Journal of Hepatology
Volume 40, Issue 4 , Pages 702-709, April 2004

Intestinal absorption of iron in HFE-1 hemochromatosis: local or systemic process?

Service des Maladies du Foie and Inserm Unit U-522, University Hospital Pontchaillou, Rennes, France

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1. Introduction 

The most common form of Genetic Haemochromatosis (GH)—also named HFE-1 haemochromatosis—is an iron overload autosomal recessive disease affecting, in caucasian adults, many organs, especially the liver, pancreas and heart [1], [2]. The genetic basis of this disease is constituted by mutations in the HFE (or HFE-1) gene which is located on the short arm of chromosome 6 [3]. The C282Y mutation (replacement of cystein by tyrosine in position 282) is by far the major involved mutation. Indeed, in northern Europe, northern America and Australia more than 90% of affected patients carry this mutation at the homozygous state (C282Y/C282Y). A number of other HFE-1 mutations have been reported, most of them associated with C282Y heterozygosity and therefore corresponding to various types of compound heterozygosities [4]. The most frequent of these mutations is H63D (histidine replaced by aspartate in position 63), which—whenever associated to C282Y heterozygosity (C282Y/H63D)—leads inconstantly to mild forms of iron overload. In HFE-1 haemochromatosis, iron overload is classically related to intestinal hyperabsorption of iron. Major advances have recently been achieved in the knowledge of the molecular mechanisms involved in this excessive iron input of digestive origin. However, a number of issues remain to be solved, such as the exact role of the various transmembrane and circulating iron transporters, the precise nature of the cascade of events linking HFE-1 mutations and the dysregulation of iron absorption, the real importance of this enhanced digestive absorption as compared to other putative mechanisms implicated in the development of iron overload, and finally the role of environmental and genetic co-factors as possible modulators of iron absorption and consequently also of the phenotypic expression of the disease.

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2. Iron is hyperabsorbed at the duodenal level in HFE-1 haemochromatosis 

A number of in vivo human isotopic studies [5], [6], [7], [8] and an ex vivo study of human duodenal biopsies [9] have documented an increased iron absorption in GH, of the order of 4 mg/day, i.e. 2–3 times the normal level [10]. After iron depletion by phlebotomy, absorption is increased to 8–10 mg/day as compared to 5 mg/day in iron-depleted healthy individuals [11]. This enhancement in iron absorption concerns both non-haem (=inorganic) and haem (=organic) iron [12]. Hfe knock-out mice, a model mimicking human hemochromatosis [13], also develop increased intestinal iron absorption [14], [15].

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3. Expression of key membrane non-haem iron transporters is enhanced in haemochromatosis at the level of duodenal enterocytes 

The expression of proteins involved in luminal iron uptake by the enterocytes is maximal at the upper part of the duodenal villus where cells are fully differentiated (Fig. 1).

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  • Fig. 1. 

    Schematic representation of the hypothesis implicating a local effect of HFE gene product on the regulation of digestive iron absorption. A: Representation of the enterocyte maturation along the villus during the differentiation process: from the cryptic to the villus enterocyte. B: The cryptic enterocyte expresses Transferrin Receptor 1 which enables the cell to take up iron from the plasmatic transferrin by an endocytic process. The HFE molecule, associated with the beta2-microglobulin, could interact directly with Transferrin Receptor 1 and thus regulate the level of iron-transferrin uptake. This phenomenon would be necessary for intracellular iron of plasmatic origin to represent adequately the body iron status. C: After differentiation, the enterocyte expresses adequate levels of proteins involved in enterocyte iron uptake at apical and basolateral membranes.

The expressions of DMT1 mRNAs, the iron transporter located at the luminal membrane [16], [17] and of ferroportin1 (Ireg1), the iron exporter at the basolateral membrane [18], are increased in HFE-1 haemochromatosis [19], [20], [21], [22], [23].

The expression of duodenal cytochrome b (Dcytb), a ferric reductase located on the luminal surface which is required to reduce dietary free ferric iron into ferrous form prior to uptake [24], has been found to be increased in GH [21] in agreement with the report of an increased luminal reductase activity in GH [25]. This result, however, has not been found by another group [26], and it should also be pointed out that Zoller et al. [21] observed that Dcytb mRNA and protein expressions were not significantly different from control subjects, suggesting that, in haemochromatosis, Dcytb activity is post-translationally upregulated (or alternatively that another type of ferric reductase exists). Hephaestin, a multi-copper oxidase with ferroxidase activity, participates to the iron efflux through the basolateral membrane [27]. No modulation in hephaestin mRNA [21], [22] or protein [22] expression has been detected in haemochromatosis. On the whole, mRNA expression of two iron transporters (DMT1 and ferroportin1) as well as Dcytb activity are increased in GH in contrast to hephaestin which seems unchanged [21].

