Iron-dependent regulation of the divalent metal ion transporter.

The first step in intestinal iron absorption is mediated by the H(+)-coupled Fe(2+) transporter called divalent cation transporter 1/divalent metal ion transporter 1 (DCT1/DMT1) (also known as natural resistance-associated macrophage protein 2). DCT1/DMT1 mRNA levels in the duodenum strongly increase in response to iron depletion. To study the mechanism of iron-dependent DCT1/DMT1 mRNA regulation, we investigated the endogenous expression of DCT1/DMT1 mRNA in various cell types. We found that only the iron responsive element (IRE)-containing form, which corresponds to one of two splice forms of DCT1/DMT1, is responsive to iron treatment and this responsiveness was cell type specific. We also examined the interaction of the putative 3'-UTR IRE with iron responsive binding proteins (IRP1 and IRP2), and found that IRP1 binds to the DCT1/DMT1-IRE with higher affinity compared to IRP2. This differential binding of IRP1 and IRP2 was also reported for the IREs of transferrin receptors, erythroid 5-aminolevulinate synthase and mitochondrial aconitase. We propose that regulation of DCT1/DMT1 mRNA by iron involves post-transcriptional regulation through the binding of IRP1 to the transporter's IRE, as well as other as yet unknown factors.

Recent advances in disorders of iron metabolism: mutations, mechanisms and modifiers.

The spectrum of known disorders of iron metabolism has expanded dramatically over the past few years. Identification of HFE, the gene most commonly mutated in patients with hereditary hemochromatosis, has allowed molecular diagnosis and paved the way for identification of other genes, such as TFR2, that are important in non-HFE-associated iron overload. There are clearly several other, unidentified, iron overload disease genes yet to be found. In parallel, our understanding of iron transport has expanded through identification of Fpn1/Ireg1/MTP1, Sfxn1 and DCYTB: Ongoing studies of Friedreich's ataxia, sideroblastic anemia, aceruloplasminemia and neurodegeneration with brain-iron accumulation are clarifying the role for iron in the nervous system. Finally, as the number of known iron metabolic genes increases and their respective functions are ascertained, new opportunities have arisen to identify genetic modifiers of iron homeostasis.

Autosomal-dominant hemochromatosis is associated with a mutation in the ferroportin (SLC11A3) gene.

Hemochromatosis is a progressive iron overload disorder that is prevalent among individuals of European descent. It is usually inherited in an autosomal-recessive pattern and associated with missense mutations in HFE, an atypical major histocompatibility class I gene. Recently, we described a large family with autosomal-dominant hemochromatosis not linked to HFE and distinguished by early iron accumulation in reticuloendothelial cells. Through analysis of a large pedigree, we have determined that this disease maps to 2q32. The gene encoding ferroportin (SLC11A3), a transmembrane iron export protein, lies within a candidate interval defined by highly significant lod scores. We show that the iron-loading phenotype in autosomal-dominant hemochromatosis is associated with a nonconservative missense mutation in the ferroportin gene. This missense mutation, converting alanine to aspartic acid at residue 77 (A77D), was not seen in samples from 100 unaffected control individuals. We propose that partial loss of ferroportin function leads to an imbalance in iron distribution and a consequent increase in tissue iron accumulation.

Expression of stimulator of Fe transport is not enhanced in Hfe knockout mice.

Hfe knockout (-/-) mice recapitulate many of the biochemical abnormalities of hereditary hemochromatosis (HH), but the molecular mechanisms involved in the etiology of iron overload in HH remain poorly understood. It was found previously that livers of patients with HH contained 5-fold higher SFT (stimulator of Fe transport) mRNA levels relative to subjects without HH. Because this observation suggests a possible role for SFT in HH, we investigated SFT mRNA expression in Hfe(-/-) mice. The 4- and 10-wk-old Hfe(-/-) mice do not have elevated levels of hepatic SFT transcripts relative to age-matched Hfe(+/+) mice, despite having 2.2- and 3.3-fold greater hepatic nonheme iron concentrations, respectively. Northern blot analyses of various mouse tissues revealed that SFT is widely expressed. The novel observation that SFT transcripts are abundant in brain prompted a comparison of SFT transcript levels and nonheme iron levels in the brains of Hfe(+/+) and Hfe(-/-) mice. Neither SFT mRNA levels nor nonheme iron levels differed between groups. Further comparisons of Hfe(-/-) and Hfe(+/+) mouse tissues revealed no significant differences in SFT mRNA levels in duodenum, the site of increased iron absorption in HH. Important distinctions between Hfe(-/-) mice and HH patients include not only differences in the relative rate and magnitude of iron loading but also the lack of fibrosis and phlebotomy treatment in the knockout animals.

