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Divalent metal transporter 1 (DMT1) is a transmembrane protein crucial for duodenal iron absorption and erythroid iron transport. DMT1 function has been elucidated largely in studies of the mk mouse and the Belgrade rat, which have an identical single nucleotide mutation of this gene that affects protein processing, stability, and function. These animals exhibit hypochromic microcytic anemia due to impaired intestinal iron absorption, and defective iron utilization in red cell precursors. We report here the first human mutation of DMT1 identified in a female with severe hypochromic microcytic anemia and iron overload. This homozygous mutation in the ultimate nucleotide of exon 12 codes for a conservative E399D amino acid substitution; however, its pre-dominant effect is preferential skipping of exon 12 during processing of pre–messenger RNA (mRNA). The lack of full-length mRNA would predict deficient iron absorption in the intestine and deficient iron utilization in erythroid precursors; however, unlike the animal models of DMT1 mutation, the patient is iron overloaded. This does not appear to be due to up-regulation of total DMT1 mRNA. DMT1 protein is easily detectable by immunoblotting in the patient's duodenum, but it is unclear whether the protein is properly processed or targeted.

A mutation of the iron transporter Nramp2 (DMT1, Slc11a2) causes microcytic anemia in mk mice and in Belgrade rats by impairing iron absorption in the duodenum and in erythroid cells, causing severe iron deficiency. Both mk and Belgrade animals display a glycine-to-arginine substitution at position 185 (G185R) in the fourth predicted transmembrane domain of Nramp2. To study the molecular basis for the loss of function of Nramp2G185R, we established cell lines stably expressing extracellularly tagged versions of wild-type (WT) or mutated transporters. Like WT Nramp2, the G185R mutant was able to reach the plasmalemma and endosomal compartments, but with reduced efficiency. Instead, a large fraction of Nramp2G185R was detected in the endoplasmic reticulum, where it was unstable and was rapidly degraded by a proteasome-dependent mechanism. Moreover, the stability of the mutant protein that reached the plasma membrane was greatly reduced, further diminishing its surface density at steady state. Last, the specific metal transport activity of plasmalemmal Nramp2G185R was found to be significantly depressed, compared with its WT counterpart. Thus, a singlepoint mutation results in multiple biosynthetic and functional defects that combine to produce the impaired iron deficiency that results in microcytic anemia.

Iron regulatory proteins (IRP1 and IRP2) are RNA-binding proteins that affect the translation and stabilization of specific mRNAs by binding to stem-loop structures known as iron responsive elements (IREs). IREs are found in the 5′-untranslated region (UTR) of ferritin (Ft) and mitochondrial aconitase (m-Aco) mRNAs, and in the 3′-UTR of transferrin receptor (TfR) and divalent metal transporter-1 (DMT1) mRNAs. Our previous studies show that besides iron, IRPs are regulated by hypoxia. Here we describe the consequences of IRP regulation and show that iron homeostasis is regulated in 2 phases during hypoxia: an early phase where IRP1 RNA-binding activity decreases and iron uptake and Ft synthesis increase, and a late phase where IRP2 RNA-binding activity increases and iron uptake and Ft synthesis decrease. The increase in iron uptake is independent of DMT1 and TfR, suggesting an unknown transporter. Unlike Ft, m-Aco is not regulated during hypoxia. During the late phase of hypoxia, IRP2 RNA-binding activity increases, becoming the dominant regulator responsible for decreasing Ft synthesis. During reoxygenation (ReO2), Ft protein increases concomitant with a decrease in IRP2 RNA-binding activity. The data suggest that the differential regulation of IRPs during hypoxia may be important for cellular adaptation to low oxygen tension.

Mutations at natural resistance-associated macrophage protein 1(Nramp1) impair phagocyte function and cause susceptibility to infections while mutations at Nramp2 (divalent metal transporter 1 [DMT1]) affect iron homeostasis and cause severe microcytic anemia. Structure-function relationships in the Nramp superfamily were studied by mutagenesis, followed by functional characterization in yeast and in mammalian cells. These studies identify 3 negatively charged and highly conserved residues in transmembrane domains (TM) 1, 4, and 7 as essential for cation transport by Nramp2/DMT1. The introduction of a charged residue (Gly185Arg) in TM4 found in the naturally occurring microcytic anemiamk (mouse) and Belgrade (rat) mutants is shown to cause a partial or complete loss of function in mammalian and yeast cells, respectively. A pair of mutation-sensitive and highly conserved histidines (His267, His272) was identified in TM6. Surprisingly, inactive His267 and His272 mutants could be rescued by lowering the pH of the transport assay. This indicates that His267/His272 are not directly involved in metal binding but, rather, play an important role in pH regulation of metal transport by Nramp proteins.

Iron is an essential nutrient required for the function of all cells, most notably for the production of hemoglobin in red blood cells. Defects in the mechanisms of iron absorption, storage, or utilization can lead to disorders of iron-limited erythropoiesis or iron overload. In an effort to further understand these processes, we have used the zebrafish as a genetic system to study vertebrate iron metabolism. Here we characterized the phenotype of chardonnay (cdy), a zebrafish mutant with hypochromic, microcytic anemia, and positioned the mutant gene on linkage group 11. The cdy gene was isolated by a functional genomics approach in which we used a combination of expression studies, sequence analyses, and radiation hybrid panel mapping. We identified erythroid-specific genes using a whole embryo mRNA in situ hybridization screen and placed these genes on the zebrafish genomic map. One of these genes encoded the iron transporter divalent metal transporter 1 (DMT1) and colocalized with the cdy gene. We identified a nonsense mutation in the cdy allele and demonstrated that, whereas wild-type zebrafish DMT1 protein can transport iron, the truncated protein expressed in cdy mutants is not functional. Our studies further demonstrate the conservation of iron metabolism in vertebrates and suggest the existence of an alternative pathway of intestinal and red blood cell iron uptake.

Divalent metal transporter 1 (DMT1) is the major transferrin-independent iron uptake system at the apical pole of intestinal cells, but it may also transport iron across the membrane of acidified endosomes in peripheral tissues. Iron transport and expression of the 2 isoforms of DMT1 was studied in erythroid cells that consume large quantities of iron for biosynthesis of hemoglobin. In mk/mk mice that express a loss-of-function mutant variant of DMT1, reticulocytes have a decreased cellular iron uptake and iron incorporation into heme. Interestingly, iron release from transferrin inside the endosome is normal in mk/mkreticulocytes, suggesting a subsequent defect in Fe++ transport across the endosomal membrane. Studies by immunoblotting using membrane fractions from peripheral blood or spleen from normal mice where reticulocytosis was induced by erythropoietin (EPO) or phenylhydrazine (PHZ) treatment suggest that DMT1 is coexpressed with transferrin receptor (TfR) in erythroid cells. Coexpression of DMT1 and TfR in reticulocytes was also detected by double immunofluorescence and confocal microscopy. Experiments with isoform-specific anti-DMT1 antiserum strongly suggest that it is the non–iron-response element containing isoform II of DMT1 that is predominantly expressed by the erythroid cells. As opposed to wild-type reticulocytes, mk/mk reticulocytes express little if any DMT1, despite robust expression of TfR, suggesting a possible effect of the mutation on stability and targeting of DMT1 isoform II in these cells. Together, these results provide further evidence that DMT1 plays a central role in iron acquisition via the transferrin cycle in erythroid cells.