Compared to these human studies it should be pointed out that data obtained with Hfe-knock-out mice have provided somewhat discordant results. (i) For DMT1. An increase in duodenal DMT1 m-RNA was reported by several authors [28], [29] and increased immunoreactive mucosal DMT1 protein has been documented [30]. In contrast, Cannone-Hergaux et al. [31], Muckenthaler et al. [32] and Herrmann et al. [33] did not find an increased mRNA DMT1 expression in their transgenic mice. (ii) For ferroportin 1. It has been found enhanced by Dupic et al. [29] but decreased by Muckenthaler et al. [32], and unchanged by Herrmann et al. [33]. (iii) For Dcytb. Dcytb RNA messenger level was reported enhanced by Dupic et al. (DBA/2 Hfe−/− mice) [29], Muckenthaler et al. [32] and Herrmann et al. [33] but unchanged in C57BL/6Hfe−/− mice [29]. These data emphasize that one should be careful in extrapolating from the transgenic animal model to the human situation. Moreover, one should admit that the data obtained in human remains partial and needs further documentation.

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4. Paradoxical iron deficiency status of cryptic enterocytes in haemochromatosis is thought to be the sensor leading to iron hyperabsorption at the level of villus enterocytes (Fig. 2)
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  • Fig. 2. 

    Schematic representation of the hypothesis favouring a putative local duodenal effect of C282Y HFE mutation (C, D) versus normal situation (A, B). C282Y HFE mutation could induce a modification of the interaction between HFE-beta2 microglobulin complex with Transferrin Receptor 1 and then alter its affinity for the transferrin receptor or its recycling (C). Such a situation could induce a low level of iron ingress in the enterocyte leading to a paradoxical iron deficiency of the enterocyte, leading, in turn, to an increase of digestive iron uptake by the apical enterocyte (D).

 

The phenotype of duodenal cells in GH is identical to that of iron deficiency, as indicated by sustained IRP (iron regulatory protein) activity, low expression of ferritin mRNA, low accumulation of ferritin, lack of TfR1 (=Transferrin Receptor 1) gene down-regulation [34], [35] and even increased TfR1 expression [21], [36]. Crypt cells are known to be precursor cells for the absorptive enterocytes according to a differentiation process which takes place in a gradient along the crypt/villus axis [37], [38] (Fig. 1). As to iron metabolism [39], [40], [41], crypt cells have been considered as sensors of body iron needs and to be programmed so that, once becoming absorptive cells, they express appropriate levels of iron transport proteins in order to ensure adequate levels of iron absorption. This regulatory process [42] would involve successively changes in the labile iron pool within the crypt cells and modifications in the mRNA-binding activities of IRPs (iron regulatory proteins). DMT1 and ferroportin1 transcripts contain IREs (iron responsive elements), their corresponding proteins can be modulated by such changes in IRP-binding activities. Therefore, iron deficiency of the crypt cells would lead to stabilization of DMT1-mRNA (through the interaction of IRPs with the IRE in the 3′ untranslated region of the transcript). This would lead to an increased DMT1 protein expression and to subsequent enhanced iron flux from the lumen into the villus enterocytes. The resulting increased labile iron pool within the enterocytes would decrease IRP binding activity and therefore (via the interaction of IRP with the IRE in the 5′ untranslated region of the iron efflux transporter ferroportin1) increase the translation of ferroportin1 at the basolateral level of the villus enterocytes. The enhanced iron transport through the enterocyte, as observed in genetic haemochromatosis, would therefore be the result of an increased iron transport both at the luminal and at the basolateral levels. The alternative view, proposed by Roy and Enns [40], is that duodenal iron deficiency would be the consequence of a dominant increased activity of ferroportin1 as compared to DMT1. In the latter hypothesis, the trigger for the increased ferroportin activity would be iron excess (and not iron deficiency) within the cryptic enterocyte. However, this mechanism -implicating primary enterocyte iron excess- seems unlikely given that ferroportin1-mRNA is only highly expressed in villus cells. Moreover, ferroportin1 has been shown to be increased by iron deficiency and is therefore not expected to be found enhanced in iron excess [43]. In addition, iron uptake from plasma transferrin by the duodenal epithelium in Hfe knock-out mice was impaired as compared with wild-type animals [44].

