Regulation of intracellular iron levels in iron-acceptor and iron-donor cells

Transfusion Science 23 (2000) 225±235

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Review

Regulation of intracellular iron levels in iron-acceptor and iron-donor cells Manuela Santos a, Maria de Sousa b, J.J.M. Marx c,* a Department of Medicine, Notre-Dame Hospital, Montreal, QC, Canada, H2L 4M1 Molecular Immunology Laboratory, Institute of Molecular and Cellular, Biology, 4150 Porto, Portugal Department of Internal Medicine, Room G02.228, Eijkman-Winkler Institute, University Medical Centre, P.O. Box 85500, 3584 GA Utrecht, Netherlands b

c

Abstract In recent years many new genes and proteins were identi®ed with crucial functions in iron metabolism. This gave an explosion of our knowledge and understanding of iron related disorders. Mutations have been found that are responsible for disturbances in iron transport, leading to either iron overload or iron de®ciency. For experts in the ®eld, these new ®ndings clarify the sky and open new routes for exploring hitherto hidden ®elds of research. For the physician, however, iron metabolism may become even more complicated. In this review, we have tried to assemble all new iron related genes into the context of pathophysiology. Important results from animal experiments, mainly derived from knockout mouse models, are included in this review as they often explain the phenotype of human disease. Ó 2000 Elsevier Science Ltd. All rights reserved. Keywords: Haemochromatosis; Iron absorption; Iron transport

1. New relevant genes in the understanding of iron metabolism pathways A number of important roles of speci®c components of iron metabolism associated with gross iron maldistribution have been clari®ed in recent years by identifying mutations in relevant genes related to human diseases or by generating and phenotyping genetically altered mice. By dissecting the mechanisms and identifying the elements involved in iron metabolism, a comprehensive picture is beginning to emerge. A list of new genes involved in iron metabolism that have been iden* Corresponding author. Tel.: +31-30-2507398; fax: +31-302518328. E-mail address: [email protected] (J.J.M. Marx).

ti®ed, cloned and characterized in recent years is presented in Table 1.

2. Iron absorption of low molecular weight iron (LMW Fe) Although iron is an abundant element, its availability is reduced because the oxidized form of the metal, Fe(III), is extremely insoluble at neutral pH. Thus, complex mechanisms for its acquisition, utilization and preservation have evolved in even the most primitive organisms. The process of intestinal iron absorption in mammals occurs in steps (Fig. 1). In the initial uptake step, iron is transported into the intestinal epithelial cell. Ferric iron in the

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Table 1 Homologous genes that a€ect iron metabolism in humans and animal models Gene

Function

Phenotype of animal disruption

Phenotype of human disease

DCT1/Nramp2 [1]

Transports iron across microvillus membrane; endosomal iron transport

mk mice [2]; microcytic anemia; low intestinal uptake of iron; b rat[3] failure of iron transport out of endosomes

? autosomal recessive ironde®ciency anemia, unresponsive to iron therapy [4,5]

SFT [45]

Endosomal iron transport

?

? ÿ=ÿ

HFE/MS2 [6,7]

Associates with transferrin receptor [8,9]; ? regulates intracellular iron levels

b2m Mice [10±12]; increased iron absorption; parenchymal iron overload HFEÿ=ÿ mice [13]; iron overload

Hereditary hemochromatosis [14]; increased iron absorption; parenchymal iron overload

sla [15] Hephaestin, [16]

Transport of iron across intestinal basolateral membrane; ferroxidase activity

sla mice [17,18]; sex-linked anemia microcytic anemia; decreased intestinal iron transfer

?

IREG1 [19]

Transport of iron across intestinal basolateral membrane

?

?

Ceruloplasmin

Ferroxidase activity

Fet3p ± Yeast homologue;de®cient growth on low Fe [20,21] Cpÿ=ÿ mice [46] progressive iron accumulation

Aceruloplasminemia [22,23] de®cient iron mobilization; low serum iron; tissue iron overload

Hemox1

Heme oxygenase: catabolysis Hemox1ÿ=ÿ mice [24]; anemia; tissue of cellular heme to bilirubin, iron overload; chronic in¯ammation carbon monoxide and free iron; iron recycling Hemox2ÿ=ÿ mice[25]; lung iron accumulation

?

