Iron Transfer Across the Mucosal Cell

The transfer step involves the passage of iron that has been taken up at the apical, brush border membrane across the mucosal epithelial cell to the basolateral membrane, where it is transferred to the circulation. However, not all the iron taken up from the lumen into the cell is transferred. As a function of the body's requirements for iron (essentially determined by the rate of erythropoiesis) a variable proportion could be sequestered within the mucosal cell, eventually to be discarded into the gastrointestinal tract when the cell exfoliates. It was suggested by Hahn et al. (1943) that iron absorbed from the gut in excess of body requirements might be incorporated into mucosal cell ferritin, where it would somehow function as a 'mucosal block' against unnecessary assimilation of dietary iron. The mucosal cell would thus function as a gatekeeper, preventing the onward transfer of unwanted dietary iron. We will return to this notion later when we consider the regulation of iron absorption, which clearly implies some kind of signal coupling the amount of iron entering the mucosa from the lumen as well as the proportion that is subsequently transferred into the plasma, for the body's iron requirements. What then, are the different forms of iron that we can observe within the mucosal cell?

Current opinion is that both haem and non-haem iron arrive within the enterocyte as Fe2+, and it is concluded that this constitutes the 'labile' cytosolic iron pool. While in the graphical representation at the end of this section, we will refer to this 'transit' pool, its nature is not as well established as the other LIPs referred to in Chapter 6.

There is no doubt that ferritin represents the best characterized pool of iron within the mucosal cell. However, as we pointed out above, it is important to distinguish between enterocytes at various stages of their development. Immunohistochemical localization of ferritin protein in rats showed that in iron-deficient animals no ferritin was seen in any epithelial cells of the crypts or villi. In iron-loaded animals and in control animals, ferritin protein was also absent from the crypt epithelial cells, but it was seen in the cytoplasm of enterocytes in increasing amounts, commencing in the midvillus region and reaching its highest levels at the villus tips (Oates and Morgan, 1997). Using in situ hybridization techniques it was shown that in all iron status groups, ferritin mRNA was seen at highest levels in epithelial cells of the crypt and in macrophages within the lamina propria and at lowest levels in villus epithelial cells. These authors concluded that in undifferentiated crypt cells, ferritin genes are transcribed but not translated, whereas after differentiation, ferritin mRNA translation is controlled by cellular iron stores. Another protein which has made regular appearances in mucosal cell literature is transferrin, together with its receptor. As pointed out in Chapter 6, neither of these proteins ever gets into the cytosol, being restricted within endosomal and recycling vesicles. We will, however, return to ferritin and the basolateral membrane transferrin receptor later in our consideration of how iron absorption might be regulated.

There are other candidates for a role in cytosolic iron transport within the enterocyte, notably the previously mentioned small protein called mobilferrin (Conrad et al., 1990). Another cytosolic complex labelled during iron transport was called paraferritin on account of its high molecular weight - it appears to consist of at least four polypeptides, closely associated with one another. Antibody studies have identified the presence of integrin, mobilferrin, and flavin monooxygenase, while biochemical studies indicate that it has an NADPH-dependent monoxygenase ferrireductase activity (Umbreit et al., 1996). It is suggested that its role is to make ferrous iron available for production of iron-containing proteins (Uzel and Conrad, 1998). Precisely what the requirement for this large ferrireductase within the mucosal cell might be, particularly when iron appears to be delivered as Fe2+, is not yet clear.

Molecular Mechanisms of Mucosal Iron Absorption 8.3.3 Release of Iron at the Basolateral Membrane and Uptake by Apotransferrin

While we may have savoured the final characterization of a long awaited ferrire-ductase to accompany DCT1 at the enterocyte's apical pole, and long appreciated the patient wait of mucosal ferritin to be crowned star of mucosal cellular iron homeostasis, it is at the basolateral membrane that there has been the greatest flurry of activity in the last few years. It was no surprise to find transferrin receptors at the basolateral membrane, which, as we inferred above, would supply the preente-rocyte with the iron from the plasma that would be necessary for its division and differentiation during its 48-hour Odyssey from the crypts of Lieberkuhn up to the tips of the villi. However, we did not expect to find that the gene product of genetic haemochromatosis, HFE, discovered in 1996 (Feder et al., 1996) associates with the transferrin receptor (Feder et al., 1998; Parkkila et al., 1997) and decreases five to ten fold its affinity for its natural ligand, diferrictransferrin, (Feder et al., 1998). Not only was the X-ray crystal structure of HFE determined (Lebron et al., 1998), but a mere two years later, the crystal structure of the HFE-transferrin receptor complex was also established (Bennett et al., 2000). We have also discovered a new member of the pantheon of classical Greek mythology, in the form of a GPI-anchored multicopper ferroxidase, called hephaestin (Vulpe etal., 1999) that appears to be involved in iron egress from intestinal enterocytes into the circulation. Finally, the long-sought duodenal iron-export protein, was identified independently by three groups, and called respectively IREG1 (McKie et al., 2000), ferroportin (Donovan et al., 2000) or MTP (Abboud and Haile, 2000).