For this issue also, the transgenic mouse model provided discordant data, Muckenthaler et al. [32] and Herrmann et al. [33] reporting no duodenal phenotype of iron deficiency in their Hfe knock-out mice.

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5. Is HFE involved in determining the amount of iron within the cryptic enterocyte? 

The major argument supporting this role is that HFE associates with the TfR1 [45] and therefore is implicated in the transferrin-mediated iron uptake pathway (Fig. 1(B)). Indeed, it is well known that isolated cryptic cells possess basolateral transferrin receptors and that, in vivo, crypt cells are able to take up transferrin-bound iron in amounts proportional to plasma iron concentration [46]. Furthermore HFE is located in endosomes which participate to the intracellular transferrin-iron cycle [47]. Initial studies, based on cultured HeLa or CaCo2 cells, concluded that wild-type HFE negatively modulated TfR1-mediated iron uptake, which rendered very difficult a logical explanation for the iron-deficient phenotypic status of enterocytes observed in case of human HFE1-haemochromatosis (i.e. with mutated HFE). More recently, a series of experiments favored a positive modulation. At the enterocyte level: (i) Concomitant overexpression of HFE and β2-microglobulin (an obligatory partner protein in the normal functioning of HFE) increased transferrin-receptor1-dependent iron uptake in cultured Chinese hamster ovary cells [48], and (ii) The already mentioned data by Trinder et al. showing a diminished transferrin iron uptake by the duodenum in Hfe knockout mice [44]. At the macrophagic level: (i) The fact that, in HFE-1 haemochromatosis, macrophagic cells are relatively iron-spared despite marked mutated HFE expression, and (ii) The demonstration that transfection of haemochromatotic macrophages with wild-type HFE increased the ferritin-iron pool within the transfected cells, which strongly suggests that the HFE mutation directly leads to the iron-deficient phenotype of macrophagic cells in HFE-1 haemochromatosis [49].

The finding by Zuccon et al. [50], using antisera which were different from those of Parkkila et al. [51], that HFE was also found in the villi could open the possibility of HFE-enterocyte interactions elsewhere than at the sole cryptic level.

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6. Deficient production of hepcidin by the haemochromatotic liver is a key factor accounting for increased intestinal iron absorption in this disease 

Following the demonstration by Pigeon et al. [52] of the link between hepcidin and iron metabolism, a number of studies have demonstrated the crucial importance of this small antimicrobial peptide (also named HAMP for hepcidin antimicrobial peptide) in modulating the intestinal absorption of iron [53], [54]. The main findings are the following: (i) Hepatic hepcidin mRNA is increased in mice overloaded by carbonyl iron or by iron-dextran, whereas β2-microglobulin knock-out mice, which spontaneously develop iron excess, show decreased expression of hepcidin when placed on a low-iron diet, indicating that hepcidin levels could reflect body iron store modulation [52]. (ii) Absence of hepcidin expression in Usf2 knock-out mice is associated with hemochromatotic phenotype [55]. (iii) Decreased hepcidin production in CEBPα knock-out mice is associated with (slight) hemochromatotic phenotype [56]. (iv) Hepcidin mutations in humans lead to a severe picture of juvenile haemochromatosis [57], [58]. (v) Overexpression of hepcidin in transgenic mice is associated with severe iron deficiency [59]. (vi) Anemia and hypoxia, conditions associated with increased intestinal absorption of iron, lead to a pronounced decrease in liver hepcidin gene expression [60]. (vii) Hepcidin expression inversely correlates with expression of duodenal iron transporters and iron absorption in rats [61]. From all these data, emerged the view that hepcidin modulates negatively intestinal absorption of iron.

As to the specific role of the C282Y mutation, most of the data obtained both in Hfe knock-out mice [62], [63], [64] and in HFE-1 haemochromatosis [63], [65] converge to indicate that mutated HFE leads to decreased hepatic hepcidin production, which could explain the increased intestinal absorption of iron observed in C282Y/C282Y haemochromatosis.

