Physiology of iron transport and the hemochromatosis gene

Iron is essential for fundamental cell functions but is also a catalyst for chemical reactions involving free radical formation, potentially leading to oxidative stress and cell damage. Cellular iron levels are therefore carefully regulated to maintain an adequate substrate while also minimizing the pool of potentially toxic "free iron." The main control of body iron homeostasis in higher organisms is placed in the duodenum, where dietary iron is absorbed, whereas no controlled means of eliminating unwanted iron have evolved in mammals. Hereditary hemochromatosis, the prototype of deregulated iron homeostasis in humans, is due to inappropriately increased iron absorption and is commonly associated to a mutated HFE gene. The HFE protein is homologous to major histocompatibility complex class I proteins but is not an iron carrier, whereas biochemical and cell biological studies have shown that the transferrin receptor, the main protein devoted to cellular uptake of transferrin iron, interacts with HFE. This review focuses on recent advances in iron research and presents a model of HFE function in iron metabolism.     

Iron-independent specific protein expression pattern in the liver of HFE-deficient mice

Hereditary hemochromatosis type I is an autosomal-recessive iron overload disease associated with a mutation in HFE gene. The most common mutation, C282Y, disrupts the disulfide bond necessary for the association of HFE with beta-2-microglobulin and abrogates cell surface HFE expression. HFE-deficient mice develop iron overload indicating a central role of the protein in the pathogenesis of hereditary hemochromatosis type I. However, despite significant effort, the role of the HFE protein in iron metabolism is still unknown. To shed a light on the molecular mechanism of HFE-related hemochromatosis we studied protein expression changes elicited by HFE-deficiency in the liver which is the organ critical for the regulation of iron metabolism. We undertook a proteomic study comparing protein expression in the liver of HFE deficient mice with control animals. We compared HFE-deficient animals with control animals with identical iron levels obtained by dietary treatment to identify changes specific to HFE deficiency rather than iron loading. We found 11 proteins that were differentially expressed in the HFE-deficient liver using two-dimensional electrophoresis and mass spectrometry identification. Of particular interest were urinary proteins 1, 2 and 6, glutathione-S-transferase P1, selenium binding protein 2, sarcosine dehydrogenase and thioredoxin-like protein 2. Our data suggest possible involvement of lipocalins, TNF-alpha signaling and PPAR alpha regulatory pathway in the pathogenesis of hereditary hemochromatosis and suggest future targeted research addressing the roles of the identified candidate genes in the molecular mechanism of hereditary hemochromatosis.     

Hemochromatosis: a genetic defect in iron metabolism

Hemochromatosis (HC), the common inherited disorder in iron metabolism, affects at least 1 in 300 Caucasians. The disorder causes inappropriately high iron absorption and accumulation of excess iron in the parenchymal cells of the major organs of the body. The gene responsible for HC has recently been cloned and is termed HFE; two missense mutations have been reported in the gene, both cause amino acid substitutions (H63D and C282Y), but to date only the C282Y mutation has been found to clearly correlate with HC in all affected populations. HFE is highly homologous to genes in the major histocompatibility complex (MHC) class I family; all of these genes encode a heterodimeric protein which is complexed to β₂-microglobulin, a coupling essential for cell surface expression of a functional molecule. The first important step toward establishing the role of HFE in the pathogenesis of HC came with the recent observation that the C282Y mutation disrupts the binding of β₂-microglobulin to the HFE protein and as a result the mutant molecule is not expressed on the cell surface. BioEssays 20: 562–568, 1998. © 1998 John Wiley & Sons Inc.     

Mutant HFE H63D protein is associated with prolonged endoplasmic reticulum stress and increased neuronal vulnerability

A specific polymorphism in the hemochromatosis (HFE) gene, H63D, is over-represented in neurodegenerative disorders such as amyotrophic lateral sclerosis and Alzheimer disease. Mutations of HFE are best known as being associated with cellular iron overload, but the mechanism by which HFE H63D might increase the risk of neuron degeneration is unclear. Here, using an inducible expression cell model developed from a human neuronal cell line SH-SY5Y, we reported that the presence of the HFE H63D protein activated the unfolded protein response (UPR). This response was followed by a persistent endoplasmic reticulum (ER) stress, as the signals of UPR sensors attenuated and followed by up-regulation of caspase-3 cleavage and activity. Our in vitro findings were recapitulated in a transgenic mouse model carrying Hfe H67D, the mouse equivalent of the human H63D mutation. In this model, UPR activation was detected in the lumbar spinal cord at 6 months then declined at 12 months in association with increased caspase-3 cleavage. Moreover, upon the prolonged ER stress, the number of cells expressing HFE H63D in early apoptosis was increased moderately. Cell proliferation was decreased without increased cell death. Additionally, despite increased iron level in cells carrying HFE H63D, it appeared that ER stress was not responsive to the change of cellular iron status. Overall, our studies indicate that the HFE H63D mutant protein is associated with prolonged ER stress and chronically increased neuronal vulnerability.     

