Characterization of three novel pathogenic SLC40A1 mutations and genotype/phenotype correlations in 7 Italian families with type 4 hereditary hemochromatosis

Mutations of SLC40A1 encoding ferroportin (Fpn), the unique cellular iron exporter, severely affect iron homeostasis causing type 4 hereditary hemochromatosis, an autosomal dominant iron overload condition with variable phenotypic manifestations. This disease can be classified as type 4A, better known as “ferroportin disease”, which is due to “loss of function” mutations that lead to decreased iron export from cells, or as type 4B hemochromatosis, which is caused by “gain of function” mutations, conferring partial or complete resistance to hepcidin-mediated Fpn degradation.In this work, we discuss clinical and molecular findings on a group of patients in whom a SLC40A1 single copy missense variant was identified. Three novel variants, p.D181N, p.G204R and p.R296Q were functionally characterized. Fpn D181N and R296Q mutants can be classified as full or partial loss of function, respectively. Replacement of G204 with arginine appears to cause a more complex defect with impact both on iron export function and hepcidin sensitivity. This finding confirms the difficulty of predicting the effect of a mutation on the molecular properties of Fpn in order to provide an exhaustive explanation to the wide variability of the phenotype in type 4 hereditary hemochromatosis.     

A compartmental model of iron regulation in the mouse

A simple compartmental model is developed for investigating the mechanism of iron homeostasis. In contrast to previous mathematical models of iron metabolism, the liver is included as a key site of iron regulation. Compartments for free iron in blood, diferric transferrin (Tf) in blood, hepatocytes, red blood cells, and macrophages are included, and their roles in iron regulation are explored. The function of hepcidin in regulating iron absorption is modeled through an inverse relationship between hepatocyte transferrin receptor 2 (TfR2) levels and the rate of iron export processes mediated by ferroportin (Fpn). Simulations of anemia and erythropoiesis stimulation support the idea that the iron demands of the erythroid compartment can be communicated through diferric Tf. The iron-responsive element of Fpn is found to be important for stabilizing intracellular iron stores in response to changing iron demands and allowing proper iron regulation through diferric Tf. The contribution of iron dysregulation to the pathogenesis of iron overload disorders is also investigated. It is shown that the characteristics of HFE hemochromatosis can be reproduced by increasing the setpoint of iron absorption in the duodenum to a level where the system cannot downregulate iron absorption to meet the iron excretion rate.     

sAPP modulates iron efflux from brain microvascular endothelial cells by stabilizing the ferrous iron exporter ferroportin

A sequence within the E2 domain of soluble amyloid precursor protein (sAPP) stimulates iron efflux. This activity has been attributed to a ferroxidase activity suggested for this motif. We demonstrate that the stimulation of efflux supported by this peptide and by sAPPα is due to their stabilization of the ferrous iron exporter, ferroportin (Fpn), in the plasma membrane of human brain microvascular endothelial cells (hBMVEC). The peptide does not bind ferric iron explaining why it does not and thermodynamically cannot promote ferrous iron autoxidation. This peptide specifically pulls Fpn down from the plasma membrane of hBMVEC; based on these results, FTP, for ferroportin‐targeting peptide, correctly identifies the function of this peptide. The data suggest that in stabilizing Fpn via the targeting due to the FTP sequence, sAPP will increase the flux of iron into the cerebral interstitium. This inference correlates with the observation of significant iron deposition in the amyloid plaques characteristic of Alzheimer's disease.     

The iron regulatory hormone hepcidin inhibits expression of iron release as well as iron uptake proteins in J774 cells

The mechanism by which hepcidin controls cellular iron release protein ferroportin 1 (Fpn1) in macrophages has been well established. However, little is known about the effects of hepcidin on cellular iron uptake proteins. Here, we demonstrated for the first time that hepcidin can significantly inhibit the expression of transferrin receptor 1 (TfR1) and divalent metal transporter 1 in addition to Fpn1, and therefore reduce transferrin-bound iron and non-transferrin-bound iron uptake and also iron release in J774 macrophages. Analysis of mechanisms using the iron-depleted cells showed that hepcidin has a direct inhibitory effect on all iron transport proteins we examined. Further studies demonstrated that the down-regulation of TfR1 induced by hepcidin is associated with cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA), probably being mediated by the cAMP–PKA pathway in J774 macrophages.     