A mutation in a mitochondrial transmembrane protein is responsible for the pleiotropic hematological and skeletal phenotype of flexed-tail (f/f) mice.

We have studied the flexed-tail (f) mouse to gain insight into mammalian mitochondrial iron metabolism. Flexed-tail animals have axial skeletal abnormalities and a transient embryonic and neonatal anemia characterized by pathologic intramitochondrial iron deposits in erythrocytes. Mitochondrial iron accumulation is the hallmark of sideroblastic anemias, which typically result from defects in heme biosynthesis or other pathways that lead to abnormal erythroid mitochondrial iron utilization. To clone the f gene, we used positional cloning techniques, and identified a frameshift mutation in a mitochondrial transmembrane protein. The mutated gene, Sfxn1, is the prototype of a novel family of evolutionarily conserved proteins present in eukaryotes.

Uroporphyria in Hfe mutant mice given 5-aminolevulinate: a new model of Fe-mediated porphyria cutanea tarda.

Porphyria cutanea tarda (PCT), a liver disease with skin lesions caused by excess liver production of uroporphyrin (URO), is associated with consumption of alcoholic beverages or estrogens, and moderate iron overload. Recently, it has been shown that many PCT patients carry mutations in the HFE gene, which is responsible for hereditary hemochromatosis. Mice homozygous for either the null mutation in the Hfe gene or the C282Y missense mutation rapidly accumulate hepatic parenchymal iron similar to patients with hemochromatosis. Here we investigated whether disruption of the murine Hfe gene would result in hepatic uroporphyria. Mice homozygous for the Hfe-null mutation accumulated high levels of hepatic URO when fed 5-aminolevulinate (ALA). Hfe (+/-) mice also accumulated hepatic URO when fed ALA, but at a much slower rate. The amount of accumulated URO in the null mutant mice was similar to that in wild-type mice treated with iron carbonyl in the diet, or injected with iron dextran. Iron in both wild-type and Hfe (+/-) mice was mostly in Kupffer cells. In contrast, Hfe (-/-) mice had considerable parenchymal iron deposition as well, in a pattern similar to that observed in wild-type mice treated with iron carbonyl. URO accumulation was accompanied by 84% and 33% decreases in hepatic uroporphyrinogen decarboxylase activities in Hfe (-/-) and Hfe (+/-) mice, respectively. No increases in CYP1A2 or other cytochrome P450s were detected in the Hfe-null mutant mice. We conclude that this experimental model of uroporphyria is a valid model for further investigations into the mechanism of PCT.

Expression of the DMT1 (NRAMP2/DCT1) iron transporter in mice with genetic iron overload disorders.

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 (Trf(hpx)) mice, Hfe knockout mice, and B2m knockout mice. Interestingly, in Trf(hpx) 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.

A mouse model of familial porphyria cutanea tarda.

