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Iron is essential for erythropoiesis and other biological processes, but is toxic in excess. Dietary absorption of iron is a highly regulated process and is a major determinant of body iron levels. Iron excretion, however, is considered a passive, unregulated process, and the underlying pathways are unknown. Here we investigated the role of metal transporters SLC39A14 and SLC30A10 in biliary iron excretion. While SLC39A14 imports manganese into the liver and other organs under physiological conditions, it imports iron under conditions of iron excess. SLC30A10 exports manganese from hepatocytes into the bile. We hypothesized that biliary excretion of excess iron would be impaired by SLC39A14 and SLC30A10 deficiency. We therefore analyzed biliary iron excretion in Slc39a14-and Slc30a10-deficient mice raised on iron-sufficient and -rich diets. Bile was collected surgically from the mice, then analyzed with nonheme iron assays, mass spectrometry, ELISAs, and an electrophoretic assay for iron-loaded ferritin. Our results support a model in which biliary excretion of excess iron requires iron import into hepatocytes by SLC39A14, followed by iron export into the bile predominantly as ferritin, with iron export occurring independently of SLC30A10. To our knowledge, this is the first report of a molecular determinant of mammalian iron excretion and can serve as basis for future investigations into mechanisms of iron excretion and relevance to iron homeostasis.

The bone morphogenetic protein (BMP) type I receptors ALK2 and ALK3 are essential for expression of hepcidin, a key iron regulatory hormone. In mice, hepatocyte-specific Alk2 deficiency leads to moderate iron overload with periportal liver iron accumulation, while hepatocyte-specific Alk3 deficiency leads to severe iron overload with centrilobular liver iron accumulation and a more marked reduction of basal hepcidin levels. The objective of this study was to investigate whether the two receptors have additive roles in hepcidin regulation. Iron overload in mice with hepatocyte-specific Alk2 and Alk3 (Alk2/3) deficiency was characterized and compared to hepatocyte-specific Alk3 deficient mice. Co-immunoprecipitation studies were performed to detect the formation of ALK2 and ALK3 homodimer and heterodimer complexes in vitro in the presence and absence of ligands. The iron overload phenotype of hepatocyte-specific Alk2/3-deficient mice was more severe than that of hepatocyte-specific Alk3-deficient mice. In vitro co-immunoprecipitation studies in Huh7 cells showed that ALK3 can homodimerize in absence of BMP2 or BMP6. In contrast, ALK2 did not homodimerize in either the presence or absence of BMP ligands. However, ALK2 did form heterodimers with ALK3 in the presence of BMP2 or BMP6. ALK3-ALK3 and ALK2-ALK3 receptor complexes induced hepcidin expression in Huh7 cells. Our data indicate that: (I) ALK2 and ALK3 have additive functions in vivo, as Alk2/3 deficiency leads to a greater degree of iron overload than Alk3 deficiency; (II) ALK3, but not ALK2, undergoes ligand-independent homodimerization; (III) the formation of ALK2-ALK3 heterodimers is ligand-dependent and (IV) both receptor complexes functionally induce hepcidin expression in vitro.

Excessive iron increases the incidence of diabetes and worsens diabetic complications. Reciprocally, diabetes induces iron loading, partially attributable to elevated intestinal iron export according to a recent report. Herein, we show that iron uptake and the mRNA expression of iron importer divalent metal transporter 1 (DMT1) were significantly increased in the duodenum of streptozotocin-induced diabetic mice. Immunofluorescence staining of human intestinal biopsies revealed increased brush border membrane (BBM) and decreased cytoplasmic DMT1 expression in patients with diabetes, suggesting translocation of DMT1. This pattern of DMT1 regulation was corroborated by immunoblotting results in diabetic mice showing that BBM DMT1 expression was increased by 210%, in contrast to a 60% increase in total DMT1. PKC mediates many diabetic complications, and PKCα activity was increased in diabetic mouse intestine. Intriguingly, diabetic mice with PKCα deficiency did not show increases in iron uptake and BBM DMT1 expression. High-glucose treatment increased plasma membrane DMT1 expression via the activation of PKCα in cultured IECs. Inhibition of PKCα potentiated the ubiquitination and degradation of DMT1 protein. We further showed that high glucose suppressed membrane DMT1 internalization. These findings demonstrate that PKCα promotes microvillus membrane DMT1 expression and intestinal iron uptake, contributing to diabetic iron loading.—Zhao, L., Bartnikas, T., Chu, X., Klein, J., Yun, C., Srinivasan, S., He, P. Hyperglycemia promotes microvillus membrane expression of DMT1 in intestinal epithelial cells in a PKCα-dependent manner.

