Mechanisms of HFE-induced regulation of iron homeostasis: Insights from the W81A HFE mutation

The mechanisms by which the hereditary hemochromatosis protein, HFE, decreases transferrin-mediated iron uptake were examined. Coimmunoprecipitation studies using solubilized cell extracts demonstrated that transferrin (Tf) competed with HFE for binding to the transferrin receptor (TfR) similar to previous in vitro studies using soluble truncated forms of HFE and the TfR. At concentrations of Tf approaching those found in the blood, no differences in Tf binding to cells were detected, which is consistent with the lower binding constant of HFE for TfR versus Tf. However, cells expressing HFE still showed a decrease in Tf-mediated iron uptake at concentrations of Tf sufficient to dissociate HFE from the TfR. These results indicate that the association of HFE with TfR is not essential for its ability to lower intracellular iron stores. To test the effect of HFE on lowering intracellular iron levels independently of its association with TfR, a mutated HFE (fW81AHFE) that shows greatly reduced affinity for the TfR was transfected into tetracycline-controlled transactivator HeLa cells. HeLa cells expressing fW81AHFE behaved in a similar manner to cells expressing wild-type HFE with respect to decreased intracellular iron levels measured by iron regulatory protein gel-shift assays and ferritin levels. The results indicate that HFE can lower intracellular iron levels independently of its interaction with the TfR.     

The hereditary hemochromatosis protein, HFE, lowers intracellular iron levels independently of transferrin receptor 1 in TRVb cells

Hereditary hemochromatosis (HH) is an autosomal recessive disease that leads to parenchymal iron accumulation. The most common form of HH is caused by a single amino acid substitution in the HH protein, HFE, but the mechanism by which HFE regulates iron homeostasis is not known. In the absence of transferrin (Tf), HFE interacts with transferrin receptor 1 (TfR1) and the 2 proteins co-internalize, and in vitro studies have shown that HFE and Tf compete for TfR1 binding. Using a cell line lacking endogenous transferrin receptors (TRVb cells) transfected with different forms of HFE and TfR1, we demonstrate that even at low concentrations Tf competes effectively with HFE for binding to TfR1 on living cells. Transfection of TRVb cells or the derivative line TRVb1 (which stably expresses human TfR1) with HFE resulted in lower ferritin levels and decreased Fe2+ uptake. These data indicate that HFE can regulate intracellular iron storage independently of its interaction with TfR1. Earlier studies found that in HeLa cells, HFE expression lowers Tf-mediated iron uptake; here we show that HFE lowers non-Tf-bound iron in TRVb cells and add to a growing body of evidence that HFE may play different roles in different cell types.     

The human counterpart of zebrafish shiraz shows sideroblastic-like microcytic anemia and iron overload

Inherited microcytic-hypochromic anemias in rodents and zebrafish suggest the existence of corresponding human disorders. The zebrafish mutant shiraz has severe anemia and is embryonically lethal because of glutaredoxin 5 (GRLX5) deletion, insufficient biogenesis of mitochondrial iron-sulfur (Fe/S) clusters, and deregulated iron-regulatory protein 1 (IRP1) activity. This leads to stabilization of transferrin receptor 1 (TfR) RNA, repression of ferritin, and ALA-synthase 2 (ALAS2) translation with impaired heme synthesis. We report the first case of GLRX5 deficiency in a middle-aged anemic male with iron overload and a low number of ringed sideroblasts. Anemia was worsened by blood transfusions but partially reversed by iron chelation. The patient had a homozygous (c.294A>G) mutation that interferes with intron 1 splicing and drastically reduces GLRX5 RNA. As in shiraz, aconitase and H-ferritin levels were low and TfR level was high in the patient's cells, compatible with increased IRP1 binding. Based on the biochemical and clinical phenotype, we hypothesize that IRP2, less degraded by low heme, contributes to the repression of the erythroblasts ferritin and ALAS2, increasing mitochondrial iron. Iron chelation, redistributing iron to the cytosol, might relieve IRP2 excess, improving heme synthesis and anemia. GLRX5 function is highly conserved, but at variance with zebrafish, its defect in humans leads to anemia and iron overload.     

