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.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 RNAbinding proteins called iron-regulatory proteins (IRP1 and IRP2) (1). The IRE is a well conserved RNA stem-loop found in the 5Ј-UTR of iron-regulated genes such as ferritin and the 3Ј-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 man...

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The translation of ferritin mRNA and degradation of transferrin receptor mRNA are regulated by the interaction of an RNA-binding protein, the iron-responsive element binding protein (ERE-BP), with RNA stem-oop structures known as iron-responsive elements (IREs) contained within these transcripts. IRE-BP produced in iron-replete cells has aconitase (EC 4.2.1.3) activity. The protein shows extensive sequence homology with mitochondrial aconitase, and sequences of peptides prepared from cytosolic aconitase are identical with peptides of IRE-BP. As an active aconitase, IRE-BP is expected to have an Fe-S duster, in analogy to other aconitases. This Fe-S cluster has been implicated as the region of the protein that senses intracellular iron levels and accordingly modifies the ability of the IRE-BP to interact with IREs. Expression of the IRE-BP in cultured cells has revealed that the IRE-BP functions either as an active aconitase, when the cells are iron-replete, or as an active RNA-binding protein, when the cells are iron-depleted. We compare properties of purified authentic cytosolic aconitase from beef liver with those of IRE-BP from tissue culture cells and establish that characteristics of the physiologically relevant form of the protein from iron-depleted cells resemble those of cytosolic aconitase apoprotein. We demonstrate that loss of the labile fourth iron atom of the Fe-S cluster results in loss of aconitase activity, but that more extensive cluster alteration is required before the IRE-BP acquires the capacity to bind RNA with the affinity seen in vivo. These results are consistent with a model in which the cubane Fe-S cluster is disassembled when intracellular iron is depleted.

The expression of ferroportin1 (FPN1) in reticuloendothelial macrophages supports the hypothesis that this iron-export protein participates in iron recycling from senescent erythrocytes. To gain insight into FPN1's role in macrophage iron metabolism, we examined the effect of iron status and erythrophagocytosis on FPN1 expression in J774 macrophages. Northern analysis indicated that FPN1 mRNA levels decreased with iron depletion and increased on iron loading. The ironinduced induction of FPN1 mRNA was blocked by actinomycin D, suggesting that transcriptional control was responsible for this effect. After erythrophagocytosis, FPN1 mRNA levels were also upregulated, increasing 8-fold after 4 hours and returning to basal levels by 16 hours. Western analysis indicated corresponding increases in FPN1 protein levels, with maximal induction after 10 hours. Iron chelation suppressed FPN1 mRNA and protein induction after erythrophagocytosis, suggesting that FPN1 induction results from erythrocyte-derived iron. Comparative Northern analyses of iron-related genes after erythrophagocytosis revealed a 16-fold increase in FPN1 levels after 3 hours, a 10-fold increase in heme oxygenase-1 (HO-1) after 3 hours, a 2-fold increase in natural resistance macrophageassociated protein 1 (Nramp1) levels after 6 hours, but no change in divalent metal ion transporter 1 (DMT1) levels. The rapid and strong induction of FPN1 expression after erythrophagocytosis suggests that FPN1 plays a role in iron recycling. (Blood. 2003;102:4191-4197)

Copper deprivation of Saccharomyces cerevisiae induces transcription of the FRE1 and CTR1 genes. FRE1 encodes a surface reductase capable of reducing and mobilizing copper chelates outside the cell, and CTR1 encodes a protein mediating copper uptake at the plasma membrane. In this paper, the protein encoded by MAC1 is identified as the factor mediating this homeostatic control. A novel dominant allele of MAC1, MAC1 up2 , is mutated in a Cys-rich domain that may function in copper sensing (a G to A change of nucleotide 812 resulting in a Cys-271 to Tyr substitution). This mutant is functionally similar to the MAC1 up1 allele in which His-279 in the same domain has been replaced by Gln. Both mutations confer constitutive copper-independent expression of FRE1 and CTR1. A sequence including the palindrome TTTGCTCA . . . TGAGCAAA, appearing within the 5-flanking region of the CTR1 promoter, is necessary and sufficient for the copper-and MAC1-dependent CTR1 transcriptional regulation. An identical sequence appears as a direct repeat in the FRE1 promoter. The data indicate that the signal resulting from copper deprivation is transduced via the Cys-rich motif of MAC1 encompassing residues 264 -279. MAC1 then binds directly and specifically to the CTR1 and FRE1 promoter elements, inducing transcription of those target genes. This model defines the homeostatic mechanism by which yeast regulates the cell acquisition of copper in response to copper scarcity or excess.

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Acute and chronic inflammation cause many changes in total body iron metabolism including the sequestration of iron in phagocytic cells of the reticuloendothelial system. This change in iron metabolism contributes to the development of the anemia of inflammation. MTP1, the duodenal enterocyte basolateral iron exporter, is also expressed in the cells of the reticuloendothelial system (RES) and is likely to be involved in iron recycling of these cells. In this study, we use a lipopolysaccharide model of the acute inflammation in the mouse and demonstrate that MTP1 expression in RES cells of the spleen, liver, and bone marrow is down-regulated by inflammation. The down-regulation of splenic expression of MTP1 by inflammation was also observed in a Leishmania donovani model of chronic infection. The response of MTP1 to lipopolysaccharide (LPS) requires signaling through the LPS receptor, Toll-like receptor 4 (TLR4). In mice lacking TLR4, MTP1 expression is not altered in response to LPS. In addition, mice lacking tumor necrosis factor-receptor 1a respond appropriately to LPS with down-regulation of MTP1, despite hyporesponsiveness to tumor necrosis factor-␣ signaling, suggesting that this cytokine may not be required for the LPS effect. We hypothesize that the iron sequestration in the RES system that accompanies inflammation is because of down-regulation of MTP1.Iron is an essential nutrient for growth and development of eucaryotes and most prokaryote species. A normal individual will absorb ϳ1 mg of elemental iron a day through the duodenum, to match an equivalent daily physiologic loss. The plasma turnover of iron is ϳ10 -20 mg a day and one source of this pool is iron released from the reticuloendothelial system (RES).