In iron overload conditions such as thalassemia major and hereditary hemochromatosis, the iron-carrying capacity of plasma transferrin is exceeded, giving rise to non-transferrin-bound iron (NTBI). NTBI is taken up preferentially by the liver, and to a lesser extent, the kidney, pancreas, and heart. How NTBI is taken up by various tissues has been elusive. We recently demonstrated that the plasma membrane metal-ion transporter SLC39A14 (ZIP14) mediates NTBI uptake and iron loading of the liver and pancreas, but not the kidney, heart or most other tissues¹ Given that the heart is particularly susceptible to iron-related toxicity, we are currently investigating the contribution of other iron transporters to iron loading of this organ. Possible alternative cardiac iron importers include L-type and T-type calcium channels, divalent metal transporter 1 (DMT1), and SLC39A8 (ZIP8). To examine the role of DMT1 and ZIP8 in cardiac iron metabolism, we generated mice with cardiomyocyte-specific disruption of DMT1 (Dmt1heart/heart) or ZIP8 (Zip8heart/heart). The mice were then crossed with hemojuvelin knockout (Hjv-/-) mice, a model of juvenile hemochromatosis characterized by high circulating levels of NTBI. Dmt1heart/heart mice were found to have cardiac non-heme iron concentrations that were 30% lower (P Reference:Jenkitkasemwong S, Wang C, Coffey R, et al. SLC39A14 is required for the development of hepatocellular iron overload in murine models of hereditary hemochromatosis.Cell Metabolism.2015; 22(1):138-150. Disclosures No relevant conflicts of interest to declare.

Genetic studies in animal models of microcytic anemia and biochemical studies of transport have implicated the Nramp2gene in iron transport. Nramp2 generates two alternatively spliced mRNAs that differ at their 3′ untranslated region by the presence or absence of an iron-response element (IRE) and that encode two proteins with distinct carboxy termini. Antisera raised against Nramp2 fusion proteins containing either the carboxy or amino termini of Nramp2 and that can help distinguish between the two Nramp2 protein isoforms (IRE: isoform I; non-IRE: isoform II) were generated. These antibodies were used to identify the cellular and subcellular localization of Nramp2 in normal tissues and to study possible regulation by dietary iron deprivation. Immunoblotting experiments with membrane fractions from intact organs show that Nramp2 is expressed at low levels throughout the small intestine and to a higher extent in kidney. Dietary iron starvation results in a dramatic upregulation of the Nramp2 isoform I in the proximal portion of the duodenum only, whereas expression in the rest of the small intestine and in kidney remains largely unchanged in response to the lack of dietary iron. In proximal duodenum, immunostaining studies of tissue sections show that Nramp2 protein expression is abundant under iron deplete condition and limited to the villi and is absent in the crypts. In the villi, staining is limited to the columnar absorptive epithelium of the mucosa (enterocytes), with no expression in mucus-secreting goblet cells or in the lamina propria. Nramp2 expression is strongest in the apical two thirds of the villi and is very intense at the brush border of the apical pole of the enterocytes, whereas the basolateral membrane of these cells is negative for Nramp2. These results strongly suggest that Nramp2 is indeed responsible for transferrin-independent iron uptake in the duodenum. These findings are discussed in the context of overall mechanisms of iron acquisition by the body.

Introduction: In a previous study, we demonstrated that dietary iron uptake and mobilization of stored iron are both up-regulated through suppression of serum hepcidin levels during erythropoietic stimulation by administration of Epoetin beta pegol (C.E.R.A.), a long-acting erythropoiesis-stimulating agent. It was also demonstrated that up-regulation of ferroportin (FPN) in reticuloendothelial macrophages and up-regulation of divalent metal transporter 1 (DMT1) and FPN in enterocytes are followed by hepcidin suppression; however, the quantitative contribution of dietary iron for erythropoiesis were undetermined. In this study, we investigated how utilization of dietary iron for erythropoiesis is regulated under erythropoietic stimulation by C.E.R.A. in mice with different body iron status. To quantitatively estimate utilization of dietary iron for hemoglobin synthesis, we used a dietary iron tracing method using the stable iron isotope 57Fe. Methods: To assess dietary iron-derived hemoglobin synthesis, a diet containing 200 ppm of 57Fe instead of natural iron (57Fe-diet) was used. A diet containing 200 ppm of natural iron (native Fe diet) was used as a control. C57BL/6NCrl mice were fed the native Fe diet and were intravenously administered 0.5 or 1.0 mg/mouse of iron dextran (iron-loaded condition) or dextran (control). Five days after iron loading, the diet was switched to the 57Fe-diet immediately after intravenous injection of 10 µg/kg of C.E.R.A. or vehicle. On Day 5 and 8 after C.E.R.A. treatment, mice were euthanized by exsanguination under anesthesia with isoflurane, and hemoglobin levels were measured. Expression levels of DMT1 and FPN in control and iron-loaded mice (1.0 mg/mouse) on Day 5 were estimated by immunohistochemistry. Serum hepcidin levels on Day 5 were also measured by liquid column chromatography-tandem mass spectrometry (LC-MS/MS). To quantify dietary iron-derived hemoglobin synthesis, the content of hemoglobin containing 57Fe (57Fe-hemoglobin) was measured on Day 8 by inductively coupled plasma mass spectrometry (ICP-MS). Results: Hemoglobin levels on Day 8 were significantly higher in the C.E.R.A.-treated groups than in the vehicle-treated groups for each iron conditions. In the C.E.R.A.-treated groups, although iron loading did not affect hemoglobin levels, 57Fe-hemoglobin levels were significantly decreased with iron loading. The serum hepcidin levels were significantly suppressed in each of the C.E.R.A.-treated groups. However, iron loading increased serum hepcidin levels on Day 5 in both the vehicle- and C.E.R.A.-treated groups. The expression levels of hepatic and splenic iron exporter FPN were not significantly changed by iron loading in the C.E.R.A.-treated group. In contrast, the expression levels of intestinal iron transporters DMT1 and FPN were significantly reduced by iron loading in the C.E.R.A.-treated group. Conclusion: Iron loading reduced utilization of dietary iron for hemoglobin synthesis under erythropoietic stimulation by C.E.R.A. treatment. However, iron loading did not affect total hemoglobin levels, indicating that the contribution of dietary iron and stored iron for erythropoiesis is properly controlled in response to body iron status. This was attributed to the tissue-specific regulatory mechanisms of iron transporters in iron absorptive tissue (intestine) and iron storage tissue (liver and spleen) in response to iron loading even FPN on both tissues is known to be commonly down-regulated by hepcidin-binding. Sensitive inactivation of iron importers and exporters in the duodenum under conditions of iron loading may effectively contribute to iron not being excessively incorporated under erythropoietic stimulation. Disclosures Noguchi-Sasaki: Chugai Pharmaceutical Co., Ltd.: Employment. Kurasawa:Chugai Pharmaceutical Co., Ltd.: Employment. Yorozu:Chugai Pharmaceutical Co., Ltd.: Employment. Shimonaka:Chugai Pharmaceutical Co., Ltd.: Employment.

Iron overload is highly prevalent, but its molecular pathogenesis is poorly understood. Recently, DMT1 was shown to be a major apical iron transporter in absorptive cells of the duodenum. In vivo, it is the only transporter known to be important for the uptake of dietary non-heme iron from the gut lumen. The expression and subcellular localization of DMT1 protein in 3 mouse models of iron overload were examined: hypotransferrinemic (Trfhpx) mice, Hfeknockout mice, and B2m knockout mice. Interestingly, in Trfhpx homozygotes, DMT1 expression was strongly induced in the villus brush border when compared to control animals. This suggests that DMT1 expression is increased in response to iron deficiency in the erythron, even in the setting of systemic iron overload. In contrast, no increase was seen in DMT1 expression in animals with iron overload resembling human hemochromatosis. Therefore, it does not appear that changes in DMT1 levels are primarily responsible for iron loading in hemochromatosis.

