A disease-causing mutation K240E disrupts ferroportin trafficking by SUMO (ferroportin SUMOylation)

Ferroportin (Fpn/IREG1/MTP1) is the only known transporter mediating iron efflux from epithelial cells and macrophages, and thus regulates how much iron is released into the circulation. Consequently, Fpn mutations are associated with haemochromatosis. Fpn itself is post-translationally regulated by hepcidin (Hepc) which induces its redistribution and degradation in a ubiquitin-dependent process. Together, the two proteins appear to be the nexus for iron homeostasis. Here we show that a rare gain-of-function mutation (K240E) that is associated with iron overload, impedes Fpn binding and subcellular trafficking by the small ubiquitin-like modifier (SUMO). Whereas wild-type Fpn is ensconced within vesicular bodies, the FpnK240E mutant appeared diffused within the cell when co-expressed with SUMO. Furthermore, compared with wild type Fpn, the sumoylation-defective mutant was constitutively-active, resulting in a lower intracellular labile iron pool than the former. These findings suggest that SUMO may regulate iron homeostasis by controlling Fpn trafficking.     

Fasting up‐regulates ferroportin 1 expression via a Ghrelin/GHSR/MAPK signaling pathway

The significant positive correlation between ghrelin and iron and hepcidin levels in the plasma of children with iron deficiency anemia prompted us to hypothesize that ghrelin may affect iron metabolism. Here, we investigated the effects of fasting or ghrelin on the expression of hepcidin, ferroportin 1 (Fpn1), transferrin receptor 1 (TfR1), ferritin light chain (Ft‐L) proteins, and ghrelin, and also hormone secretagogue receptor 1 alpha (GHSR1α) and ghrelin O‐acyltransferase (GOAT) mRNAs in the spleen and/or macrophage. We demonstrated that fasting induces a significant increase in the expression of ghrelin, GHSR1α, GOAT, and hepcidin mRNAs, as well as Ft‐L and Fpn1 but not TfR1 proteins in the spleens of mice in vivo. Similar to the effects of fasting on the spleen, ghrelin induced a significant increase in the expression of Ft‐L and Fpn1 but not TfR1 proteins in macrophages in vitro. In addition, ghrelin was found to induce a significant enhancement in phosphorylation of ERK as well as translocation of pERK from the cytosol to nuclei. Furthermore, the increased pERK and Fpn1 induced by ghrelin was demonstrated to be preventable by pre‐treatment with either GHSR1α antagonist or pERK inhibitor. Our findings support the hypothesis that fasting upregulates Fpn1 expression, probably via a ghrelin/GHSR/MAPK signaling pathway.     

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.     

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.     

Duodenal Ferroportin Is Up-Regulated in Patients with Chronic Hepatitis C

Hepatitis C virus (HCV) infection is a leading cause of liver-related mortality. Chronic hepatitis C (CHC) is frequently associated with disturbances in iron homeostasis, with serum iron and hepatic iron stores being elevated. Accumulating evidence indicates that chronic HCV infection suppresses expression of hepatic hepcidin, a key mediator of iron homeostasis, leading to iron overload conditions. Since hepcidin mediates degradation of ferroportin, a basolateral transporter involved in the release of iron from cells, diminished hepcidin expression probably leads to up-regulation of ferroportin-1 (Fpn1) in patients with CHC. In this study, we determined the protein levels of duodenal Fpn1, and found that its expression was significantly up-regulated in patients with CHC. The expression of duodenal Fpn1 is negatively correlated with mRNA levels of hepcidin, and positively correlated with serum iron parameters. Although iron is a critical factor for growth of a variety of pathogenic bacteria, our results suggest that iron overload in blood does not increase the infection rate of bacteria in patients with CHC.     

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.     

The role of ubiquitination in hepcidin-independent and hepcidin-dependent degradation of ferroportin

The iron exporter ferroportin (Fpn) is essential to transfer iron from cells to plasma. Systemic iron homeostasis in vertebrates is regulated by the hepcidin-mediated internalization of Fpn. Here, we demonstrate a second route for Fpn internalization; when cytosolic iron levels are low, Fpn is internalized in a hepcidin-independent manner dependent upon the E3 ubiquitin ligase Nedd4-2 and the Nedd4-2 binding protein Nfdip-1. Retention of cell-surface Fpn through reductions in Nedd4-2 results in cell death through depletion of cytosolic iron. Nedd4-2 is also required for internalization of Fpn in the absence of ferroxidase activity as well as for the entry?of hepcidin-induced Fpn into the multivesicular?body. C.?elegans lacks hepcidin genes, and C.?elegans Fpn expressed in mammalian cells is not internalized by hepcidin but is internalized in response to iron deprivation in a Nedd4-2-dependent manner, supporting the hypothesis that Nedd4-2-induced internalization of Fpn is evolutionarily conserved.     

