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The Journal of Nutrition Vol. 128 No. 11 November 1998,
pp. 1841-1844
Mineral Bioavailability Laboratory, USDA Human Nutrition Research Center on Aging at Tufts University, Boston MA 02111
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ABSTRACT |
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Abstract
Introduction
References
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Gene mapping techniques to identify gene mutations in humans and animals with phenotypic abnormalities in iron metabolism are providing important insights into the probable molecular mediators of intestinal iron absorption. Positional gene cloning in humans with hereditary hemochromatosis has identified a mutation in a novel major histocompatibility complex (MHC) gene called HFE that is likely to be involved in regulating intestinal iron absorption. In addition, recent observations based on positional cloning strategies in the mk/mk mouse and the Belgrade (b/b) rat rodent models of hypochromic, microcytic anemia have shown that the phenotypic abnormality in iron metabolism is associated with a mutation in the Nramp2 gene. Functional cloning studies in Xenopus oocytes have characterized DCT1 (Nramp2) as an iron-regulated proton-coupled divalent cation transporter. Nramp2 is likely to be the membrane transporter that functions in controlling iron entry across the apical membrane and in the export of iron out of endosomal vesicles. The observation that the expression of both HFE and Nramp2 mRNAs are reciprocally regulated by cellular iron status in Caco-2 cells, a human intestinal cell line, lends additional credence to the notion that these proteins may work in concert to regulate intestinal iron absorption.
KEY WORDS: HFE · Nramp2 · DCT1 · hemochromatosis
membrane transporters
The continued progress of the Human Genome Project will offer a plethora of new genes that undoubtedly will have relevance to our understanding of mineral absorption and metabolism. The impact of new investigational techniques in molecular biology and functional genomics for identifying and characterizing genes of nutritional relevance is already evident. It is incumbent on the community of nutritional scientists to embrace this new world of the 21st century so that we may deepen our current understanding of nutritional biochemistry, as well as be able to ponder the new and offer our own unique perspective.
Yeast Iron Transport.
Intestinal Iron Absorption.
INTRODUCTION
Abstract
Introduction
References
, Zhou and Gitschier 1997). Recent advances in the molecular genetics of metal transport in yeast have been reviewed in detail (Askwith et al. 1996) and will be summarized here only briefly because homologues of at least some of these genes will likely play an important role in cellular iron transport in mammals. At least eight different gene products (FRE1, FRE2, FET3, FET4, FTR1, CTR1, CCC2 and AFT1) are involved in iron metabolism in the yeast S. cerevisiae (Fig. 1). Yeast utilize at least two different iron uptake systems depending on the bioavailability of iron. A specific, two-component, iron transport system, composed of a high-affinity ferric (Fe+3) iron transporter (FTR1) and a multi-copper oxidase (FET3) is brought into play when environmental iron is limiting. This oxidase is a membrane protein that displays high sequence and functional homology to ceruloplasmin. Proper functioning of the yeast iron transport system requires the involvement of three additional gene products: CTR1, which is a plasma membrane copper transporter; CCC2, an intracellular copper transporting protein homologous to the human Menkes copper ATPase; and AFT1, a putative transcription factor that regulates transcription of iron-dependent genes. Under iron-replete environmental conditions, a low-affinity ferrous (Fe+2) iron transporter (FET4) is apparently sufficient to acquire iron. This low-affinity membrane transporter is not inducible by iron deprivation and can also transport other metals, including cobalt, cadmium and nickel. The cell surface is supplied with Fe+2 by the action of the ferric reductases FRE1 and FRE2. Iron deprivation increases ferrireductase activity in S. cerevisiae. Additional genes involved in iron transport in yeast are likely to be identified and characterized in the future. Moreover, it is clear that the process of iron metabolism is quite complicated even in simple eukaryotic organisms, such as yeast. Undoubtedly, the orchestration of cellular iron transport and metabolism in higher organisms will reveal a similar level of complexity.
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Fig 1.
Iron transport in the yeast Saccharomyces cerevisiae. Iron is taken up into yeast under iron-replete conditions by a low-affinity ferrous (Fe+2) iron transporter (FET4). Ferrous iron is supplied to yeast through the action of cell surface ferrireductases (FRE1 and FRE2). When iron is limiting, uptake occurs via a two-component, high-affinity iron transport system. The latter system consists of an iron-inducible high-affinity ferric (Fe+3) iron transporter (FTR1) and a multi-copper oxidase (FET3). Additional cellular components necessary for iron transport in yeast include a plasma membrane copper transporter (CTR1), an intracellular Menkes-like copper ATPase (CCC2) and the transcription factor AFT1.
). The movement of iron through the enterocyte likely involves proteins that act as membrane carriers or channels and intracellular transport proteins or "chaperones" that deliver iron to specific cellular locations, including the sites of iron exit from the enterocyte on the basolateral membrane.
Apical iron entry.
