Wild-type HFE protein normalizes transferrin iron accumulation in macrophages from subjects with hereditary hemochromatosis

Hereditary hemochromatosis (HC) is one of the most common single-gene hereditary diseases. A phenotypic hallmark of HC is low iron in reticuloendothelial cells in spite of body iron overload. Most patients with HC have the same mutation, a change of cysteine at position 282 to tyrosine (C282Y) in the HFE protein. The role of HFE in iron metabolism and the basis for the phenotypic abnormalities of HC are not understood. To clarify the role of HFE in the phenotypic expression of HC, we studied monocytes-macrophages from subjects carrying the C282Y mutation in the HFE protein and clinically expressing HC and transfected them with wild-type HFE by using an attenuated Salmonella typhimurium strain as a gene carrier. The Salmonella system allowed us to deliver genes of interest specifically to monocytes-macrophages with high transduction efficiency. The accumulation of (55)Fe delivered by (55)Fe-Tf was significantly lower in macrophages from patients with HC than from controls expressing wild-type HFE. Transfection of HC macrophages with the HFE gene resulted in a high level of expression of HFE protein at the cell surface. The accumulation of (55)Fe delivered by (55)Fe-Tf was raised by 40% to 60%, and this was reflected by an increase in the (55)Fe-ferritin pool within the HFE-transfected cells. These results suggest that the iron-deficient phenotype of HC macrophages is a direct effect of the HFE mutation, and they demonstrate a role for HFE in the accumulation of iron in these cells.     

HIV-1 Nef down-regulates the hemochromatosis protein HFE, manipulating cellular iron homeostasis

The multifunctional Nef protein of HIV-1 is important for the progression to AIDS. One action of Nef is to down-regulate surface MHC I molecules, helping infected cells to evade immunity. We found that Nef also down-regulates the macrophage-expressed MHC 1b protein HFE, which regulates iron homeostasis and is mutated in the iron-overloading disorder hemochromatosis. In model cell lines, Nef reroutes HFE to a perinuclear structure that overlaps the trans-Golgi network, causing a 90% reduction of surface HFE. This activity requires a Src-kinase-binding proline-rich domain of Nef and a conserved tyrosine-based motif in the cytoplasmic tail of HFE. HIV-1 infection of ex vivo macrophages similarly down-regulates naturally expressed surface HFE in a Nef-dependent manner. The effect of Nef expression on cellular iron was explored; iron and ferritin accumulation were increased in HIV-1-infected ex vivo macrophages expressing wild-type HFE, but this effect was lost with Nef-deleted HIV-1 or when infecting macrophages from hemochromatosis patients expressing mutated HFE. The iron accumulation in HIV-1-infected HFE-expressing macrophages was paralleled by an increase in cellular HIV-1-gag expression. We conclude that, through Nef and HFE, HIV-1 directly regulates cellular iron metabolism, possibly benefiting viral growth.     

Regulation of transferrin-mediated iron uptake by HFE, the protein defective in hereditary hemochromatosis

The protein defective in hereditary hemochromatosis, called HFE, is similar to MHC class I-type proteins and associates with beta2-microglobulin (beta2M). Its association with beta2M was previously shown to be necessary for its stability, normal intracellular processing, and cell surface expression in transfected COS cells. Here we use stably transfected Chinese hamster ovary cell lines expressing both HFE and beta2M or HFE alone to study the effects of beta2M on the stability and maturation of the HFE protein and on the role of HFE in transferrin receptor 1 (TfR1)-mediated iron uptake. In agreement with prior studies on other cell lines, we found that overexpression of HFE, without overexpressing beta2M, resulted in a decrease in TfR1dependent iron uptake and in lower iron levels in the cells, as evidenced by ferritin and TfR1 levels measured at steady state. However, overexpression of both HFE and beta2M had the reverse effect and resulted in an increase in TfR1-dependent iron uptake and increased iron levels in the cells. The HFE-beta2M complex did not affect the affinity of TfR1 for transferrin or the internalization rate of transferrin-bound TfR1. Instead, HFE-beta2M enhanced the rate of recycling of TfR1 and resulted in an increase in the steady-state level of TfR1 at the cell surface of stably transfected cells. We propose that Chinese hamster ovary cells provide a model to explain the effect of the HFE-beta2M complex in duodenal crypt cells, where the HFE-beta2M complex appears to facilitate the uptake of transferrin-bound iron to sense the level of body iron stores. Impairment of this process in duodenal crypt cells leads them to be iron poor and to signal the differentiating enterocytes to take up iron excessively after they mature into villus cells in the duodenum of hereditary hemochromatosis patients.     