Transferrin

Iron transport in plasma and into cells

hpx mice [27]; hypotransferrinaemic; microcytic anemia; parenchymal iron overload; increased iron absorption [28,29]

Hypotransferinemia [30,31]; anemia; parenchymal iron overload

Transferrin receptor

Iron transport into cells

Trfrÿ=ÿ mice [32]; more severe phenotyoe tha hpx mice; impaired erythroid and neurologic development, and abnormal iron homeostasis

Transferrin receptor-2

Iron transport into cells

In development

Hereditary hemochromatosis [47]; increased iron absorption; parenchymal iron overload

Frataxin [33]

Mitochondrial iron transport

Yfh1p ± yeast homologue; de®cient growth on non-fermentable carbon sources due to mitochondrial iron overload [34,35]; frataxinÿ=ÿ mice [36] early embryonic lethality

Friedreich's ataxia [37±39]; neurodegeneration and cardiac myopathy

L-ferritin

Iron storage

?

Hereditary hyperferritinemiacataract syndrome [40±43]

H-ferritin

Iron storage

H-ferritinÿ=ÿ _mice [44]; early embryonic lethality

Hemox2

? idiopathicpulmonary siderosis [26,31]

M. Santos et al. / Transfusion Science 23 (2000) 225±235

Fig. 1. Schematic diagram of intestinal iron absorption. Gene products involved in iron absorption are schematically represented and relevant animal models in which a particular gene is mutated are represented in italic. Reduction of ferric (Fe(III)) iron complexes to the ferrous (Fe(II)) form is achieved by the action of a brush border ferric-reductase. The ferrous form is transported via the proton-coupled divalent cation transporter (DCT1 or Nramp2) to intra-cellular iron pool. Ferrous iron is then transported across the basolateral membrane by IREG1, where the membrane-associated copper oxidase hephaestin (Heph) promotes release and binding of Fe(III) to transferrin. Mouse models: mk ± Microcytic anaemia; sla ± sex-linked anaemia; hpx - hypotransferrinemia; b2mÿ=ÿ ± b2-microglobulin knockout; HFEÿ=ÿ ± HFE knockout; LMW Fe ± low molecular weight iron; FeRed ± ferric reductase; Trf ± transferrin; Trf-R ± transferrin receptor.

lumen of the gut is reduced to ferrous iron by a ferric reductase. Next, the ferrous iron product is transported into the cell by a ferrous transporter, recently identi®ed as DCT1/Nramp2 [1], which is mutated in microcytic anemic (mk) mice [2]. Mucosal uptake is in¯uenced by many intraluminal factors: the state of the iron in the test dose (ferric or ferrous, heme or non-heme), the amount of iron, the composition of the test dose, gastric and intestinal secretions and the state of the brush border of the mucosal cells. After uptake, the iron becomes part of a cellular iron pool and may be stored as ferritin or transported across the basolateral membrane of the cell (mucosal transfer) to complete the process of absorption [48]. The basolateral ferrous transporter has been recently identi®ed as IREG1 [19]. Next, ferrous iron is oxidised to ferric iron by hephaestin [16], a membrane associated copper oxidase similar to the serum protein ceruloplasmin. Ceruloplasmin is a multicopper oxidase with ferroxidase activity, which presumably promotes the release of iron from cells [21] and facilitates iron binding to