The role of cellular iron deficiency in controlling iron export

BackgroundIron export via the transport protein ferroportin (Fpn) plays a critical role in the regulation of dietary iron absorption and iron recycling in macrophages. Fpn plasma membrane expression is controlled by the hepatic iron-regulated hormone hepcidin in response to high iron availability and inflammation. Hepcidin binds to the central cavity of the Fpn transporter to block iron export either directly or by inducing Fpn internalization and lysosomal degradation. Here, we investigated whether iron deficiency affects Fpn protein turnover.MethodsWe ectopically expressed Fpn in HeLa cells and used cycloheximide chase experiments to study basal and hepcidin-induced Fpn degradation under extracellular and intracellular iron deficiency.Conclusions/General significanceWe show that iron deficiency does not affect basal Fpn turnover but causes a significant delay in hepcidin-induced degradation when cytosolic iron levels are low. These data have important mechanistic implications supporting the hypothesis that iron export is required for efficient targeting of Fpn by hepcidin. Additionally, we show that Fpn degradation is not involved in protecting cells from intracellular iron deficiency.     

The iron regulatory hormone hepcidin reduces ferroportin 1 content and iron release in H9C2 cardiomyocytes

Iron plays a key pathophysiological role in a number of cardiac diseases. Studies on the mechanisms of heart iron homeostasis are therefore crucial for understanding the causes of excessive heart iron. In addition to iron uptake, cellular iron balance in the heart also depends on iron export. We provided evidence for the existence of iron exporter ferroportin 1 (Fpn1) in the heart in a recent study. The presence of hepcidin, a recently discovered iron regulatory hormone, was also confirmed in the heart recently. Based on these findings and the inhibiting role of hepcidin on Fpn1 in other tissues, we speculated that hepcidin might be able to bind with, internalize and degrade Fpn1 and then decrease iron export in heart cells, leading to an abnormal increase in heart iron and iron mediated cell injury. We therefore investigated the effects of hepcidin on the contents of Fpn1 and iron release in H9C2 cardiomyocyte cell line. We demonstrated that hepcidin has the ability to reduce Fpn1 content as well as iron release in this cell. The similar regulation patterns of hepcidin on the Fpn1 and iron release suggested that the decreased iron release resulted from the decreased content of Fpn1 induced by hepcidin. We also found that hepcidin has no significant effects on ceruloplasmin (CP) and hephaestin (Heph) — two proteins required for iron release from mammalian cells. The data imply that Fpn1, rather than Heph and CP, is the limited factor in the regulation of iron release from heart cells under physiological conditions.     

Iron imports. III. Transfer of iron from the mucosa into circulation

Transfer of iron from the mucosa is a critical step in dietary iron assimilation that is tightly regulated to ensure the appropriate amount of iron is absorbed to meet the body's demands. Too much iron is highly toxic, and failure to properly control intestinal iron export causes iron overload associated with hereditary forms of hemochromatosis. One form of genetic iron overload, ferroportin disease, originates due to defects in ferroportin, the membrane iron exporter. Ferroportin acts in conjunction with the intestinal ferroxidase hephaestin to mediate release of iron from the enterocyte. How iron is then acquired by transferrin and released into circulation remains an unknown step in this process.     

利用FRET技术研究Hepcidin 和Fpn 相互作用

铁是生命必需的微量元素,ferroportin(Fpn)是小肠吸收细胞铁释放的重要蛋白。新近发现肝脏分泌的抗菌多肽 hepcidin 具有调节肠铁吸收的重要作用,但目前尚缺少Fpn和hepcidin发生作用的实验依据。应用荧光共振能量转移技术(fluorescence resonance energy transfer ,FRET)对hepcidin和Fpn之间的作用关系进行了深入研究。首先进行了hepcidin-CF P融合蛋白表达载体的构建及表达鉴定;然后对含YFP,Fpn-YFP基因动物细胞表达载体的构建、表达和FRET检测。实验结果证实hepcidin和Fpn之间存在直接的相互作用,并发现两种蛋白发生相互作用后hepcidin也在细胞质中有分布。为临床治疗铁代谢紊乱性疾病提供了新的治疗策略和重要理论依据。     