Function of the hemochromatosis protein HFE： Lessons from animal models

Hereditary hemochromatosis （HH） is caused by chronic hyperabsorption of dietary iron. Progressive accumulation of excess iron within tissue parenchymal cells may lead to severe organ damage. The most prevalent type of HH is linked to mutations in the HFE gene, encoding an atypical major histocompatibility complex class I molecule. Shortly after its discovery in 1996, the hemochromatosis protein HFE was shown to physically interact with transferrin receptor 1 （TfR1） and impair the uptake of transferrin-bound iron in cells. However, these findings provided no clue why HFE mutations associate with systemic iron overload. It was later established that all forms of HH result from misregulation of hepcidin expression. This liverderived circulating peptide hormone controls iron efflux from duodenal enterocytes and reticuloendothelial macrophages by promoting the degradation of the iron exporter ferroportin. Recent studies with animal models of HH uncover a crucial role of HFE as a hepatocyte iron sensor and upstream regulator of hepcidin. Thus, hepatocyte HFE is indispensable for signaling to hepcidin, presumably as a constituent of a larger ironsensing complex. A working model postulates that the signaling activity of HFE is silenced when the protein is bound to TfR1. An increase in the iron saturation of plasma transferrin leads to displacement of TfR1 from HFE and assembly of the putative iron-sensing complex. In this way, iron uptake by the hepatocyte is translated into upregulation of hepcidin, reinforcing the concept that the liver is the major regulatory site for systemic iron homeostasis, and not merely an iron storage depot.     

HFE and non-HFE hemochromatosis

Hereditary hemochromatosis (HH) is a disorder of iron metabolism in which enhanced absorption of dietary iron causes increased iron accumulation in the liver, heart, and pancreas. Most individuals with HH are homozygous for a point mutation in the HFE gene, leading to a C282Y substitution in the HFE protein. The function of HFE protein is unknown, but the available evidence suggests that it acts in association with beta2-microglobulin and transferrin receptor 1 to regulate iron uptake from plasma transferrin by the duodenum, the proposed mechanism by which body iron levels are sensed. The identification of HFE has established the foundation for a better understanding of the molecular and cellular biology of iron homeostasis and its altered regulation in HH. Additionally, the ability to accurately diagnose iron overload disorders has been strengthened, family screening has been improved, and evaluation of patients with other forms of liver disease complicated by moderate-to-severe iron overload is now possible. However, the role of HFE testing in generalized population screening for HH is still controversial. Recently, other forms of HH have been described that are not related to HFE but are due to mutations in genes coding iron transport proteins.     

Hepatic iron metabolism gene expression profiles in HFE associated hereditary hemochromatosis