Protective role of calreticulin in HFE hemochromatosis

HFE gene mutations are associated with over 80% of cases of hereditary hemochromatosis (HH), an iron-overload disease in which the liver is the most frequently affected organ. Research on HFE has traditionally focused on its interaction with the transferrin receptor. More recent studies have suggested a more complex function for this nonclassical MHC-I protein. The aim of this study was to examine how HFE and its two most common mutations affect the expression of selected genes in a hepatocyte-like cell line. Gene expression was analyzed in HepG2 cells overexpressing wild-type and mutant HFE. The effect of HFE in iron import and oxidative stress levels was assessed. Unfolded protein response (UPR)-activated gene expression was analyzed in peripheral blood mononuclear cells from characterized HH patients. C282Y HFE down-regulated hepcidin and enhanced calreticulin mRNA expression. Calreticulin levels correlated with intracellular iron increase and were associated with protection from oxidative stress. In C282Y(+/+) patients calreticulin levels correlated with the expression of the UPR marker BiP and showed a negative association with the number of hereditary hemochromatosis clinical manifestations. The data show that expression of C282Y HFE triggers a stress-protective response in HepG2 cells and suggest a role for calreticulin as a modifier of the clinical expression of HH.     

Hemochromatosis gene sequence deviations in German patients with porphyria cutanea tarda

Patients with porphyria cutanea tarda (PCT) reveal a susceptibility to reversible inactivation of hepatic uroporphyrinogen decarboxylase, which might be triggered by alcohol, hepatitis C virus infection, and iron overload. Inherited factors that may predispose to clinically overt PCT also include sequence deviations in the HFE gene that is mutated in classical hemochromatosis. Here, we studied the prevalence of both common and rare hemochromatosis gene variations in 51 PCT patients and 54 healthy controls of German origin. The frequency of the common HFE gene mutation C282Y was 15.7 % in PCT patients and 2.8 % in healthy control individuals (P < 0.001). By contrast, the frequencies of the common H63D mutation did not differ, and the allele frequencies of the less frequently observed sequence deviations as substitution S65C in the HFE gene and mutation Y250X in the TFR2 gene underlying hemochromatosis type 3 (HFE3) were < 0.02 both in PCT patients and controls. Our results comprise the first molecular studies of both common and rare hemochromatosis gene variants in German PCT patients, indicating a significant role of the C282Y mutation in the pathogenesis of PCT.     

Possible roles of the hereditary hemochromatosis protein, HFE, in regulating cellular iron homeostasis

Hereditary hemochromatosis (HH) is the most common inherited disorder in people of Northern European descent. Over 83% of the cases of HH result from a single mutation of a Cys to Tyr in the HH protein. HFE. This mutation causes a recessive disease resulting in an accumulation of iron in selected tissues. Iron overload damages these organs leading to cirrhosis of the liver, diabetes, cardiomyopathy, and arthritis. The mechanism by which HFE influences iron homeostasis in cells and in the body remains elusive. Lack of functional HFE in humans produces the opposite effects in different cell types in the body. In the early stages of the disease. Kupffer cells in the liver and enterocytes in the intestine cells are iron depleted and have low intracellular ferritin levels, whereas hepatocytes in the liver are iron overloaded and have high intracellular iron levels. This review gives the background and a model as to possible mechanisms of how HFE could exert different effects on iron homeostasis in different cell types.     

