HFE Expression Research Articles

Localisation of proteins of iron metabolism in the human placenta and liver

Two anatomical sites that are important in human iron metabolism are the liver and placenta. Liver macrophages recycle iron from erythrocytes, and the placenta transfers iron from the mother to the fetus. The cellular distribution of proteins involved in iron transport in these two sites was studied. Transferrin receptor-1 (TfR1) and Ferroportin (FPN) expression was found on the placental syncytiotrophoblast (STB) and were polarised such that TfR1 was on the apical maternal-facing membrane and FPN was on the basal fetal-facing membrane, consistent with unidirectional iron transport from mother to fetus. Ferritin was strongly expressed in the stroma, suggesting that fetal tissue can store and accumulate iron. HFE was on some parts of the basal STB and, where present, HFE clearly colocalised with FPN but not TfR1. In the stroma, both HFE and FPN were present on CD68+ Hofbauer macrophage cells. In liver, the location of HFE is controversial. Using four mouse monoclonals and two polyclonal sera we showed that the pattern of HFE expression mirrored the distribution of CD68+ macrophage Kupffer cells. FPN was also most strongly expressed by CD68+ Kupffer cells. These findings contribute to understanding how iron is transported and stored in the human placenta and liver.

Expression of the hemochromatosis gene modulates the cytotoxicity of doxorubicin in breast cancer cells

The antineoplastic agent doxorubicin inhibits cell growth through mechanisms that include an interaction with iron, resulting in the generation of cytotoxic reactive oxygen species (ROS). Prior studies have shown that the wild-type hemochromatosis gene (wt HFE) may downregulate iron uptake and alter iron homeostasis in cells. We therefore tested the hypothesis that expression of wt HFE would affect the cytotoxicity of doxorubicin. Human breast cancer MCF-7 cells were transfected with an expression plasmid for a FLAG-tagged wt HFE gene [fwtHFE(+) cells], to examine the impact of wt HFE expression on doxorubicin-induced apoptosis. Our results show that, in MCF-7 cells, fwtHFE expression resulted in a reduction in cellular iron uptake and a decrease in the growth inhibitory effects of doxorubicin. Two micromolar doxorubicin inhibited the growth of fwtHFE(+) and fwtHFE(-) MCF-7 cells by 34% and 61%, respectively. In parallel, doxorubicin induced caspase-3-like activity in fwtHFE(-) cells, but not in fwtHFE(+) cells. On analysis with a DCF fluorescence assay, ROS could be detected in fwtHFE(-) cells but not in fwtHFE(+) cells exposed to doxorubicin. Western blot analysis of breast biopsy samples from patients revealed immunoreactive HFE and transferrin receptor proteins in both normal and malignant breast tissues. Our studies suggest that HFE expression and its consequent effect on cellular iron homeostasis may modulate doxorubicin-induced oxidative stress and apoptosis in breast cancer cells. Further investigation is warranted to determine whether HFE expression in tumor cells impacts on the clinical efficacy of doxorubicin.

Haemochromatosis protein is expressed on the terminal web of enterocytes in proximal small intestine of the rat

The haemochromatosis protein (HFE) is an important regulator of body iron stores. In the liver, HFE is required for appropriate expression of hepcidin, a humoral mediator of iron absorption. HFE is also present in enterocytes, though its function in the intestine is unknown; it is not intrinsically required for iron absorption, but can augment iron absorption when over-expressed-independent of hepcidin regulation by the liver. In this study, an antibody was raised against rat HFE and validated by enzyme-linked immunosorbent assay, Western blot and quenching of antibody function by the immunising peptide. The sub-cellular location of HFE in enterocytes of iron-deficient and control rats was determined by double-labelling experiments with markers for the microvillus membrane, terminal web, early endosomes, lysosomes and the transferrin receptor. Parallel studies were performed for the primary iron absorption protein, divalent metal transporter 1 (DMT1). HFE co-localised exclusively with the terminal web of intestinal enterocytes. HFE expression was increased in iron deficiency, consistent with a second regulatory role for HFE in iron absorption, independent of hepcidin from the liver. DMT1 was localised primarily on the microvillus membrane, but did partially co-localise with HFE raising the possibility that the two proteins may interact to regulate iron absorption.