In this issue of Blood,Nai et al demonstrate that the serine protease matriptase-2 mediates erythropoietin (EPO)-dependent suppression of hepcidin, a central regulator of iron homeostasis with causal or contributing roles in iron-loading diseases, hemoglobinopathies, and infectious, inflammatory, and neoplastic disorders.1

Many past and recent advances in the field of iron metabolism have relied upon the use of mouse models of disease. These models have arisen spontaneously in breeder colonies or have been engineered for global or conditional ablation or overexpression of select genes. Full phenotypic characterization of these models typically involves maintenance on iron-loaded or -deficient diets, treatment with oxidative or hemolytic agents, breeding to other mutant lines or other stresses. In this review, we focus on systemic iron biology and the contributions that mouse model-based studies have made to the field. We have divided the field into three broad areas of research: dietary iron absorption, regulation of hepcidin expression and cellular iron metabolism. For each area, we begin with an overview of the current understanding of key molecular and cellular determinants then discuss recent advances. Finally, we conclude with brief comments on prospects for future study. This article is part of a Special Issue entitled: Cell Biology of Metals.

Transferrin is an abundant serum metal-binding protein best known for its role in iron delivery. The human disease congenital atransferrinemia and animal models of this disease highlight the essential role of transferrin in erythropoiesis and iron metabolism. Patients and mice deficient in transferrin exhibit anemia and a paradoxical iron overload attributed to deficiency in hepcidin, a peptide hormone synthesized largely by the liver that inhibits dietary iron absorption and macrophage iron efflux. Studies of inherited human disease and model organisms indicate that transferrin is an essential regulator of hepcidin expression. In this paper, we review current literature on transferrin deficiency and present our recent findings, including potential overlaps between transferrin, iron and manganese in the regulation of hepcidin expression.

Here we investigate the regulation of hepcidin, a hormone that inhibits dietary iron absorption and macrophage iron recycling, by the serum iron-binding protein transferrin. Mice deficient in transferrin (Tf(hpx/hpx)) and hemojuvelin (Hjv(-/-)), a gene mutated in juvenile hemochromatosis, a disease of hepcidin deficiency and iron overload, were generated. While Tf(hpx/hpx) Hjv(+/+) and Tf(hpx/hpx) Hjv(-/-) phenotypes did not differ markedly, transferrin treatment and RBC transfusions robustly increased hepcidin levels in Tf(hpx/hpx) Hjv(+/+) but not Tf(hpx/hpx) Hjv(-/-)mice. These results suggest that, while hemojuvelin is not essential for the establishment or maintenance of hepcidin deficiency in transferrin-deficient mice, hemojuvelin is essential for transferrin-dependent and transferrin-independent hepcidin expression in conditions of iron overload.

As a central regulator of iron metabolism, hepcidin inhibits dietary iron absorption and macrophage iron recycling. Its expression is regulated by multiple factors including iron availability and erythropoietic activity. To investigate the role of transferrin (Tf) in the regulation of hepcidin expression by these factors in vivo, we employed the hypotransferrinemic (hpx) mouse. These Tf-deficient mice have severe microcytic anemia, tissue iron overload, and hepcidin deficiency. To determine the relationship of Tf levels and erythropoiesis to hepcidin expression, we subjected hpx mutant and control mice to a number of experimental manipulations. Treatment of hpx mice with Tf injections corrected their anemia and restored hepcidin expression. To investigate the effect of erythropoiesis on hepcidin expression, we suppressed erythropoiesis with blood transfusions or myeloablation with chemotherapeutic drugs. Transfusion of hpx animals with wild-type red blood cells led to increased hepcidin expression, while hepcidin expression in myeloablated hpx mice increased only if Tf was administered postablation. These results suggest that hepcidin expression in hpx mice is regulated both by Tf-restricted erythropoiesis and by Tf through a mechanism independent of its role in erythropoiesis.

