Iron Efflux Research Articles

A polymorphic miR-155 binding site in AGTR1 is associated with cardiac hypertrophy in Friedreich ataxia

Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative condition with a heterogeneous cardiac phenotype caused primarily by an expanded GAA trinucleotide repeat in the frataxin gene ( FXN). FXN is important in mitochondrial iron efflux, sensitivity to oxidative stress, and cell death. The number of GAA repeats on the smaller FXN allele (GAA1) only accounts for a portion of the observed variability in cardiac phenotype. Genetic modifying factors, such as single nucleotide polymorphisms (SNPs) in genes of the Renin–Angiotensin–Aldosterone system (RAAS), may contribute to phenotype variability. This study investigated genetic variability in the angiotensin-II type-1 receptor ( AGTR1), angiotensin-converting enzyme ( ACE), and ACE2 genes as cardiac phenotype modifying factors in FRDA patients. Comprehensive review of the AGTR1, ACE and ACE2 genes identified twelve haplotype tagging SNPs. Correlation of these SNPs with left ventricular internal diameter in diastole (LVIDd), interventricular septal wall thickness (SWT) and left ventricular mass (LVM) was examined in a large Australian FRDA cohort (n = 79) with adjustments performed for GAA repeats, age, sex, body surface area and diastolic blood pressure. A significant inverse relationship was observed between GAA1 and LVIDd (p = 0.010) but not with SWT or LVM after adjustment for covariates. The AGTR1 polymorphism rs5186 was more common in FRDA patients than in a control population (p = 0.002). Using a recessive model of inheritance, the C allele of rs5186 was associated with a significant increase in SWT (p = 0.003) and LVM (p = 0.001). This functional polymorphism increases expression of AGTR1 by altering the binding site for miR-155, a regulatory microRNA. No significant associations with left ventricular structure were observed for the remaining RAAS polymorphisms. The AGTR1 polymorphism rs5186 appears to modify the FRDA cardiac phenotype independently of GAA1. This study supports the role of RAAS polymorphisms as modifiers of cardiac phenotype in FRDA patients.

* Host iron metabolism gene expression in ins-gas mouse model of helicobacter infection

IntroductionIron deficiency is the most common nutritional disorder globally. There is increasing evidence from clinical and population studies for a role of H pylori infection in the aetiology of iron...

Splenic iron overload does not correlate with reduced expression of GPI‐ceruloplasmin following dietary copper deficiency

Many believe anemia following copper deficiency is due to impairment in iron efflux from intestine and storage sites in liver and spleen because copper is necessary for ferroxidase activity of multicopper oxidases hephaestin and ceruloplasmin (sCp). Prior work in brain discovered a splice variant of Cp anchored by glycosylphosphatidylinositol (GPI-Cp). Studies were conducted in Sprague Dawley rats to investigate GPI-Cp. Robust mRNA levels of GPI-Cp were detected in multiple rodent tissues in addition to brain. Purified membrane preparations detected immunoreactive Cp protein, especially enriched in spleen and kidney. Plasma from anemic dietary copper-deficient (CuD) rats contained no detectable sCp oxidase activity and reductions (60–90%) of sCp protein. Purified spleen membranes of CuD rats contained marked (>50%) reductions in GPI-Cp abundance. Surprisingly, however, lower non-heme iron concentrations compared to copper-adequate controls were detected. Perhaps the augmentation in ferroportin, the iron exporter, in CuD rats compensates for lower levels of the multicopper oxidase GPI-Cp. Livers of CuD rats also contained lower GPI-Cp levels but no iron augmentation. Additional research is needed to determine the mechanism for copper deficiency induced anemia but reduced sCp and GPI-Cp do not appear to be main factors in this pathology. Supported by NIH HD-39708 and USDA NIFA 2006-35200-17378.

