Extensive evidence shows that in erythrocyte precursors and other cell types dependent on transferrin for their iron supply, DMT1 fulfills a critically important and nonredundant role by transporting to the cytoplasm iron delivered by transferrin 73 , Furthermore, DMT1 and a related molecule, natural resistance associated macrophage protein-1 Nramp1 , are also involved in iron transport in macrophages — , but their specific function in these cells remains uncertain.
Thus, despite its name, the transporter seems to be essential for normal iron homeostasis but less important for that of the other divalent metals. Initial studies of DMT1 were greatly facilitated by the discovery of mice microcytic anemia or Mk mouse and rats Belgrade rat with hypomorphic mutations in DMT1 73, More recently, human mutations giving rise to microcytic anemia have also been identified reviewed in Ref.
These hypomorphic defects in mice, rats, and humans affect iron homeostasis in a complex manner, usually causing severe microcytic anemia due to decreased ability of erythrocyte precursors to utilize transferrin-bound iron, as well as causing demonstrable deficits in intestinal iron absorption, accompanied by paradoxical hyperabsorption of iron through residual iron transport activity in the intestine 19 , , Animal models with complete tissue-specific ablation of DMT1 helped clarify the role of DMT1 in intestinal iron transport. Conditional intestine-specific knockout mice generated by crossing floxed DMT1 and villin-Cre mice develop postnatal anemia and systemic iron deficiency but can be rescued by parenteral iron administration bypassing the intestinal tract.
This finding establishes the essential role of DMT1 in intestinal iron transport. Human DMT1 is found in at least 4 isoforms which share a core of amino acids but differ in the NH 2 or COOH termini as a result of differing transcription initiation sites and exon splicing Most importantly, the mRNAs for some of the isoforms contain a 3' iron-responsive element 3'IRE that binds iron-regulatory proteins 1 and 2 to stabilize the mRNA and increase DMT1 synthesis when cellular iron concentrations are low.
Effective iron transport by DMT1 depends on the concentration of ferrous iron and on the cotransport of protons.
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DMT1 function therefore requires the conversion of dietary ferric iron to ferrous iron prior to its transport and an acid microenvironment in the brush border of enterocytes. Duodenal cytochrome B dcytB contributes to the reduction of luminal ferric iron but is not required for DMT1 function 42 , , perhaps because other ferric reductase activities contribute as well. The distinct role of dcytB is highlighted by its effect as a genetic modifier of iron overload in hereditary hemochromatosis Ascorbate, a known potentiator of dietary iron absorption, increases the reductase activity of dcytB, likely by acting as its preferred intracellular electron donor There are currently two candidates for heme transporters that move heme into the cytoplasm.
Heme carrier protein 1 HCP1 was isolated by subtractive hybridization of duodenal versus ileal genes from hypotransferrinemic mice where iron-related transporters are expected to be highly induced. Although the molecule is clearly capable of transporting heme, it later turned out to transport folate and to be mutated in patients with genetic folate deficiency , indicating that its nonredundant function was folate uptake in the duodenum.
Morever, mice with ablated HCP1 developed folate deficiency anemia and could be rescued by parenteral folate derivatives but not by parenteral iron or heme. The contribution of HCP1 to heme transport remains to be determined but is clearly not essential. The second mammalian heme uptake transporter, heme responsive gene-1, was cloned by homology to heme transporters identified in the heme auxotroph Caenorhabditis elegans It localizes to the phagolysosomes of macrophages 57 , , suggesting that it could be involved in the transport of heme recovered from senescent erythrocytes and its recycling for iron Figure 2.
Ferritin is a spherical heteropolymeric protein composed of 24 subunits of heavy H or light L type. Relevant to systemic iron homeostasis, cytoplasmic ferritin can store large amounts of iron in its interior. Targeted deletion of the H-subunit in the intestine caused systemic iron dysregulation with increased intestinal iron absorption and mild systemic iron overload manifested by increased plasma and hepatic iron concentrations The ability of the ferritin compartment to store iron in enterocytes may be required for controlled delivery of iron to the basolateral iron exporters.
Ferrous iron is delivered to ferritin by cytoplasmic chaperones, chiefly poly rC -binding protein 1 PCBP1 Exit of iron from ferritin may occur through gated pores or by autophagy and lysosomal degradation of ferritin , How iron transits from ferritin to iron exporters is not known. A soluble, relatively iron-poor form of ferritin is found in blood plasma. This form is a subunit polymer containing mostly l -ferritin, and is derived primarily from macrophages Serum concentrations of ferritin correlate with iron stores in most but not all physiological and pathological conditions 46 , , , with exceptions reflecting pathological situations in which the macrophages are much less or much more iron-loaded than parenchymal tissue, or situations where ferritin synthesis is primarily driven by inflammation.