Blunted erythropoietin production and decreased erythropoiesis in early pregnancy

After decreasing in the first trimester of pregnancy, the total red blood cell mass increases in the second and third trimesters to peak at term at about 120% to 125% of nonpregnant values, but how this is brought about by changes in the rate of erythropoiesis is not known. We evaluated erythropoiesis by measuring serum transferrin receptor (TfR) levels in 406 women during normal pregnancy (N = 317), at delivery (N = 63), or in the early postpartum (N = 27). Despite the presence of the placenta and the frequent occurrence of iron deficiency, TfR levels remained low in the first two trimesters and increased in the third trimester and at delivery. To explain why erythropoiesic activity was relatively low in early pregnancy, we also measured serum immunoreactive erythropoietin (Epo) in relation to the degree of anemia. There was a very strong correlation between serum TfR and Epo levels in the entire group (r = .59, P less than .0001) as well as in each period of pregnancy. Epo levels remained low for the degree of anemia and did not correlate with hematocrit in the first two trimesters, but recovered afterwards. In the early postpartum, Epo production and erythropoiesis were normal. We conclude that: (1) erythropoiesis is decreased in the first part of pregnancy but increases afterwards; and (2) blunted Epo production in early pregnancy could be responsible for that observation.     

Reticulocyte transferrin receptor (TfR) expression and contribution to soluble TfR levels

BACKGROUND AND OBJECTIVES: Transferrin receptor (TfR) expression in erythroid cells is regulated by a number of factors, including iron status and erythropoietin (Epo) stimulation. However, the impact of these factors on reticulocyte TfR expression in vivo has never been studied. A soluble form of TfR (sTfR) is present in serum in proportion to the mass of cellular TfR. Although sTfR shedding by reticulocytes and erythroblasts has been demonstrated in vitro, the contribution of reticulocyte TfR to serum sTfR has never been evaluated in vivo. DESIGN AND METHODS: We measured directly the total number of reticulocyte TfR in normal rats of different age and iron status, as well as in animals experiencing various conditions and treatments aimed at altering erythropoietic activity and iron status, including rHuEpo therapy, hemolytic anemia, phlebotomies, hypertransfusions, thiamphenicol-induced red cell aplasia or inflammation. In addition, we examined the impact of repeated hypertransfusions with normal, reticulocyte-poor and reticulocyte-rich blood on serum sTfR levels. RESULTS: The number of TfR molecules per reticulocyte was around 50,000 in young rats but was around 100,000 in older animals. These values remained constant in most conditions and in particular were not influenced by iron supplementation or iron overload. However, functional iron deficiency as well as rHuEpo therapy resulted in increased reticulocyte TfR expression. In addition, TfR numbers in reticulocytes were elevated in the early phase of recovery after acute hemolysis or red cell aplasia but normalized soon after. Hypertransfusion experiments clearly demonstrated that reticulocytes can contribute substantially to sTfR levels in vivo. INTERPRETATION AND CONCLUSIONS: TfR numbers are regulated in vivo by the same factors as in vitro, in particular iron deficiency and erythropoietin stimulation. Circulating reticulocytes contribute significantly to serum sTfR levels.     

Noncanonical interactions between serum transferrin and transferrin receptor evaluated with electrospray ionization mass spectrometry

The primary route of iron acquisition in vertebrates is the transferrin receptor (TfR) mediated endocytotic pathway, which provides cellular entry to the metal transporter serum transferrin (Tf). Despite extensive research efforts, complete understanding of Tf-TfR interaction mechanism is still lacking owing to the complexity of this system. Electrospray ionization mass spectrometry (ESI MS) is used in this study to monitor the protein/receptor interaction and demonstrate the ability of metal-free Tf to associate with TfR at neutral pH. A set of Tf variants is used in a series of competition and displacement experiments to bracket TfR affinity of apo-Tf at neutral pH (0.2–0.6 μM). Consistent with current models of endosomal iron release from Tf, acidification of the protein solution results in a dramatic change of binding preferences, with apo-Tf becoming a preferred receptor binder. Contrary to the current models implying that the apo-Tf/TfR complex dissociates almost immediately upon exposure to the neutral environment at the cell surface, our data indicate that this complex remains intact. Iron-loaded Tf displaces apo-Tf from TfR, making it available for the next cycle of iron binding, transport and delivery to tissues. However, apo-Tf may still interfere with the cellular uptake of engineered Tf molecules whose TfR affinity is affected by various modifications (e.g., conjugation to cytotoxic molecules). This work also highlights the great potential of ESI MS as a tool capable of providing precise details of complex protein-receptor interactions under conditions that closely mimic the environment in which these encounters occur in physiological systems.     