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transferrin. Hephaestin is the defective gene in mice with sex-linked anemia (sla) mice [15±18]. Mucosal transfer is less dependent on intraluminal factors and it re¯ects better the iron status of the body. To allow discrimination between mucosal uptake into intestinal epithelial cells and mucosal transfer into the plasma during the process of iron absorption, the use of a non-absorbable indicator …51 CrCl3 † has proved to be useful in iron absorption measurements [50]. This method has been successfuly adapted for use in small animal models [51,52]. 3. The b2mÿ=ÿ mouse as a model for hereditary hemochromatosis Interest in the b2-microglobulin knockout mouse as a model for human hemochromatosis had two roots: one derived from the clinic [53] and another from an interest in duodenum associated molecules possibly involved in the regulation of iron absorption [11]. The availability of appropriate animal models can greatly aid the understanding of the mechanism(s) of iron absorption. In a previous study, we have revealed the existence of hepatic iron overload in b2-microglobulin (b2m)-de®cient mice similar to that found in hereditary hemochromatosis (HH), with pathologic iron depositions occurring predominantly in liver parenchymal cells [10]. In these mice, cell surface expression of major histocompatibility complex (MHC) class I molecules is severely decreased, and as a consequence they lack CD8‡ lymphocytes. This observation was later reported by Rothenberg and Voland [11]. They extended these ®ndings, documenting progressive iron deposition in the liver of b2-microglobulin de®cient mice that increased with supplemental dietary iron. In addition, these authors reported an increased incidence of hepatocellular carcinoma in b2m knockout mice. More recently, we demonstrated that b2m-de®cient mice have a fourfold increase in plasma iron concentrations, increased transferrin saturation …> 80%† and increased hepatic iron when compared with normal control mice [12]. Furthermore, mucosal

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uptake of ferric, but not ferrous iron, and the subsequent mucosal transfer into the plasma is inappropriately increased in b2-microglobulin knockout mice [54]. Importantly, mice in which the CD8 molecule has been disrupted, (CD8ÿ=ÿ ) and mice that lacked the endoplasmic reticulum transporter for class I associating peptides (TAP1ÿ=ÿ ) had a normal iron metabolism [12]. Taken together, these observations strongly suggest the involvement of a MHC class I-like, b2mdependent gene product in iron absorption. The ®nding of a novel gene of the MHC class I family, HFE, that is mutated in the majority of HH patients provides further independent support for the proposed causative role of a b2m-dependent gene in HH and its involvement in iron absorption [6]. The recently characterized HFEÿ=ÿ mouse [13] decisively demonstrates that mutations that disrupt the function of the HFE gene product result in HH. 4. Adaptive response of iron absorption Although body iron content is the principal factor in the regulation of iron absorption, there are other physiological variables, such as erythropoietic rate [55], hypoxia [56] and in¯ammation [57]. In normal individuals, if the rate of erythropoiesis is stimulated by blood loss, dyserythropoiesis or acute hemolysis, the eciency of iron absorption is increased. Conversely, if erythropoiesis is inhibited by hypertransfusion, starvation or descent from high altitude to sea level, then iron absorption falls. The adaptive response of iron absorption to an increase in the rate of erythropoiesis, stimulated either by blood loss or acute hemolysis, remains intact in b2mÿ=ÿ mice and indistinguishable from control wild type mice [54]. This suggests that the regulatory mechanism(s) of iron absorption operating in these situations are independent of the expression of HFE. In contrast, when iron stores are altered through dietary manipulations or parenteral iron injections, both steps of mucosal uptake and mucosal transfer are quantitatively a€ected in b2mÿ=ÿ mice [54]. Therefore, the expression of the defect in

