signal or localization sequences. Several N- and O-linked glycosylation sites were identified. The homology to known metal transporters supports the hypothesis that MTP1 is a metal transporter.

MTP1 Protein Localizes to Cells Implicated in Iron Kinetics/Turnover in the Body-- Northern blot analysis of the tissue distribution of the mouse and human MTP1 mRNA, using commercially available multiple tissue RNA blots, demonstrated highest abundance in the spleen, liver, kidney, heart, and duodenum in mouse and abundance in the placenta, intestine, muscle, and spleen in human blots (data not shown).

A rabbit polyclonal anti-MTP1 peptide antibody was made and confirmed by both Western blot and immunohistochemistry to recognize specifically MTP1 protein produced by overexpression in NIH3T3 and COS7 cells (data not shown). MTP1 expression was assayed by immunohistochemistry in several mouse tissues (Fig. 2a). MTP1 immunostaining was detected in the mature duodenal epithelial cells and not in the crypt cells (Fig. 2a, panels A-D). The staining pattern from a mouse on a normal diet demonstrated a predominantly cytoplasmic distribution with no discernible basolateral membrane (Fig. 2a, panels A and B). Duodenal anti-MTP1 staining from an iron-deficient mouse demonstrated concentration at the basolateral membrane of the cells, but cytoplasmic staining was also evident (Fig. 2a, panels C and D). Immunostaining was also evident in the splenic macrophage and megakaryocytes in the red pulp (Fig. 2a, panels E and F) and the Kupffer cells of the liver (Fig. 2a, panels G and H). Expression was noted in glomerular cells and weaker staining in the proximal tubular cells of the kidney (Fig. 2a, panels I and J) and in spindle-shaped cells in heart muscle (Fig. 2a, panels K and L). These data support the Northern blot expression data and indicate that MTP1 is expressed in tissues of known importance in iron metabolism.

View larger version (110K): [in this window] [in a new window]   Fig. 2.   Tissue distribution of MTP1 protein in adult and embryonic mice. a, bright field photographs of mouse tissues immunostained using an affinity purified anti-MTP1 antibody. Panel A, duodenum from mouse on normal diet (× 200); panel C, duodenum from iron-deprived mouse (× 400); panel E, spleen (× 100); panel G, liver (× 100); panel I, kidney (× 400); panel K, heart muscle (× 400). Panels B, D, F, H, J, and L are corresponding controls, and immunostaining was done with the same concentration of normal rabbit IgG. Antibody concentrations were 2.5 µg/ml for panels A, B, E, F, G, and H; 0.6 µg/ml was for the others. Panels A-H are paraffin-embedded, and others are frozen sections. Chromogen for G and H was diaminobenzidine. b, Brightfield photographs of immunostained mouse embryo tissues using the anti-MTP1 antibody were described above. Panel A, day 10 pregnant uterus. Day 16 embryos: panel C, whole embryo (× 4), panel E, brain; panel F, spinal cord; panel G, heart and great vessels (arrows); panel H, brain (arrowhead) and tongue, (thin arrow). Day 14 embryos: panel I, liver; J, section through tongue; k, surface of brain; L, spinal cord. Panels B and D are controls immunostained with normal rabbit IgG. Sections are paraffin-embedded, and anti-MTP1 and control rabbit IgG concentrations used in immunostaining were 2.5 µg/ml.

Expression of MTP1 was also examined in mouse embryos (Fig. 2b). In early embryogenesis (days 8 and 10), MTP1 expression was localized to the decidua of the pregnant uterus (Fig. 2b, panels A and B). Later in development (days 14 and 16), MTP1 expression was noted throughout the embryo (Fig. 2b, panels C and D). Notably, in later embryos, staining was present in the brain (Fig. 2b, panels E and K) and spinal cord (Fig. 2b, panels F and L), myocytes in developing vessels and heart (Fig. 2b, panel G), tongue (Fig. 2b, panels H and J), and other muscles in the embryo and Kupffer cells of the embryonic liver (Fig. 2b, panel I).

Iron-dependent Regulation of MTP1 Expression-- Total body iron-dependent regulation of MTP1 was examined in frozen sections of the liver and duodenum from iron-replete or iron-deprived mice (Fig. 3a). Immunohistochemical staining using the anti-MTP1 antibody demonstrated strong duodenal epithelial cell staining in iron-deprived mice and weaker staining in iron-replete mice. In contrast, the pattern of expression in the Kupffer cells of the liver showed strong expression in iron-replete mice and less expression in iron-deprived mice. These results demonstrate that MTP1 expression is iron-regulated in these tissues but in a reciprocal manner.