BACKGROUND: Individuals with pathogenic mutations in HFE, hemojuvelin (HJV) and transferrin receptor 2 (TfR2) have low levels of hepcidin, but little is known about the hepatic expression of these molecules in patients with physiological iron overload or HFE associated Hemochromatosis (HH). AIMS: To examine the hepatic mRNA expression of iron homeostasis genes in patients with HH, physiological iron overload and healthy controls. PATIENTS: Untreated C282Y homozygous HH patients (n=20) with elevated serum ferritin (SF) and patients with physiological iron overload (n=12) with positive hepatocellular iron staining and negative HFE mutation analysis were evaluated. The control cohort (n=10) had normal iron parameters, negative HFE mutation analysis and negative hepatocellular iron staining. METHODS: Hepcidin, HJV (hemojuvelin), TfR2 (transferrin receptor 2), HFE, IL6 (interleukin 6) and ferroportin mRNA expression patterns were evaluated using quantitative real-time PCR. RESULTS: Physiological iron overload led to significantly upregulated hepcidin, HJV and ferroportin mRNA expression while TfR2 expression was not significantly different to controls. In contrast, HFE associated iron overload failed to induce hepcidin or HJV. TfR2 mRNA expression was significantly reduced when compared to controls. Ferroportin expression in HH was comparable to that found in physiological iron overload. Neither HFE nor IL6 expression was altered by variation in iron status. CONCLUSIONS: These findings suggest that patients with HH, in contrast to those with physiological iron overload, have a weakened TfR2 sensing mechanism that leads to the lack of induction of hepcidin and HJV. The C282Y HFE mutation does not appear to impede the hepatocellular iron export function of ferroportin.     

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.     

Transferrin receptor is negatively modulated by the hemochromatosis protein HFE: implications for cellular iron homeostasis.

Hereditary hemochromatosis is a common autosomal recessive disorder of iron metabolism. Recent demonstration of an association between transferrin receptor (TfR) and HFE, a major histocompatibility complex class I-like molecule that has been implicated to play a role in hereditary hemochromatosis, further strengthens the notion that HFE is involved in iron metabolism. Herein we show that TfR is required for and controls the assembly and the intracellular transport and surface expression of HFE. Because surface-expressed HFE and TfR remain firmly associated physically, only the fraction of TfR that is associated with HFE during biosynthesis is affected functionally. Moreover, we show that HFE binding reduces the number of functional transferrin binding sites and impairs TfR internalization, thus reducing the uptake of transferrin-bound iron. Thus, iron homeostasis is indirectly regulated by HFE, a negative modulator of TfR.     

HFE hemochromatosis in African Americans: Prevalence estimates of iron overload and iron overload-related disease

BackgroundLittle is known about the prevalence of HFE (homeostatic iron regulator) hemochromatosis in African Americans (AA).MethodsWe defined AA as self-identified AA, blacks, or non-Hispanic blacks. We defined hemochromatosis-associated HFE genotypes as p.C282Y/p.C282Y and p.C282Y/p.H63D. We compiled prevalences of these genotypes in AA using published population and cohort data and numbers of men and women ≥18 y? in 2018 U.S. Census estimates. We defined iron overload (IO) and IO-related disease by genotype as previously reported in population and cohort studies of hemochromatosis in whites of European ancestry. We used these definitions to estimate prevalences and numbers of AA with IO and IO-related disease associated with hemochromatosis-associated HFE genotypes.ResultsThere were ～16,287,599 men and ～17,644,898 women. HFE genotypes and their respective prevalences were: p.C282Y/p.C282Y, 0.00017 (6/34,905) [95% confidence interval 0.000034, 0.00031] and p.C282Y/p.H63D, 0.0012 (41/33,596) [0.000084, 0.0016]. IO prevalences were: men 0.000076 [0.000072, 0.000081] and women 0.0000061 [0.0000050, 0.0000073]. IO-related disease prevalences were: men 0.000063 [0.000059, 0.000067] and women 0.0000021 [0.0000014, 0.0000027]. There were ～1021 [961, 1091] men and ～36 [25, 48] women with IO-related disease.ConclusionsWe conclude that ～1/25,061 AA >18 y have a hemochromatosis-associated HFE genotype and IO and that ～1/32,103 AA >18 y have a hemochromatosis-associated HFE genotype and IO-related disease.     