The divalent metal ion transporter DMT1 is critical for nonheme iron import. We have previously shown that DMT1 is regulated in vitro by ubiquitination that is facilitated by the adaptor proteins Ndfip1 and Ndfip2. Here we report that in Ndfip1−/− mice fed a low- iron diet, DMT1 expression and activity in duodenal enterocytes are significant higher than in the wild-type animals. This correlates with an increase in serum iron levels and transferrin saturation. Liver and spleen iron stores were also increased in Ndfip1−/− mice fed a normal diet. Counterintuitive to the increase in iron uptake, Ndfip1−/− mice fed a low iron diet develop severe microcytic, hypochromic anemia. We demonstrate that this is due to a combination of iron deficiency and inflammatory disease in Ndfip1−/− mice, because Ndfip1−/−/Rag1−/− immunodeficient mice fed a low iron diet did not develop anemia and showed an iron overload phenotype. These data demonstrate that Ndfip1 is a critical mediator of DMT1 regulation in vivo, particularly under iron restricted conditions.

To the editor: Ludwiczek et al[1][1] reported that nifedipine and other dihydropyridine-class calcium-channel blockers can reverse iron overload by stimulating the activity of divalent metal-ion transporter-1 (DMT1)[2][2],[3][3] and consequently mobilizing tissue iron. Since their paper disagreed

Several distinct congenital disorders can lead to tissue-iron overload with anemia including β-thalassemia and sickle cell disease. We show that intestinal absorption of iron is highly increased and significantly contributes to tissue iron accumulation in these disorders. The present work describes a novel pathway by which oxygen sensing transcription factors are highly upregulated in iron overload anemias and are subsequently essential for the increase intestinal iron absorption. Oxygen signaling is mediated through well-conserved hypoxia driven transcription factors, hypoxia-inducible factor (HIF)1a and HIF2a. In the intestine, HIF2a directly activates divalent metal transporter 1 (DMT1), duodenal ferric reductase (DcytB), and Fpn1, which are iron transporters critical for adaptive changes in iron absorption. We demonstrate that HIF2a and its downstream target gene, DMT1 are essential for iron accumulation in mouse models of β-thalassemia and sickle cell disease. Furthermore, studies of thalassemic mouse model with established iron overload demonstrated that loss of intestinal HIF2a and DMT1 signaling led to decreased tissue iron accumulation in the livers. Interestingly, disrupting intestinal HIF2a not only improves tissue iron accumulation, but a marked improvement of anemia was also observed. These novel findings suggests that inhibition of HIF2a signaling pathway could be a novel and robust treatment strategy for several conditions that cause iron overload with anemia. Disclosures No relevant conflicts of interest to declare.

We have previously described a case of severe hypochromic microcytic anemia caused by a homozygous mutation in the divalent metal transporter 1 (DMT1 1285G > C). This mutation encodes for an amino acid substitution (E399D) and causes preferential skipping of exon 12 during processing of the DMT1 mRNA. To examine the functional consequences of this mutation, full-length DMT1 transcript with the patient's point mutation or a DMT1 transcript with exon 12 deleted was expressed in Chinese hamster ovary (CHO) cells. Our results demonstrate that the E399D substitution has no effect on protein expression and function. In contrast, deletion of exon 12 led to a decreased expression of the protein and disruption of its subcellular localization and iron uptake activity. We hypothesize that the residual protein in hematopoietic cells represents the functional E399D DMT1 variant, but because of its quantitative reduction, the iron uptake activity of DMT1 in the patient's erythroid cells is severely suppressed.

Evidence is accumulating that hepcidin, a liver regulatory peptide, could be the common pathogenetic denominator of all forms of iron overload syndromes including HFE-related hemochromatosis, the most prevalent genetic disorder characterized by inappropriate iron absorption. To understand the mechanisms whereby hepcidin controls iron homeostasis in vivo, we have analyzed the level of iron-related proteins by Western blot and immunohistochemistry in hepcidin-deficient mice, a mouse model of severe hemochromatosis. These mice showed important increased levels of duodenal cytochrome b (Dcytb), divalent metal transporter 1 (DMT1), and ferroportin compared with control mice. Interestingly, the level of ferroportin was coordinately up-regulated in the duodenum, the spleen, and the liver (predominantly in the Kupffer cells). Finally, we also evidenced a decrease of ceruloplasmin in the liver of hepcidin-deficient mice. We hypothesized that the deregulation of these proteins might be central in the pathogenesis of iron overload, providing key therapeutic targets for iron disorders. (Blood. 2005;105:4861-4864)

We investigated the effects of the iron regulatory peptide hepcidin on iron transport by the human intestinal epithelial Caco-2 cell line. Caco-2 cells were exposed to hepcidin for 24 hours prior to the measurement of both iron transport and transporter protein and mRNA expression. Incubation with hepcidin significantly decreased apical iron uptake by Caco-2 cells. This was accompanied by a decrease in both the protein and the mRNA expression of the iron-response element containing variant of the divalent metal transporter (DMT1[+IRE]). In contrast, iron efflux and iron-regulated gene1 (IREG1) expression were unaffected by hepcidin. Hepcidin interacts directly with a model intestinal epithelium. The primary effect of this regulatory peptide is to modulate the apical membrane uptake machinery, thereby controlling the amount of iron absorbed from the diet. (Blood. 2004;104:2178-2180)

Hereditary hemochromatosis (HH) is an iron-overload disorder caused by a C282Y mutation in the HFE gene. In HH, plasma nontransferrin-bound iron (NTBI) levels are increased and NTBI is bound mainly by citrate. The aim of this study was to examine the importance of NTBI in the pathogenesis of hepatic iron loading in Hfe knockout mice. Plasma NTBI levels were increased 2.5-fold in Hfe knockout mice compared with control mice. Total ferric citrate uptake by hepatocytes isolated from Hfe knockout mice (34.1 ± 2.8 pmol Fe/mg protein/min) increased by 2-fold compared with control mice (17.8 ± 2.7 pmol Fe/mg protein/min; P < .001; mean ± SEM; n = 7). Ferrous ion chelators, bathophenanthroline disulfonate, and 2′,2-bipyridine inhibited ferric citrate uptake by hepatocytes from both mouse types. Divalent metal ions inhibited ferric citrate uptake by hepatocytes, as did diferric transferrin. Divalent metal transporter 1 (DMT1) mRNA and protein expression was increased approximately 2-fold by hepatocytes from Hfe knockout mice. We conclude that NTBI uptake by hepatocytes from Hfe knockout mice contributed to hepatic iron loading. Ferric ion was reduced to ferrous ion and taken up by hepatocytes by a pathway shared with diferric transferrin. Inhibition of uptake by divalent metals and up-regulation of DMT1 expression suggested that NTBI uptake was mediated by DMT1.