The sole known mammalian iron exporter is ferroportin [also called Scl40a1, iron-regulated gene 1 IREG1 , or metal transporter protein 1 MTP1 ] 1 , 59 , It is expressed at all sites involved in iron transfer to plasma Figure 1 , i. Like DMT1, ferroportin is thought to be a transmembrane domain protein with both termini in the cytoplasm , , but the exact boundaries of the exposed segments of its extracellular and cytoplasmic faces are not certain , FPN1B is highly expressed in the duodenum and in erythroid precursors, allowing perhaps for altruistic export of iron by these cells even when they sense iron deficiency A mutation in the 5'IRE causes transient polycythemia in mice by a mechanism that is not well understood.
Cellular iron export is dependent on members of a family of copper-containing ferroxidases , including ceruloplasmin, hephaestin and perhaps also Zyklopen 37 , 38 , , that use molecular oxygen to oxidize ferrous to ferric iron Figure 2.
Ceruloplasmin is a kDa copper-containing protein highly expressed in the liver and the retina. Alternative splicing generates a membrane GPI-linked form and a soluble plasma form. Hephaestin and zyklopen are related and kDa transmembrane proteins expressed predominantly in enterocytes and the placenta, respectively.
All three ferroxidases are found in the brain. Hephaestin-deficient mice sex-linked anemia or sla manifest iron deficiency anemia with accumulation of iron in enterocytes , indicating that the basolateral transfer of iron to plasma is defective. Ceruloplasmin deficiency impedes both intestinal iron absorption and the release of iron from macrophages 37 , 38 , , , and causes accumulation of iron in the brain and in hepatocytes , It remains to be established how the four known ferroxidase forms cooperate to provide ferroxidase function for enterocytes, macrophages, hepatocytes, and the placenta.
Feline leukemia virus, type C, receptor 1 FLVCR1 is a transmembrane domain kDa protein and the sole known heme exporter whose ablation in mice causes a severe fetal anemia lethal in mid-gestation FLVCR1b is required for erythroid development and differentiation, presumably because without it heme does not reach the cytoplasm and is not incorporated into hemoglobin. The full-length form of FLVCR1a is found in the plasma membrane and is not required for erythroid development. Selective disruption of FLVCR1a causes embryonic lethality by interfering with vascular and skeletal development and causing hemorrhages.
Thus FLVCR1b is essential for heme export from mitochondria to the cytoplasm but does not appear to be involved in systemic iron homeostasis. Under normal circumstances, ferric iron exported from cells becomes bound to the plasma iron carrier transferrin, a to kDa glycosylated protein that can carry up to two ferric ions, and deliver them to target tissues for uptake by the transferrin receptor-1 TfR1.
The essential and nonredundant role of transferrin in delivering iron for erythropoiesis is revealed by the severe anemia in genetic hypotransferrinemia or atransferrinemia in humans and in mice reviewed in Refs.
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Paradoxically, the disorder results in systemic iron overload, showing that other tissues can take up non-transferrin-bound iron NTBI in quantities that meet or exceed their requirements. In atransferrinemia, or if iron enters plasma in excess of the carrying capacity of transferrin, iron becomes complexed to citrate, acetate, and albumin, and these NTBI forms are taken up by tissues by alternative mechanisms reviewed elsewhere 12 , 24 , 49 , In addition to the NTBI, plasma ferritin may also deliver iron to some tissues 66 , , but the relative physiological contribution of this process is not understood.
In addition to carriers that bind inorganic iron, hemopexin and haptoglobin are plasma proteins that bind free heme and free hemoglobin, respectively, limiting their toxic effects and scavenging them for recycling into iron , , Hemopexin and haptoglobin have an important homeostatic role during hemolytic stress and diseases Moreover, iron absorption is increased in mice or humans during periods of iron deficiency, and absorption is decreased by parenteral iron overload reviewed in Ref. These observations have led to the expectation that one or more systemically acting hormones regulate the major flows of iron and are in turn regulated by iron Surprisingly, the hormone and its function in iron homeostasis were only discovered during the last decade history reviewed in Ref.
The iron-regulatory hormone hepcidin is a 2.
The NH 2 -terminal hepcidin segment of six amino acids is highly conserved, unstructured, and essential for the iron-regulatory function of hepcidin and its interaction with its receptor ferroportin The mature amino acid peptide is generated from an amino acid prepropeptide containing a characteristic NH 2 -terminal amino acid signal sequence which is cleaved to yield the cellular intermediate prohepcidin , , , , There is no evidence that this intermediate has any function other than as a precursor of mature hepcidin.
The generation of the mature amino acid form requires furin-like prohormone convertases that cleave prohepcidin at the COOH-terminal peptide bond after a characteristic polybasic sequence In addition, NH 2 -terminally truncated shorter forms 22 and 20 amino acids are also found in human urine and the amino acid form in human plasma, generally at much lower concentrations than the full-length amino acid hepcidin 27 , Human hepcidin is encoded by a single three exon gene on chromosome 19 , , , and a similar three exon structure is conserved among other vertebrate hepcidin genes.