Erythroblast iron metabolism and serum soluble transferrin receptor values in the anemia of rheumatoid arthritis

OBJECTIVES: We have investigated in vitro erythroblast iron metabolism in the anemia of rheumatoid arthritis (RA). We also have examined the results in relation to bone marrow iron status in an attempt to explain the reported difference between serum soluble transferrin receptor (sTfR) values in anemia of chronic disease (ACD) and iron deficiency anemia (IDA) in patients with RA. METHODS: Bone marrow was examined in 29 anemic patients with RA, 9 healthy volunteers, and 6 patients with simple IDA. High purity erythroblast fractions were prepared from these bone marrow samples. Erythroblast surface TfR expression and iron uptake was assessed in vitro using (125)I-transferrin (Tf) and (59)Fe-Tf, respectively. The efficiency of erythroblast surface TfR function for Tf-iron uptake was determined by relating total iron uptake at 4 hours to surface TfR number. Serum sTfR values were measured for the RA anemia group, which was subdivided as RA-ACD (marrow iron present) or RA-IDA (marrow iron absent) on the basis of visible reticuloendothelial (RE) marrow iron stores. RESULTS: High purity (87 +/- 5%) erythroblast fractions were obtained from 35 of the 44 marrow samples. Erythroblasts obtained from patients with simple IDA showed a significant increase in surface TfR expression (P = 0.0003) and Tf-iron uptake (P = 0.001). RA anemia also led to a significant increase in erythroblast Tf-iron uptake (P = 0.016). This increase was not associated with an increase in surface TfR expression (P = 0.5), but was seen to occur as a result of a significant increase in the efficiency of surface TfR for Tf-iron uptake (P = 0.027). Within the RA anemia group, the increase in erythroblast Tf- iron uptake at 4 hours was more evident for RA-IDA (3.96 +/- 1.73 versus 1.66 +/- 0.66; P = 0.03) than for RA-ACD (2.69 +/- 1.18 versus 1.66 +/- 0.66; P = 0.057). This additional erythroblast response to absent RE iron stores led to a highly significant difference in serum sTfR values between RA-IDA and RA-ACD (40.2 +/- 14.0 versus 23.9 +/- 5.3 nmoles/liter; P = 0.001) CONCLUSIONS: An increase in erythroblast surface TfR efficiency for Tf-iron uptake compensates for the low plasma iron levels associated with anemia in RA and helps to maintain RA erythroblast iron uptake. With adequate RE iron stores, this increased efficiency limits intracellular iron deprivation and consequently reduces the need to increase surface TfR expression. As a result, serum sTfR levels in RA-ACD remain within the normal range. RA erythroblasts, however, are still able to respond to any additional worsening of the iron supply caused by absent RE iron stores. This additional response causes the highly significant increase in serum sTfR values seen between RA-IDA and RA-ACD.     

In vitro binding of HFE to the cation-independent mannose-6 phosphate receptor

Hereditary hemochromatosis is most frequently associated with mutations in HFE, which encodes a class Ib histocompatibility protein. HFE binds to the transferrin receptor-1 (TfR1) in competition with iron-loaded transferrin (Fe–Tf). HFE is released from TfR1 by increasing concentrations of Fe–Tf, and free HFE may then regulate iron homeostasis by binding other ligands. To search for new HFE ligands we expressed recombinant forms of HFE in the human cell line 293T. HFE protein was purified, biotinylated and made into fluorescently labelled tetramers. HFE tetramers bound to TfR1 in competition with Tf, but in addition we detected a binding activity on some cell types that was not blocked by Fe–Tf or by mutations in HFE that prevent binding to TfR1. We identified this second HFE ligand as the cation independent mannose-6-phosphate receptor (CI-MPR, also known as the insulin-like growth factor-2 receptor, IGF2R). HFE:CI-MPR binding was mediated through phosphorylated mannose residues on HFE. Recombinant murine Hfe also bound to CI-MPR. HFE bound to TfR1 was prevented from binding CI-MPR until released by increasing concentrations of Fe–Tf, a feature consistent with an iron sensing mechanism. However, it remains to be determined whether endogenous HFE in vivo also acquires the mannose-6 phosphate modification and binds to CI-MPR.     