Pitfalls in the genetic diagnosis of hereditary hemochromatosis

The widespread use of the genotype assay that identifies the common C282Y mutation in the HFE gene has allowed an earlier diagnosis to be made in many subjects. A significant number of these patients may have no evidence of phenotypic disease and have a normal serum ferritin level. This phenomenon is more common when the genotype assay is used to screen populations rather than higher-risk groups such as family members of a proband with hereditary hemochromatosis. Moreover, patients with significant iron overload may be wild type for the C282Y mutation and have no other demonstrable mutation of the HFE gene. The HFE genotype assay has recently been found to give a false-positive C282Y homozygous result in half of the subjects in one population screening study due to the presence of a single nucleotide polymorphism (SNP) that interfered with primer binding in the PCR assay. The problem may be overcome by using alternate primers. A number of other groups have confirmed the finding but in a much smaller number of subjects, whereas others found that their assays were not affected by the SNP. The use of the HFE genotype assay as the sole diagnostic criterion for hereditary hemochromatosis is not recommended. The genotype assay should be used as an adjunct to the established methods of demonstrating iron overload and be viewed as a predictor of either the presence of iron overload or the subsequent development of iron overload during an individual's lifetime.     

Regulatory networks for the control of body iron homeostasis and their dysregulation in HFE mediated hemochromatosis

Although the recent identification of several genes has extended our knowledge on the maintenance of body iron homeostasis, their tissue specific expression patterns and the underlying regulatory networks are poorly understood. We studied C57black/Sv129 mice and HFE knockout (HFE -/-) variants thereof as a model for hemochromatosis, and investigated the expression of iron metabolism genes in the duodenum, liver, and kidney as a function of dietary iron challenge. In HFE +/+ mice dietary iron supplementation increased hepatic expression of hepcidin which was paralleled by decreased iron regulatory protein (IRP) activity, and reduced expression of divalent metal transporter-1 (DMT-1) and duodenal cytochrome b (Dcytb) in the enterocyte. In HFE -/- mice hepcidin formation was diminished upon iron challenge which was associated with decreased hepatic transferrin receptor (TfR)-2 levels. Accordingly, HFE -/- mice presented with high duodenal Dcytb and DMT-1 levels, and increased IRP and TfR expression, suggesting iron deficiency in the enterocyte and increased iron absorption. In parallel, HFE -/- resulted in reduced renal expression of Dcytb and DMT-1. Our data suggest that the feed back regulation of duodenal iron absorption by hepcidin is impaired in HFE -/- mice, a model for genetic hemochromatosis. This change may be linked to inappropriate iron sensing by the liver based on decreased TfR-2 expression, resulting in reduced circulating hepcidin levels and an inappropriate up-regulation of Dcytb and DMT-1 driven iron absorption. In addition, iron excretion/reabsorption by the kidneys may be altered, which may aggravate progressive iron overload.     

The enigmatic role of the hemochromatosis protein (HFE) in iron absorption

The HFE gene, a member of the class-I transplantation antigen gene family, is responsible for hereditary hemochromatosis, one of the most common inherited diseases in individuals of European descent. Patients exhibit predictable changes in iron homeostasis, including elevations in both transferrin saturation and serum ferritin levels. A subset of patients progress to overt clinical sequelae, resulting from iron overload. A hallmark of the disease is increased absorption of iron by the intestine. Although the HFE protein appears to modulate the function of the transferrin receptor in vitro, its precise role in vivo remains obscure. With multiple cell types involved in iron metabolism, the function of HFE is likely to be complex.     

Hemochromatosis: As a conformational disorder

Hereditary hemochroamtosis (HH) refers to a unique clinicopathologic subset of iron overload syndromes that includes the disorder related to C282Y homozygous mutation of the hemochromatosis protein (HFE), the most common form of hereditary hemochromatosis. Recent reports have highlighted analogies with the class of disorders, known as the conformational diseases whereby HFE C282Y mutant protein forms aggregates and is subsequently retained in the endoplasmic reticulum (ER). In conformational disorders, accumulation of unfolded or misfolded proteins in the ER can activate a complex cascade linked to the regulation of diverse physiologic processes, disease onset and progression. To-date, reviews on HFE C282Y HH have largely dealt with the end-stage consequence of this disorder (iron overload). However, our review focuses on upstream molecular events resulting from the mislocalization of the aggregation-prone HFE C282Y protein leading to potential advances in treatment and diagnosis.     

Iron Imports. VI. HFE and regulation of intestinal iron absorption

The majority of clinical cases of iron overload is caused by mutations in the HFE gene. However, the role that HFE plays in the physiology of intestinal iron absorption remains enigmatic. Two major models have been proposed: 1) HFE exerts its effects on iron homeostasis indirectly, by modulating the expression of hepcidin; and 2) HFE exerts its effects directly, by changing the iron status (and therefore the iron absorptive activity) of intestinal enterocytes. The first model places the primary role of HFE in the liver (hepatocytes and/or Kupffer cells). The second model places the primary role in the duodenum (crypt cells or villus enterocytes). These models are not mutually exclusive, and it is possible that HFE influences the iron status in each of these cell populations, leading to cell type-specific downstream effects on intestinal iron absorption and body iron distribution.     