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7. Remaining issues 

7.1. The place of haem iron absorption 

It is noteworthy that almost all the studies dealing with disturbed intestinal iron absorption in haemochromatosis have focused on non-haem iron absorption. In fact, haem iron, although representing only 10–15% of dietary iron, may provide as much as one-third to one-half of absorbed dietary iron in iron-replete subjects due to its much higher bioavailability than the non-haem iron pool [66]. Taking into account that haem-iron absorption is increased in patients with genetic haemochromatosis and, unlike non-haem iron, is not negatively modulated by increased iron stores [7], it is essential to pay attention to this haem-iron pool when trying to understand the basis for the development of iron overload in haemochromatosis. Haem iron is considered to enter the duodenal cells, as an intact porphyrin ring, via a vesicular transport process. A candidate haem tranporter, highly expressed in duodenum, has recently been characterized [67]. It will be obviously most important to study its expression in haemochromatosis.

7.2. The exact cascade of events linking the mutated HFE and increased intestinal absorption of non-haem iron requires further characterization (Fig. 3)
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  • Fig. 3. 

    Schematic representation of local and systemic hypotheses linking HFE gene product to iron metabolism. A: Cell types potentially involved during HFE-1 disease. B: Putative molecules playing a role in the development of iron overload during HFE-1 hemochromatosis. HFE normal gene is expressed in numerous cell types including duodenal enterocytes, macrophages and hepatocytes. All these cells are likely implicated at various levels in HFE-1 hemochromatosis. The first mechanism evoked to explain the development of iron overload in HFE-1 hemochromatosis was a local one at the duodenal enterocytic level (1). Complementary local mechanisms involved in the development of iron overload in HFE-1 GH could be: (1b) a direct effect of HFE mutation on macrophages leading to an abnormal iron egress from these cells which play quantitatively a critical role in iron bioavailability and metabolism, or (1c) interaction with hepatocyte proteins involved in iron uptake and storage. Recent data demonstrates that hepcidin, a peptide secreted in plasma by the hepatocytes, is involved in the development of iron overload during GH, suggesting that an HFE mutation may lead to the development of iron overload through an hormonal signal (2). Mechanisms leading to a decrease of hepcidin expression when HFE is C282Y mutated as well as the cellular targets of hepcidin are not known. Whether hepcidin expression is always dysregulated in case of C282Y mutation remains to be determined. Other genes may play a role. Thus, it has been demonstrated that hemojuvelin, which is to date not fully characterized, may modulate hepcidin expression by an unknown mechanism (3). Mechanisms leading to the development of iron overload in case of Transferrin Receptor 2 gene mutation (4) are not known. Interaction between HFE and Transferrin Receptor 2 as well as hemojuvelin must be investigated.

 

7.2.1. Cellular target of HFE 

(i) At the duodenal level. As previously seen, crypt cells were considered as the preferential strategic site of action for HFE because HFE was localized at this site and especially because its interaction with TfR1 is also taking place there [68]. However, in C282Y/C282Y patients, HFE protein has been uniformly detected throughout enterocytes of crypts and villi [50], so that some HFE functioning at the villus level cannot be totally excluded. (ii) The macrophage, either hepatic (Kupffer cell) or extrahepatic, is a very likely site of action for HFE. Two main studies support this interaction: (i) The work by Montosi et al. showing that expression of wild-type HFE in hemochromatotic macrophages increased iron retention [49]; (ii) The study by Drakesmith et al. [69] showing that wild-type HFE inhibits the release of iron from ex vivo hemochromatotic macrophages, in accordance with the report by Moura et al. [70] indicating that monocytes from hemochromatotic patients released more low-molecular weight iron in vitro than control monocytes. (iii) At the hepatocyte level. A direct role of the HFE protein was until recently considered unlikely due to its apparently scanty localisation [51]. However, the recent reports that HFE mRNA [71], [72] and protein [72] were detected in rat hepatocytes at high levels should lead to reconsider this view.

7.2.2. Source and nature of the hepcidin regulatory signal at the liver site 

Considering on one hand that the macrophage is an important cellular target for HFE, on the other hand that hepcidin is essentially produced by the hepatocytes, it is tempting to admit that the macrophagic system represents a significant source for the signal which reaches the liver, modulates hepcidin hepatic synthesis and subsequently the amount of released peptide into the circulation. The nature of this signal remains to de determined. Likewise, whether other cellular sources (especially the enterocytes and the hepatocytes) are involved in this signalling process needs further studies.

