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.     

The ferroportin-ceruloplasmin system and the mammalian iron homeostasis machine: regulatory pathways and the role of lactoferrin

In the last 20 years, several new genes and proteins involved in iron metabolism in eukaryotes, particularly related to pathological states both in animal models and in humans have been identified, and we are now starting to unveil at the molecular level the mechanisms of iron absorption, the regulation of iron transport and the homeostatic balancing processes. In this review, we will briefly outline the general scheme of iron metabolism in humans and then focus our attention on the cellular iron export system formed by the permease ferroportin and the ferroxidase ceruloplasmin. We will finally summarize data on the role of the iron binding protein lactoferrin on the regulation of the ferroportin/ceruloplasmin couple and of other proteins involved in iron homeostasis in inflamed human macrophages.     

6-Hydroxydopamine upregulates iron regulatory protein 1 by activating certain protein kinase C isoforms in the dopaminergic MES23.5 cell line

Iron-induced oxidative stress is thought to play a crucial role in the pathogenesis of Parkinson's disease. Our previous studies demonstrated that decreased expression of ferroportin 1 contributes to 6-hydroxydopamine induced intracellular iron accumulation and that decreased ferroportin 1 expression is caused by increased expression of iron regulatory protein 1. Iron regulatory protein 1 is a central regulator of iron homeostasis and is a likely target of extracellular agents to program changes in cellular iron metabolism. Therefore, the mechanism of iron regulatory protein 1 upregulation induced by 6-hydroxydopamine has become a significant focus of research. Iron regulatory protein 1 is regulated by protein kinase C, although this regulation is tissue specific. Therefore, in the present study, we aimed to determine whether alteration of protein kinase C activity modified iron regulatory protein 1 expression in the dopaminergic MES23.5 cell line, Furthermore, we investigated whether 6-hydroxydopamine induced iron regulatory protein 1 upregulation is mediated by protein kinase C, thus achieving regulation of cellular iron levels. The results showed that iron regulatory protein 1 was upregulated by phorbol 12-myristate-13-acetate, the PKC activator in dopaminergic MES23.5 cells, and ferroportin 1 expression and iron efflux were decreased as a result of iron regulatory protein 1 upregulation. The protein kinase C inhibitor bisindolylmaleimide I hydrochloride abolished the effect of phorbol 12-myristate-13-acetate. Protein kinase C-δ and protein kinase C-ζ, but not protein kinase C-? were activated by 6-hydroxydopamine. The protein kinase C-δ inhibitor rottlerin inhibited protein kinase C-δ phosphorylation and abolished iron regulatory protein 1 upregulation induced by 6-hydroxydopamine. The protein kinase C-ζ pseudo-substrate inhibitor inhibited protein kinase C-ζ phosphorylation and abolished iron regulatory protein 1 upregulation induced by 6-hydroxydopamine. These data indicate that iron regulatory protein 1 is regulated by protein kinase C in dopaminergic MES23.5 cells and that protein kinase C activated by 6-hydroxydopamine regulates iron regulatory protein 1 expression, thus achieving regulation of cellular iron levels.     

The iron exporter ferroportin/Slc40a1 is essential for iron homeostasis

Ferroportin (SLC40A1) is an iron transporter postulated to play roles in intestinal iron absorption and cellular iron release. Hepcidin, a regulatory peptide, binds to ferroportin and causes it to be internalized and degraded. If ferroportin is the major cellular iron exporter, ineffective hepcidin function could explain manifestations of human hemochromatosis disorders. To investigate this, we inactivated the murine ferroportin (Fpn) gene globally and selectively. Embryonic lethality of Fpn(null/null) animals indicated that ferroportin is essential early in development. Rescue of embryonic lethality through selective inactivation of ferroportin in the embryo proper suggested that ferroportin has an important function in the extraembryonic visceral endoderm. Ferroportin-deficient animals accumulated iron in enterocytes, macrophages, and hepatocytes, consistent with a key role for ferroportin in those cell types. Intestine-specific inactivation of ferroportin confirmed that it is critical for intestinal iron absorption. These observations define the major sites of ferroportin activity and give insight into hemochromatosis.     