THE METABOLIC FATE OF THE PEPTIDE HORMONE HEPCIDIN

Hepcidin (Hep), a 25-amino acid peptide hormone, is the principal regulator of plasma iron concentrations. Hepcidin's receptor is the iron channel ferroportin (Fpn), the only known cellular iron exporter in vertebrates. Fpn exports iron from duodenal enterocytes, macrophages and hepatocytes into plasma. Hep binding induces Fpn internalization and degradation, thus blocking iron efflux from cells into plasma. The aim of this study was to characterize the fate of Hep after binding to Fpn and determine its cellular catabolism and the mechanism of clearance. It has been known that Hep is rapidly excreted through the kidney, but we now describe that Hep is taken up by ferroportin-expressing cells and degraded together with its receptor. When Texas red-labeled Hep was added to Fpn-GFP expressing cells, confocal microscopy showed co-localization of red Hep and green Fpn. We assessed Hep degradation by flow cytometry and showed that Hep is almost completely degraded by 24h after addition. Lysosomal inhibitors and to a lesser extent the proteosomal inhibitor MG132 prevented degradation of both Fpn and Hep suggesting that the receptor-ligand complex is degraded by both degradation pathways. In addition, using radio-labeled Hep and HPLC analysis we show that Hep is not recycled, and that only degradation products are released from the cells. Together these results show that the hormone Hep after binding to its receptor Fpn, is internalized together with Fpn and trafficked to lysosomes where both are degraded. We confirmed this finding in vivo using PET/CT mouse imaging studies. In conditions of iron deficiency when Fpn is upregulated, I124-Hep accumulated in ferroportin-expressing tissues more than when animals were iron replete and Fpn expression was lower. These data suggest that both receptor-mediated endocytosis as well as renal clearance contribute to the removal of hecidin from the circulation.

Glycosylphosphatidylinositol-linked ceruloplasmin is expressed in multiple rodent organs and is lower following dietary copper deficiency

Ceruloplasmin (Cp), a multicopper ferroxidase, is expressed as both a secreted (sCp) plasma enzyme from the liver and a membrane-bound glycosylphosphatidylinositol-anchored (GPI-Cp) splice variant protein. Cp is thought to be essential for iron mobilization as selective iron overload occurs in aceruloplasminemia in humans and in Cp null mice. Dietary copper-deficient (CuD) rodents have near total loss of Cp activity, severe loss of Cp protein and develop anemia. Hepatic iron augmentation is often observed, suggesting that loss of Cp function may be correlated with anemia. The impact of CuD treatment on GPI-Cp has not previously been evaluated. Our hypothesis was that CuD rodents would have lower levels of GPI-Cp and this would correlate with higher tissue iron retention. In these studies, GPI-Cp was detected in purified membranes of multiple organs of rats and mice but not Cp -/- mice. Immunoreactive Cp protein was released with phosphatidylinositol phospholipase C treatment and expressed ferroxidase activity. Following perinatal and postnatal copper restriction, GPI-Cp was markedly lower in the spleen and modestly lower in the liver of CuD rats and mice, when compared with copper-adequate (CuA) rodents. However, spleen non-heme iron (NHI) was lower in CuD than CuA rats, and not different in CuD mice. Hepatic iron was higher only in CuD mice. Spleen and liver membranes of CuD rats expressed augmented levels of ferroportin, the iron efflux transporter, which may explain lower NHI content in the spleen of CuD rats despite a greater than 50% lower level of the multicopper ferroxidase GPI-Cp. Spleen and liver levels of GPI-Cp mRNA were not impacted in CuD rats, suggesting that turnover rather than biosynthesis may explain the lower steady-state levels of GPI-Cp following dietary copper restriction. Lower GPI-Cp did not correlate with tissue iron retention and thus the role, if any, of Cp in anemia of copper deficiency is unknown.

Nrf2 regulates ferroportin 1-mediated iron efflux and counteracts lipopolysaccharide-induced ferroportin 1 mRNA suppression in macrophages

Iron is an essential element of hemoglobin, and efficient iron recycling from senescent erythrocytes by splenic macrophages is required for erythrocyte hemoglobin synthesis during erythropoiesis. Ferroportin 1 (Fpn1) is the sole iron exporter in mammals, and it also regulates iron reutilization. In this study, we demonstrated genetically that a redox-sensitive transcription factor, Nrf2, regulates Fpn1 mRNA expression in macrophages. Nrf2 activation by several electrophilic compounds commonly resulted in the upregulation of Fpn1 mRNA in bone marrow-derived and peritoneal macrophages obtained from wild-type mice but not from Nrf2 knockout mice. Further, Nrf2 activation enhanced iron release from the J774.1 murine macrophage cell line. Previous studies showed that inflammatory stimuli, such as LPS, downregulates macrophage Fpn1 by transcriptional and hepcidin-mediated post-translational mechanisms leading to iron sequestration by macrophages. We showed that two Nrf2 activators, diethyl maleate and sulforaphane (SFN; a natural Nrf2 activator found in broccoli), restored the LPS-induced suppression of Fpn1 mRNA in human and mouse macrophages, respectively. Furthermore, SFN counteracted the LPS-induced increase of Hepcidin mRNA by an Nrf2-independent mechanism in mouse peritoneal macrophages. These results demonstrate that Nrf2 regulates iron efflux from macrophages through Fpn1 gene transcription and suggest that Nrf2 may control iron metabolism during inflammation.