Growth hormone releasing substances: types and their receptors

A series of structurally diverse growth hormone (GH) releasing substances have been synthesized that are distinct from the naturally occurring GH releasing hormone (GHRH). These synthetic molecules range from the family of GH releasing peptides and mimetics such as MK-0677. The physiological importance of these molecules and their receptor is exemplified by studies in the elderly. For example, when MK-0677 was administered chronically to 70- to 90-year-old subjects, once daily, the age-related reduced amplitude of GH pulses was reversed to that of the physiological profile typical of young adults. In 1996, the synthesis of (35)S-MK-0677 was reported and used as a ligand to characterize a common receptor (GH secretagogue receptor [GHS-R]) for the GH releasing substances. The GHS-R is distinct from the GHRH receptor. Subsequently, the GHS-R gene was cloned and shown to encode a unique G-protein coupled receptor with a deduced protein sequence that was 96% identical in human and rat. Because of the physiological importance of the GHS-R, a search for family members (FMs) was initiated and its molecular evolution investigated. Three FMs GPR38, GPR39 and FM3 were isolated from human genomic libraries. To accelerate the identification of other FMs, a vertebrate organism with a compact genome distant in evolutionary terms from humans was exploited. The pufferfish (Spheroides nephelus) genome provides an ideal model for the discovery of human genes. Three distinct full-length clones encoding proteins of significant sequence identity to the human GHS-R were cloned from the pufferfish. Remarkably, the pufferfish gene with highest sequence homology to the human receptor was activated by the hexapeptide and non-peptide ligands. These intriguing results show that the structure and function of the ligand binding pocket of the human GHS-R has been highly conserved in evolution ( approximately 400 million years) and strongly suggests that an endogenous natural ligand has been conserved. This new information is consistent with a natural ligand for the GHS-R playing a fundamentally important and conserved role in physiology.     

Overexpression of hemochromatosis protein, HFE, alters transferrin recycling process in human hepatoma cells

HFE is a MHC class 1-like protein that is mutated in hereditary hemochromatosis. In order to elucidate the role of HFE protein on cellular iron metabolism, functional studies were carried out in human hepatoma cells (HLF) overexpressing a fusion gene of HFE and green fluorescent protein (GFP). The expression of HFE-GFP was found to be localized on cell membrane and perinuclear compartment by fluorescent microscopy. By co-immunoprecipitation and Western blotting, HFE-GFP protein formed a complex with endogenous transferrin receptor and beta(2)-microglobulin, suggesting that this fusion protein has the function of HFE reported previously. We then examined the (59)Fe uptake and release, and internalization and recycling of (125)I-labeled transferrin in order to elucidate the functional roles of HFE in the cell system. In the transfectants, HFE protein decreased the rate of transferrin receptor-dependent iron ((59)Fe) uptake by the cells, but did not change the rate of iron release, indicating that HFE protein decreased the rate of iron influx. Scatchard analysis of transferrin binding to HFE-transfected cells showed an elevation of the dissociation constant from 1.9 to 4. 3 nM transferrin, indicating that HFE protein decreased the affinity of transferrin receptor for transferrin, while the number of transferrin receptors decreased from 1.5x10(5)/cell to 1. 2x10(5)/cell. In addition, the rate of transferrin recycling, especially return from endosome to surface, was decreased in the HFE-transfected cells by pulse-chase study with (125)I-labeled transferrin. Our results strongly suggest an additional role of HFE on transferrin receptor recycling in addition to the decrease of receptor affinity, resulting in the reduced cellular iron.     