Excessive iron absorption is one of the main features of β-thalassemia and can lead to severe morbidity and mortality. Serial analyses of β-thalassemic mice indicate that while hemoglobin levels decrease over time, the concentration of iron in the liver, spleen, and kidneys markedly increases. Iron overload is associated with low levels of hepcidin, a peptide that regulates iron metabolism by triggering degradation of ferroportin, an iron-transport protein localized on absorptive enterocytes as well as hepatocytes and macrophages. Patients with β-thalassemia also have low hepcidin levels. These observations led us to hypothesize that more iron is absorbed in β-thalassemia than is required for erythropoiesis and that increasing the concentration of hepcidin in the body of such patients might be therapeutic, limiting iron overload. Here we demonstrate that a moderate increase in expression of hepcidin in β-thalassemic mice limits iron overload, decreases formation of insoluble membrane-bound globins and reactive oxygen species, and improves anemia. Mice with increased hepcidin expression also demonstrated an increase in the lifespan of their red cells, reversal of ineffective erythropoiesis and splenomegaly, and an increase in total hemoglobin levels. These data led us to suggest that therapeutics that could increase hepcidin levels or act as hepcidin agonists might help treat the abnormal iron absorption in individuals with β-thalassemia and related disorders.

Mice have been essential for distinguishing the role of hepcidin in iron homeostasis. Currently, investigators monitor levels of murine hepatic hepcidin-1 mRNA as a surrogate marker for the bioactive hepcidin protein itself. Here, we describe and validate a competitive, enzyme-linked immunosorbent assay that quantifies hepcidin-1 in mouse serum and urine. The assay exhibits a biologically relevant lower limit of detection, high precision, and excellent linearity and recovery. We also demonstrate correlation between serum and urine hepcidin-1 values and validate the competitive enzyme-linked immunosorbent assay by analyzing plasma hepcidin response of mice to physiological challenges, including iron deficiency, iron overload, acute blood loss, and inflammation. Furthermore, we analyze multiple murine genetic models of iron dysregulation, including β-thalassemia intermedia (Hbb(th3/+)), hereditary hemochromatosis (Hfe(-/-), Hjv(-/-), and Tfr2(Y245X/Y245X)), hypotransferrinemia (Trf(hpx/hpx)), heterozygous transferrin receptor 1 deficiency (Tfrc(+/-)) and iron refractory iron deficiency anemia (Tmprss6(-/-) and Tmprss6(hem8/hem8)). Novel compound iron metabolism mutants were also phenotypically characterized here for the first time. We demonstrate that serum hepcidin concentrations correlate with liver hepcidin mRNA expression, transferrin saturation and non-heme liver iron. In some circumstances, serum hepcidin-1 more accurately predicts iron parameters than hepcidin mRNA, and distinguishes smaller, statistically significant differences between experimental groups.

Copper/zinc superoxide dismutase (SOD1) is an abundant intracellular enzyme with an essential role in antioxidant defense. The activity of SOD1 is dependent upon the presence of a bound copper ion incorporated by the copper chaperone for superoxide dismutase, CCS. To elucidate the cell biological mechanisms of this process, SOD1 synthesis and turnover were examined following 64Cu metabolic labeling of fibroblasts derived from CCS +/+ and CCS –/– embryos. The data indicate that copper is rapidly incorporated into both newly synthesized SOD1 and preformed SOD1 apoprotein, that each process is dependent upon CCS and that once incorporated, copper is unavailable for cellular exchange. The abundance of apoSOD1 is inversely proportional to the intracellular copper content and immunoblot and gel filtration analysis indicate that this apoprotein exists as a homodimer that is distinguishable from SOD1. Despite these distinct differences, the abundance and half-life of SOD1 is equivalent in CCS +/+ and CCS –/– fibroblasts, indicating that neither CCS nor copper incorporation has any essential role in the stability or turnover of SOD1 in vivo. Taken together, these data provide a cell biological model of SOD1 biosynthesis that is consistent with the concept of limited intracellular copper availability and indicate that the metallochaperone CCS is a critical determinant of SOD1 activity in mammalian cells. These kinetic and biochemical findings also provide an important framework for understanding the role of mutant SOD1 in the pathogenesis of familial amyotrophic lateral sclerosis.