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 iron-induced 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 up-regulated, 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 macrophage-associated 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)

Hephaestin is a membrane-bound multicopper ferroxidase necessary for iron egress from intestinal enterocytes into the circulation. Mice with sex-linked anemia (sla) have a mutant form of Hephaestin and a defect in intestinal basolateral iron transport, which results in iron deficiency and anemia. Ireg1 (SLC11A3, also known as Ferroportin1 or Mtp1) is the putative intestinal basolateral iron transporter. We compared iron levels and expression of genes involved in iron uptake and storage in sla mice and C57BL/6J mice fed iron-deficient, iron-overload, or control diets. Both iron-deficient wild-type mice and sla mice showed increased expression of Heph and Ireg1 mRNA, compared to controls, whereas only iron-deficient wild-type mice had increased expression of the brush border transporter Dmt1. Unlike iron-deficient mice, sla mouse enterocytes accumulated nonheme iron and ferritin. These results indicate that Dmt1 can be modulated by the enterocyte iron level, whereas Hephaestin and Ireg1 expression respond to systemic rather than local signals of iron status. Thus, the basolateral transport step appears to be the primary site at which the small intestine responds to alterations in body iron requirements.

Nramp2 (DMT1) is a pH-dependent divalent cation transporter that acts as the transferrin-independent iron uptake system at the intestinal brush border and also transports iron released from transferrin across the membrane of acidified endosomes. In this study, RAW264.7 macrophages and 2 independently derived murine Sertoli cells lines, TM4 and 15P-1, were used to further study the subcellular localization of Nramp2/DMT1 in phagocytic cells, including possible recruitment to the phagosomal membrane. Nramp2/DMT1 was localized primarily to the EEA1-positive recycling endosome compartment, with some overlapping staining with Lamp1-positive late endosomes. After phagocytosis, immunofluorescence analysis and in vitro biochemical studies using purified latex bead-containing phagosomes indicated Nramp2/DMT1 recruitment to the membrane of Lamp1, cathepsin D, and rab7-positive phagosomes. Nramp2/DMT1 was also found associated with erythrocyte-containing phagosomes in RAW macrophages and with the periphery of sperm-containing phagosomes in Sertoli cells. These results suggest that, as for the macrophage-specific Nramp1 protein, Nramp2/DMT1 may transport divalent metals from the phagosomal space.

In a recent Blood article, Mims and colleagues[1][1] reported the phenotype of a patient with anemia and iron overload who was homozygous for a novel mutation in the iron transporter SLC11A2 (DMT1). SLC11A2 (solute carrier family 11, member 2) is the only known transporter involved in cellular iron

Comment on Mims et al, page [1337][1] A new human genetic model of hypochromic microcytic anemia is due to DMT1 mutations. At variance with rodent models, the first identified patient is also iron loaded. Mims and colleagues have identified the first human example of the iron transporter

Abstract 3178 Delivery of iron (Fe) to most cells occurs following the binding of diferric transferrin (Tf) to its cognate receptors on the cell membrane. The Tf-receptor complexes are then internalized via endocytosis, and iron is released from Tf by a process involving endosomal acidification and reduction by Steap3. Iron is then transported across the endosomal membrane by the divalent metal transporter, DMT1. Unfortunately, the post-endosomal path of iron within cells remains elusive or is, at best, controversial. It has been commonly accepted that a low molecular weight intermediate chaperones iron in transit from endosomes to mitochondria and other sites of utilization; however, this much sought iron binding intermediate has never been identified. In erythroid cells, more than 90% of iron has to enter mitochondria where ferrochelatase, the final enzyme in the heme biosynthetic pathway that inserts Fe2+ into protoporphyrin IX, resides. Indeed, strong evidence exists for specific targeting of Fe toward mitochondria in developing red blood cells in which iron acquired from Tf continues to flow into mitochondria even when the synthesis of protoporphyrin IX is suppressed. Based on this, we have formulated the hypothesis that, in erythroid cells, a transient mitochondrion-endosome interaction is involved in Fe translocation to its final destination and have collected experimental support for this proposition (Zhang et al. Blood 105:368, 2005; Sheftel et al. Blood 110: 125, 2007). We have previously shown, using 3D live confocal imaging, that the iron delivery pathway in reticulocytes involves a transient interaction of endosomes with mitochondria. Moreover, we have demonstrated the interaction of these organelles by a novel method exploiting flow cytometry to analyze reticulocyte lysates labeled with Alexa Green Transferrin (AGTf) and MitoTracker Deep Red (MTDR). By using this new technique of flow subcytometry, we identified a double-labeled population representing endosomes interacting with mitochondria. The dynamic nature of this interaction was shown by chase experiments in which a time-dependent decrease of the double-labeled population was observed when reticulocytes were washed and re-incubated with unlabeled Fe2-Tf. Furthermore, we have shown that the iron status of endosomes governs the efficacy of endosome-mediated iron delivery to mitochondria. Experiments with heme, which feedback inhibited the release of iron from Tf in endosomes, slows the generation of the double-labeled population. Additionally, treatment of cells with heme in chase experiments retarded the dissociation of endosomes from mitochondria. In this study, we provide further evidence for an interaction between mitochondria with endosomes. Fluorescently-labeled mitochondria isolated from mouse reticulocytes with MTDR and AGTf were analyzed using 2D confocal microscopy. Results from these experiments confirmed that mitochondria indeed come in physical contact with endosomes. In addition, we have used different constructs of fluorescently labeled, recombinant human Tf, which either remain permanently bound to iron (recombinant diferric-transferrin; rTf) or cannot bind to iron (recombinant apotransferrin; rapoTf), in flow subcytometry studies. As expected, these studies showed that reticulocytes incubated with MTDR and rapoTf failed to produce a double-labeled population in uptake experiments. Interestingly, when reticulocyte lysates were incubated with MTDR and rTf, compared to controls using wild type human Tf, the size of the double-labeled population was decreased. This suggests that failure of iron release from rTf may interfere with the process of endocytosis or endosomal trafficking. Disclosures: No relevant conflicts of interest to declare.

Abstract 1994 Poster Board I-1016 Divalent metal transporter 1 (DMT1) and natural resistance-associated macrophage protein 1 (Nramp1) are iron transporters that localize, respectively, to the early and late endosomal compartments. DMT1 is ubiquitously expressed, while Nramp1 is found only within macrophages and neutrophils. Our previous studies have identified a role for Nramp1 during macrophage erythrophagocytosis; however, little is known about the function of DMT1 during this process. Wild-type RAW264.7 macrophages (Nramp1-/-), and those stably transfected with Nramp1 (Nramp1+/+) were treated with either DMT1-siRNA, or with ebselen, a selective inhibitor of DMT1. While macrophages lacking either functional DMT1 or Nramp1 experienced a moderate reduction in iron recycling efficiency, the ability of macrophages lacking both functional DMT1 and Nramp1 to recycle hemoglobin-derived iron was severely compromised. Compared to macrophages singly deficient in either DMT1 or Nramp1 transport ability, macrophages where DMT1 and Nramp1 were both compromised exhibited an abrogated increase in labile iron pool content, released less iron, and experienced diminished upregulation of ferroportin and heme-oxygenase 1 levels following erythrophagocytosis. These results suggest that while the loss of either Nramp1 or DMT1 transport ability results in minor impairment following erythrophagocytosis, the simultaneous loss of both Nramp1 and DMT1 iron transport activity is detrimental to the iron recycling capacity of the macrophage. Disclosures: No relevant conflicts of interest to declare.