Approximately one-third of patients with porphyria cutanea tarda (PCT), the most common porphyria in humans, inherit a single mutant allele of the uroporphyrinogen decarboxylase (URO-D) gene. PCT associated with URO-D mutations is designated familial PCT. The phenotype is characterized by a photosensitive dermatosis with hepatic accumulation and urinary excretion of uroporphyrin and hepta-carboxylic porphyrins. Most heterozygotes for URO-D mutations do not express a porphyric phenotype unless hepatic siderosis is present. Hemochromatosis gene (HFE) mutations are frequently found when the phenotype is expressed. We used homologous recombination to disrupt one allele of murine URO-D. URO-D(+/-) mice had half-wild type (wt) URO-D protein and enzymatic activity in all tissues but did not accumulate hepatic porphyrins, indicating that half-normal URO-D activity is not rate limiting. When URO-D(+/-) mice were injected with iron-dextran and given drinking water containing delta-aminolevulinic acid for 21 days, hepatic porphyrins accumulated, and hepatic URO-D activity was reduced to 20% of wt. We bred mice homozygous for an HFE gene disruption (HFE(-/-)) to URO-D(+/-) mice, generating mice with the URO-D(+/-)/HFE(-/-) genotype. These animals developed a porphyric phenotype by 14 weeks of age without ALA supplementation, and URO-D activity was reduced to 14% of wt. These data indicate that iron overload alone is sufficient to reduce URO-D activity to rate-limiting levels in URO-D(+/-) mice. The URO-D(+/-) mouse serves as an excellent model of familial PCT and affords the opportunity to define the mechanism by which iron influences URO-D activity.

Inherited iron overload disorders.

Iron is an essential nutrient that is highly toxic in excess. Normal iron balance is maintained primarily by regulation of intestinal absorption of the metal from the diet. Iron overload generally results from a chronic increase in intestinal absorption. During the past 5 years, it has become apparent that there are at least eight inherited disorders of iron metabolism characterized by the toxic accumulation of iron. This review provides an update for pediatricians on the clinical features and pathogenesis of these disorders.

The Nramp2/DMT1 iron transporter is induced in the duodenum of microcytic anemia mk mice but is not properly targeted to the intestinal brush border.

Microcytic anemia (mk) mice and Belgrade (b) rats are severely iron deficient because of impaired intestinal iron absorption and defective iron metabolism in peripheral tissues. Both animals carry a glycine to arginine substitution at position 185 in the iron transporter known as Nramp2/DMT1 (divalent metal transporter 1). DMT1 messenger RNA (mRNA) and protein expression has been examined in the gastrointestinal tract of mk mice. Northern blot analysis indicates that, by comparison to mk/+ heterozygotes, mk/mk homozygotes show a dramatic increase in the level of DMT1 mRNA in the duodenum. This increase in RNA expression is paralleled by a concomitant increase of the 100-kd DMT1 isoform I protein expression in the duodenum. Immunohistochemical analyses show that, as for normal mice on a low-iron diet, DMT1 expression in enterocytes of mk/mk mice is restricted to the duodenum. However, and in contrast to normal enterocytes, little if any expression of DMT1 is seen at the apical membrane in mk/mk mice. These results suggest that the G185R mutation, which was shown to impair the transport properties of DMT1, also affects the membrane targeting of the protein in mk/mk enterocytes. This loss of function of DMT1 is paralleled by a dramatic increase in expression of the defective protein in mk/mk mice. This is consistent with a feedback regulation of DMT1 expression by iron stores. (Blood. 2000;96:3964-3970)

Comparison of the interactions of transferrin receptor and transferrin receptor 2 with transferrin and the hereditary hemochromatosis protein HFE.

The transferrin receptor (TfR) interacts with two proteins important for iron metabolism, transferrin (Tf) and HFE, the protein mutated in hereditary hemochromatosis. A second receptor for Tf, TfR2, was recently identified and found to be functional for iron uptake in transfected cells (Kawabata, H., Germain, R. S., Vuong, P. T., Nakamaki, T., Said, J. W., and Koeffler, H. P. (2000) J. Biol. Chem. 275, 16618-16625). TfR2 has a pattern of expression and regulation that is distinct from TfR, and mutations in TfR2 have been recognized as the cause of a non-HFE linked form of hemochromatosis (Camaschella, C., Roetto, A., Cali, A., De Gobbi, M., Garozzo, G., Carella, M., Majorano, N., Totaro, A., and Gasparini, P. (2000) Nat. Genet. 25, 14-15). To investigate the relationship between TfR, TfR2, Tf, and HFE, we performed a series of binding experiments using soluble forms of these proteins. We find no detectable binding between TfR2 and HFE by co-immunoprecipitation or using a surface plasmon resonance-based assay. The affinity of TfR2 for iron-loaded Tf was determined to be 27 nm, 25-fold lower than the affinity of TfR for Tf. These results imply that HFE regulates Tf-mediated iron uptake only from the classical TfR and that TfR2 does not compete for HFE binding in cells expressing both forms of TfR.