Copper-mediated oxidative damage is proposed to play a critical role in the pathogenesis of Cu/Zn superoxide dismutase (SOD1)-linked familial amyotrophic lateral sclerosis (FALS). We tested this hypothesis by ablating the gene encoding the copper chaperone for SOD1 (CCS) in a series of FALS-linked SOD1 mutant mice. Metabolic 64Cu labeling in SOD1-mutant mice lacking the CCS showed that the incorporation of copper into mutant SOD1 was significantly diminished in the absence of CCS. Motor neurons in CCS-/- mice showed increased rate of death after facial nerve axotomy, a response documented for SOD1-/- mice. Thus, CCS is necessary for the efficient incorporation of copper into SOD1 in motor neurons. Although the absence of CCS led to a significant reduction in the amount of copper-loaded mutant SOD1, however, it did not modify the onset and progression of motor neuron disease in SOD1-mutant mice. Hence, CCS-dependent copper loading of mutant SOD1 plays no role in the pathogenesis of motor neuron disease in these mouse models.

Ferritins are highly conserved multi-subunit proteins that detoxify and reversibly store intracellular iron(1). Mice lacking the cytosolic ferritin heavy chain (H) exhibit embryonic lethality; mice with a conditional ferritin H deletion exhibit severe tissue damage(2,3). Mitochondrial ferritin (Ftmt) is structurally and functionally homologous to ferritin H chain(4,5). Elevated Ftmt expression in erythroblasts from sideroblastic anemia patients suggests a role for Ftmt in these anemias, a heterogeneous group of inherited and acquired disorders characterized by mitochondrial iron deposition(6). While cytosolic ferritin H and light (L) chains are ubiquitous in mammals, Ftmt mRNA is found largely in the testes(7,8). The lack of an iron responsive element in the Ftmt mRNA, while present in ferritin H and L mRNAs, suggests that Ftmt may function independently of iron metabolism. Studies to date have focused on protective effects of Ftmt overexpression against iron overload and oxidative stress in tissue culture, yeast and flies(9–15). To investigate the in vivo role of Ftmt, we deleted the Ftmt gene in mice and examined baseline hematology, iron metabolism and male fertility phenotypes. In addition, we subjected them to vitamin B6 (pyridoxine) deprivation to induce sideroblast/siderocyte formation. In all cases, we observed no significant defects in mice lacking mitochondrial ferritin. To construct Ftmt-deficient mice, we replaced the entire open reading frame (ORF) of the intronless Ftmt gene with a neomycin-resistance cassette (Fig. 1A). Southern blotting using probes flanking the region encompassed by the targeting construct indicated successful recombination at the Ftmt locus in embryonic stem (ES) cells (data not shown). We confirmed Ftmt deletion using PCR and primers specific to the flanking regions and the neomycin cassette (Fig. 1B). To measure Ftmt expression, we used reverse transcriptase-polymerase chain reaction (RT-PCR) and primers specific to β-actin and Ftmt; control reactions without reverse transcription were performed to ensure that PCR products did not represent amplification of genomic DNA. As Ftmt is most abundantly expressed in the testis, we measured expression in RNA isolated from testes of wild-type and homozygous littermates; no expression was detected in homozygous mutant animals (Fig. 1C). We could not detect Ftmt expression in bone marrow from either genotype, even after pyridoxine deprivation (data not shown; see below). Figure 1 Construction and analysis of Ftmt-deficient mice Ftmt−/− animals on the C57BL/6J genetic background at backcross generation greater than N7 were obtained at expected Mendelian frequencies from heterozygous pairings and no gross anatomic or histological abnormalities, including iron staining, were present in the mutant offspring (data not shown). Given the testis-specific expression, we assessed male fertility by pairing mutant and wild type males with two wild type females for 14 days and counted the number of pregnancies and total