Leishmania donovani inhibits ferroportin translation by modulating FBXL5‐IRP2 axis for its growth within host macrophages

Hepcidin mediated ferroportin (Fpn) degradation in macrophages is a well adopted strategy to limit iron availability towards invading pathogens. Leishmania donovani (LD), a protozoan parasite, resides within macrophage and competes with host for availing iron. Using in vitro and in vivo model of infection, we reveal that LD decreases Fpn abundance in host macrophages by hepcidin independent mechanism. Unaffected level of Fpn‐FLAG in LD infected J774 macrophage confirms that Fpn down‐regulation is not due its degradation. While increased Fpn mRNA but decreased protein expression in macrophages suggests blocking of Fpn translation by LD infection that is confirmed by ³⁵S‐methionine labelling assay. We further reveal that LD blocks Fpn translation by induced binding of iron regulatory proteins (IRPs) to the iron responsive element present in its 5′UTR. Supershift analysis provides evidence of involvement of IRP2 particularly during in vivo infection. Accordingly, a significant increase in IRP2 protein expression with simultaneous decrease in its stability regulator F‐box and leucine‐rich repeat Protein 5 (FBXL5) is detected in splenocytes of LD‐infected mice. Increased intracellular growth due to compromised expressions of Fpn and FBXL5 by specific siRNAs reveals that LD uses a novel strategy of manipulating IRP2‐FBXL5 axis to inhibit host Fpn expression.     

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.     

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.     

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.     

Suppressed hepcidin expression correlates with hypotransferrinemia in copper-deficient rat pups but not dams

Copper deficiency leads to anemia but the mechanism is unknown. Copper deficiency also leads to hypoferremia, which may limit erythropoiesis. The hypoferremia may be due to limited function of multicopper oxidases (MCO) hephaestin in enterocytes or GPI-ceruloplasmin in macrophages of liver and spleen whose function as a ferroxidase is thought essential for iron transfer out of cells. Iron release may also be limited by ferroportin (Fpn), the iron efflux transporter. Fpn may be lower following copper deficiency because of impaired ferroxidase activity of MCO. Fpn is also dependent on the liver hormone hepcidin as Fpn is degraded when hepcidin binds to Fpn. Anemia and hypoferremia both down regulate hepcidin by separate mechanisms. Current studies confirmed and extended earlier studies with copper-deficient (CuD) rats that suggested low hepicidin resulted in augmented Fpn. However, current studies in CuD dams failed to confirm a correlation that hepcidin expression was associated with low transferrin receptor 2 (TfR2) levels and also challenged the dogma that holotransferrin can explain the correlation with hepcidin. CuD dams exhibited hypoferremia, low liver TfR2, anemia in some rats, yet no depression in Hamp expression, the hepcidin gene. Normal levels of GDF-15, the putative erythroid cytokine that suppresses hepcidin, were detected in plasma of CuD and iron-deficient (FeD) dams. Importantly, FeD dams did display greatly lower Hamp expression. Normal hepcidin in these CuD dams is puzzling since these rats may need extra iron to meet needs of lactation and the impaired iron transfer noted previously.     

Splenic microenvironment of the CBA/N mouse: immunohistochemical analysis using monoclonal antibodies against lymphocytes and nonlymphoid cells

CBA/N mice carry an X-linked immune-deficiency gene, leading to a defect in the ability to form antibodies against T-independent type 2 antigens. By using immunohistochemistry, the organization of the spleen of the immune-deficient male (xid) CBA/N F1 and the normal female F1 were compared. Staining with antilymphocyte markers showed that the total number of cells in the various T- and B-cell areas was smaller in the xid mouse, resulting in very small white pulp compartments. Fewer B cells were seen in the marginal zone. When the spleens of the F1 mice were examined for macrophage markers, the rings of marginal-zone macrophages and the ring of marginal metallophilic macrophages were much thinner in the xid mouse. In particular, the marginal-zone macrophages are thought to play a role in the response against thymus-independent type 2 antigens, and their small numbers in the xid mouse are suggestive of a role for the microenvironment in the defects in these mice.     

Loss of ferroportin induces memory impairment by promoting ferroptosis in Alzheimer’s disease

Iron homeostasis disturbance has been implicated in Alzheimer’s disease (AD), and excess iron exacerbates oxidative damage and cognitive defects. Ferroptosis is a nonapoptotic form of cell death dependent upon intracellular iron. However, the involvement of ferroptosis in the pathogenesis of AD remains elusive. Here, we report that ferroportin1 (Fpn), the only identified mammalian nonheme iron exporter, was downregulated in the brains of APPswe/PS1dE9 mice as an Alzheimer’s mouse model and Alzheimer’s patients. Genetic deletion of Fpn in principal neurons of the neocortex and hippocampus by breeding Fpn^fl/fl mice with NEX-Cre mice led to AD-like hippocampal atrophy and memory deficits. Interestingly, the canonical morphological and molecular characteristics of ferroptosis were observed in both Fpn^fl/fl/NEXcre and AD mice. Gene set enrichment analysis (GSEA) of ferroptosis-related RNA-seq data showed that the differentially expressed genes were highly enriched in gene sets associated with AD. Furthermore, administration of specific inhibitors of ferroptosis effectively reduced the neuronal death and memory impairments induced by Aβ aggregation in vitro and in vivo. In addition, restoring Fpn ameliorated ferroptosis and memory impairment in APPswe/PS1dE9 mice. Our study demonstrates the critical role of Fpn and ferroptosis in the progression of AD, thus provides promising therapeutic approaches for this disease.Subject terms: Neural ageing, Ageing