Common variants in the BMP2, BMP4, and HJV genes of the hepcidin regulation pathway modulate HFE hemochromatosis penetrance

Most cases of genetic hemochromatosis (GH) are associated with the HFE C282Y/C282Y (p.Cys282Tyr/p.Cys282Tyr) genotype in white populations. The symptoms expressed by C282Y homozygotes are extremely variable. Only a few suffer from an overt disease. Several studies have suggested that, in addition to environmental factors, a genetic component could explain a substantial part of this phenotypic variation, although very few genetic factors have been identified so far. In the present study, we tested the association between common variants in candidate genes and hemochromatosis penetrance, in a large sample of C282Y homozygotes, using pretherapeutic serum ferritin level as marker of hemochromatosis penetrance. We focused on two biologically relevant gene categories: genes involved in non-HFE GH (TFR2, HAMP, and SLC40A1) and genes involved in the regulation of hepcidin expression, including genes from the bone morphogenetic protein (BMP) regulatory pathway (BMP2, BMP4, HJV, SMAD1, SMAD4, and SMAD5) and the IL6 gene from the inflammation-mediated regulation pathway. A significant association was detected between serum ferritin level and rs235756, a common single-nucleotide polymorphism (SNP) in the BMP2 genic region (P=4.42x10-5). Mean ferritin level, adjusted for age and sex, is 655 ng/ml among TT genotypes, 516 ng/ml in TC genotypes, and 349 ng/ml in CC genotypes. Our results further suggest an interactive effect on serum ferritin level of rs235756 in BMP2 and a SNP in HJV, with a small additive effect of a SNP in BMP4. This first reported association between common variants in the BMP pathway and iron burden suggests that full expression of HFE hemochromatosis is linked to abnormal liver expression of hepcidin, not only through impairment in the HFE function but also through functional modulation in the BMP pathway. Our results also highlight the BMP regulation pathway as a good candidate for identification of new modifier genes.     

Molecular cloning of growth hormone encoding cDNA of Indian major carps by a modified rapid amplification of cDNA ends strategy

A modified rapid amplification of cDNA ends (RACE) strategy has been developed for cloning highly conserved cDNA sequences. Using this modified method, the growth hormone (GH) encoding cDNA sequences ofLabeo rohita, Cirrhina mrigala andCatla catla have been cloned, characterized and overexpressed inEscherichia coli. These sequences show 96–98% homology to each other and are about 85% homologous to that of common carp. Besides, an attempt has been made for the first time to describe a 3-D model of the fish GH protein.     

A primer for predicting risk of disease in HFE-linked hemochromatosis

Since the discovery of the hemochromatosis gene (HFE) in 1996, there has been increasing interest in diagnostic testing for the C282Y and H63D mutations. The high frequency of these two alleles and their incomplete penetrance in homozygotes and compound heterozygotes make genetic counseling for hemochromatosis different from some other autosomal recessive conditions in that parents and children may also be at risk for iron overload, while homozygotes may remain asymptomatic. We provide a guideline for genetic counseling in HFE-linked hemochromatosis based on the genetic probability of inheriting HFE mutations and known information about expression of iron overload in various HFE genotypes. Genetic probabilities were based on allele frequencies derived from large population studies and Hardy-Weinberg equilibrium estimates. Expression of iron overload in those of various genotypes was based on available estimates of serum ferritin from population screening studies. Estimates for the likelihood of clinical iron overload requiring follow-up screening or treatment are provided by gender and genotype. The probability of inheriting HFE mutations and developing iron overload can be estimated in family members of a proband with HFE mutations. Many C282Y homozygotes will not have clinical iron overload. The risk is highest in men and their C282Y homozygous brothers and significantly lower in homozygous women. Iron overload is uncommon in compound heterozygotes and H63D homozygotes.     

Serum ferritin and transferrin saturation levels in β⁰ and β(+) thalassemia patients

There have been few studies on the mutations that cause heterozygous beta-thalassemia and how they affect the iron profile. One hundred and thirty-eight individuals were analyzed, 90 thalasemic β? and 48 thalasemic β(+), identified by classical and molecular methods. Mutations in the hemochromatosis (HFE) gene, detected using PCR-RFLP, were found in 30.4% of these beta-thalassemic patients; heterozygosity for H63D (20.3%) was the most frequent. Ferritin levels and transferrin saturation were similar in beta-thalassemics with and without mutations in the HFE gene. Ferritin concentrations were significantly higher in men and in individuals over 40 years of age. Transferrin saturation also was significantly higher in men, but only in those without HFE gene mutations. There was no significant difference in the iron profile among the β? and β(+) thalassemics, with and without HFE gene mutations. The frequency of ferritin values above 200 ng/mL in women and 300 ng/mL in men was also similar in β? and β(+) thalassemics (P > 0.72). Our conclusion is that ferritin levels are variable in the beta-thalassemia, trait regardless of the type of beta-globin mutation. Furthermore, HFE gene polymorphisms do not change the iron profile in these individuals.