Intestinal iron absorption: current concepts circa 2000.

Iron is essential for all mammalian cells, and particularly needed for the production of erythrocyte haemoglobin. However, excess iron is toxic, and tissue iron concentrations must be strictly regulated. This regulation occurs at the sites of entrance of iron into the body: in the placenta before birth, and the small intestine after birth. Although iron homeostasis has been intensively studied for half a century, the molecular details have only recently begun to emerge. This review will cover current information on intestinal iron absorption.

The molecular defect in hypotransferrinemic mice.

Hypotransferrinemic (Trf(hpx/hpx)) mice have a severe deficiency in serum transferrin (Trf) as the result of a spontaneous mutation linked to the murine Trf locus. They are born alive, but before weaning, die from severe anemia if they are not treated with exogenous Trf or red blood cell transfusions. We have determined the molecular basis of the hpx mutation. It results from a single point mutation, which alters an invariable nucleotide in the splice donor site after exon 16 of the Trf gene. No normal Trf messenger RNA (mRNA) is made from the hpx allele. A small amount of mRNA results from the usage of cryptic splice sites within exon 16. The predominant cryptic splice site produces a Trf mRNA carrying a 27-base pair (bp), in-frame deletion. Less than 1% of normal levels of a Trf-like protein is found in the serum of Trf(hpx/hpx) mice, most likely resulting from translation of the internally deleted mRNA. Despite their severe Trf deficiency, however, Trf(hpx/hpx) mice initially treated with transferrin injections can survive after weaning without any further treatment. They have massive tissue iron overload develop in all nonhematopoietic tissues, while they continue to have severe iron deficiency anemia. Their liver iron burden is 100-fold greater than that of wild-type mice and 15- to 20-fold more than that of mice lacking the hemochromatosis gene, Hfe. Trf(hpx/hpx) mice thus provide an additional model with a defined molecular defect for the study of genetic iron disorders.

Ectopic expression of transcription factor NF-E2 alters the phenotype of erythroid and monoblastoid cells.

In this study, regulation of transcription factor NF-E2 was examined in differentiating erythroid and myeloid cells, and the impact of raising NF-E2 concentrations within these cell types was assessed. NF-E2 was expressed in the J2E erythroid cell line, but the levels increased only marginally during erythropoietin-induced differentiation. In contrast, rare myeloid variants of J2E cells did not express NF-E2. Although NF-E2 was present in M1 monoblastoid cells, it was undetectable as these cells matured into macrophages. Compared with erythroid cells, transcription of the NF-E2 gene was reduced, and the half-life of the mRNA was significantly shorter in monocytoid cells. Ectopic expression of NF-E2 had a profound impact upon the J2E cells; morphologically mature erythroid cells spontaneously emerged in culture, but the cells failed to synthesize hemoglobin, even in the presence of erythropoietin. Although proliferation and viability increased in the NF-E2-transfected J2E cells, their responsiveness to erythropoietin was severely diminished. Strikingly, increasing the expression of NF-E2 in M1 cells produced sublines that contained erythroid or immature megakaryocytic cells. Finally, overexpression of NF-E2 in primary hemopoietic progenitors from fetal liver increased erythroid colony formation in the absence of erythropoietin. These data demonstrate that elevated NF-E2 (i) had a dominant effect on the phenotype and maturation of J2E erythroid cells, (ii) was able to reprogram the M1 monocytoid line, and (iii) promoted the development of erythroid colonies by normal progenitors.

Genes that modify the hemochromatosis phenotype in mice.