In the mouse, there are two hepcidin genes, but only hepcidin-1 is involved in iron homeostasis Among other vertebrate species, some fish also have two or more hepcidin genes 61 , , some of which are not liver-specific and appear to be transcribed only during infections These findings suggest that hepcidin may have evolved in early vertebrates, perhaps as an antimicrobial peptide that became secondarily involved in iron regulation.
Figure 3. Hepcidin: the amino acid sequence and structure. The NH 2 -terminal segment known to interact with ferroportin is shaded in light red. The characteristic cysteines and their disulfide bonds are shown in yellow. Hepcidin acts by posttranslationally controlling the membrane concentration of its receptor, the sole known cellular iron exporter ferroportin Figure 4.
As ferroportin is the transporter that delivers dietary, stored, or recycled iron to blood plasma, the hepcidin-ferroportin interaction effectively controls the flux of iron into plasma and the iron supply available to all the iron-consuming tissues. Chronic transgenic overexpression of hepcidin causes iron deficiency anemia , , both by inhibiting iron absorption and restricting the release of stored iron.
Hepcidin overexpression during fetal life can impair iron transfer to the fetus sufficiently to cause severe iron deficiency anemia at birth with most mice dying perinatally At the other extreme, hepcidin deficiency in mice or humans causes hyperabsorption of iron and iron overload in parenchymal organs including the liver, pancreas, and the heart, coupled with the paradoxical loss of macrophage iron stores , , These effects of hepcidin excess or deficiency are evidence of the fundamental role of hepcidin in the control of iron absorption and the release of recycled iron from macrophages.
Importantly, the phenotype of hepcidin deficiency is mimicked by heterozygous human ferroportin mutations that interfere with hepcidin binding , , confirming the critical role of the hepcidin-ferroportin interaction in iron homeostasis, and suggesting that ferroportin may be the sole target of hepcidin. Figure 4. Human ferroportin topology and functional domains. Both termini are in the cytoplasm, and transmembrane helices are shaded in gray.
The extracellular hepcidin-binding loop is in red, with boundaries as determined by cysteine-scanning mutagenesis and cysteine-directed biotinylation Key residues required for hepcidin binding are shown in dark red. The cytoplasmic loop that undergoes hepcidin-dependent ubiquitination is shown in blue. The AP-2 adapter-binding motif for clathrin-dependent endocytosis is shown in green. An axis of approximate symmetry is shown by a dashed red line. Although ferroportin is evolutionarily ancient with conserved sequences down to plants, worms, and other multicellular animals, its ligand hepcidin is found only in vertebrates, with the possible exception of birds The absence of hepcidin in invertebrates suggests that alternative mechanisms for systemic regulation of ferroportin may exist in invertebrates and persist in vertebrates, although they may not be sufficiently effective to compensate for pathological situations in which hepcidin is deficient or excessive.
The two factors that may affect iron homeostasis in a hepcidin-independent manner are hypoxia and cellular iron deficiency, when affecting cells and tissues involved in systemic iron transport. In the mouse, duodenal ferroportin mRNA is increased by hypoxia and iron deficiency 39 , , , , and hypoxia and anemia increase ferroportin mRNA in macrophages but not hepatocytes It should be noted that a potential counterregulatory mechanism could decrease ferroportin during cellular iron deficiency.
It is possible that direct regulation of ferroportin by cellular hypoxia and cellular iron deficiency functions best in small animals where the oxygen tension and iron content of iron-transporting cells are representative of the organisms as a whole.
Iron metabolism: current facts and future decisions
Tissue differences in iron content and oxygen tension in larger animals may have favored a more centralized model of iron regulation with a systemically acting hormone, hepcidin, produced by the principal iron storage organ, the liver. It remains to be determined whether hypoxic regulation represents the evolutionarily more ancient form of iron homeostasis.
If so, hepcidin may have initially evolved as a vertebrate host defense mediator see sect.
IV that overrode the more primitive iron homeostatic responses during infection, and hepcidin may then have been secondarily adopted and evolved for iron homeostasis. Hepcidin binds directly to ferroportin, inducing the endocytosis of ferroportin and its proteolysis in lysosomes The concentrations of hepcidin required for this effect are in the 10— nM range , and these correspond approximately to the upper range of hepcidin concentrations in human blood plasma under pathological conditions causing hypoferremia The identification of the binding interface between hepcidin and ferroportin was achieved by studying the effects of natural and experimental mutations in both partners 43 , , For hepcidin, the NH 2 -terminal five amino acids were necessary for bioactivity , and the NH 2 -terminal nine amino acids Figure 3 were sufficient for bioactivity provided that the C7 cysteine involved in disulfide bonding was replaced by a thiol cysteine.