Helicobacter pylori infection and HFE hemochromatosis

Helicobacter pylori infections are associated with iron deficiency, even in the absence of bleeding. To determine whether H. pylori infection plays a role in modifying the phenotype of patients homozygous for the c.845 G > A (C282Y) mutation of the HFE gene we studied 79 homozygous women and 76 homozygous men, comparing the pretreatment hemoglobin, MCV, serum ferritin, transferrin saturation of those who were seropositive and seronegative for H. pylori. No difference between seropositive and seronegative homozytoes was found. There was also no difference between seropositive and seronegative control subjects. We also compared the total iron of 56 of the male and 32 of the female homozygotes as determined by serial phlebotomy. No significant difference was found.     

Molecular aspects of iron absorption: Insights into the role of HFE in hemochromatosis

Hereditary hemochromatosis is the most common genetic disorder occurring in persons of northern European descent, and the clinical hallmark of the disease is the gradual accumulation of iron in internal organs, especially the liver, heart, and pancreas, which ultimately leads to organ failure. HFE, the gene that is defective in the majority of cases, was identified in 1996 and, although the exact role that HFE plays in the uptake and utilization of iron is not yet clear, important aspects of HFE function are emerging. Identification and studies of new proteins involved in the absorption of iron in the gut and in somatic cells has led to a clearer picture of how humans absorb iron from the diet and regulate this absorption to meet metabolic needs and to balance body iron stores. This review focuses on the molecular aspects of iron absorption and the role that HFE may play in these processes.     

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

OBJECTIVES: 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. METHODS: The expression and localization of HFE, TfR1 and TfR2 proteins in human platelets were examined using Western blotting and immunocytochemistry. RESULTS: 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. CONCLUSIONS: 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.     

Alternate splicing produces a soluble form of the hereditary hemochromatosis protein HFE

HFE is a non-typical MHC class 1-type protein that is mutated in hereditary hemochromatosis. The purpose of this study was to identify possible splice variants of HFE mRNA and investigate the regulation of these isoforms in duodenum and liver of patients with normal and altered iron stores. RT-PCR was performed using HFE specific primers and duodenal RNA obtained from patients with hemochromatosis, iron deficiency, secondary iron overload and normal controls. The reaction products were visualized by Southern blot and identified by DNA sequence analysis. Additional studies were performed on RNA isolated from liver and a range of human tissues. A truncated (soluble) form of HFE protein was identified that lacks the transmembrane domain and occurs as a result of alternative splicing. Soluble HFE was found predominantly in the duodenum, spleen, breast, skin and testicle. In hereditary hemochromatosis full length HFE was the predominant isoform present in the duodenum similar to iron deficiency. Alternate splicing produces soluble HFE that may have a unique function to regulate cellular iron transport.     

Modifying factors of the HFE hemochromatosis phenotype

C282Y homozygosity is the only common HFE genotype able to produce a complete hemochromatosis phenotype. However, its biochemical penetrance is incomplete (75% in men and 50% in women) and its clinical penetrance is low, especially in women (1 vs 25% in men). Environmental (e.g., diet, alcohol, drugs and metabolic syndrome) and genetic (digenism, common polymorphisms in the bone morphogenetic protein pathway involved in the regulation of hepcidin synthesis) explain a part of the variability of the C282Y homozygous phenotype. All other common HFE genotypes – including C282Y–H63D compound heterozygosity – are not associated with significant biochemical and clinical expression in the absence of comorbid factors (e.g., alcohol, diabetes or steatohepatitis). Better identification of acquired and genetic modifiers of iron burden and iron-related organ damage is needed to improve the preventive, diagnostic and therapeutic management of HFE hemochromatosis.     

欧洲肝病学会(EASL)2010年HFE基因相关性血色病临床诊疗指南要点

1关于基因检测的建议1．1对普通人群不推荐进行HFE基因相关性血色病基因检测,因为本病的外显率低且只有少数C282Y纯合子会发展为铁超负荷（1B）。1．2对于病因不明的肝病患者,如果转铁蛋白饱和度升高,则应考虑测定血色病基因。