iron absorption in the b2mÿ=ÿ mice is clearly quantitative, and suggests an involvement of HFE-b2m complexes in delivering information to intestinal epithelial cells about the iron status of the body. Delivery of the signal for adaptive regulation of intestinal absorption is dependent on plasma transferrin, since in human transferrin de®ciency, as in iron-loaded hypotransferrinemic (hpx) mice, increased iron absorption occurs despite the presence of systemic iron overload [29]. The recent ®nding by others that HFE associates with the transferrin receptor (TfR), forming a ternary HFE/transferrin receptor/iron-saturated transferrin complex, further supports this notion [9]. 5. Regulation of iron absorption The most favourite concept concerning the regulation of iron absorption is that of pre-programming of crypt intestinal cells according to the state of body iron stores and previous exposure to iron. The programming would involve the setting of constitutive synthesis of several proteins involved in iron transport and storage such as Land H-ferritin, transferrin receptor and DCT1, most likely via IRP/IRE regulation. An important observation is the ability of HFE gene product to associate with the transferrin receptor (TfR; [9]). Thus, HFE protein may play a quantitative role in iron absorption by participating in the pre-programming of intestinal epithelial cells, through the modulation of cellular iron levels. Once programmed, the cells would migrate to the villus and become functional absorptive cells. Several observations may support this concept. (a) Delayed response of 2±3 days of iron absorption to acute changes in body iron status, whether overload or de®ciency [58,59]. This lag period in iron absorption could be related to the time taken for new cells, with altered capacity to absorb iron, to become functional. (b) Correlation between the amount of ferritin in the intestinal cell, which is high in patients with anemia of chronic disease (ACD; [60]) and low in HH patients [61±63], and the level of iron absorption, which is low in patients with ACD

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[60,64] and high in HH patients [50,65,66]. Of note, although the levels of several iron proteins are clearly inappropriate in the intestine of HH, they are still all coordinately regulated. The absence of a functional HFE-b2m complex, as in b2m-de®cient mice, may result in a situation where decreasing amounts of iron gain access to the mucosal cell via TfR [9], leading to low ferritin expression and increased expression of ferrous transporters at both microvillus and basolateral membranes. The overall expression of this cascade of events would lead to the observed paradox, i.e., despite elevated iron body stores, intestinal cells would behave as iron-de®cient cells. 6. Iron storage in cells from the mononuclear phagocytic system After the completion of the process of iron absorption, diferric transferrin is taken up by receptor-mediated endocytosis via the transferrin receptor. Transport of iron out of the transferrin cycle endosome requires a functional Nramp2/ DCT1, as recently shown in the Belgrade (b) rat. The Belgrade rat has an autosomal recessively inherited, hypochromic, microcytic anemia associated with defective cellular iron uptake [3]. Although diferric transferrin is taken up into b reticulocytes, the iron is only poorly retained, and much is inappropriately recycled to the extracellular space along with transferrin [67]. Linkage between the b phenotype and Nramp2, the ®rst mammalian iron transporter to be characterized at a molecular level [1], led to the identi®cation of a functionally signi®cant mutation in the b allele that is shared by the mk mouse [2,3]. These ®ndings strongly support the idea that Nramp2 is the transferrin cycle endosomal iron transporter in addition to an intestinal transporter. Although impaired, iron uptake persists in Belgrade reticulocytes, presumably via stimulator of Fe transport (SFT), which transports both Fe(III) and Fe(II) out of endosomes [45]. After exiting the endosome, iron is incorporated into ferritin, or, in the case of red blood cells iron is primarily incorporated in hemoglobin or other heme or iron-sulphur proteins (Fig. 2). Senescent red cells are phagocytosed

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Fig. 2. Schematic representation of iron uptake via transferrin receptor by erythrocytes and recycling by macrophages. Transport of iron out of the transferrin cycle endosome in the erythron requires a functional Nramp2/DCT1 [1,96] and/or SFT [45]. Transport of iron out of the phagosome with engulfed pathogens in the macrophage requires a functional Nramp1 [68,97]. Iron storage in macrophages is in¯uenced by the presence of functional HFE-b2m-complex [12,13]. Since HFE and Trf-R are both expressed in monocytes and macrophages [98], the physical association of HFE-2m-complex and Trf-R may exist in these cells as well. Possible roles for Nramp1 and Nramp2 based on phenotypes of mutant animals are represented in italic. Bcgs ± Bcg susceptible; b ± Belgrade rat (Fleming MD et al., 1998); b2mÿ=ÿ : beta2-microglobulin knockout mice; Trf ± transferrin; Trf-R ± transferrin receptor; SFT stimulator of Fe transport; IREG1 iron regulated mRNA (McKie AT et al., 2000).