Hereditary hemochromatosis (HH) is a prevalent human disease caused by a mutation in HFE, which encodes an atypical HLA class I protein involved in regulation of intestinal iron absorption. To gain insight into the pathogenesis of hemochromatosis, we have bred Hfe knockout mice to strains carrying other mutations that impair normal iron metabolism. Compound mutant mice lacking both Hfe and its interacting protein, beta-2 microglobulin (B2m), deposit more tissue iron than mice lacking Hfe only, suggesting that another B2m-interacting protein may be involved in iron regulation. Hfe knockout mice carrying mutations in the iron transporter DMT1 fail to load iron, indicating that hemochromatosis involves iron flux through DMT1. Similarly, compound mutants deficient in both Hfe and hephaestin (Heph) show less iron loading than do Hfe knockout mice, indicating that iron absorption in hemochromatosis involves the function of Heph as well. Finally, compound mutants lacking Hfe and the transferrin receptor accumulate more tissue iron than do mice lacking Hfe alone, consistent with the idea that interaction between these two proteins contributes to the control of normal iron absorption. In addition to providing insight into the pathogenesis of HH, our results suggest that each of these genes might be a candidate modifier of the human hemochromatosis phenotype.

Positional cloning of zebrafish ferroportin1 identifies a conserved vertebrate iron exporter.

Defects in iron absorption and utilization lead to iron deficiency and overload disorders. Adult mammals absorb iron through the duodenum, whereas embryos obtain iron through placental transport. Iron uptake from the intestinal lumen through the apical surface of polarized duodenal enterocytes is mediated by the divalent metal transporter, DMTi. A second transporter has been postulated to export iron across the basolateral surface to the circulation. Here we have used positional cloning to identify the gene responsible for the hypochromic anaemia of the zebrafish mutant weissherbst. The gene, ferroportin1, encodes a multiple-transmembrane domain protein, expressed in the yolk sac, that is a candidate for the elusive iron exporter. Zebrafish ferroportin1 is required for the transport of iron from maternally derived yolk stores to the circulation and functions as an iron exporter when expressed in Xenopus oocytes. Human Ferroportin1 is found at the basal surface of placental syncytiotrophoblasts, suggesting that it also transports iron from mother to embryo. Mammalian Ferroportin1 is expressed at the basolateral surface of duodenal enterocytes and could export cellular iron into the circulation. We propose that Ferroportin1 function may be perturbed in mammalian disorders of iron deficiency or overload.

Iron homeostasis: insights from genetics and animal models.

Disorders that perturb iron balance are among the most prevalent human diseases, but until recently iron transport remained poorly understood. Over the past five years, genetic studies of patients with inherited iron homeostasis disorders and the analysis of mutant mice, rats and zebrafish have helped to identify several important iron-transport proteins. With information being mined from the genomes of four species, the study of iron metabolism has benefited enormously from positional-cloning efforts. Complementing the genomic strategy, targeted mutagenesis in mice has produced new models of human iron diseases. The animal models described in this review offer valuable tools for investigating iron homeostasis in vivo.

Iron metabolism: iron deficiency and iron overload.

Iron is an essential cofactor in a variety of cellular processes. Except for a few unusual bacterial species, iron is indispensable for living organisms. However, free iron is toxic because of its propensity to induce the formation of dangerous free radicals. Consequently, iron balance is tightly regulated. Disorders of iron homeostasis are among the most common afflictions of humans. This review discusses inherited iron deficiency and iron overload disorders and recent insights into their pathophysiology.

The iron transporter DMT1.

Divalent metal transporter 1 (DMT1) is the first mammalian transmembrane iron transporter to be identified. In 1997, parallel experiments from two groups provided compelling evidence of its function. Fleming and colleagues identified mutations in DMT1 (formerly known as Nramp2 and DCT1) in mice and rats with defects in intestinal iron absorption and red blood cell iron utilization. Gunshin and co-workers (H Gunshin, B MacKenzie, UV Berger, Y Gunshin, MF Romero, WF Boron, S. Nussberger, JL Gollan, MA Hediger, Cloning and characterization of a mammalian proton-coupled metal-ion transporter, Nature 388 (1997) 482-488.) isolated DMT1 through an expression cloning strategy looking for mRNAs that stimulated iron uptake by Xenopus oocytes. Taken together, these data indicate that the twelve transmembrane domain protein DMT1 transfers iron across the apical surface of intestinal cells and out of transferrin cycle endosomes. Human DMT1 may be a good target for pharmacological intervention in patients with iron overload disorders attributable to increased iron absorption.