An exquisite relationship between iron and heme in hemoglobin-synthesizing cells makes blood red. Erythroid cells are the most avid consumers of iron (Fe) in the organism and synthesize heme at a breakneck speed. Additionally, there is virtually no free Fe or heme detectable during hemoglobin (Hb) synthesis. Developing red blood cells (RBC) can take up Fe only from the plasma glycoprotein transferrin (Tf). Delivery of iron to these cells occurs following the binding of Tf to its cognate receptors on the cell membrane. The Tf-receptor complexes are then internalized via endocytosis, and iron is released from Tf by a process involving endosomal acidification. Iron, following its reduction to Fe2+ by Steap3, is then transported across the endosomal membrane by the divalent metal transporter, DMT1. However, the post-endosomal path of Fe in the developing RBC remains elusive or is, at best, controversial. It has been commonly accepted that a low molecular weight intermediate chaperones Fe in transit from endosomes to mitochondria and other sites of utilization; however, this much sought iron-binding intermediate has never been identified. In erythroid cells, more than 90% of iron must enter mitochondria since ferrochelatase, the final enzyme in the heme biosynthetic pathway that inserts Fe2+ into protoporphyrin IX, resides in the inner part of the inner mitochondrial membrane. In fact, in erythroid cells, strong evidence does exist for specific targeting of Fe toward mitochondria. This targeting is demonstrated in Hb-synthesizing cells in which Fe acquired from Tf continues to flow into mitochondria, even when the synthesis of protoporphyrin IX is suppressed. Based on this, we have formulated a hypothesis that in erythroid cells a transient mitochondrion-endosome interaction is involved in iron translocation to its final destination. Recently, we have collected strong experimental evidence supporting this hypothesis: we have shown that Fe, delivered to mitochondria via the Tf pathway, is unavailable to cytoplasmic chelators. Moreover, we have demonstrated that Tf-containing endosomes move and contact mitochondria in erythroid cells, that vesicular movement is required for iron delivery to mitochondria, and that “free” cytoplasmic Fe is not efficiently used for heme biosynthesis. As mentioned above, the substrate for the endosomal transporter DMT1 is Fe2+, the redox form of iron that is also the substrate for ferrochelatase. These facts make the above hypothesis quite attractive, since the “chaperone”-like function of endosomes may be one of the mechanisms that keeps the concentrations of reactive Fe2+ at extremely low levels in oxygen-rich cytosol of erythroblasts, preventing ferrous ion’s participation in a dangerous Fenton reaction. In conclusion, the delivery of iron into Hb occurs extremely efficiently, since mature erythrocytes contain about 45,000-fold more heme iron (20 mM) than non-heme iron (440 nM). These facts, together with experimental data that will be discussed, indicate that the iron transport machinery in erythroid cells is an integral part of the heme biosynthetic pathway.

Iron homeostasis in mammals is maintained at the level of iron absorption by the gut. Hepcidin plays a central role in homeostasis by binding to ferroportin and regulating cellular iron export. We found that mice weaned onto diets ranging from 35–350 mg Fe/kg for a period of 4 wk did not change body iron levels as measured by organ iron content and hematological parameters. Direct measure of absorption of 59Fe administered by gavage revealed an inverse correlation between dietary iron content and absorption. Gavage experiments were done following a 4h fast when the stomach and proximal small bowel were free of dietary content. Although iron absorption changed, liver expression of hepcidin mRNA did not. We measured the absorptive response in mice weaned onto diets containing 35 mg Fe/kg for 4 wk and abruptly changed to 350 mg Fe/kg. There was no change in iron absorption at day 1 but by day 3 absorption was reduced nearly 3-fold compared to controls and remained at this level for at least 7d. During this time neither liver nor spleen iron content changed but transferrin saturation increased approximately 1.5-fold. Most importantly, serum hepcidin levels, measured by a competitive binding assay (De Domenico et al. Cell Metab.2008, 8:146–156), were unchanged. Mice were then changed from diets containing 350 mg Fe/kg to diets containing 35 mg Fe/kg. Within 24h mice increased absorption of 59Fe 3-fold. Elevated absorption continued for at least 3d, declined by 7d and at 14d was at a level found in mice maintained on a diet containing 35 mg Fe/kg. During this period there was no change in organ iron content or in transferrin saturation. Serum hepcidin did not change on day 1, but was reduced by approximately 40% on days 3 through 7. Increased iron absorption could be attributed in part to increased expression of Dmt1 but no change in ferroportin message was detected. Enterocyte ferritin levels doubled on day 1 but returned to control levels on days 3 through 7. Finally, mice with a targeted disruption of the hepcidin gene were challenged with an abrupt reduction in dietary iron content and absorption increased approximately 3-fold. These data suggest that iron absorption can respond to changes in dietary iron content independent of hepcidin and that response to changes in luminal iron content are at the level of uptake and storage in a manner intrinsic to the enterocyte.

[Introduction and aim] Iron chelation therapy has been applied for iron overload. Desferrioxamine (DFO) must have been the most used iron chelator in the world. DFO has been thought to chelate iron from the liver and reticuloendotherial systems in that iron is accumulated, and accelerate iron excretion from the body. On the other hand, many new molecules involved in iron metabolism have been discovered in these 10 years, and the understanding of the molecular mechanisms of iron metabolism has progressed. However, the changes of the genes involved in iron metabolism in iron chelating therapy have not been fully investigated. Therefore, the aim of this study is to clarify the responses of the body to the iron chelation therapy and the mechanisms of iron removal by iron chelator DFO. We investigated the changes of genes involved in iron metabolism in the important organs, such as the liver and the gastrointestinal tract, at the early stage of iron chelation therapy by treating the iron overloaded mouse with DFO. [Methods] Iron overloaded mouse model was made by giving iron dextran interperitonealy (the iron-loaded group). DFO was given for the iron overloaded mice regularly by the interperitoneal (The iron-loaded + DFO treated group). The iron chelation by DFO had continued for 4 weeks. Mice without any treatment were participated as the control. After 4 weeks, mice were sacrificed, and the liver, small intestine, heart, and blood were collected from all mice. Total RNA was then purified, and quantitative RT-PCR (qRT-PCR) was performed for genes involved in iron metabolism. The genes investigated in this study were HFE, transferrin receptor 1 (TfR1), transferrin receptor 2 (TfR2), hepcidin-1 (HAMP1), hepcidin-2 (HAMP2), ferroportin 1 (FPN1), divalent metal transporter 1 (DMT1), duodenal cytochrome b (Dcytb), hephasetin and ferritin. Protein isolated from collected organ was applied for western blot. [Results] There was no significant difference in the physique, the weight of the liver and spleen, hemoglobin, serum iron concentration among all groups of mice. The serum ferritin was remarkably increased in iron-loaded group, and there was no decline of serum ferritin in the iron-loaded + DFO treated group. Although the expressions of HFE, TfR2, HAMP1, HAMP2 in the liver didn’t show significant difference among each of the groups by qRT-PCR, the ferritin mRNA and FPN1 mRNA were decreased significantly in the iron-loaded + DFO treated group compared to the iron-loaded group. In the duodenum, there was no difference in the expressions of DMT1, Dcytb, hephasetin, but the expressions of ferritin mRNA and FPN1 mRNA were significantly decreased in the iron-loaded + DFO treated group. [Discussion and conclusions] In addition to removal of iron from the organ that iron is accumulated, our data showed that iron chelation therapy had the effect on the expressions of the genes involved in iron metabolism. The decreases of ferritin mRNA at both of the liver and the duodenum might imply that intracellular free iron was chelated by DFO. Concerning the decline of FPN1 mRNA, there might be a possibility that intracellular iron depletion by DFO cause the inhibition of translation of FPN1 mRNA via iron responsive element which exists in 5’-untranslated region of FPN1 mRNA. FPN1 in duodenum functions for iron transfer from the enterocyte to blood, so that the decrease of body iron likely increase the expression of FPN1 in the duodenum because body would like to increase iron uptake from duodenum. However, our result indicated that iron chelation therapy did not upregulate FPN1 mRNA expression. Our data rather showed that FPN1 is downregurated, and this indicated that iron chelation would lead to decreased iron uptake from the duodenum, and this would be preferable effect of DFO in the view of iron chelation therapy for iron overload.