Quercetin–iron chelates are transported via glucose transporters

Flavonoids are well-known antioxidants and free radical scavengers. Their metal-binding activity suggests that they could be effective protective agents in pathological conditions caused by both extracellular and intracellular oxidative stress linked to metal overload. Quercetin is both a permeant ligand via glucose transport proteins (GLUTs) and a high-affinity inhibitor of GLUT-mediated glucose transport. Chelatable “free iron” at micromolar concentrations in body fluids is a catalyst of hydroxyl radical (OH •) production from hydrogen peroxide. A number of flavonoids, e.g., quercetin, luteolin, chrysin, and 3,6-dihydroxyflavone, have been demonstrated to chelate intracellular iron and suppress OH • radical production in Madin Darby canine kidney cells. The most effective chelation comes from the flavonone B ring catechol found in both quercetin and luteolin. We show here that quercetin concentrations of < 1 μM can facilitate chelatable iron shuttling via GLUT1 in either direction across the cell membrane. These siderophoric effects are inhibited by raised quercetin concentrations (> 1 μM) or GLUT inhibitors, e.g., phloretin or cytochalasin B, and iron efflux is enhanced by impermeant extracellular iron chelators, either desferrioxamine or rutin. This iron shuttling property of quercetin might be usefully harnessed in chelotherapy of iron-overload conditions.

Environmental adaptability and stress tolerance of Laribacter hongkongensis: a genome-wide analysis

BackgroundLaribacter hongkongensis is associated with community-acquired gastroenteritis and traveler's diarrhea and it can reside in human, fish, frogs and water. In this study, we performed an in-depth annotation of the genes in its genome related to adaptation to the various environmental niches.ResultsL. hongkongensis possessed genes for DNA repair and recombination, basal transcription, alternative σ-factors and 109 putative transcription factors, allowing DNA repair and global changes in gene expression in response to different environmental stresses. For acid stress, it possessed a urease gene cassette and two arc gene clusters. For alkaline stress, it possessed six CDSs for transporters of the monovalent cation/proton antiporter-2 and NhaC Na+:H+ antiporter families. For heavy metals acquisition and tolerance, it possessed CDSs for iron and nickel transport and efflux pumps for other metals. For temperature stress, it possessed genes related to chaperones and chaperonins, heat shock proteins and cold shock proteins. For osmotic stress, 25 CDSs were observed, mostly related to regulators for potassium ion, proline and glutamate transport. For oxidative and UV light stress, genes for oxidant-resistant dehydratase, superoxide scavenging, hydrogen peroxide scavenging, exclusion and export of redox-cycling antibiotics, redox balancing, DNA repair, reduction of disulfide bonds, limitation of iron availability and reduction of iron-sulfur clusters are present. For starvation, it possessed phosphorus and, despite being asaccharolytic, carbon starvation-related CDSs.ConclusionsThe L. hongkongensis genome possessed a high variety of genes for adaptation to acid, alkaline, temperature, osmotic, oxidative, UV light and starvation stresses and acquisition of and tolerance to heavy metals.

Role of iron in pathogenesis of Parkinson disease

Parkinson’s disease is one of the most frequent human neurodegenerations. Motor symptoms of Parkinson’s disease are the consequence of the destruction of nervous cells in the substantia nigra (SN), a small (about 500 mg) structure located deep in human brain. The concentration of iron in SN is comparable to that in liver and is equal to about 180 ± 60 ng/mg of wet tissue and the iron in SN is mostly bound to ferritin. For many years it has been believed that the degeneration of nervous cells in SN in Parkinson’s disease is related to an important increase in the concentration of iron. Our own studies based on Mossbauer spectroscopy and other studies conducted with the use of various techniques have not confirmed this finding. The ratio of the concentration of iron in PD vs. control SN evaluated by Mossbauer spectroscopy was found to be equal 1.00 ± 0.13. We also confirmed that most of iron in SN is located within ferritin. ELISA studies demonstrated a significant decrease in L ferritin in parkinsonian SN compared to the control group. As L-ferritin is related to safe keeping of iron within the ferritin shell, its decrease may lead to an efflux of iron and increase in the concentration of labile iron. Indeed our studies did show a difference in the concentration of labile iron between PD and control SN (135 ± 10 ng/g vs. 76 ± 5 ng/g). This labile iron, which may initiate Fenton reaction, may be the cause of the oxidative stress leading to the death of nervous cells in PD.