Erythroblast transferrin receptors and transferrin kinetics in iron deficiency and various anemias

To clarify the role of transferrin receptors in cases of altered iron metabolism in clinical pathological conditions, we studied: number of binding sites; affinity; and recycling kinetics of transferrin receptors on human erythroblasts. Since transferrin receptors are mainly present on erythroblasts, the number of surface transferrin receptors was determined by assay of binding of 125I-transferrin and the percentage of erythroblasts in bone marrow mononuclear cells. The number of binding sites on erythroblasts from patients with an iron deficiency anemia was significantly greater than in normal subjects (p less than 0.01). Among those with an aplastic anemia, hemolytic anemia, myelodysplastic syndrome, and polycythemia vera compared to normal subjects, there were no considerable differences in the numbers of binding sites. The dissociation constants (Kd) were measured using Scatchard analysis. The apparent Kd was unchanged (about 10 nmol/L) in patients and normal subjects. The kinetics of endocytosis and exocytosis of 125I-transferrin, examined by acid treatment, revealed no variations in recycling kinetics among the patients and normal subjects. These data suggest that iron uptake is regulated by modulation of the number of surface transferrin receptors, thereby reflecting the iron demand of the erythroblast.     

Analyses for binding of the transferrin family of proteins to the transferrin receptor 2

Transferrin receptor 2 alpha (TfR2 alpha), the major product of the TfR2 gene, is the second receptor for transferrin (Tf), which can mediate cellular iron uptake in vitro. Homozygous mutations of TfR2 cause haemochromatosis, suggesting that TfR2 alpha may not be a simple iron transporter, but a regulator of iron by identifying iron-Tf. In this study, we analysed the ligand specificity of TfR2 alpha using human transferrin receptor 1 (TfR1) and TfR2 alpha-stably transfected and expressing cells and flow-cytometric techniques. We showed that human TfR2 alpha interacted with both human and bovine Tf, whereas human TfR1 interacted only with human Tf. Neither human TfR1 nor TfR2 alpha interacted with either lactoferrin or melanotransferrin. In addition, by creating point mutations in human TfR2 alpha, the RGD sequence in the extracellular domain of TfR2 alpha was shown to be crucial for Tf-binding. Furthermore, we demonstrated that mutated TfR2 alpha (Y250X), which has been reported in patients with hereditary haemochromatosis, also lost its ability to interact with both human and bovine Tf. Although human TfR1 and TfR2 alpha share an essential structure (RGD) for ligand-binding, they have clearly different ligand specificities, which may be related to the differences in their roles in iron metabolism.     

Effects of cell proliferation on the uptake of transferrin-bound iron by human hepatoma cells

The effects of cellular proliferation on the uptake of transferrin-bound iron (Tf-Fe) and expression of transferrin receptor-1 (TfR1) and transferrin receptor-2 (TfR2) were investigated using a human hepatoma (HuH7) cell line stably transfected with TfR1 antisense RNA expression vector to suppress TfR1 expression. At transferrin (Tf) concentrations of 50 nmol/L and 5 micromol/L, when Tf-Fe uptake occurs by the TfR1- and TfR1-independent (NTfR1)-mediated process, respectively, the rate of Fe uptake by proliferating cells was approximately 250% that of stationary cells. The maximum rate of Fe uptake by the TfR1- and NTfR1-mediated process by proliferating cells was increased to 200% and 300% that of stationary cells, respectively. The maximum binding of Tf by both TfR1- and NTfR1-mediated processes by proliferating cells was increased significantly to 160% that of stationary cells. TfR1 and TfR2-alpha protein levels expressed by proliferating cells was observed to be approximately 300% and 200% greater than the stationary cells, respectively. During the proliferating growth phase, expression of TfR1 messenger RNA (mRNA) increased to 300% whereas TfR2-alpha mRNA decreased to 50% that of stationary cells. In conclusion, an increase in Tf-Fe uptake by TfR1-mediated pathway by proliferating cells was associated with increased TfR1 mRNA and protein expression. An increase in Tf-Fe uptake by NTfR1-mediated pathway was correlated with an increase in TfR2-alpha protein expression but not TfR2-alpha mRNA. In conclusion, TfR2-alpha protein is likely to have a role in the mediation of Tf-Fe uptake by the NTfR1 process by HuH7 hepatoma cell in proliferating and stationary stages of growth.     