Natural resistance-associated macrophage protein 1 (Nramp1) is a divalent metal transporter expressed exclusively in phagocytic cells such as macrophages and neutrophils. Based on our earlier in vitro study (Soe-Lin et al. Exp Hematol.2008;36:929–937), we hypothesize that Nramp1 may participate in the recycling of iron acquired through the phagocytosis of senescent red blood cells by macrophages. In order to examine the effect of Nramp1 on iron recycling in vivo, the iron parameters of wildtype (Nramp1+/+) and Nramp1 knockout mice (Nramp1−/−) were analyzed following both acute and chronic induction of hemolytic anemia by phenylhydrazine treatment. We observe that untreated Nramp1−/− mice exhibited greater serum transferrin saturation and splenic iron content, higher duodenal ferroportin (Fpn) and divalent metal transporter 1 (DMT1) expression, and dramatically lower hepcidin mRNA levels than untreated Nramp1+/+ mice. Significant iron loading of the reticuloendothelial organs was found to increase with age in knockout mice. Following acute treatment with the hemolytic agent phenylhydrazine, Nramp1−/− mice experienced a significant decrease in serum iron levels and hematocrit, while their Nramp1+/+ counterparts were relatively unaffected. Following a month-long phenylhydrazine regimen, Nramp1−/− mice retained markedly increased quantities of iron within the liver and spleen, and exhibited greater splenomegaly and reticulocytosis than wild-type mice. Furthermore, while hepcidin mRNA levels decreased following chronic phenylhydrazine treatment in both Nramp1+/+ and Nramp1−/− mice, this effect was significantly more pronounced in Nramp1−/− mice. The data presented in this report suggest that in the absence of Nramp1, iron accumulates to a greater degree within reticuloendothelial organs such as the liver and spleen following acute and chronic hemolytic anemia. We hypothesize that the low hepcidin mRNA levels seen in Nramp1−/− mice are a response to a diminished availability of iron for erythropoiesis resulting from the aberrant increase in iron retention within their splenic reticuloendothelial macrophages. Our observation of increased DMT1 and ferroportin within the duodenums of the Nramp1−/− animals imply that the increase in transferrin saturation despite the impaired iron release from erythrophagocytosing macrophages occurs due to a compensatory increase in iron absorption from the diet. These findings are consistent with our hypothesis that Nramp1 promotes the efficient recycling of iron in erythrophagocytosing macrophages.

While iron is essential for cell growth and survival excess iron through oxidative stress may produce hepatitic cirrhosis and hepatocellular carcinoma, diabetes mellitus, and cardiomyopathy. Iron is absorbed across the duodenum with transport across the brush border mediated by DMT1 and across the basolateral surface by ferroportin with mechanisms that are inversely regulated by body iron concentrations. We have identified in rat intestine DAP, a novel protein that binds to the C-terminus of DMT1 (IRE) but not to the C-terminus of the non-IRE isoform (Blood, Nov 2004; 104: 53). DAP is a 526 amino acid protein that has been previously described as binding to the peripheral benzodiazepine receptor, an intrinsic mitochondrial protein involved in steriodogenesis and possibly in protoporphyrin IX transport into the mitochrondria. To investigate if DAP may have a role in regulation of intracellular iron transport DAP expression was down regulated using a vector containing a siRNA for DAP transfected into K562 cells by electroporation. Expression levels of DAP, transferrin receptor 1 (TfR1), divalent metal transporter 1 (DMT1) and ferritin were examined by western blot and quantitative quantitivative PCR assays from days 1 to 6 after transfection. Following transfection with the DAP siRNA DAP mRNA levels were decreased 50% by day 1 with DAP protein levels decreasing by 50% at day 3. The DAP siRNA also decreased DMT1 protein expression by about 50% for the DMT1 (IRE) protein but had no effect on the protein derived from the non-IRE isoform. The leels of DMT1 mRNA were not affected by DAP siRNA. The decrease of DAP expression was not associated with any change in TfR1 or ferritin expression, suggesting that altered levels of DAP did not affect intracellular iron pools. Transfection with the DAP siRNA resulted also in more protean effects decreasing cell proliferation, the transition from S-phase to G2 in cell cycle, and protein synthesis. These data are consistent with DAP regulating DMT1 expression in K562 cells by modulating turnover of DMT1 (IRE) protein and also having more global effects on cellular metabolism.

The final steps of heme biosynthesis include the transport of coproporphyrin with the transport step probably mediated by the peripheral benzodiazepine receptor (PBR). Within the mitochondria copropoprhyrin is then converted to protoporphyrin IX (PPIX) which in turn is converted to hemin with insertion of iron by ferrochelatase. PBR is ubiquitously expressed and has been implicated in steriodogenesis, apoptosis, erythroid differentiation, and inflammation. Interestingly, PPIX is among several high affinity ligands for PBR. Various cytosolic proteins that interact with PBR have also been defined including PBR associated protein 7 (PAP7). The various PBR ligands including PPIX may affect the binding of these proteins to PBR. We have demonstrated (Blood, Nov 2004; 104: 53) that DAP, a protein highly homologous to PAP7, binds to the C-terminus of DMT1 and may have a role in regulation of intracellular iron transport. We, therefore, examined the effects of PPIX on the functions of DAP and other proteins that affect cellular iron metabolism. DAP is 526 amino acid protein with a nuclear localization signal domain (aa 212–229) and a Golgi localization domain (aa 380–524), and is distributed in the cytoplasm, Golgi apparatus, and nuclei of K562 cells. K562 cells were grown in the presence of 5 μM PPIX for 24 hours and then the expression of DAP, transferrin receptor 1 (TfR1), and ferritin examined by western blot analysis. In addition, cells were grown in medium of either normal iron content (3.5 μM from ferri-transferrin), high iron content (217 μM from the addition of ferric ammonium citrate), or low iron content (by the addition of 50 mM desferroxamine). Under all three iron conditions PPIX induced differentiation but down-regulated ferritin expression and up-regulated TfR1 expression. Additionally, PPIX had a striking effect on DAP expression markedly decreasing DAP levels but only in cells grown either in normal or low iron medium. In addition, PPIX affected the expression of the iron transporter DMT1in parallel with DAP. As PPIX induced erythroid differentiation of K562 cells we examined the effects of hemin which can also induce differentiation of K562 cells. In contrast to PPIX, hemin caused strong down-regulation of TfR and up-regulation of ferritin and DAP. The down-regulation of DAP induced by PPIX was restored by the addition of hemin. These results indicate that PPIX affects DAP expression and other important elements involved in cellular iron metabolism and that these effects are partially modified by the iron status of the cell.