We begin with the transferrin receptor (TfR). Developing enterocytes in the crypts of Lieberkuhn express transferrin receptors at their basolateral membranes (Anderson etal., 1991, 1994; McKie etal., 1996). Radioiron administered from the blood side is restricted to enterocytes in the crypt compartment in rats (Conrad and Crosby, 1963; Schumann et al., 1999), where it enables the cells to acquire the quantities of iron from the blood that will be necessary for their differentiation and division during their progress along the pathway from crypt to apical villus. Likewise, in human duodenal biopsies, the crypt cell fraction showed dramatically higher transferrin-bound iron uptake than villus cells (Waheed et al., 1999). The possible role of TfRs during the absorptive phase of the enterocyte's progress towards the tip of the villus and its ultimate demise remain unclear, although it seems unlikely that they play a major role in active iron uptake by the villus enterocytes. In rats and guinea pigs, duodenal iron uptake from the gut lumen, on the other hand, is restricted to enterocytes in the upper part of the villi (O'Riordan et al, 1997; Chowrimootoo et al., 1992), while in human duodenum, the villus cells showed an uptake of ionic iron (from the apical side) two or three times higher than crypt cells (Waheed et al., 1999).

Genetic haemochromatosis (GH) is an autosomal recessive disease in which patients chronically absorb a slightly greater amount of dietary iron than normal from the gastrointestinal tract, resulting in deposition of excess iron particularly in the parenchymal cells of the liver, pancreas and heart (reviewed in the next chapter). The gene mutated in GH codes for a transmembrane glycoprotein, HFE, homologous to Class I Major Histocompatibility Complex (MHC), which associates with a Class I light chain b2-microglobulin. Most GH patients are homozygous for a mutation that converts Cys-260 to Tyr (Feder et al., 1996), eliminating a disulfide bond in HFE and preventing its association with b2-microglobulin and its expression at the cell surface in cell-culture models (Feder et al., 1997; Waheed et al., 1997). Both HFE and TfR are expressed in crypt enterocytes of human duodenum, and Western blots show that HFE is physically associated with both TfR and b2-microglobulin in crypt enterocytes (Waheed etal., 1999). In vitro it was observed that the HFE associates with TfR (Parkkila et al., 1997; Feder et al., 1998) and decreases its affinity for diferric transferrin by a factor of 5 to 10 (Feder et al., 1998). Further details of the interaction of HFE and Tfr (Lebron et al., 1998) together with the crystal structure of the HFE-TfR (Bennett et al., 2000) are presented in Chapter 5. It has been postulated that HFE protein modulates the uptake of transferrin-bound iron by crypt enterocytes, and is involved in the mechanism by which crypt enterocytes sense the level of body iron stores. The classical GH mutation, Cys292Tyr, could in some way provide a paradoxical signal in crypt enterocytes that programmes them to absorb more dietary iron when they mature to villus enterocytes (Waheed et al., 1999).

While both TfR and HFE are important in regulating iron influx into the enterocyte across the basolateral membrane during its crypt phase, other proteins have recently been identified which are implicated in iron egress from the villus enterocyte into the circulation where the natural receptor is assumed to be apotransferrin. The first of these was discovered by a genetic approach to identify the gene mutant in sla mice. These mice which have a genetically inherited sex-linked anaemia (hence sla), have a block in intestinal iron transport and develop a severe microcytic hypochromic anaemia. Their mature epithelial cells take up iron normally from the intestinal lumen, but fail to transfer it to the circulation. The iron accumulated in the entero-cytes is lost during subsequent turnover of the epithelial epithelium. The mutant gene in sla mice was identified and designated Heph - it encodes a transmembrane-bound ceruloplasmin homologue, which is highly expressed in intestine (Vulpe et al., 1999). The hephaestin* protein is a multicopper ferroxidase which appears to be necessary for iron egress from intestinal enterocytes into the circulation, and represents a major link between iron and copper metabolism in mammals (see Chapter 13). Hephaestin appears to be inserted into the basolateral membrane by a GPI (Glyco-Phospho-Inositol)-anchor, where it may intervene together with the basolateral iron transporter to load iron onto apotransferrin (see below).