Contribution of Hfe expression in macrophages to the regulation of hepatic hepcidin levels and iron loading

AbstractHereditary hemochromatosis (HH), an iron overload disease associated with mutations in the HFE gene, is characterized by increased intestinal iron absorption and consequent deposition of excess iron, primarily in the liver. Patients with HH and Hfe-deficient (Hfe-/-) mice manifest inappropriate expression of the iron absorption regulator hepcidin, a peptide hormone produced by the liver in response to iron loading. In this study, we investigated the contribution of Hfe expression in macrophages to the regulation of liver hepcidin levels and iron loading. We used bone marrow transplantation to generate wild-type (wt) and Hfe-/- mice chimeric for macrophage Hfe gene expression. Reconstitution of Hfe-deficient mice with wt bone marrow resulted in augmented capacity of the spleen to store iron and in significantly decreased liver iron loading, accompanied by a significant increase of hepatic hepcidin mRNA levels. Conversely, wt mice reconstituted with Hfe-deficient bone marrow had a diminished capacity to store iron in the spleen but no significant alterations of liver iron stores or hepcidin mRNA levels. Our results suggest that macrophage Hfe participates in the regulation of splenic and liver iron concentrations and liver hepcidin expression. (Blood. 2005;106:2189-2195)

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

AbstractHereditary 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 non-classical HLA class I molecule HFE does not influence the NK-like activity contained in fresh human PBMCs and does not interact with NK cells

In humans, four beta2-microglobulin-associated non-classical class I molecules are encoded in the MHC: HLA-E, -F, -G and -H. Three of them (HLA-E, -F and -G) were shown to inhibit NK activity. On the contrary, the fourth one, HLA-H, named HFE after it was found to be mutated in patients suffering from inherited hemochromatosis, has been shown to be involved only in the regulation of iron uptake. We tested the capacity of HFE to affect (enhance or reduce) specifically the NK activity contained in non-manipulated fresh human PBMCs. We showed that HFE expression by target cells does not affect their killing by the NK-like activity contained in PBMCs. Moreover, using fluorescent HFE tetramers, we could confirm that blood NK cells as well as blood gammadelta T cells do not bind HFE. Altogether, our data indicate that HFE does not affect the NK activity contained in the PBMCs.

Regulation of Hfe by stress factors in BV-2 cells

Mutations in the Hfe gene can be associated with the iron overload disorder known as hemochromatosis. A number of recent studies suggest that carrying an Hfe mutation is a risk factor or genetic modifier for Alzheimer's disease (AD). In AD, Hfe protein expression is induced on cells associated with neuritic plaques and on neurons in the periplaque area. In this study, the factors that may be responsible for induction of Hfe in AD brain were determined using BV-2 cells. Hfe expression was induced by serum deprivation, menadione and β-amyloid. The labile iron pool was consistently decreased when Hfe expression increased. However, the changes in expression of Hfe appeared independent of the expression of transferrin receptor and ferritin. These data provide insight into the induction of Hfe in AD and indicate that Hfe expression may be a protective function to limit cellular iron exposure during cell stress. These results are the first in a series of studies to understand how mutations in Hfe can be a risk factor for AD.

Deletion of Murine Smn Exon 7 Directed to Liver Leads to Severe Defect of Liver Development Associated with Iron Overload

Spinal muscular atrophy (SMA) is characterized by degeneration of lower motor neurons caused by mutations of the survival motor neuron 1 gene (SMN1). SMN is involved in various processes including the formation of the spliceosome, pre-mRNA splicing and transcription. To know whether SMN has an essential role in all mammalian cell types or an as yet unknown specific function in the neuromuscular system, deletion of murine Smn exon 7, the most frequent mutation found among SMA patients, has been restricted to liver. Homozygous mutation results in severe impairment of liver development associated with iron overload and lack of regeneration leading to dramatic liver atrophy and late embryonic lethality of mutant mice. These data strongly suggest an ubiquitous and essential role of full-length SMN protein in various mammalian cell types. In SMA patients, the residual amount of SMN allows normal function of various organs except motor neurons. However, data from mouse and human suggest that other tissues might be involved in severe form of SMA or during prolonged disease course which reinforce the need of therapeutic approaches targeted to all tissues. In addition, liver function of patients should be carefully investigated and followed up before and during therapeutic trials.