Hemojuvelin在铁稳态平衡中的关键作用

铁调素（hepcidin）是由肝脏分泌的一种肽类激素,它通过改变细胞膜上ferroportin的水平而调节全身铁代谢。Ferroportin是唯一已知的哺乳动物中的铁外排通道,它表达在小肠细胞的基底外侧膜和巨噬细胞的质膜上。铁调素结合ferroportin导致其在溶酶体内降解,从而减少铁从饮食的吸收和巨噬细胞铁的释放。Hemojuvelin（HJV）是一种glycosylphosphatidylinositol（GPI）相连的膜蛋白,它作为骨形态发生蛋白（BMP）的共受体可以激活肝细胞Smad信号通路和铁调素表达。除了表达在细胞膜上,hemojuvelin还可以被切割并分泌到胞外,形成可溶性蛋白。由furin切割产生的可溶性HJV可以选择性地结合到BMP配体,抑制内源性BMP诱导的铁调素表达。TMPRSS6也被认为可以切割细胞膜上HJV并影响铁调素的表达。最近的研究表明,HJV还可能参与脂肪组织对铁代谢的调控。综述了近期对细胞膜HJV和可溶性HJV如何调节铁调素的表达与铁代谢的研究结果,并对这一研究领域需要填补的空白进行了初步探讨。     

Reactive oxygen species are involved in ferroportin degradation induced by ceruloplasmin mutant Arg701Trp

The ceruloplasmin mutant R701W, that causes a dramatic phenotype in the young heterozygous patient carrying this mutation, has been shown to have profound effects also in cell culture models. Here we show that Golgi rearrangement and degradation of the iron exporter ferroportin, that follow transfection of cells with this mutant, are accompanied by the massive production of reactive oxygen species (ROS) in the cell. Scavenging ROS production with different antioxidants, including reduced glutathione and zinc, restores Golgi morphology and rescues ferroportin on the cell membrane.     

Hepcidin-induced endocytosis of ferroportin is dependent on ferroportin ubiquitination

Ferroportin exports iron into plasma from absorptive enterocytes, erythrophagocytosing macrophages, and hepatic stores. The hormone hepcidin controls cellular iron export and plasma iron concentrations by binding to ferroportin and causing its internalization and degradation. We explored the mechanism of hepcidin-induced endocytosis of ferroportin, the key molecular event in systemic iron homeostasis. Hepcidin binding caused rapid ubiquitination of ferroportin in cell lines overexpressing ferroportin and in murine bone marrow-derived macrophages. No hepcidin-dependent ubiquitination was observed in C326S ferroportin mutant which does not bind hepcidin. Substitutions of lysines between residues 229 and 269 in the third cytoplasmic loop of ferroportin prevented hepcidin-dependent ubiquitination and endocytosis of ferroportin, and promoted cellular iron export even in the presence of hepcidin. The human ferroportin mutation K240E, previously associated with clinical iron overload, caused hepcidin resistance in vitro by interfering with ferroportin ubiquitination. Our study demonstrates that ubiquitination is the functionally relevant signal for hepcidin-induced ferroportin endocytosis.     

Differing expression of genes involved in non-transferrin iron transport across plasma membrane in various cell types under iron deficiency and excess

We studied the effect of iron deficiency, i.e., 24-h preincubation in iron-free medium, and the effect of high level of non-transferrin iron, i.e., the preincubation in ferric citrate medium containing 500 muM ferric citrate, on the expression of DMT1, Dcytb, ferroportin, hephaestin, and ceruloplasmin in various functional types of human cells. The expression of these proteins potentially involved in non-transferrin iron transport across cell membranes was tested on mRNA level by quantitative real-time PCR as well as on protein level by western blot analysis in Caco-2 (colorectal carcinoma), K562 (erythroleukemia), and HEP-G2 (hepatocellular carcinoma) cells. We found that changes in non-transferrin iron availability, i.e., iron deficiency and high level of non-transferrin iron, affect the expression of tested proteins in a cell type-specific manner. We also demonstrated that changes in the expression on mRNA level do not often correlate with relevant changes on protein level.     