Role of extracellular Hydrogen peroxide in regulation of iron homeostasis genes in neuronal cells: Implication in iron accumulation

Iron accumulation and oxidative stress are associated with neurodegenerative disease. Labile iron is known to catalyze free radical generation and subsequent neuronal damage, whereas the role of oxidative stress in neuronal iron accumulation is less well understood. Here, we examined the effect of hydrogen peroxide (H₂O₂) treatment on cellular iron-uptake, -storage, and -release proteins in the neuroblastoma cell line SH-SY5Y. We found no detectable change in the iron-uptake proteins transferrin receptor-1 and divalent metal ion transporter. In contrast, H₂O₂ treatment resulted in significant degradation of the iron-exporter ferroportin (Fpn). A decrease in Fpn is expected to increase the labile iron pool (LIP), reducing the iron-regulatory protein (IRP)–iron-responsive element interaction and increasing the expression of ferritin-H (Ft-H) for iron storage. Instead, we detected IRP1 activation, presumably due to oxidative stress, and a decrease in Ft-H translation. A reduction in Ft-H mRNA was also observed, probably dependent on an antioxidant-response element present in the Ft-H enhancer. The decrease in Fpn and Ft-H upon H₂O₂ treatment led to a time-dependent increase in the cellular LIP. Our study reveals a complex regulation of neuronal iron-release and iron-storage components in response to H₂O₂ that may explain iron accumulation detected in neurodegenerative diseases associated with oxidative stress.     

Iron imports. IV. Hepcidin and regulation of body iron metabolism

Hepcidin, a small peptide synthesized in the liver, controls extracellular iron by regulating its intestinal absorption, placental transport, recycling by macrophages, and release from stores. Hepcidin inhibits the cellular efflux of iron by binding to and inducing the degradation of ferroportin, the sole iron exporter in iron-transporting cells. In turn, hepcidin synthesis is increased by iron loading and decreased by anemia and hypoxia. Hepcidin is markedly induced during inflammation, trapping iron in macrophages, decreasing plasma iron concentrations, and contributing to the anemia of inflammation. Hepcidin deficiency due to the dysregulation of its synthesis causes most known forms of hemochromatosis.     

The Ferroxidase Hephaestin But Not Amyloid Precursor Protein is Required for Ferroportin-Supported Iron Efflux in Primary Hippocampal Neurons

Iron efflux in mammalian cells is mediated by the ferrous iron exporter ferroportin (Fpn); Fpn plasma membrane localization and function are supported by a multicopper ferroxidase and/or the soluble amyloid precursor protein (sAPP). Fpn and APP are ubiquitously expressed in all cell types in the central nervous system including neurons. In contrast, neuronal ferroxidase(s) expression has not been well characterized. Using primary cultures of hippocampal neurons, we examined the molecular mechanism of neuronal Fe efflux in detail. Developmental increases of Fpn, APP, and the ferroxidase hephaestin (Hp) were observed in hippocampal neurons. Iron efflux in these neurons depended on the level of Fpn localized at the cell surface; as noted, Fpn stability is supported by ferroxidase activity, an enzymatic activity that is required for Fe efflux. Iron accumulation increases and iron efflux decreases in Hp knockout neurons. In contrast, suppression of endogenous APP by RNAi knockdown does not affect surface Fpn stability or Fe efflux. These data support the model that the neuronal ferroxidase Hp plays a unique role in support of Fpn-mediated Fe efflux in primary hippocampal neurons. Our data also demonstrate that Hp ferroxidase activity relies on copper bioavailability, which suggests neuronal iron homeostasis will be modulated by cellular copper status.     

Copper repletion enhances apical iron uptake and transepithelial iron transport by Caco-2 cells

The influence of copper status on Caco-2 cell apical iron uptake and transepithelial transport was examined. Cells grown for 7-8 days in media supplemented with 1 microM CuCl(2) had 10-fold higher cellular levels of copper compared with control. Copper supplementation did not affect the integrity of differentiated Caco-2 cell monolayers grown on microporous membranes. Copper-repleted cells displayed increased uptake of iron as well as increased transport of iron across the cell monolayer. Northern blot analysis revealed that expression of the apical iron transporter divalent metal transporter-1 (DMT1), the basolateral transporter ferroportin-1 (Fpn1), and the putative ferroxidase hephaestin (Heph) was upregulated by copper supplementation, whereas the recently identified ferrireductase duodenal cytochrome b (Dcytb) was not. These results suggest that DMT1, Fpn1, and Heph are involved in the iron uptake process modulated by copper status. Although a clear role for Dcytb was not identified, an apical surface ferrireductase was modulated by copper status, suggesting that its function also contributes to the enhanced iron uptake by copper-repleted cells. A model is proposed wherein copper promotes iron depletion of intestinal Caco-2 cells, creating a deficiency state that induces upregulation of iron transport factors.     