by cells from the mononuclear phagocytic system (MPS), such as spleen macrophages and Kup€er cells in the liver, and iron is scavenged from heme. A homologous protein Nramp1, which can also transport iron [1], is likely to transport iron scavenged from red blood cells out of phagosomes in macrophages. Finally, the subsequent transfer of iron back to the plasma would require a still unidenti®ed protein. Contrary to Nramp2, that is expressed in many tissues, including bone marrow, Nramp1 is selectively expressed in macrophages, and has been originally identi®ed as a gene that determines resistance to intracellular pathogens by murine macrophages [69]. The Nramp1 gene, also known as Bcg, Ity and Lsh, determines natural resistance or susceptibility to infection with antigenically unrelated intracellular microbes. This gene has two alleles in inbred strains (Bcg r , resistant; Bcg s , susceptible), which are phenotypically expressed

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either as rapid (Bcg s ) or very poor (Bcg r ) microbial replication in MPS cells (e.g. spleen and liver) during the early phase of infection [69]. Professional phagocytes, the ecological niche of mycobacteria, might thus have selected a defence mechanism (phagocyte-speci®c Nramp1) that could act to antagonize the transport of a common substrate such as iron, essential for the intracellular survival of the bacterium. One of the distinguishing features of the pathology in HH in humans is that the initial deposition of iron is predominantly in the parenchymal cells of a€ected tissue, with insigni®cant early involvement of macrophages of the MPS. Accordingly, HH patients have been reported to have a defect in iron storage in MPS cells [70±72]. Reconstitution of lethally irradiated b2-microglobulin de®cient mice with normal hematopoietic donor cells redistributes the iron from parenchymal cells to Kup€er cells in the liver but does not correct the inappropriate iron absorption [12]. In addition, both b2mÿ=ÿ and HFEÿ=ÿ mice have a signi®cantly lower capacity to store iron in the spleen when compared with B6 control mice on the same diet [12,13]. These observations suggest that the lack of appropriately formed HFE-b2m complexes leads to a defective iron storage in these cells. Thus, ultimately, the lower iron loading capacity of the spleen may be related to a lower capacity of MPS cells to store iron, which may be caused by decreased erythrophagocytosis [73] and increased release of low-molecular-weight iron [74]. Iron handling and storage in MPS cells and epithelial intestinal cells may thus be similar, in that they may be regarded as iron-donor cells and because in HH they both behave as iron-de®cient cells.

ated peroxidative damage, leading to organ dysfunction. Parenchymal cells may acquire iron by several mechanism, involving Nramp2, transferrin-, hemoglobin-, and ferritin- receptor (Fig. 3). HFE may interfere with iron uptake in these cells through its interaction with the transferrin receptor (TfR) and reducing TfR anity for transferrin [8]. In addition, overexpression of HFE has been shown to drastically reduce ferritin levels [49]. We have demonstrated that increased amounts of intestinally absorbed iron are directed to the liver in b2mÿ=ÿ mice compared with wild type mice [54]. Taking into account that b2mÿ=ÿ mice have a higher transferrin saturation and reduced amounts of apo-transferrin in plasma, this indicates that absorbed iron is released from mucosal cells independent of the availability of transferrin in plasma and is deposited in the liver. This is in agreement with previous ®ndings in hypotransferrinemic mice, characterized by a heritable reduction in circulating transferrin, leading to anaemia but also to increased storage of iron in the liver [27,28]. In these mice the absorbed iron not bound to transferrin is deposited in the liver, suggesting the existence of an uptake system for non-transferrin-bound iron [29]. Thus, HFE in¯uence on intracellular iron levels in parenchymal cells could involve (1) cellular iron uptake: via its

7. Parenchymal iron overload in b2mÿ=ÿ and in b2mRAG1-double knockout mice Contrary to MPS cells and epithelial intestinal cells, parenchymal cells will function primarily as iron-acceptor cells. While iron storage in MPS cells is considered to be innocuous, excess iron in parenchymal cells is known to induce iron-medi-

Fig. 3. Schematic diagram of normal hepatocyte iron metabolism and e€ects of hereditary hemochromatosis. LMW Fe ± low molecular weight iron; Trf ± transferrin; Trf-R ± transferrin receptor; Hb ± hemoglobin; FR ± ferritin receptor.