Decreased hephaestin expression and activity leads to decreased iron efflux from differentiated Caco2 cells

Iron is transported across intestinal brush border cells into the circulation in at least two distinct steps. Iron can enter the enterocyte via the apical surface through several paths. However, iron egress from the basolateral side of enterocytes converges on a single export pathway requiring the iron transporter, ferroportin1, and hephaestin, a ferroxidase. Copper deficiency leads to reduced hephaestin protein expression and activity in mouse enterocytes and intestinal cell lines. We tested the effect of copper deficiency on differentiated Caco2 cells grown in transwells and found decreased hephaestin protein expression and activity as well as reduced ferroportin1 protein levels. Furthermore, the decrease in hephaestin levels correlates with a decrease of ⁵⁵Fe release from the basolateral side of Caco2 cells. Presence of ceruloplasmin, apo‐transferrin or holo‐transferrin did not significantly alter the results observed. Repletion of copper in Caco2 cells leads to reconstitution of hephaestin protein expression, activity, and transepithelial iron transport. J. Cell. Biochem. 107: 803–808, 2009. © 2009 Wiley‐Liss, Inc.     

低氧与铁代谢相关蛋白

氧和铁这两种元素对生命活动十分重要. 低氧诱导因子(hypoxia-inducible factors, HIFs)作为转录因子,参与一系列靶基因的表达调控以适应低氧. 铁参与 DNA合成、氧气运输、代谢反应等多种细胞活动,过量游离铁会通过Haber-Weiss或 Fenton反应产生毒性自由基. 细胞通过与铁吸收、存储和利用有关的多种铁代谢相 关蛋白之间的协同作用来维持铁稳态. 与铁稳态相关的一些基因是HIFs的靶基因或 者间接受低氧调控,包括转铁蛋白、转铁蛋白受体、二价金属转运体1、铁调素、膜 铁转运蛋白、血浆铜蓝蛋白、铁蛋白等,而胞内铁浓度的改变能影响HIFs的表达. 本文就低氧与铁代谢相关蛋白的关系,尤其是低氧对铁代谢相关蛋白的调节作一综 述.     

小肠铁释放机制及相关疾病研究进展

铁是生物体必需的微量元素。铁缺乏和铁过载均会导致铁代谢紊乱相关疾病,因此有关机体铁水平稳态的调节机制已成为了目前铁代谢领域的研究热点。小肠吸收细胞是调节肠铁吸收、肠铁释放,以及维持机体铁稳态的重要部位。最新的研究表明,铁从小肠吸收细胞基底端释放入血液循环,主要是由膜铁转运蛋白（ferroportin1,Fp1）介导,并在膜铁转运辅助蛋白（haphaestin,Hp）和铜蓝蛋白（ceruloplasmin,Cp）的参与下完成。其中Fp1在小肠铁释放过程中起着至关重要的作用。本文重点阐述铁释放相关蛋白Fp1的作用机制及其调节机制,并详细介绍Fp1基因突变导致的铁代谢相关疾病方面的最新研究讲展。     

A physiological model to study iron recycling in macrophages

Following erythrophagocytosis (EP) of senescent red blood cells (RBCs), heme iron is recycled to the plasma by tissue macrophages. This process is critical for mammalian iron homeostasis but remains elusive. We characterized a cellular model using artificially-aged murine RBCs and murine bone marrow-derived macrophages (BMDMs) and study mRNA and protein expression of HO-1, ferroportin and ferritin after EP. In vitro ageing of RBCs was obtained by raising intracellular calcium concentration. These RBCs exhibit several features of erythrocyte senescence including externalization of phosphatidyl-serine, specific binding and phagocytosis by BMDMs. During the first hours of EP, we observed a rapid increase of HO-1 and ferroportin mRNAs and proteins, whereas ferritin protein expression was progressively induced with no major changes in RNA levels. At later stages after EP, a different pattern of expression was observed with a net decrease of ferroportin, a sustained high level of HO-1, and a strong increase in ferritins. Taken together, these results suggest that after EP, iron is rapidly extracted from heme and exported by ferroportin. Surprisingly, the gene expression profile at late stages after EP, which is indicative of iron storage, is reminiscent of what is observed in inflammation. However, phagocytosis of artificially-aged red blood cells seems to repress the proinflammatory response of macrophages.     