P2-277 Expression and regulation of HFE in the brain: a risk factor for AD

P2-277 Expression and regulation of HFE in the brain: a risk factor for AD

Expression of the Hereditary Hemochromatosis Protein HFE Increases Ferritin Levels by Inhibiting Iron Export in HT29 Cells

Iron is essential for life in almost all organisms and, in mammals, is absorbed through the villus cells of the duodenum. Using a human colonic carcinoma cell line that has many duodenal characteristics, HT29, we show that genes involved in intestinal iron transport are endogenously expressed. When stably transfected to express the hereditary hemochromatosis protein HFE these cells have increased ferritin levels. We demonstrate that this is not due to an effect on the transferrin (TF)-mediated iron uptake pathway but rather due to inhibition of iron efflux from the cell. The effect of HFE was independent of its interaction with TF receptor 1 as indicated by similar results using both the wild type HFE and the W81A mutant that binds TF receptor 1 with greatly reduced affinity. HFE expression did not affect the mRNA levels of most of the genes involved in iron absorption that were tested; however, it did correspond to a decrease in hephaestin message levels. These results point to a role for HFE in inhibition of iron efflux in HT29 cells. This is a distinct role from that in HeLa and human embryonic kidney 293 cells where HFE has been shown to inhibit TF-mediated iron uptake resulting in decreased ferritin levels. Such a distinction suggests a multifunctional role for HFE that is dependent upon expression levels of proteins involved in iron transport.

Duodenal HFE expression and hepcidin levels determine body iron homeostasis: modulation by genetic diversity and dietary iron availability.

HFE affects the interaction of transferrin bound iron with transferrin receptors (TfR) thereby modulating iron uptake. To study genetically determined differences in HFE expression we examined individual HFE levels in C57BL/Sv129 mice and assessed their relationship to the regulation of iron homeostasis in the duodenum and the liver, and their regulation by diet. We found an up to 14-fold variation in inter-individual expression of HFE mRNA in the duodenum. Mice with high duodenal HFE mRNA expression presented with significantly higher levels of TfR and DMT-1 mRNAs and an increased IRP-1 binding affinity as compared to mice with low HFE levels. Duodenal HFE expression was positively associated with serum iron and liver HFE levels. Dietary iron supplementation decreased HFE in the duodenum but not in the liver. This was paralleled by reduced amounts of DMT-1 and FP-1 in the duodenum while the expression of DMT-1, FP-1, and hepcidin in the liver were increased with dietary iron overload. Duodenal and liver HFE levels are regulated by divergent penetration of as yet unelucidated modifier genes and to a much lesser extent by dietary iron. These measures control duodenal iron transport and liver iron homeostasis by modulating HFE expression either directly or via stimulation of iron sensitive regulatory molecules, such as hepcidin, which then exert their effects on body iron homeostasis.

A study of some hepatic immunological markers, iron load and virus genotype in chronic hepatitis C

Background/Aims Host factors that may influence progression of hepatitis C infection to chronic hepatitis include T-cell responses and iron accumulation. We evaluated the hepatic expression of immunological markers relevant for a cytotoxic response in relation to viral and HFE genotype. Methods Frozen liver biopsies were obtained at diagnosis from 28 HFE genotyped patients. Sections stained for CD8, MHC-I, β 2m, HFE and CD68 were analyzed blind by morphometry. Response to therapy was available in 12 cases. Results A negative correlation was found between the number of CD8 + cells and fibrosis. CD8 + cells localized as clusters in portal tracts and sinusoids and were seen interacting with MHC-I positive lining cells. MHC-I and β 2m were expressed mainly in the endothelial and Kupffer cells. HFE was expressed in most, but not all, round and dendritic CD68 + cells. Patients with virus genotype 3a had higher hepatic MHC-I and HFE expression, and a better-sustained response to IFN therapy than other patients. Conclusions In chronic hepatitis C virus infection MHC-I expression in the liver seems to relate to viral-genotype. In addition, the expression of MHC-I molecules by Kupffer cells places them as probable important players in the host response to HCV.