Dietary iron absorption by enterocytes is mediated by a ferrous transporter (DMT1) and possibly a ferric reductase (Cbyrd1). The role of Cbyrd1 is uncertain, as a knockout mouse has no defect in absorption (Gunshin et al. Blood, 2005, June 16, Epub). Transfer of iron to plasma is mediated by ferroportin (FPN). FPN’s residence on the basolateral membrane is regulated by hepcidin (Nemeth et al. Science 2004, 306:2090–3). Iron absorption responds to erythropoiesis, hypoxia and iron stores. A dietary iron content of 120 mg/kg consumed will maintain hepatic iron stores near that of mice found in the wild. Mice will grow and breed given diets containing 35 mg/kg. Commercial mouse chow iron content ranges from 200–350 mg/kg. We studied the effects of diets containing 2, 35, 120, 350 and 2000 mg iron/kg on iron absorption, liver and spleen iron content and transcriptional levels of DMT1, FPN and hepcidin in A/J mice. Mice were weaned at 3 weeks of age and groups of 8 animals were placed on one of the 5 diets for 4 weeks. No differences between groups were noted in hematocrit, hemoglobin and MCV. Mean hepatic iron content was 52.7 ±3.7 ug/g (wet wt) in mice fed the 2 mg/kg diet. Mean hepatic iron content was 560 ±23.7 ug/g in mice fed the 2000 mg/kg diet. There was no difference in hepatic iron content in mice fed intermediate iron diets (35–350 mg/kg). Mean hepatic iron concentration in these groups was 110 ±3 ug/g. Mean spleen iron content was 132 ±12.2 ug/g (wet wt) in mice maintained on 2 mg/kg. Mean spleen iron content was 598 ±49 ug/g in mice fed the 2000 mg/kg diet. There was no difference in spleen iron content in mice maintained on intermediate iron diets (mean 359 ±10 ug/g). These data indicate that mice maintain constant levels of hepatic and splenic iron over a ten-fold range in dietary iron content. Iron absorption was measured as percent of a measured dose of 59Fe (5 ug total) remaining in the carcass (minus the GI tract) 24 h after administration by gavage. Absorption was inversely proportional to dietary iron content. Mean absorption was 86% ±4 in the group on the 2 mg/kg diet, 42% ±3 on the 35 mg/kg diet, 26% ±7 on the 120 mg/kg diet, 19% ±4 on the 350 mg/kg diet and 6% ±1 on the 2000 mg/kg diet. Transcript levels of hepcidin, DMT1 and FPN were measured by real-time PCR and normalized to beta actin mRNA. Liver hepcidin expression was 20-fold greater in mice on the 2000 mg/kg diet than in mice on the 2 mg/kg diet (3900 ±1021 copies/actin copy vs. 198 ±47). Hepcidin expression did not differ in mice on intermediate diets (745 ±147 copies). Enterocytes were isolated from everted gut explants by elution in EDTA. Transcript levels of enterocyte DMT1 and FPN were 4566 ±SEM and 236 ±SEM copies respectively in mice on the 2 mg/kg diet. No detectable transcripts were found in mice on the 2000 mg/kg diet. Enterocyte transcript levels for DMT1 and FPN were no different in groups on intermediate iron diets (17 ±2 copies and 20 ±9 copies respectively). These data indicate that tissue iron content, hepcidin, DMT1 and FPN remain constant over a ten-fold range in dietary iron and only vary at extremes, while iron absorption is inversely proportional to dietary iron. The data also suggest that dietary iron, within defined limits, regulates iron absorption by a mechanism intrinsic to the enterocyte.

Divalent metal transporter 1 (DMT1) is involved in dietary iron uptake on the luminal side of duodenal enterocytes and transfers iron from the endosome to the cytosol in the marrow erythroblasts. Spontaneous (mk mice and Belgrade rats) or acquired (DMT1 -/- mice) inactivation of DMT1 in rodents produces a severe microcytic anemia at birth, caused by inefficient intestinal iron absorption and defective iron utilization in erythroid cells. The first reported patient with DMT1 mutations had microcytic anemia and iron overload in adult life. We here report the hematological phenotype of a newborn with a severe mycrocytic anemia (Hb 4 g/dL, MCV 71 fL) at birth and during the first months of life. Serum iron, transferrin saturation and serum ferritin were 160 microg/L, 100% and 846 ng/ml respectively at 3 months of age. Hepatic iron overload wad documented at the age of 5 years by both non invasive SQUID and liver biopsy. Sequence analysis of genomic DNA of the family revealed that the child was compound heterozygote for two novel DMT1 mutations, inherited by the asymptomatic parents. The first change deleted 3 bp (c.310 - 3_5del CTT) in intron 4 resulting in a splicing abnormality and the skipping of exon 5. The second was C>T 1246 substitution that causes arginine > cysteine replacement at position 416 (p. R416C) in the protein. This missense affects an highly conserved residue in one of the putative transmembrane domains. A striking reduction of the protein in peripheral blood cells of the proband was demonstrated by western blot using an anti-DMT1 antibody. The child required blood transfusions at birth and in the first two months of life. Thereafter, treatment with subcutaneous erythropoietin mantained hemoglobin levels between 7.5–9.5 g/dL, allowing transfusion-independence. The haematological phenotype of this patient highlights the essential role of DMT1 in erythropoiesis. The early and significant hepatic iron accumulation indicates that, as in animal models, DMT1 is dispensable for liver iron uptake. Finally DMT1 inactivation in the gut is likely bypassed by other pathways of iron absorption.

We have previously described a case of severe hypochromic microcytic anemia caused by a homozygous mutation in the divalent metal transporter 1 ( DMT1 1285G>C ). This mutation encodes for an amino acid substitution (E399D) and causes preferential skipping of exon 12 during processing of the DMT1 mRNA. To examine the functional consequences of this mutation, full length DMT1 transcript with the patient’s point mutation or a DMT1 transcript with exon 12 deleted was expressed in Chinese hamster ovary (CHO) cells. Our results demonstrate that the E399D substitution has no effect on protein expression and function. In contrast, deletion of exon 12 led to a decreased expression of the protein and disruption of its subcellular localization and iron uptake activity. We hypothesize that the residual protein in hematopoietic cells represents the functional E399D DMT1 variant, but because of its quantitative reduction, the iron uptake activity of DMT1 in the patient’s erythroid cells is severely suppressed. Because of the positive effect of erythropoietin (EPO) on the growth of patient’s BFU-Es in our in vitro studies, we have treated the patient with recombinant human EPO. The dose of 1.2 μg/kg given once a week did not lead to any improvement in hemoglobin level. However, doubling the dose of EPO (2.4 μg/kg) led to an increase in hemoglobin level from 75 to 91 g/L and to an improvement in the patient’s clinical condition.