Studies in humans have shown that absorption of ferrous iron salts is superior to ferric salts. However, this observation may only reflect the relatively greater solubility of ferrous compared to ferric forms of iron in the intestinal lumen, rather than reflecting two distinct types of iron transporters. Ferrireductase activity has been demonstrated in the intestine of humans (Riedel et al. 1995) and in apical membrane preparations of Caco-2 cells, a human colon adenocarcinoma cell line (Ekmekcioglu et al. 1996). Moreover, the importance of reducing ferric iron to ferrous iron for optimal iron transport in the enterocyte is supported by the observation that inhibition of ferrireductase activity in Caco-2 cells reduces iron transport (Han et al. 1995, Nunez et al. 1994).
reported the presence of a saturable, temperature-dependent, iron uptake process in apical membrane vesicles prepared from human upper small intestine. These investigators were also able to isolate from these membranes a putative iron transport protein, which they characterized as a 160-kD iron-binding glycoprotein composed of three 54-kD monomers. Based on its ability to bind iron, Conrad and colleagues (1993) have suggested that integrin, consisting of a 150- and a 90-kD protein chain, found on intestinal microvilli in rat duodenum is an important cell-surface mediator of iron absorption.
Intracellular iron trafficking.
Important molecular details of transferrin-mediated iron uptake and processing within the endosomal compartment in various cell types have been elucidated. However, the molecular details of intracellular trafficking of iron that has been absorbed across the apical membrane of the enterocyte and is destined for absorption into the blood stream remains a mystery. Intestinal proteins that can bind iron have been described and may be involved in the vectorial transfer of iron across the intestine or may serve as chaperone proteins to deliver iron to specific cellular organelles or to other intracellular proteins. For example, Conrad and colleagues have proposed a sequential relay of iron, from mucin found in the intestinal lumen to integrin on the apical cell surface to the intracellular protein mobilferrin, as a pathway of iron absorption in the intestine (Conrad et al. 1993). However, in general, the regulatory function, if any, of these iron-binding proteins in iron trafficking has yet to be adequately described.
Basolateral iron exit. Very little is known about the processes involved in iron exit from the enterocyte. Iron appears to be released from the basolateral membrane in the reduced ferrous state and by a temperature-dependent mechanism. The subsequent sojourn of iron across the interstitial space and through endothelial cells of the portal capillary system may be mediated by small molecular weight molecules, such as bicarbonate, that eventual deliver iron to circulating transferrin.
Molecular Aspects of Iron Absorption.
Our increasing understanding of the molecular basis of genetic disorders of iron metabolism in mammalian species is providing new insights into genes involved in mediating intestinal iron absorption. For example, two genes, HFE and Nramp2, have been identified by positional cloning strategies from humans or animals with known genetic defects in intestinal iron absorption. A new model of intestinal iron transport is shown in Figure 2.
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HFE.
Hereditary hemochromatosis is a common autosomal recessive genetic disease in humans characterized by a defective regulation of iron absorption, an increase of plasma transferrin saturation and progressive systemic iron overload resulting in tissue damage. Iron absorption is not appropriately regulated in hereditary hemochromatosis because of a defect in one or more steps of mucosal iron transport. In hereditary hemochromatosis an altered set-point of intestinal iron absorption is evident such that the level of iron absorption is not appropriate to the body's iron stores. However, the molecular mechanisms underlying this intestinal defect are unclear. The recent report of an unexpected candidate gene mutation for hereditary hemochromatosis (Feder et al. 1996) has opened up exciting new avenues of investigation into the molecular mechanisms underlying the homeostatic regulation of intestinal iron absorption. This new candidate gene involved in hereditary hemochromatosis was originally designated as HLA-H, but has since been renamed HFE. The HFE gene is expressed at low levels in most, if not all, tissues, including the intestine. While the precise regulatory function of HFE in the intestine is unknown, HFE is presumably required in some way as a critical part of the cellular machinery that leads to the appropriate down-regulation of intestinal iron absorption that normally occurs with high body iron stores. The HFE protein has 343 amino acids and is similar to major histocompatibility complex (MHC) class I molecules, such as HLA-A2, and nonclassical MHC class-I-like molecules, such as HLA-G and the human Fc receptor (Feder et al. 1996). The primary HFE mutation responsible for hereditary hemochromatosis causes a cysteine-to-tyrosine substitution at amino acid 282 (C282Y). The C282Y missense mutation affects one of the highly conserved cysteine residues involved in intramolecular disulfide bridging in MHC class I proteins. Subsequent investigation has shown that the C282Y mutation causes an abnormality in HFE protein trafficking and cell surface expression due to an inability of the mutated HFE to bind 
2-microglobulin (Feder et al. 1997). The importance of the HFE interaction with 2-microglobulin in the regulation of iron absorption is supported by the observation of progressive iron overload in 2-microglobulin-deficient knockout mice (Rothenberg and Voland 1996) who are unable to express MHC class I gene products on the cell surface. A more direct demonstration of the importance of the wild-type HFE gene in maintaining iron homeostasis is observed in the recent report of a HFE knockout mouse (Zhou et al. 1998). HFE knockout mice have increased plasma transferrin saturation and increased hepatocellular iron deposition
an important stigmata characteristic of human hereditary hemochromatosis. Furthermore, the phenotype of the HFE knockout mouse suggests that the HFE C282Y mutation that produces hereditary hemochromatosis in humans does so by causing a deficiency of the functional HFE protein rather than by changing the characteristics or localization of the mutated HFE protein in the cell.