A study of some hepatic immunological markers, iron load and virus genotype in chronic hepatitis C

<h2>Abstract</h2><h3>Background/Aims</h3> Host factors that may influence progression of hepatitis C infection to chronic hepatitis include T-cell responses and iron accumulation. We evaluated the hepatic expression of immunological markers relevant for a cytotoxic response in relation to viral and HFE genotype. <h3>Methods</h3> Frozen liver biopsies were obtained at diagnosis from 28 HFE genotyped patients. Sections stained for CD8, MHC-I, β₂m, HFE and CD68 were analyzed blind by morphometry. Response to therapy was available in 12 cases. <h3>Results</h3> A negative correlation was found between the number of CD8⁺ cells and fibrosis. CD8⁺ cells localized as clusters in portal tracts and sinusoids and were seen interacting with MHC-I positive lining cells. MHC-I and β₂m were expressed mainly in the endothelial and Kupffer cells. HFE was expressed in most, but not all, round and dendritic CD68⁺ cells. Patients with virus genotype 3a had higher hepatic MHC-I and HFE expression, and a better-sustained response to IFN therapy than other patients. <h3>Conclusions</h3> In chronic hepatitis C virus infection MHC-I expression in the liver seems to relate to viral-genotype. In addition, the expression of MHC-I molecules by Kupffer cells places them as probable important players in the host response to HCV.

Relationships and distinctions in iron-regulatory networks responding to interrelated signals

Specialized cDNA-based microarrays (IronChips) were developed to investigate complex physiological gene-regulatory patterns in iron metabolism. Approximately 115 human cDNAs were strategically selected to represent genes involved either in iron metabolism or in interlinked pathways (eg, oxidative stress, nitric oxide [NO] metabolism, or copper metabolism), and were immobilized on glass slides. HeLa cells were treated with iron donors or iron chelators, or were subjected to oxidative stress (H(2)O(2)) or NO (sodium nitroprusside). In addition, we generated a stable transgenic HeLa cell line expressing the HFE gene under an inducible promoter. Gene-response patterns were recorded for all of these interrelated experimental stimuli, and analyzed for common and distinct responses that define signal-specific regulatory patterns. The resulting regulatory patterns reveal and define degrees of relationship between distinct signals. Remarkably, the gene responses elicited by the altered expression of the hemochromatosis protein HFE and by pharmacological iron chelation exhibit the highest degree of relatedness, both for iron-regulatory protein (IRP) and non-IRP target genes. This finding suggests that HFE expression directly affects the intracellular chelatable iron pool in the transgenic cell line. Furthermore, cells treated with the iron donors hemin or ferric ammonium citrate display response patterns that permit the identification of the iron-loaded state in both cases, and the discrimination between the sources of iron loading. These findings also demonstrate the broad utility of gene-expression profiling with the IronChip to study iron metabolism and related human diseases.

Co-localization of the mammalian hemochromatosis gene product (HFE) and a newly identified transferrin receptor (TfR2) in intestinal tissue and cells.