number of offspring obtained(16); based on this assessment, there was no difference in male fertility between mutants and controls (data not shown). To determine the effect of Ftmt deletion on baseline murine iron metabolism and hematology, we measured serum, spleen and liver iron concentrations, liver hepcidin expression and red blood cell parameters. There were no significant differences between wild-type and homozygous animals (Fig. 1D, 2A–G). To assess the role of genetic background, we bred the Ftmt deletion onto 129/SvEvTac for five generations (N5); we identified no significant differences between wild-type and homozygous animals on this background as well(data not shown). Given that baseline parameters of iron metabolism differ between these strains, this result suggests that alterations in serum or liver iron levels do not impact the steady state phenotype of Ftmt-deficient mice. Figure 2 Analysis of Ftmt-deficient mice on a pyridoxine-deficient diet We next placed a cohort of wild-type and homozygous mice on a pyridoxine-deficient diet for four months beginning at one month of age to induce formation of sideroblast or siderocyte (iron-granule positive nucleated or enucleated erythrocytes) (Fig. 2A–2G). Similar to observations made in C57BL/6J x C3H (B6C3F1) mice maintained on a pyridoxine-deficient diet(17), we noted a decreased MCV and hematocrit, along with decreased MCH, reticulocyte counts and CHR. The lack of decrease in HGB levels in our mice, while noted in the study in B6C3F1 mice, may have been due to strain-specific differences. While a small but statistically significant increase in red cell distribution width and an increase in number of siderocytes were noted in the homozygous animals early in the course of the experiment, these differences did not persist. Notably, the number of siderocytes was greater in homozygous than wild-type animals after three months on a pyridoxine-deficient diet; the decrease in siderocyte number thereafter in the homozygous animals may reflect an unknown mechanism of chronic adaptation to the pyridoxine-deficient diet (Fig. 2H). Siderocytes had similar appearance by light microscopy in wild-type and homozygous mutant animals (Fig. 3A–B), as did reticulocyte mitochondria by electron microscopy (Fig. 3C–D). As is true of most mouse sideroblastic anemia models, bone marrow ringed sideroblasts were not present in wild type or mutant animals (data not shown). Overall, there were no pronounced differences in red cell parameters between wild-type and homozygous mice maintained on the pyridoxine-deficient diet. Figure 3 Microscopic evaluation of Ftmt-deficient erythrocytes Our results do not contradict the relatively small body of Ftmt literature. Most, if not all, functional studies to date have relied on overexpression to demonstrate that Ftmt can alter the cellular effects of challenges such as oxidative stress under iron-loaded conditions. We have shown that Ftmt is not essential for viability or male fertility and that Ftmt deletion does not alter several parameters of iron metabolism under baseline conditions, nor does it affect the hematological response to a pyridoxine-deficient diet. Intriguingly, even in the absence of Ftmt, iron-rich deposits can be formed in erythroid cells, suggesting that Ftmt is not essential for siderocyte formation in mice. Furthermore, there may be redundant systems in mice to compensate for the lack of Ftmt function, as is the case with endothelial nitric synthase- or myoglobin-deficient mice in which adaptive mechanisms thoroughly compensate for the respective gene defects(18). One can also speculate that a failure to induce Ftmt expression on a pyridoxine-deficient diet might account for the fact that bone marrow ringed sideroblasts are not typically seen in mice. This demonstrates some of the limitations of using the mouse as a model for sideroblastic anemia—a failure to induce sideroblast formation in mice prevents us from effectively determining if Ftmt plays a role in detoxification of erythroid mitochondrial iron.