Copper deficiency has minimal impact on ferroportin expression or function

Interactions between copper and iron homeostasis have been known since the nineteenth century when anemia in humans was first described due to copper limitation. However, the mechanism remains unknown. Intestinal and liver iron concentrations are usually higher following copper deficiency (CuD). This may be due to impaired function of the multicopper oxidases hephaestin or ceruloplasmin (Cp), respectively. However, iron retention could be due to altered ferroportin (Fpn), the essential iron efflux transporter in enterocytes and macrophages. Fpn mRNA is controlled partially by intracellular iron and IRE dependence. CuD should augment Fpn based on iron level. Some argue that Fpn stability is controlled partially by membrane ferroxidase (GPI-Cp). CuD should result in lower Fpn since GPI-Cp expression and function is reduced. Fpn turnover is controlled by hepcidin. CuD results in variable Hamp (hepcidin) expression. Fpn mRNA and protein level were evaluated following dietary CuD in rats and mice. To correlate with Fpn expression, measurements of tissue iron were conducted in several rodent models. Following CuD there was little change in Fpn mRNA. Previous work indicated that under certain circumstances Fpn protein was augmented in liver and spleen following CuD. Fpn levels in CuD did not correlate with either total iron or non-heme iron (NHI), as iron levels in CuD liver were higher and in spleen lower than copper adequate controls. Fpn steady state levels appear to be regulated by a complex set of factors. Changes in Fpn do not explain the anemia of CuD.     

Hepcidin: a direct link between iron metabolism and immunity

Hepcidin, originally discovered in urine as a bactericidal peptide synthesized by hepatocytes was later proved to be a key regulator of iron metabolism at the whole body level, namely, in conditions of altered iron demand such as the increased or decreased total amount of body iron, inflammation, hypoxia and anemia. The major mechanism of hepcidin function seems to be the regulation of transmembrane iron transport. Hepcidin binds to its receptor, protein ferroportin, which serves as a transmembrane iron channel enabling iron efflux from cells. The hepcidin-ferroportin complex is then degraded in lysosomes and iron is locked inside the cells (mainly enterocytes, hepatocytes and macrophages). This leads to lowering of iron absorption in the intestine and to a decrease in serum iron concentration. Utilizing this mechanism, hepcidin regulates serum iron levels during inflammation, infection and possibly also in cancer. Under these conditions iron is shifted from circulation into cellular stores in hepatocytes and macrophages, making it less available for invading microorganisms and tumor cells. In anemia and hypoxia, hepcidin regulates the availability of iron for erythropoiesis. Hepcidin or hepcidin-related therapeutics could find a place in the treatment of various diseases such as hemochromatosis and anemia of chronic disease.     

Substantial changes of cellular iron homeostasis during megakaryocytic differentiation of K562 cells

We investigated the remodeling of iron metabolism during megakaryocytic development of K562 cells. Differentiation was successfully verified by increase of the megakaryocytic marker CD61 and concomitant decrease of the erythroid marker γ-globin. The reduction of erythroid properties was accompanied by changes in the cellular iron content and in the expression of proteins regulating cellular iron homeostasis. Independent of available inorganic or transferrin-bound extracellular iron, total intracellular iron increases while the iron-to-protein ratio decreases. The iron exporter ferroportin is downregulated within 1-6 h, followed by downregulation of transferrin receptor-1 (TfR1) and ferritin heavy chain (H-ferritin) mainly after 24-48 h. The hemochromatosis protein-1, a ligand of TfR1, peaked after 24 h. All effects were independent of iron supply with the exception of H-ferritin, which was restored by excess iron. While alterations of CD61, TfR1 and ferritin expression were revoked by a protein kinase C inhibitor, downregulation of ferroportin remained unaffected.