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interaction with transferrin receptor [8,9], and possibly via regulation of Nramp2 expression levels for LMW Fe; and (2) cellular iron storage: through the regulation of ferritin levels [49]. Quantitative regulation of intracellular iron levels by HFE could be the result of a direct interaction with iron-binding-proteins, or/and indirect, via IRE/IRP regulation. Presumably, regulation of intracellular iron levels mediated by HFE is different in iron-acceptor and iron-donor cells, depending on interactions with other cell-speci®c proteins. Supporting this concept, it was demonstrated that the normal protein product of the HFE gene has an intracellular and perinuclear localization in crypt cells in the duodenum, as opposed to its membrane localization in other celltypes [75]. The evidence so far suggests that the mutations in the recently discovered HFE gene in HH patients cannot per se explain the heterogeneity of the disease [76,77]. Therefore, other genetic and environmental factors must certainly a€ect the severity of iron overload. In this context, the role of lymphocyte subpopulations has been suggested from previous observations of lymphocyte abnormalities in HH patients [53,78]. Moreover, a linkage of cell-mediated immunity to iron metabolism involving cytokines and nitric oxide in the pathogenesis of ACD has been suggested [79±81]. To directly test the role of lymphocytes in cellular iron handling, we have generated a double knockout mouse by crossing RAG1ÿ=ÿ and b2mÿ=ÿ mice (in preparation). RAG1-single de®cient mice, that lack mature lymphocytes [82], have a normal capacity to regulate iron absorption, but iron depositions in the liver when dietary iron loaded occur in a pattern similar to that seen in b2mÿ=ÿ mice. The b2 mRAG1-double knockout mice develop a more severe phenotype involving increased iron absorption, iron accumulation in the liver, heart and pancreas. Iron deposition in the heart deserves special interest, because heart failure is the most frequent cause of death in untreated HH and post-transfusional secondary hemosiderosis [83,84]. Iron excess in myocytes could cause oxidative stress and the alteration of myocyte functions through the iron-catalyzed Fenton chemistry [85,86]. We observed that b2mRAG1-

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double knockout mice develop heart ®brosis, which could be prevented by reconstitution with normal hematopoietic cells. The possible in¯uence of lymphocytes in cellular iron storage and ironmediated cellular damage may include cytokine regulation [87±90] and/or a role in macrophage di€erentiation and functions [91,92]. More recently, the generation of HFE b2mdouble mutant mice, which deposit more tissue iron than mice lacking HFE, only [93], suggest that other b2m-interacting proteins may be involved in iron regulation and may be the causative gene in non-HFE hemochromatosis [94] or in Juvenile hemochromatosis [95]. 8. Summary and conclusions With the identi®cation of an increasing number of new elements involved in iron metabolism, a comprehensive picture of its regulation in mammals is beginning to emerge. Iron is an essential nutrient for nearly all organisms. Because of its ability to both donate and accept electrons, iron participates in a wide variety of cellular oxidation± reduction reactions. The chemical properties of iron place two limitations on the biological behaviour of this element. First, although iron is abundant, the metal is most commonly found in nature as the insoluble Fe(III) hydroxide. Therefore, organisms have evolved complex mechanisms to obtain iron from their environment. Second, iron is potentially toxic by participating in the generation of toxic oxygen radicals. Consequently, at the same time that iron is accumulated in amounts sucient for metabolism, organisms must ensure that their intracellular concentration of ``free'' iron does not reach toxic levels. This is ensured, at a cellular level, by coordinate regulation of iron binding proteins for storage, uptake and transport of iron. Cellular iron levels may be di€erentially regulated in iron-acceptor cells (parenchymal cells) and in iron-donor cells (macrophages and intestinal epithelial cells in the duodenum). This di€erential regulations may be achieved through HFE interaction with other cellspeci®c components, leading also to cell-speci®c pattern of expression.

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