Tanshinone IIA Prevented Brain Iron Dyshomeostasis in Cerebral Ischemic Rats

Background: Tanshinone IIA is a lipid-soluble compound extracted from Chinese herb Danshen which was commonly used in the treatment of cerebrovascular diseases. Brain iron homeostasis is very essential for normal physiological functions of neurons, and brain iron accumulation contributes to many neurological disorders. The present study was aimed to determine whether Tanshinone IIA protects against cerebral ischemic injury via maintaining brain iron homeostasis. Methods: Wistar Rats were orally administered with Tanshinone IIA (4, 20 and 100 mg/kg/d) for one week, and then subjected to cerebral ischemia by middle cerebral artery occlusion (MCAO) for 2 h. In vitro, cultured neurons were pretreated with Tanshinone IIA (2, 10 and 50 uM) for one week, and then exposed to hypoxia for 24 h. Brain iron content was determined by Flame Atomic Absorption Spectrophotometer and intracellular free iron level was measured by laser scanning Confocal microscopy. Expression levels of iron transport proteins including DMT1, TfR, Fpn1 and Heph were assessed by Western blot technique. Results: Tanshinone IIA pretreatment resulted in a significant reduction of cerebral infarct volume in MCAO rats. Compared with control rats, cerebral ischemia considerably augmented total iron content in the brain of MCAO rats, which was also effectively restricted by Tanshinone IIA pretreatment. MCAO rats exhibited the increased expression of iron influx proteins DMT1 and TfR, and the decreased expression of iron efflux proteins Fpn1 and Heph compared with control rats, which was responsible for elevated iron content in the ischemic brain. Tanshinone IIA pretreatment prevented the dysregulation of these four iron transport proteins and maintained brain iron homeostasis. In vitro studies also confirmed that Tanshinone IIA alleviated the hypoxia-induced decline of cell viability and the overload of intracellular free iron level in neurons through downregulating the expression of DMT1 and TfR, and upregulating the expression of Fpn1 and Heph. Conclusion: Tanshinone IIA protected brain tissues against ischemic and hypoxic damage in vivo and in vitro by mediating brain iron homeostasis which was associated with the downregulation of DMT1 and TfR, and the upregulation of Fpn1 and Heph. These results provide new insights into molecular mechanisms of ischemia-induced brain iron abnormalities and suggest maintaining brain iron homeostasis may be a novel therapeutic strategy for ischemic cerebrovascular diseases.

Adipocyte Hypoxia Increases Hepatocyte Hepcidin Expression

Hepcidin plays a key role in regulating iron metabolism by blocking iron efflux from macrophages and enterocytes. Hepcidin is synthesized primarily in the liver, and its expression is increased by iron overload and inflammation. Obesity is associated with chronic inflammation as well as poor iron status. Central obesity causes adipocyte hypoxia resulting in chronic inflammation. Therefore, the objective of the present study was to determine if adipocyte hypoxia and associated inflammation signal hepatocyte hepcidin expression. The effect of adipocyte hypoxia on hepcidin expression was modeled using a 3T3-L1 adipocyte/Huh7 hepatocyte co-culture model. Adipocytes were cultured at either standard conditions (19% O2) or hypoxic conditions (1% O2). Compared to standard conditions, hypoxic 3T3-L1 cells had significantly higher IL-6 and leptin expression. Treatment of Huh7 cells with media from hypoxic or LPS-treated 3T3-L1 adipocytes significantly increased hepcidin promoter activity and mRNA compared to cells treated with normoxic 3T3-L1 media or control media. When the hepcidin STAT3 binding site was mutated, promoter activation by hypoxic media was abrogated. These data suggest that adipocyte hypoxia (a feature of central obesity) may increase hepcidin expression and plays a role in the association between obesity and poor iron status.

Iron chelation and neuroprotection in neurodegenerative diseases

Iron is an essential element for multiple functions of the brain. Maintenance of iron homeostasis involves regulation of iron influx, iron efflux and iron storage. Mismanagement of brain iron has been implicated in neuronal injury and death in several neurodegenerative diseases, such as Parkinson's disease (PD), Alzheimer's disease (PD) and Amyotrophic lateral sclerosis (ALS). Multiple iron chelators have been shown neuroprotective and neurorestorative in these diseases, suggesting that iron chelation might be a promising therapeutics. In this paper, we briefly review the new findings of biological function of several molecules that regulate iron homeostasis in the brain, the possible role of iron mismanagement in the pathogenesis of PD, AD and ALS, and then discuss the putative mechanisms for current available iron chelators as potential therapeutics for neurodegenerative diseases.