The divalent metal transporter 1 (DMT1) is essential for cellular iron uptake both in the intestine and in erythroid cells. We have previously shown that with iron feeding the DMT1 expressed on the brush border membrane (BBM) of the intestine undergoes endocytosis (Am. J. Physiol. 283, G965, 2002). Using the yeast two-hybrid system we have isolated a cDNA clone encoding a protein of 526-amino acid residues with a calculated molecular mass of 60 kDa, which interacts with the C-terminus of DMT1 expressed from the IRE containing mRNA (Blood ,100, 7a, 2002). The ORF of the rat protein has been fully sequenced (Genbank #AY336075) and is now designated DMT1 associated protein (DAP). DAP is ubiquitously expressed and is especially abundant in the rat intestine and colon. In rat duodenum DAP is colocalized with DMT1 in the BBM. Both by salt and pH elution DAP was demonstrated to be a peripheral membrane protein. With iron feeding both DMT1 and DAP translocate: DAP moves from the BBM to basolateral membrane (BLM) with DMT1 and some of DMT1 undergoes endocytosis and is found in cytopasmic vesicles. Immunoprecipitation and pull-down assays confirm the interaction of DAP and DMT1 in the BBM vesicles (MMBV). We have analyzed the function of DAP by exploring the role of various consensus sequences in the DAP ORF in the cellular localization of the protein. By sequence motif analysis DAP has a nuclear localization signal, Glutamic acid-rich region, Glutamine-rich region, Arginine-rich region, PKC phosphorylation sites and GOLD domain (Golgi dynamics). The region of DAP protein interacting with the COOH-terminal cytoplasmic domain of DMT1(IRE) was found to be from 171aa to 331aa which contains Glutamic acid-rich region, Glutamine-rich region and Arginine-rich region. Immunocytochemistry confirmed that DAP is localized in the nuclei and the Golgi complex of K562, MDCK, Hela, Cos1 cells, and Caco2 (where DAP is found also in BBM). GFP-fusion constructs containing the DAP nuclear localization signal (amino acids 171–277) or GOLD domain (amino acid 278–526) were transfected into COS-1 and K562 cells and specificity of intracellular localization confirmed by fluorescence confocal microscopy. DAP expression was controlled by cellular iron content: Cells which were iron depleted had increased levels of DAP protein while cells which were iron replete had decreased DAP protein. The regulation by iron was post-transcriptional as iron levels had no affect on DAP mRNA. The levels of DAP expression was also seen to affect iron uptake. Over expression of the region of DAP which binds to DMT1 by transfection of the appropriate construct into K562 cells decreased iron uptake as measured by an increase of transferrin receptor expression and decreased levels of ferritin. Elevated DAP had no affect on endogenous DMT1 expression. Conversely, when siRNAs were used to decrease DAP mRNA in K562 cells there was increased iron uptake with decreased expression of transferrin receptor and increased ferritin expression. In these experiments siRNAs reduced DAP expression by about 60%. This is the first demonstration that a protein which interacts with DMT1 can regulate the uptake of iron into the cell and suggests that DAP may act in a regulatory pathway for iron homeostasis.

Disorders of iron metabolism and homeostatis belong to the most common diseases in humans; however, neither the regulation of iron metabolism in erythroid cells nor the pathophysiology of these diseases are completely understood. Last year we reported at this meeting a case of severe hypochromic microcytic anemia caused by a homozygous mutation in the divalent metal transporter 1 (DMT1) gene (DMT1 1285G>C). The defective growth of this patient's erythroid progenitors in vitro was rescued by the addition of 10 μM iron-salicylaldehyde isonicotinoyl hydrazone (Fe-SIH), an iron chelate that has been shown to deliver iron for heme synthesis without involving the transferrin receptor/DMT1 pathway. To further characterize the impact of the DMT1 mutation on the in vitro growth of erythroid progenitors, 10 μM Fe-SIH, 25 μM hemin or 10 μM zinc chloride (ZnCl2) were added to the erythropoietin (Epo) containing cultures. All substances increased the plating efficiency in normal control as well as mutant BFU-Es. Moreover, all substances led to a 1.4 to 1.6 fold increase in hemoglobin (Hb) content in normal BFU-Es. Although hemin did not significantly increase Hb content in the patient's BFU-E, the addition of Fe-SIH to the Epo/hemin-containing cultures increased Hb content 2.2 fold. Since Hb synthesis in heme-deficient erythroid cells is inhibited by heme-regulated inhibitor (HRI) kinase, which specifically phosphorylates the α subunit of the eukaryotic initiation factor 2 (eIF2α), we have analyzed the level of eIF2α phosphorylation in cytospine-harvested BFU-Es. Immunofluorescence analysis of erythroid cells with phospo-eIF2α (Ser51) antibody revealed increased phosphorylation of eIF2α in mutant BFU-Es compared to control BFU-Es grown under all aforementioned conditions. The most striking differences were observed in Epo and Epo/hemin containing cultures, in which the fractions of phospho-eIF2α-positive cells were significantly higher in mutant BFU-Es. These data suggest that the block of terminal differentiation/hemoglobinization in the DMT1-mutant erythroid cells is only partially caused by translational arrest of globin chain synthesis; the delivery of iron may be required for other cellular functions apart from Hb synthesis. Interestingly, the most prominent positive effect on the proliferation (i.e., BFU-E cellularity) as well as hemoglobinization of mutant BFU-Es had a combination of Epo/Fe-SIH and ZnCl2; this combination led to the highest increase in Hb content that was associated with almost complete lack of eIF2α phosphorylation. This suggests that the addition of Zn2+ to Fe-SIH containing cultures further stimulates processes that are initially rescued in DMT1-mutant erythroid cells by the addition of iron. We conclude, that the DMT1 defect in erythroid cells is complex, probably involving the combination of transcriptional and translational defects that cannot be rescued by hemin alone. Download : Download high-res image (244KB) Download : Download full-size image Figure

During differentiation, immature erythroid cells acquire vast amounts of iron at a breakneck rate. Proper coordination of iron delivery and utilization in heme synthesis is essential and disruption of this process likely underlies iron loading disorders such as sideroblastic anemia and myelodysplastic syndrome with ringed sideroblasts. Iron is taken up by the cells via receptor mediated endocytosis, a process whereby diferric transferrin (Tf) binds to its cognate receptor (TfR) on the erythroid cell plasma membrane, followed by internalization of the Tf-TfR complex. Subsequent to endocytosis, the endosome is acidified by a H+-ATPase, allowing the release of iron from Tf. Through an unknown mechanism, iron is targeted to the inner membrane of the mitochondria, where the enzyme that inserts Fe into protoporphyrin IX, ferrochelatase, resides. Although it has been demonstrated that the divalent metal transporter, DMT1, is responsible for the egress of reduced Fe from the vesicle, the immediate fate of the iron atoms after their transport across the vesicular membrane remains unknown. Because reduced iron is a strong pro-oxidant, contributing to free radical formation through Fenton chemistry, it has been predicted that an iron binding molecule shuttles Fe from the endosome to mitochondria. However, this much sought iron binding intermediate, that would constitute the labile iron pool (LIP), has yet to be identified. Thus, we hypothesize that, in Hb-producing cells, there is a direct relaying of Fe from the endosomal machinery to that of the mitochondria. By loading the cytoplasm of reticulocytes with an impermeant form of the iron chelator, desferrioxamine, (hDFO) which would intercept Fe that traversed the cytosol bound to the putative LIP intermediate, we show that 59Fe, delivered via Tf, can bypass the cytosol. Importantly, iron delivered to these cells in a form that freely diffuses across the membrane, iron-salicylaldehyde isonicotinoyl hydrazone (59FeSIH2), was significantly prevented from being used for heme synthesis in hDFO-laden reticulocytes. We have also used time-lapse confocal microscopy to track iron-loaded endosomes and mitochondria with high spatial and temporal resolutions. Using this approach, we have found that endosomes are very mobile organelles that continuously traverse the cytosol and touch a number of mitochondria multiple times. Addition of N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7), a calmodulin antagonist that is known to block the myosin motor, to cells that had been treated with fluorescent holo-Tf, inhibited endosomal movement. Interestingly, W-7 treatment of cells whose endosomes were loaded with 59Fe-Tf blocked both incorporation of that iron into heme or mobilization of the iron by chelators. Together, these data are congruent with our hypothesis that iron is directly delivered to mitochondria by endosomes in a “kiss and run” paradigm.