). Recent studies have shown that HFE can bind to transferrin receptor in human embryonic kidney cells, fibroblast cells, as well as human placenta (Feder et al. 1998, Parkkila et al. 1997). Because transferrin receptors on the cell membrane control cellular iron status by influencing diferric transferrin uptake into cells, it is conceivable that the association of HFE with transferrin receptors may indicate a critical HFE-mediated step in the homeostatic control of intestinal iron absorption. Moreover, it has also been observed that the level of HFE expression is responsive to the iron status of the enterocyte (Han et al. 1998).
Nramp2.
Hypochromic, microcytic anemia is found in mk/mk mice and has been associated with a defect of iron absorption due to reduced iron entry into intestinal cells. However, the exit of iron from mk/mk cells is normal, and bypassing the intestinal block in iron absorption by parenteral iron administration does not cure the anemia. Collectively, these observations are consistent with a generalized defect of iron entry into cells of this mutant mouse strain (Andrews 1997). Similar to mk/mk mice, homozygous (b/b) Belgrade rats have an inherited hypochromic, microcytic anemia, which is known to be associated with impaired iron transport into immature erythrocytes and intestine (Fleming et al. 1998). Studies using reticulocytes from b/b rats have demonstrated a defect in a putative iron carrier involved in transport of iron released from transferrin within endocytic vesicles. However, binding and uptake of nontransferrin-bound Fe+2 by reticulocytes from b/b rats is also impaired, suggesting that an iron transport defect is also present on the cell membrane. Therefore, it is likely that the same iron carrier is responsible for iron transport defects in erythroid and mucosal cell membranes of the b/b rat (Fleming et al. 1998).
). In addition, functional expression cloning studies in Xenopus oocytes have identified a homologous rat gene (DCT1) that functions as a proton-coupled divalent cation (Fe, Zn, Mn, Co, Cd, Cu, Ni and Pb) transporter (Gunshin et al. 1997). DCT1 codes for a protein with 12 putative membrane-spanning domains that appears to preferentially transport iron, but may be relevant to the transport of other essential cations such as zinc (McMahon and Cousins 1998). The expression of this gene is up-regulated by iron depletion in rats and in human intestinal cells (Han et al. 1998). Recently, characterization of the genetic mutation in the anemic Belgrade rat revealed a missense mutation that results in a glycine-to-arginine substitution at amino acid 185 (G185R) in Nramp2, which is identical to the gene mutation identified in mk/mk mice. Since endosomal iron transport is also defective in the Belgrade rat, mounting evidence suggests that Nramp2 is likely responsible for both endosomal iron transport and iron transport across the apical membrane (Fleming et al. 1998). Alternatively, it has been suggested that Nramp2 may be involved with the transport of another metal that is necessary for optimal iron processing (Fleming et al. 1997), analogous to the role of copper transport in the control of iron metabolism in yeast (Dancis et al. 1994).
Future Developments.
The identity of the protein involved in the exit step of intestinal iron absorption is unknown. However, nutritional observations first made in the 1950s in copper-deficient swine identified a hypochromic, microcytic anemia despite the presence of sufficient dietary iron. Moreover, oral administration of additional iron failed to correct the anemia and instead resulted in accumulation of iron in duodenal enterocytes. Presumably, copper deficiency was blocking the efflux of iron out of the intestinal cells. The proposed mechanisms implicated ceruloplasmin, a serum copper-containing protein with ferroxidase activity. In this regard, it is intriguing that a glycosylphosphatidylinositol-anchored ceruloplasmin has been recently reported to be expressed in mammalian astrocytes (Patel and David 1997), which may offer a paradigm to be investigated of ceruloplasmin activity in the intestine. In addition, iron transport by everted duodenal gut sacs from sla (sex-linked anemia) mice indicates that their mucosal cells can take up iron normally but have a defect in the exit step of iron transport (Bannerman 1976). These observations suggest that identification of the genetic defect in sla mice (Anderson et al. 1998) may uncover a protein involved in the exit of iron out of the enterocyte.
). This gene product may be able to transport both Fe+2 and Fe+3. When this gene was overexpressed in transfected HeLa cervical carcinoma cells, SFT stimulated uptake of both transferrin-bound and nontransferrin-bound iron. Interestingly, stimulation of iron uptake by SFT is impaired in copper-deficient HeLa cells (Yu et al. 1998). Little is currently known about the role of this novel iron transport gene in intestinal iron transport.
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FOOTNOTES |
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LITERATURE CITED |
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Abstract
Introduction
References
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a paradigm for mineral absorption.
J. Nutr.
1998;
128:917-920the mucin-mobilferrin-integrin pathway. A competitive pathway for metal absorption.
Am. J. Hematol.
1993;
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