Mutations in the HFE gene and a newly identified second transferrin receptor gene, TfR2, cause hemochromatosis. The cognate proteins, HFE and TfR2, are therefore of key importance in human iron homeostasis. HFE is expressed in small intestinal crypt cells where transferrin-iron entry may determine subsequent iron absorption by mature enterocytes, but the physiological function of TfR2 is unknown. Using specific peptide antisera, we examined the duodenal localization of HFE and TfR2 in humans and mice, with and without HFE deficiency, by confocal microscopy. We also investigated potential interactions of these proteins in human intestinal cells in situ. Duodenal expression of HFE and TfR2 (but not TfR1) in wild-type mice and humans was restricted to crypt cells, in which they co-localized. HFE deficiency disrupted this interaction, altering the cellular distribution of TfR2 in human crypts. In human Caco-2 cells, HFE and TfR2 co-localized to a distinct CD63-negative vesicular compartment showing marked signal enhancement on exposure to iron-saturated transferrin ligand, indicating that HFE preferentially interacts with TfR2 in a specialized early endosomal transport pathway for transferrin-iron. This interaction occurs specifically in small intestinal crypt cells that differentiate to become iron-absorbing enterocytes. Our immunohistochemical findings provide evidence for a novel mechanism for the regulation of iron balance in mammals.

The discovery of the new haemochromatosis gene: Feder JN, Gnirke A, Thomas W, Tsuchihashi Z, Ruddy DA, Basava A, et al. A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis [Nat Genet 1996;13:399–408

The discovery of the new haemochromatosis gene: Feder JN, Gnirke A, Thomas W, Tsuchihashi Z, Ruddy DA, Basava A, et al. A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis [Nat Genet 1996;13:399–408

A single viral protein HCMV US2 affects antigen presentation and intracellular iron homeostasis by degradation of classical HLA class I and HFE molecules

HFE is a nonclassical class I molecule that associates with beta 2-microglobulin (beta 2m) and with the transferrin receptor. HFE accumulates in transferrin-containing endosomes, and its overexpression in human cell lines correlates with decreased transferrin receptor (TFR)-mediated iron uptake and decreased intracellular iron pools. A mutation that interferes with proper folding and assembly of HFE complexes results in a severe iron-overload disease hereditary hemochromatosis. We previously suggested that viruses could also interfere with iron metabolism through the production of proteins that inactivate HFE, similarly to classical class I proteins. In particular, we demonstrated in a transient expression system that human cytomegalovirus (HCMV) US2 targeted HFE for proteasomal degradation. Here we demonstrate that the stable expression of HCMV US2 in HEK 293 cells constitutively expressing HFE leads to loss of HFE expression both intracellularly and on the cell surface, and the significant reduction of classical class I expression. Both HFE and classical class I molecules are targeted to degradation via a similar pathway. This HCMV US2-mediated degradation of HFE leads to increased intracellular iron pools as indicated by reduced synthesis of TfR and increased ferritin synthesis. Whether this interference with regulation of iron metabolism potentiates viral replication and/or promotes damage of HCMV-infected tissues remains to be determined. Nevertheless, the deleterious effect of US2 on the expression of HFE and classical class I major histo-compatibility complexes (MHC) provides HCMV with an efficient tool for altering cellular metabolic functions, as well as supporting the escape of virus-infected cells from cytotoxic T lymphocyte (CTL)-mediated immune responses.

Duodenal expression of HFE by real time PCR is not increased in hereditary hemochromatosis or iron deficiency anaemia

Duodenal expression of HFE by real time PCR is not increased in hereditary hemochromatosis or iron deficiency anaemia

Human platelets express hemochromatosis protein (HFE) and transferrin receptor 2.

While body iron status may influence platelets, little information is available about platelet expression of proteins regulating iron homeostasis. HFE, the protein defective in hereditary hemochromatosis, and transferrin receptor 2 (TfR2) are two novel protein candidates that could be involved in mechanisms of iron transport across the platelet plasma membrane. The expression and localization of HFE, TfR1 and TfR2 proteins in human platelets were examined using Western blotting and immunocytochemistry. Human platelets expressed HFE and TfR2, whereas no signal for TfR1 was found. The positive reactions for HFE and TfR2 were mainly confined to the platelet plasma membrane. Expression of HFE and TfR2 proteins in human platelets may indicate that the mutations in the corresponding genes could influence platelet count, size and/or activation. The presence of TfR2 and absence of TfR1 suggests that HFE may serve a different function in platelets compared with the other HFE-positive cell types, e.g. enterocytes, macrophages and syncytiotrophoblasts.

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.