Transepithelial heme-iron transport: effect of heme oxygenase overexpression

Heme iron is found in the diet mainly in the form of hemoglobin and myoglobin. It is known that heme iron (heme-Fe) and inorganic iron are absorbed differently. Intracellularly, heme oxygenase-1 (HO1) participates in the cleavage of the heme ring producing biliverdin, CO and ferrous iron. Iron released from heme becomes part of labile iron pool, and it can be stored in ferritin or released through the basolateral membrane. The mechanism by which heme-Fe is metabolized within cells is not completely understood. This study focused on the uptake and transport of heme iron and on the role of heme oxygenase-1 on heme iron metabolism. Caco-2 cells were incubated with different concentrations of heme-Fe. A full-length heme oxygenase-1 cDNA was expressed in Caco-2 cells and intracellular iron and heme-Fe content, heme uptake, heme and iron transport and heme oxygenase-1 immunolocalization were assessed in these cells. Heme-Fe was bioavailable and induced an intracellular increase in iron, ferritin and HO1 levels and a decrease in DMT1 expression. In cells overexpressing HO1, heme-Fe uptake and transepithelial Fe transport was higher than in controls. Most heme-Fe was metabolized to free iron, most of which was found mainly in the basolateral chamber. However, there is a fraction of heme that is delivered intact to the basolateral side. In a high heme-Fe condition, HO1 is found near the plasma membrane. These results suggest that heme oxygenase-1 catabolizes most of the heme-Fe and favors iron influx and efflux in intestinal cells.

Identification of Zyklopen, a New Member of the Vertebrate Multicopper Ferroxidase Family, and Characterization in Rodents and Human Cells1–3

We previously detected a membrane-bound, copper-containing oxidase that may be involved in iron efflux in BeWo cells, a human placental cell line. We have now identified a gene encoding a predicted multicopper ferroxidase (MCF) with a putative C-terminal membrane-spanning sequence and high sequence identity to hephaestin (Heph) and ceruloplasmin (Cp), the other known vertebrate MCF. Molecular modeling revealed conservation of all type I, II, and III copper-binding sites as well as a putative iron-binding site. Protein expression was observed in multiple diverse mouse tissues, including placenta and mammary gland, and the expression pattern was distinct from that of Cp and Heph. The protein possessed ferroxidase activity, and protein levels decreased in cellular copper deficiency. Knockdown with small interfering RNA in BeWo cells indicates that this gene represents the previously detected oxidase. We propose calling this new member of the MCF family “zyklopen.”

G80S-linked ferroportin disease: Classical ferroportin disease in an Asian family and reclassification of the mutant as iron transport defective

Hereditary iron overload associated with mutations in the ferroportin gene produces a dichotomy of phenotypes resulting from either increase or decrease in iron efflux capacity. In this study, we examined the molecular basis of iron overload in a family of Vietnamese origin, characterized the molecular and cellular defect, and correlated it with the clinical and pathological phenotype. We analyzed the ferroportin gene by DNA sequencing. The molecular characterization was performed by immunofluorescence microscopy analysis of transfected cells. We analyzed ferritin levels, in cells expressing wild-type and mutant ferroportin, to define the nature of the molecular defect in iron transport. We identified a G to A nucleotide change at position 238 in the ferroportin gene leading to the G80S substitution. Cellular analysis of the mutant protein indicates that this amino acid change does not affect the localization of the protein but does affect its ability to transport iron. The G80S mutation results in a mutated ferroportin associated with iron overload and is predicted to be defective in iron export.

Role of Ferroportin in Macrophage-Mediated Immunity

Perturbations in iron metabolism have been shown to dramatically impact host response to infection. The most common inherited iron overload disorder results from defects in the HFE gene product, a major histocompatibility complex class I-like protein that interacts with transferrin receptors. HFE-associated hemochromatosis is characterized by abnormally high levels of the iron efflux protein ferroportin. In this study, J774 murine macrophages overexpressing ferroportin were used to investigate the influence of iron metabolism on the release of nitric oxide (NO) in response to infection. Overexpression of ferroportin significantly impaired intracellular Mycobacterium tuberculosis growth during early stages of infection. When challenged with lipopolysaccharide (LPS) or M. tuberculosis infection, control macrophages increased NO synthesis, but macrophages overexpressing ferroportin had significantly impaired NO production in response to LPS or M. tuberculosis. Increased NO synthesis in control cells was accompanied by increased iNOS mRNA and protein, while upregulation of iNOS protein was markedly reduced when J744 cells overexpressing ferroportin were challenged with LPS or M. tuberculosis, thus limiting the bactericidal activity of these macrophages. The proinflammatory cytokine gamma interferon reversed the inhibitory effect of ferroportin overexpression on NO production. These results suggest a novel role for ferroportin in attenuating macrophage-mediated immune responses.