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.     

Cu loading alters expression of non-IRE regulated, but not IRE regulated, Fe dependent proteins in HepG2 cells

This paper investigates the extent to which Cu loading influences Fe levels in HepG2 cells and the effect on proteins regulated by Fe status. Cu supplementation increased Cu content 3-fold, concomitant with a decrease in cellular Fe levels. Intracellular levels of both transferrin (Tf) and ceruloplasmin (Cp) protein rose in parallel with increased secretion into the culture media. There was no increase in mRNA levels for either protein. Rather, our data suggested increased translation of the mRNA. The increase was not reflected in total protein synthesis, which actually decreased. The effect was not a generalised stress or cell damage response, since heat shock protein 70 levels and lactate dehydrogenase secretion were not significantly altered. To test whether the Cu effect could be acting though the decrease in Fe levels, we measured transferrin receptor (TfR) levels using ¹²⁵I labeled Tf and mRNA analysis. Neither protein nor mRNA levels were changed. Neither was the level of ferroportin mRNA. As a positive control, Fe chelation increased Tf and Cp secretion significantly, and TfR mRNA levels rose 2-fold. We excluded the possibility that the increased Cp or Tf could provide the required substrate to stimulate Fe efflux, and instead demonstrate that Cu can substitute for Fe in the iron regulatory protein - iron responsive element regulation mechanism.     

膜铁转运蛋白1，铁调素的靶分子？

膜铁转运蛋白1是重要的跨膜铁输出分子,主要分布于十二指肠和单核巨噬系统的细胞膜上,参与机体的肠铁吸收和巨噬细胞对铁的再循环等过程。铁调素是调节机体铁代谢平衡的激素,机体通过肝脏分泌的铁调素对铁转运相关蛋白的表达进行调控,从而实现机体自身的铁稳态。最新研究显示,铁调素的靶分子可能是膜铁转运蛋白1,它通过直接的作用引起膜铁转运蛋白1的内化(internalization)、降解,从而调节其在细胞膜上的表达量,进而控制肠铁吸收和巨噬细胞对铁的再循环过程,以维持机体的铁稳态。     

Heterogenous distribution of ferroportin-containing neurons in mouse brain

Iron is crucial for a variety of cellular functions in neuronal cells. Neuronal iron uptake is reflected in a robust and consistent expression of transferrin receptors and divalent metal transporter 1 (DMT 1). Conversely, the mechanisms by which neurons neutralize and possibly excrete iron are less clear. Studies indicate that neurons express ferroportin which could reflect a mechanism for iron export. We mapped the distribution of ferroportin in the adult mouse brain using an antibody prepared from a peptide representing amino acid sequences 223–303 of mouse ferroportin. The antibody specifically detected ferroportin in brain homogenates, whereas homogenates of cultured endothelial cells were devoid of immunoreactivity. In brain sections, ferroportin was confined to neuronal cell bodies and peripheral processes of cerebral cortex, hippocampus, thalamus, brain stem, and cerebellum. In brain stem ferroportin-labeling was particularly high in neurons of cranial nerve nuclei and reticular formation. Ferroportin was hardly detectable in striatum, pallidum, or hypothalamus. Among non-neuronal cells, ferroportin was detected in oligodendrocytes and choroid plexus epithelial cells. A comparison with previous studies on the distribution of transferrin receptors in neurons shows that many neuronal pools coincide with those expressing ferroportin. The data therefore indicate that neuronal iron homeostasis consists of a delicate balance between transferrin receptor-mediated uptake of iron-transferrin and ferroportin-related iron excretion. The findings also suggest a particular high turnover of iron in neuronal regions, such as habenula, hippocampus, reticular formation and cerebellum, as several neurons in these regions exhibit a prominent co-expression of transferrin receptors and ferroportin.