On the ferroportin side, identification of the potential receptor site was greatly aided by the discovery of an informative family which manifested resistance to hepcidin as a result of an isosteric CS substitution in ferroportin 63 , 67 , , , located in an extracellular loop Figure 4 in both of the reasonably supported ferroportin models , Alanine scanning of this ferroportin loop identified additional residues critical for hepcidin binding In addition to the critical role of the ferroportin thiol C in binding hepcidin, several aromatic amino acid side chains phenylalanine and tyrosine in the interacting segments of hepcidin and ferroportin were experimentally identified as important for hepcidin-ferroportin interactions, and were seen to interact in a docking model of hepcidin to the hepcidin-binding loop of ferroportin The binding of hepcidin to ferroportin is followed within minutes by the ubiquitination of lysines in a cytoplasmic loop of ferroportin Figure 4 which appears to be required for the subsequent endocytosis of ferroportin , An earlier report that phosphorylation of a pair of adjacent tyrosines preceded ubiquitination and was required for endocytosis 54 could not be verified despite extensive efforts It is not yet certain whether ferroportin endocytosis is mediated by clathrin 54 , one of the alternative endocytic pathways 9 or both, and there could be differences between the predominant endocytic pathways in different cell types.
The relative stability of plasma iron concentrations despite rapid turnover of iron suggests feedback regulation of hepcidin by plasma iron Figure 5. Experimentally, regulation of hepcidin by plasma iron concentrations was detected in human volunteers 84 given small doses of iron sufficient to raise plasma iron concentrations transiently but too small to contribute significantly to iron stores. On the other hand, the relatively narrow distributions of estimated body iron stores in men and women on varied diets suggest that body stores could regulate hepcidin independently of the short-term effects of plasma iron concentrations Surprisingly, experimental evidence of dual regulation of hepcidin by tissue iron stores and plasma iron concentrations was not obtained until recently 47 , 66 , , In humans, observations that support hepcidin regulation by iron stores include the correlation between hepcidin mRNA and iron stores in human liver biopsies 7 , 58 , 88 and the strong correlation between serum ferritin, a recognized marker of iron stores, and serum hepcidin Hepcidin regulation by plasma iron and by tissue iron stores appears to operate on different time scales hours vs.
Figure 5. Regulation of hepcidin synthesis in hepatocytes. The major regulatory influences include iron-transferrin and iron stores blue , inflammation green , and erythroid activity red. Hepatocytes are the predominant producers of hepcidin , , , Unlike the distribution of ceruloplasmin and ferroportin which is predominantly periportal , , hepcidin mRNA appears to be evenly distributed among hepatocytes Other cell types including macrophages and adipocytes 15 , contain much lower concentrations of hepcidin mRNA.
Although non-hepatocyte sources could in principle exert autocrine or paracrine effects, these have not yet been documented. To date, the only known mode of hepcidin regulation is transcriptional. The molecular mechanisms that mediate hepcidin regulation by iron appear to be surprisingly complicated Figure 6. Much of what we know about these mechanisms was learned through studies of human genetic diseases or mouse transgenic models in which hepcidin is dysregulated. Table 1 lists genes implicated in hepcidin regulation by iron. Figure 6. Molecular pathways regulating hepcidin transcription.
Iron-related mediators are shown in blue, and inflammatory mediators are in green. The erythroid regulator red and its transduction pathways are not known. Table 1. Genetic lesions in hepcidin regulators and their phenotypic consequences. The BMP receptor and its canonical SMAD pathway are at the core of the hepcidin-regulating complex as indicated by the strong induction of hepcidin by multiple BMPs 10 , 11 , the presence of several functional BMP-response elements in the hepcidin promoter 31 , , , and the profound effect of liver-specific SMAD4 ablation on hepcidin expression BMP6 is the essential and specific BMP receptor ligand for iron-related signaling, at least in mice, as BMP6 knockout mice have very low hepcidin and develop severe iron overload, with no evidence of any other abnormalities 6 , The GPI-linked protein hemojuvelin is an essential co-receptor for activation of the BMP receptor for iron-related signaling.
Its ablation leads to severe hepcidin deficiency and severe iron overload in humans early onset, juvenile form of hereditary hemochromatosis and in mice , , The hepatic form of hemojuvelin appears to be essential for hepcidin regulation while the muscle form is dispensable 36 , The role of muscle-associated hemojuvelin remains to be elucidated. A soluble form of hemojuvelin is generated by proteolytic cleavage by furin , , and can act as an antagonist of BMP signaling, but its physiological role is not known.
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The nature of the extracellular iron sensors and how they couple to the BMP pathway is less certain. Transferrin receptor 2 is a strong candidate as a sensor of extracellular holotransferrin concentration.