Nature of the hepcidin regulatory signal: Iron per se is probably not directly involved in this inducing effect as indicated by the absence of increased hepcidin mRNA in primary mouse hepatocytes exposed to iron-citrate [52], and furthermore by the decreased hepcidin mRNA expression observed in human hepatocytes exposed to ferric ammonium citrate or to diferric transferrin [73] and in HepG2 cells incubated with Fe-NTA [65]. Cytokines could play an important role as shown by the inducing effect of IL-6-like cytokines on hepcidin mRNA [73].

7.2.3. Cellular and molecular targets of hepcidin 

Hepcidin, a circulating peptide, is expected to be in contact with miscellaneous cellular types. Until a putative cellular hepcidin receptor is found, it will be difficult to identify the cells involved in this interaction. Whether, at the basolateral membrane of the cryptic enterocyte level, hepcidin contributes to form a quaternary complex with HFE, TfR-1 and β2-microglobulin deserves to be explored. Recent data suggest that hepcidin could act directly at the level of the villus enterocytes. Thus, in case of iron deficient diet, an inverse correlation has been found in the rat between hepcidin expression and the expression of iron transporters (DMT1, Dcytb and ferroportin), in a time frame incompatible with the duration required if hepcidin targeted (only) cryptic enterocytes [74]. Moreover, intraenterocytic iron overload has been reported to regulate the expression of luminal iron transporters, whereas the basolateral ones would respond to systemic iron needs [75], [76], [77]. Likewise, at the luminal level of the mouse villus enterocyte, the ferric reductase Dcytb could be a final hepcidin target [32], [33].

7.3. The quantitative importance of intestinal absorption of iron versus other putative mechanisms involved in the development of visceral iron overload 

The basis for the development of iron excess in genetic haemochromatosis is classically represented by increased intestinal absorption of iron and there is no doubt that this mechanism is implicated. However, one should not forget that, in the overall processing of iron by the normal body, the macrophages are by far the main cells concerned by this metabolism through the degradation process of red blood cells which corresponds to the daily release into blood of approximately 20 mg of iron versus 1–2 mg for the digestive iron [42]. Knowing that, in haemochromatosis, an increased release of iron from macrophages has been documented [69], it may well be that the macrophagic source of iron is the predominating iron overloading process compared to the duodenal source. Another possible contributing mechanism, which should not be ruled out, is represented by a relative decreased biliary iron excretion [78].

7.4. The involvement of other regulatory proteins 

At least two main proteins need to be mentioned.

7.4.1. The transferrin receptor 2 protein (TfR2) 

TfR2, discovered by Kawabata et al. [79], has been shown to be highly expressed in the hepatocytes without down-regulation of its expression either in dietary iron-overloaded or in Hfe knock-out mice [54]. Unlike TfR1, TfR2 lacks iron regulatory elements. TfR2 has been reported to co-localize with HFE in intestinal tissue and cells [80]. The demonstration and characterization of a link between hepcidin and TfR2 [81] requires further studies.

7.4.2. Hemojuvelin 

The recent cloning of the HFE2 gene responsible for chromosome 1q-linked juvenile haemochromatosis [82] (which is more frequent than the juvenile haemochromatosis form due to hepcidin mutations [57], [58]) together with the identification of the HFE2 gene product, named hemojuvelin, represents a new milestone in the understanding not only of iron overload diseases but also of normal iron physiology. It is, indeed, highly expected that the mutated protein, especially due to the G320V missense mutation, which is responsible for such a massive and early form of haemochromatosis means that the corresponding normal proteins plays a major role in the physiological inhibition of intestinal iron absorption and/or macrophagic iron retention.

In conclusion, the mechanisms accounting for the increased iron absorption in genetic haemochromatosis have amazingly benefited from continuous and rapid advances in molecular biology and genetics applied to transgenic animal models and to human pathology. Rather than a purely enterocytic phenomenon, intestinal iron absorption seems to reflect a systemic process. The time is now close when this improved pathophysiological knowledge will open the way for major diagnostic and therapeutic applications in the field of iron overload and, more generally, iron-related diseases.

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PII: S0168-8278(04)00046-7

doi:10.1016/j.jhep.2004.01.020

Journal of Hepatology
Volume 40, Issue 4 , Pages 702-709, April 2004

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