View larger version (57K): [in this window] [in a new window]   Fig. 3.   MTP1 expression in the duodenum and liver is regulated by iron. a, iron-dependent regulation of MTP1 expression in duodenal and liver tissue. Duodenum and liver frozen sections from iron-deficient or -replete mice were immunostained using the anti-MTP1 antibody (0.3 µg/ml) as indicted above: A, iron-replete duodenum; B, iron-deficient duodenum; C, iron-replete liver; D, iron-deficient liver. b, iron-dependent regulation of luciferase reporter gene containing MTP1 5'-UTR. COS7 cells were transfected with IRE-LUC or IRE-LUC plasmids and subsequently treated with hemin or desferrioxamine. Sixteen hours later, cells were assayed for luciferase using the commercially available kit. Data presented are arbitrary firefly luciferase light units that have been normalized using the Renilla luciferase measurements to account for transfection efficiency. All experiments were done in quadruplicate, and error bars represent the standard deviation.

A luciferase expression system was used to directly confirm iron-dependent translational regulation by the MTP1 5'-UTR. COS7 cells were transiently transfected with a luciferase reporter gene containing 3.4-kb 5'-flanking region and the 5'-UTR of MTP1 (IRE-LUC) or a control plasmid containing the same promoter but lacking an IRE (IRE-LUC). Twenty four hours later, the cells were treated with either hemin or desferrioxamine in order to make the cells either iron-replete or iron-deficient, respectively. Subsequently, luciferase levels of cell lysates were measured. Iron starvation of cells led to a decrease in luciferase expression in the IRE-LUC transfectants compared with IRE-LUC transfectants (Fig. 3b). These results indicate that the MTP1 5'-UTR confers iron-dependent regulation and the regulation similar to the iron-dependent regulation of ferritin, with iron deprivation inhibiting MTP1 expression. The MTP1 regulation observed in the liver may be secondary to the IRE. The regulation in the duodenum may depend upon other mechanisms.

Subcellular Localization of MTP1-- Localization of MTP1 expression was also examined in the duodenum of iron-deficient mice (Fig. 4a). Indirect immunofluorescent staining of frozen mouse duodenal sections demonstrated a distribution of MTP1 that is prominently basolateral but with staining also present throughout the apical and basal cytoplasm. Indirect immunofluorescence staining of frozen mouse liver sections (Fig. 4b) demonstrated abundant staining in the Kupffer cells with the subcellular distribution being predominantly cytoplasmic. In many cells, co-localization of MTP1 immunostaining with hemosiderin granules in the Kupffer cells was noted (Fig. 4b, panels A and C). Specific MTP1 immunostaining was also noted at the sinusoidal borders of the hepatocytes. This plasma membrane staining of the hepatocytes was not noted in the previous paraffin-embedded section staining and was evident only at higher antibody concentrations.

View larger version (24K): [in this window] [in a new window]   Fig. 4.   Indirect immunofluorescence localizes MTP1 to basolateral border of mouse intestinal duodenum, plasma membrane of hepatocyte, and intracellular compartment of Kupffer cells. a, frozen section of mouse duodenum from an iron-deficient animal was stained for indirect immunofluorescence using the following: A, anti-MTP1 antibody, or B, control rabbit IgG both at 12.5 µg/ml as described above. Images were visualized and photographed using a fluorescent microscope equipped with a fluorescent isothiocyanate (green) filter. Image is × 400. b, frozen section of mouse liver was stained for indirect immunofluorescence using anti-MTP1 antibody panels A, C, and D or panel B control rabbit IgG both at 12.5 µg/ml as described above. A, arrows indicate Kupffer cells with cytoplasmic staining, and red arrows indicate co-localization of anti-MTP1 immunofluorescence with hemosiderin fluorescence. C, enlargement of section of panel A with red arrow indicating Kupffer cells and white arrows indicting borders between cells. D, section of mouse intestine stained for MTP1 using indirect immunofluorescence as indicated above and also stained with propidium iodide to indicate location of hepatocyte nucleus. Arrows indicate borders of hepatocyte. Images were obtained as indicated above. Original magnification is × 400.