Copper is an essential trace metal which plays a fundamental role in the biochemistry of the human nervous system. Menkes disease and Wilson disease are inherited disorders of copper metabolism and the dramatic neurodegenerative phenotypes of these two diseases underscore the essential nature of copper in nervous system development as well as the toxicity of this metal when neuronal copper homeostasis is perturbed. Ceruloplasmin contains 95% of the copper found in human plasma and inherited loss of this essential ferroxidase is associated with progressive neurodegeneration of the retina and basal ganglia. Gain-of-function mutations in the cytosolic copper enzyme superoxide dismutase result in the motor neuron degeneration of amyotrophic lateral sclerosis and current evidence suggests a direct pathogenic role for copper in this process. Recent studies have also implicated copper in the pathogenesis of neuronal injury in Alzheimer's disease and the prion-mediated encephalopathies, suggesting that further elucidation of the mechanisms of copper trafficking and metabolism within the nervous system will be of direct relevance to our understanding of the pathophysiology and treatment of neurodegenerative disease.

Like iron, manganese is an essential yet potentially toxic metal. While inherited forms of iron excess were discovered long ago, the first disease of inherited manganese excess was only recently reported in 2012. Mutations in a metal transport protein, SLC30A10, were described in patients with manganese excess, polycythemia, liver failure and Parkinsonism. Manganese excess in these patients is attributed to an essential role for SLC30A10 in hepatobiliary manganese excretion. Polycythemia, liver failure and Parkinsonism are attributed to manganese excess although the underlying mechanisms have yet to be established. To explore the role of SLC30A10 in metal biology and disease, we have generated Slc30a10-deficient mice. Characterization of these mice indicates that murine Slc30a10 deficiency recapitulates the human disease. Given that manganese and iron share some transport pathways, we interrogated iron homeostasis in these mutant mice. Intriguingly, mice display hepcidin deficiency and tissue iron excess. While hepcidin deficiency may explain the tissue iron excess, the cause of hepcidin deficiency is unclear. Based on expression profiling of hypoxia-regulated genes, we propose that hepcidin deficiency reflects aberrant activation of hypoxia signaling by manganese excess in the absence of true hypoxia. Overall, this data suggests novel interactions between iron and manganese with implications for our understanding of the mechanisms by which aberrant metal homeostasis leads to human disease. Disclosures No relevant conflicts of interest to declare.

Bone morphogenetic protein (BMP) signaling induces hepatic expression of the peptide hormone hepcidin. Hepcidin reduces serum iron levels by promoting degradation of the iron exporter ferroportin. A relative deficiency of hepcidin underlies the pathophysiology of many of the genetically distinct iron overload disorders, collectively termed hereditary hemochromatosis. Conversely, chronic inflammatory conditions and neoplastic diseases can induce high hepcidin levels, leading to impaired mobilization of iron stores and the anemia of chronic disease. Two BMP type I receptors, Alk2 (Acvr1) and Alk3 (Bmpr1a), are expressed in murine hepatocytes. We report that liver-specific deletion of either Alk2 or Alk3 causes iron overload in mice. The iron overload phenotype was more marked in Alk3- than in Alk2-deficient mice, and Alk3 deficiency was associated with a nearly complete ablation of basal BMP signaling and hepcidin expression. Both Alk2 and Alk3 were required for induction of hepcidin gene expression by BMP2 in cultured hepatocytes or by iron challenge in vivo. These observations demonstrate that one type I BMP receptor, Alk3, is critically responsible for basal hepcidin expression, whereas 2 type I BMP receptors, Alk2 and Alk3, are required for regulation of hepcidin gene expression in response to iron and BMP signaling.

In this issue of Blood, Zumerle et al elegantly demonstrate that hepatocytes are the key site of synthesis of hepcidin, a central regulator of iron homeostasis deficient or aberrantly expressed in hereditary hemochromatosis, β-thalassemia, anemia of inflammation, and other iron-related conditions.1