Intestinal Ferritin H Is Required for an Accurate Control of Iron Absorption

To maintain appropriate body iron levels, iron absorption by the proximal duodenum is thought to be controlled by hepcidin, a polypeptide secreted by hepatocytes in response to high serum iron. Hepcidin limits basolateral iron efflux from the duodenal epithelium by binding and downregulating the intestinal iron exporter ferroportin. Here, we found that mice with an intestinal ferritin H gene deletion show increased body iron stores and transferrin saturation. As expected for iron-loaded animals, the ferritin H-deleted mice showed induced liver hepcidin mRNA levels and reduced duodenal expression of DMT1 and DcytB mRNA. In spite of these feedback controls, intestinal ferroportin protein and (59)Fe absorption were increased more than 2-fold in the deleted mice. Our results demonstrate that hepcidin-mediated regulation alone is insufficient to restrict iron absorption and that intestinal ferritin H is also required to limit iron efflux from intestinal cells.

Interleukin-1β induces ceruloplasmin and ferroportin-1 gene expression via MAP kinases and C/EBPβ, AP-1, and NF-κB activation

Previously, we demonstrated that IL-1β was able to increase iron efflux from glial cells through a coordinate induction of both ferroportin-1 (Fpn) and ceruloplasmin (Cp) synthesis. In this study, we have investigated the signaling pathways that are involved in the transcriptional activation of the Cp and Fpn. Our data show that the expression of Cp and Fpn in response to IL-1β requires the activation of MAP kinase pathways as a consequence of an IL-1β receptor stimulation. Moreover, we have observed that IL-1β regulates the expression of Cp and Fpn genes through (i) p38 MAPK-mediated activation of C/EBP transcription factor, (ii) ERK1/2-, JNK1- and partially p38 MAPK-dependent activation of AP-1, and through (iii) activation of NF-κB partially mediated by p38 MAPK.

Ferroportin and Iron Regulation in Breast Cancer Progression and Prognosis

An iron efflux pump is a pivotal protein in breast cancer biology and a strong and independent predictor of breast cancer prognosis.

Biological effects of mutant ceruloplasmin on hepcidin-mediated internalization of ferroportin

Ceruloplasmin plays an essential role in cellular iron efflux by oxidizing ferrous iron exported from ferroportin. Ferroportin is posttranslationally regulated through internalization triggered by hepcidin binding. Aceruloplasminemia is an autosomal recessive disorder of iron homeostasis resulting from mutations in the ceruloplasmin gene. The present study investigated the biological effects of glycosylphosphatidylinositol (GPI)-linked ceruloplasmin on the hepcidin-mediated internalization of ferroportin. The prevention of hepcidin-mediated ferroportin internalization was observed in the glioma cells lines expressing endogenous ceruloplasmin as well as in the cells transfected with GPI-linked ceruloplasmin under low levels of hepcidin. A decrease in the extracellular ferrous iron by an iron chelator and incubation with purified ceruloplasmin in the culture medium prevented hepcidin-mediated ferroportin internalization, while the reconstitution of apo-ceruloplasmin was not able to prevent ferroportin internalization. The effect of ceruloplasmin on the ferroportin stability was impaired due to three distinct properties of the mutant ceruloplasmin: namely, a decreased ferroxidase activity, the mislocalization in the endoplasmic reticulum, and the failure of copper incorporation into apo-ceruloplasmin. Patients with aceruloplasminemia exhibited low serum hepcidin levels and a decreased ferroportin protein expression in the liver. The in vivo findings supported the notion that under low levels of hepcidin, mutant ceruloplasmin cannot stabilize ferroportin because of a loss-of-function in the ferroxidase activity, which has been reported to play an important role in the stability of ferroportin. The properties of mutant ceruloplasmin regarding the regulation of ferroportin may therefore provide a therapeutic strategy for aceruloplasminemia patients.