Evidence for Iron Transport-- IRP1 is an iron-sulfur cluster containing protein that serves as an iron-dependent regulator of IRE-containing genes, including ferritin. Therefore, a perturbation in intracellular iron content secondary to MTP1 overexpression would be expected to alter both IRE binding activity of IRP1 and ferritin expression. Iron deficiency results in conversion of holoIRP1 to apoIRP1 resulting in an increase in IRE binding activity of IRP1 (1, 11). HEK293T cells were transiently transfected with the IRE-MTP1 or control plasmids (pSPORT2 CMV), and 48 h later, detergent-extracted cell lysates were examined for IRP1 binding activity using electrophoretic mobility shift assay. The IRE-MTP1 construct was chosen for the overexpression experiments in order to abolish potential iron-dependent regulation of mtp1 gene itself. As expected, transient transfection of MTP1 in these cells resulted in activation of IRP1 IRE binding, indicating that MTP1 expression leads to intracellular/cytosolic iron depletion (Fig. 5a). On treatment with ferricyanide (which destroys the iron-sulfur cluster), all lysates had similar amounts of IRP1 binding activity. Thus, the observed changes in IRP1 activity were due to a shift from the holoIRP1 to apoIRP1 in the MTP1-transfected cells. The amount of ferritin in the transfected and control cells was assessed by Western blotting of lysates using an anti-ferritin antibody. Blots were stripped and re-probed using an anti-actin antibody in order to confirm equal loading of lanes. As expected cells transiently transfected with MTP1 had diminished cellular ferritin levels compared with control transfected cells (Fig. 5b). Indirect immunofluorescence examination of transiently transfected HEK293T cells demonstrated plasma membrane localization of the MTP1 gene product (data not shown). The results of these experiments demonstrate that MTP1 overexpression depletes intracellular and cytosolic iron.

View larger version (80K): [in this window] [in a new window]   Fig. 5.   Overexpression of MTP1 results in cytosolic iron and ferritin depletion. a, HEK293T cells were transiently transfected with IRE-MTP1 (M) or a control pSPORT2 CMV (C), and 48 h later lysates were assayed for IRE binding activity of IRP1 by gel retardation assay (a) or cytosolic ferritin level by Western blotting (b). Panel a, cell lysates were assayed as described above using a 32P-labeled ferritin H-chain IRE RNA. The results of two experiments are illustrated (lanes 1-4). Ferricyanide (5 mM) treatment of experimental samples destroys the iron-sulfur cluster of IRP1 and results in the conversion of holoIRP1 to the RNA binding apoIRP1 (lanes 5-8). Probe alone, lane 9. Specificity controls consist of competition with either tRNA (t) or cold IRE (I), (lanes 10-15). b, Western blot of cell lysates noted of MTP1 transfected (M) or control transfected HEK293 (C) cells assayed for ferritin and actin content, two separate experiments are noted.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The expected pattern of expression and regulation of MTP1 in the mouse intestine is that which would be predicted for a molecule involved in iron acquisition. MTP1 is member of a family of the DMT1 (NRAMP) metal transporters, and overexpression of MTP1 in tissue culture cells results in IRP1 activation and ferritin depletion indicating a possible role as an iron exporter. Furthermore, while this manuscript was in review, other investigators have reported the cloning and characterization of this gene using a zebrafish anemia model and provided evidence for MTP1-mediated iron export in Xenopus oocytes overexpressing this protein (12). The expression of MTP1 in the decidua of the pregnant uterus is on the maternal side of the circulation indicating that MTP1 may be exporting iron from the maternal side to the fetal circulation. The duodenal expression of MTP1 is limited to the mature absorptive epithelial cells of the villus and found at the basolateral membrane. DMT1, the apical intestinal cell iron transporter, is also localized to the mature absorptive cells of the villus (2). The up-regulation of expression of MTP1 in the duodenum with iron deprivation parallels that of DMT1 (3). These data suggest that MTP1 is likely the basal surface iron transporter and is responsible for exporting iron from the duodenal epithelial cell. The recent elucidation of the molecular defect of the sex-linked anemia as a mutation in hephaestin (6), a copper-dependent ferroxidase, has provided evidence that ferrous iron transport across membranes requires a ferroxidase activity. The sex-linked anemia mouse is unable to export iron out of the intestinal epithelial cell despite total body iron depletion. It is possible that iron transport across the basolateral membrane or other membranes by MTP1 is coupled to oxidation of the iron by hephaestin.

The regulation by iron of the expression of MTP1 in the duodenum is the opposite that which would be expected of a gene with a 5'-UTR IRE. In the duodenum of iron-treated rodents, we (data not shown) and others (13) have observed up-regulation of ferritin expression in the duodenal epithelial cells of the villus. Therefore, in the duodenum, ferritin and MTP1 are reciprocally regulated by iron despite the presence of a 5'-IRE on both genes. The mechanism by which MTP1 is regulated oppositely from ferritin is not known, but other authors (3, 13) have pointed to likely IRE and non-IRE mediated post-transcriptional and transcriptional regulation of ferritin and DMT1 in duodenal epithelial cells. Other investigators (14) have determined that IREs at a distance of greater than 67 nucleotides from the cap site are not repressible by IRE/IRP1 system. For the human small intestine MTP1 mRNA, the distance from the transcription start site to the base of the IRE is longer than 67 bases suggesting that the IRE may not be functional. Transcription start site selection may play a role in the regulation of this gene.