The copper chaperone for superoxide dismutase (CCS) is an intracellular metallochaperone required for incorporation of copper into the essential antioxidant enzyme copper/zinc superoxide dismutase (SOD1). Nutritional studies have revealed that the abundance of CCS is inversely proportional to the dietary and tissue copper content. To determine the mechanisms of copper-dependent regulation of CCS, copper incorporation into SOD1 and SOD1 enzymatic activity as well as CCS abundance and half-life were determined after metabolic labeling of CCS-/- fibroblasts transfected with wild-type or mutant CCS. Wild-type CCS restored SOD1 activity in CCS-/- fibroblasts, and the abundance of this chaperone in these cells was inversely proportional to the copper content of the media, indicating that copper-dependent regulation of CCS is entirely post-translational. Although mutational studies demonstrated no role for CCS Domain I in this copper-dependent regulation, similar analysis of the CXC motif in Domain III revealed a critical role for these cysteine residues in mediating copper-dependent turnover of CCS. Further mutational studies revealed that this CXC-dependent copper-mediated turnover of CCS is independent of the mechanisms of delivery of copper to SOD1 including CCS-SOD1 interaction. Taken together these data demonstrate a mechanism determining the abundance of CCS that is competitive with the process of copper delivery to SOD1, revealing a unique post-translational component of intracellular copper homeostasis.

An abundant serum metal-binding protein, transferrin is a critical factor in mammalian iron homeostasis. As transferrin is essential for iron delivery for erythropoiesis, transferrin-deficient patients and mice develop anemia but also a paradoxical systemic iron overload. Iron overload results from deficiency in hepcidin, a peptide hormone synthesized largely by the liver that inhibits dietary iron absorption and macrophage iron efflux. Using transferrin-deficient mice, we have previously shown that transferrin deficiency leads to hepcidin deficiency via two mechanisms. First, the anemia and/or hypoxia that results from inadequate iron delivery to bone marrow actively suppresses hepcidin expression by the liver. Second, transferrin directly stimulates hepcidin expression by the liver independently of transferrin's role in erythropoiesis; this second mechanism is dependent upon hemojuvelin, a bone morphogenetic protein coreceptor essential for hepcidin expression that is mutated in juvenile hemochromatosis. Here we present data examining the reversibility of iron loading in transferrin deficiency. Measurement of tissue metal levels in adult transferrin-deficient mice demonstrated progressive and systemic iron loading yet minimal to moderate changes in levels of other metals. Treatment of adult transferrin-deficient mice with exogenous transferrin normalized red cell parameters and hepcidin levels and reduced iron load in most organs. To further investigate the reduced tissue iron burden in transferrin-treated mice, we employed iron-loaded cell lines to demonstrate that cellular iron efflux can be stimulated by transferrin and that this efflux is specific to iron but not manganese or zinc. Current studies focus on two goals. First, we are determining if the reduction in tissue iron load represents solely a redistribution of iron stores to the erythron or additionally excretion of iron via urine and/or feces by measuring whole body iron levels in mice before and after transferrin treatment. Second, we are identifying transporters required for mobilization of iron stores using expression profiling of known and putative iron transporters in mouse tissues and validating the role of transporters of interest using our cell culture model. Results of these studies and their implications will be presented. Disclosures No relevant conflicts of interest to declare.

Prion diseases are neurodegenerative disorders that result from conformational transformation of a normal cell surface glycoprotein, PrP(C), into a pathogenic isoform, PrP(Sc). Although the normal physiological function of PrP(C) has remained enigmatic, the recent observation that the protein binds copper ions with micromolar affinity suggests a possible role in brain copper metabolism. In this study, we have used mice that express 0, 1, and 10 times the normal level of PrP to assess the effect of PrP expression level on the amount of brain copper and on the properties of two brain cuproenzymes. Using mass spectrometry, we find that the amount of ionic copper in subcellular fractions from brain is similar in all three lines of mice. In addition, the enzymatic activities of Cu-Zn superoxide dismutase and cytochrome c oxidase in brain extracts are similar in these groups of animals, as is the incorporation of (64)Cu into Cu-Zn superoxide dismutase both in cultured cerebellar neurons and in vivo. Our results differ from those of another set of published studies, and they require a re-evaluation of the role of PrP(C) in copper metabolism.