It had long been expected that an iron transporter would be found at the baso-lateral membrane of the enterocyte responsible for the exportation of iron from the enterocytes into the portal vein circulation, and it turns out that three groups appear to have made this discovery almost simultaneously (McKie et al., 2000; Donovan etal., 2000; Abboud and Haile, 2000). The first of these, IREG1, was originally identified by subtractive cloning techniques (McKie et al., 1998), and isolated and

+ Hephaestus was the Greek god of fire, son of Zeus and Hera, and husband of Aphrodite. Homer called him chalkeas, the metalworker, the most skilful of all of the gods in a domain which could not be done without the use of fire. The myths tell us that Aphrodite (the Roman goddess Venus) was once unfaithful to her husband with Ares, the powerful god of war (the Roman god Mars). Skilled craftsman that he was, Hephaestus made nets of metal hammered so thin they were as invisible as spider's webs, and he spread them on the bed he shared with Aphrodite, and pretended to be called away to his forge. Returning to find Ares and Aphrodite entangled in his net, he thus made fools of the illicit couple in front of all of the gods. See Chapter 13 for more on Venus (copper) and Mars (iron).

characterized as a novel cDNA from duodenal mucosa of homozygous atrans-ferrinaemic mice which exhibit abnormally high rates of iron absorption (McKie et al., 1998,2000). IREG1 is a transmembrane protein, with ten potential membrane-spanning regions, that localizes to the basolateral membrane of polarized epithelial cells; both its mRNA and protein expression are increased under conditions of increased iron absorption, and the 5'-UTR of the Iregl mRNA contains a functional IRE. IREG1 stimulates iron efflux following expression in Xenopus oocytes. Alignment of human, rat and mouse IREF2 protein sequences shows a high degree of conservation, and IREGl-related proteins have been identified (McKie et al., 2000) both in plants (Arabidopsis thaliana) and nematode worms (Caenorhabditis elegans). Increased levels of Ireg1 mRNA levels were also found in duodenal biopsies from three GH patients. Ferrroportin (Donovan etal., 2000) was cloned as the gene responsible for the hypochromic anaemia of the zebrafish mutant weissherbst. It is also a multiple transmembrane domain protein which is required for transport of iron from maternally derived yolk stores to the circulation and it also functions as an iron exporter when expressed in Xenopus oocytes. A similar protein is found expressed both at the basolateral surface of duodenal enterocytes and of placental syncytiotrophoblasts suggesting that it not only functions in iron export from the intestine, but also in iron transfer from mother to embryo. It may also function in iron release from macrophages, since high levels of expression are found in Kupffer cells (Donovan etal., 2000). Comparison of the cDNA sequences of IREG1 and ferroportin confirms that they are the same. Yet a third group (Abboud and Haile, 2000) has reported an apparently identical gene and called it MTP1 (Metal Transporter Protein), expressed in tissues involved in body iron homeostasis, including the reticuloendothelial system, the duodenum and the pregnant uterus. MTPI is homologous to the DCT1 family of metal transporters, and is localized to the

Mobilferrin Paraferritin

Figure 8.3 A model of iron transport across the intestine. Reduction of ferric complexes to the ferrous form is achieved by the action of the brush border ferric reductase. The ferrous form is transported across the brush border membrane by the proton-coupled divalent cation transporter (DCT1) where it enters an unknown compartment in the cytosol. Ferrous iron is then transported across the basolateral membrane by IREG1, where the membrane-bound copper oxidase hephaestin (Hp) promotes release and binding of Fe3+ to circulating apotransferrin. Except for hephaestin the number of transmembrane domains for each protein is not shown in full. Reprinted from McKie et al., 2000. Copyright (2000), with permission from Elsevier Science.

Figure 8.3 A model of iron transport across the intestine. Reduction of ferric complexes to the ferrous form is achieved by the action of the brush border ferric reductase. The ferrous form is transported across the brush border membrane by the proton-coupled divalent cation transporter (DCT1) where it enters an unknown compartment in the cytosol. Ferrous iron is then transported across the basolateral membrane by IREG1, where the membrane-bound copper oxidase hephaestin (Hp) promotes release and binding of Fe3+ to circulating apotransferrin. Except for hephaestin the number of transmembrane domains for each protein is not shown in full. Reprinted from McKie et al., 2000. Copyright (2000), with permission from Elsevier Science.

basolateral membrane of duodenum epithelial cells and the cytoplasm of reticuloen-dothelial system cells. Iron deficiency induces MTP1 expression but downregulates it in the liver. Like IREG1 and ferroportin, MTP1 has a 5'-UTR IRE sequence.

The way in which these different partners may be involved in iron uptake across the intestine is presented in Figure 8.3. Reduction of ferric complexes to the Fe2+ form is achieved by the action of the brush border ferric reductase. The Fe2+ is transported across the brush border membrane by the proton-coupled divalent cation transporter (DCT1) where it enters an unknown compartment in the cytosol. Fe2+ is then transported across the basolateral membrane by IREG1, where the membrane-bound copper oxidase hephaestin (Hp) promotes release and binding of Fe3+ to circulating apotransferrin.

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