In RE cells, MTP1 is likely involved in the reutilization/storage of iron that is scavenged from erythrocytes. In our iron dextran-injected animals, the Kupffer cell expression of MTP1 was clearly regulated by iron in a ferritin-like manner with iron-inducing expression and iron derivation inhibiting the expression. This is in contrast to the duodenal regulation by iron of MTP1, which is the opposite pattern. The regulation observed in Kupffer cells parallels that of ferritin, another 5'-IRE-containing gene, suggesting that the 5'-IRE may be functional in these cells.

The subcellular distribution of MTP1 in intestinal and Kupffer cells is also distinctly different. In Kupffer cells, MTP1 is expressed predominantly in a cytoplasmic/intracellular distribution and not surface membrane-localized. Additionally, MTP1 in the tissue culture mouse monocyte cell line, RAW267.4, is also predominantly cytoplasmic with no membrane-localized staining noted (data not shown). The identity of the intracellular compartment in cells is not known. Experiments have demonstrated that COS7 cell MTP1 expression is primarily ER in distribution (data not shown). It is not likely that MTP1 is responsible for iron export across the plasma membrane in these cells. More likely, MTP1 is involved in iron export from the cytosol to an intracellular compartment and possibly export of the iron through the secretory pathway. Alternatively, MTP1 may not have a role in iron export from these cells but may simply move iron into intracellular compartments. Most experimental models of erythophagocytosis have demonstrated that iron from hemoglobin degradation is released either as low molecular weight iron that is capable of binding to transferrin or as secreted ferritin (7, 8). MTP1 may supply the iron to ER-synthesized ferritin that is bound for export. Further experiments are needed in order to determine if MTP1 is localized to the endoplasmic reticulum in RE cells and if an ER-mediated secretory pathway may play a role in iron export/storage in these cells.

If MTP1 is involved in iron export from Kupffer cells, it is not clear how these cells are able to store iron in the presence of enhanced MTP1 activity. The balance between iron storage and export may depend on differential regulation of ferritin and MTP1 due to differences in transcriptional rates or to differences in the affinity of each IRE to the IRPs. The ratio of heavy to light chain ferritin subunits may also alter this balance in particular tissues.

In the liver hepatocytes, MTP1 appears to be expressed at the cell surfaces lining the sinusoids. In our iron-deprived and iron-replete mice, no differences in MTP1 expression at the sinusoidal surface were noted, although the iron dextran injections only resulted in RE and not parenchymal liver iron loads. Therefore, it is not known whether this hepatocyte expression is hepatocyte iron-regulated. MTP1 expression on the sinusoidal surface of hepatocytes suggests a role for MTP1 in hepatocyte iron metabolism and suggests that MTP1 may export iron from these cells. Dysregulation of MTP1 expression in hepatocytes may possibly be important in hemochromatosis and other conditions.

In other cells of the body, especially in the embryo, MTP1 may have a protective function of limiting cytoplasmic iron levels. The developing nervous system may require MTP1 in order to tightly regulate intracellular or cytosolic iron levels to protect these cells against iron-mediated oxidative injury. Furthermore, MTP1 is also expressed in both adult and embryonic muscle cells indicating a role for this protein in iron metabolism in these tissues. MTP1 and DMT1 are also expressed in kidney cells with MTP1 localized to the glomerulus and proximal tubular cells of the kidney (2). These results indicate a possible role of the kidney in iron metabolism. MTP1 appears to be involved in both iron absorption and iron recycling around the body. In addition, it also has a role in the developing nervous system and in muscle metabolism. In addition, this gene is also expressed in a variety of tumor cells. The identification of MTP1 and elucidation of its role in iron metabolism will lead to a better understanding of both iron absorption and recycling.

	FOOTNOTES

^* This work was supported by a grant from the American Heart Association, Texas Affiliate, and Howard Hughes Medical Institute Pilot Project Grant, and National Institutes of Health R01DK53079 and R03DK96009.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AF215636, AF215637, and AF216834.

To whom correspondence should be addressed. Tel.: 210-567-4848; Fax: 210-567-1956; E-mail: Haile@UTHSCSA.edu.

Published, JBC Papers in Press, March 21, 2000, DOI 10.1074.jbc.M000713200

² D. J. Haile, manuscript in preparation.

	ABBREVIATIONS

The abbreviations used are: IREs, iron-responsive elements; UTRs, untranslated regions; DMT1, divalent metal transporter 1; MTP1, metal transporter protein; RE, reticuloendothelial; kb, kilobase pair; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; ER, endoplasmic reticulum; IRP, iron-regulatory protein; CMV, cytomegalovirus.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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