Recent studies in Saccharomyces cerevisiae suggest that the delivery of copper to Cu/Zn superoxide dismutase (SOD1) is mediated by a cytosolic protein termed the copper chaperone for superoxide dismutase (CCS). To determine the role of CCS in mammalian copper homeostasis, we generated mice with targeted disruption of CCS alleles ( CCS −/− mice). Although CCS −/− mice are viable and possess normal levels of SOD1 protein, they reveal marked reductions in SOD1 activity when compared with control littermates. Metabolic labeling with 64 Cu demonstrated that the reduction of SOD1 activity in CCS −/− mice is the direct result of impaired Cu incorporation into SOD1 and that this effect was specific because no abnormalities were observed in Cu uptake, distribution, or incorporation into other cuproenzymes. Consistent with this loss of SOD1 activity, CCS −/− mice showed increased sensitivity to paraquat and reduced female fertility, phenotypes that are characteristic of SOD1-deficient mice. These results demonstrate the essential role of any mammalian copper chaperone and have important implications for the development of novel therapeutic strategies in familial amyotrophic lateral sclerosis.

Studies in hpx mice, a model of inherited transferrin deficiency, have demonstrated that transferrin is essential for iron delivery to the erythron and regulation of expression of hepcidin, a hormone that inhibits dietary iron absorption. Here we address two other putative roles for s transferrin. First, we determined if transferrin is essential for metabolism of metals other than iron. Metal content analysis of fractionated mouse sera indicated that iron was the most abundant metal in transferrin-rich fractions. Organ metal content analysis demonstrated severe imbalances for iron and minimal to moderate imbalances for copper, zinc and manganese in hpx tissues. Analysis of metal-dependent gene expression and organ metal content in a mouse model of inherited hepcidin excess suggested that hpx tissue imbalances in copper, zinc and manganese levels were not all physiologic or specific to primary transferrin deficiency. Second, we tested if transferrin modulates the mobilization of tissue iron stores. Transferrin treatment of hpx mice decreased iron levels in most tissues analyzed. Iron mobilization was independent of hepcidin, as iron levels decreased in hpx mice deficient in hemojuvelin, a membrane protein essential for hepcidin expression. Iron mobilization led to decreased ferritin iron and heavy chain levels. The decrease in liver ferritin iron stores did not match the decrease in total liver iron stores, suggesting that iron was mobilized from multiple storage sites. Overall, our analysis suggests that the primary consequences of transferrin deficiency are largely specific to iron and that transferrin can modulate the mobilization of tissue iron stores. Disclosures: No relevant conflicts of interest to declare.

Abstract 612 Sporadic iron overload has been reported previously in rats but the underlying cause has not been ascertained. In this study, phenotypic analysis of a subpopulation of Wistar rats designated Hsd:HHCL revealed a low incidence of histologically detected liver iron overload. One rat out of 132 screened animals exhibited liver iron accumulation in a predominantly periportal, hepatocellular distribution; this male rat expressed low RNA levels of the iron regulatory hormone hepcidin and low protein levels of transferrin receptor 2, a membrane protein essential for hepcidin expression in humans and mice and mutated in forms of hereditary hemochromatosis, a disease of excessive intestinal iron absorption and progressive tissue iron overload. Sequencing of the transferrin receptor 2 gene in the iron-overloaded rat revealed a novel Ala679Gly polymorphism affecting a highly conserved residue. Quantitative trait locus mapping revealed that a transferrin receptor 2 polymorphism correlated strongly with serum iron and transferrin saturations in male rats. Transfection of Tfr2 expression constructs into tissue culture cell lines revealed that the Gly679 Tfr2 variant is expressed at a lower level than the Ala679 variant. Selective breeding of rats carrying this polymorphism and characterization of iron metabolism in the resulting progeny indicated that homozygosity for the Ala679Gly allele leads to a hemochromatosis phenotype. The Hsd:HHCL rat is the first genetic rat model of hereditary hemochromatosis and may prove useful for understanding the molecular mechanisms underlying the regulation of iron metabolism and the pathogenesis of hereditary hemochromatosis. Disclosures: No relevant conflicts of interest to declare.

Copper is an essential transition metal that plays a fundamental role in the biochemistry of all aerobic organisms. Recent work elucidating the structural mechanisms of copper delivery to superoxide dismutase provides insight into the cell biology of copper metabolism and serves as an example of how to understand the principles governing the incorporation of metals into proteins.