The hemochromatosis protein HFE inhibits iron export from macrophages

Hereditary hemochromatosis (HH) is a disorder of iron metabolism caused by common mutations in the gene HFE. The HFE protein binds to transferrin receptor-1 (TfR1) in competition with transferrin, and in vitro, reduces cellular iron by reducing iron uptake. However, in vivo, HFE is strongly expressed by liver macrophages and intestinal crypt cells, which behave as though they are relatively iron-deficient in HH. These latter observations suggest, paradoxically, that expression of wild-type HFE may lead to iron accumulation in these specialized cell types. Here we show that wild-type HFE protein raises cellular iron by inhibiting iron efflux from the monocytemacrophage cell line THP-1, and extend these results to macrophages derived from healthy individuals and HH patients. In addition, we find that the HH-associated mutant H41D has lost the ability to inhibit iron release despite binding to TfR1 as well as wild-type HFE. Finally, we show that the ability of HFE to block iron release is not competitively inhibited by transferrin. We conclude that HFE has two mutually exclusive functions, binding to TfR1 in competition with Tf, or inhibition of iron release.     

Heterotypic interactions between transferrin receptor and transferrin receptor 2

Cellular iron uptake in most tissues occurs via endocytosis of diferric transferrin (Tf) bound to the transferrin receptor (TfR). Recently, a second transferrin receptor, transferrin receptor 2 (TfR2), has been identified and shown to play a critical role in iron metabolism. TfR2 is capable of Tf-mediated iron uptake and mutations in this gene result in a rare form of hereditary hemochromatosis unrelated to the hereditary hemochromatosis protein, HFE. Unlike TfR, TfR2 expression is not controlled by cellular iron concentrations and little information is currently available regarding the role of TfR2 in cellular iron homeostasis. To investigate the relationship between TfR and TfR2, we performed a series of in vivo and in vitro experiments using antibodies generated to each receptor. Western blots demonstrate that TfR2 protein is expressed strongest in erythroid/myeloid cell lines. Metabolic labeling studies indicate that TfR2 protein levels are approximately 20-fold lower than TfR in these cells. TfR and TfR2 have similar cellular localizations in K562 cells and coimmunoprecipitate to only a very limited extent. Western analysis of the receptors under nonreducing conditions reveals that they can form heterodimers.     

Erythroblast iron metabolism in sideroblastic and sideropenic states

Iron appears to exert self-regulatory control over erythroblast iron uptake, iron storage and its incorporation into haem. It does this via iron regulatory proteins (IRPs) which bind reversibly to the iron responsive elements (IREs) on the mRNA of transferrin receptor (TfR), erythroid 5-aminolaevulinic acid synthase (ALA-S2) and ferritin. Iron deficiency leads to the binding of IRP to IRE. This binding inhibits the translation of mRNA for ALA-S2 and ferritin but stabilizes mRNA for TfR expression. Sideroblastic erythropoiesis is highly ineffective and characterized by mitochondrial iron loading. The study of X-linked sideroblastic anaemia has shown that the entry of iron into the mitochondria is poorly controlled and able to occur when protoporphyrin production is reduced, as is seen with the ALA-S2 mutations, or when it is increased as has been seen with ABC7 transporter mutations. Sideropenia characterises both iron deficiency anaemia (IDA) and the anaemia of chronic disease (ACD). Erythroblasts in ACD seem doubly equipped to protect their iron supply with their ability to increase the efficiency of transferrin-iron uptake as well as to activate the IRP/IRE system to increase surface TfR production. This increase in efficiency restricts the need to increase surface TfR production and maintains serum soluble TfR (sTfR) values within the normal range in iron replete ACD. The coexistence of iron deficiency with chronic disease, however, is associated with an increase in both the efficiency and number and a highly significant rise in sTfR values.