Cellular Catabolism of the Iron-Regulatory Peptide Hormone Hepcidin

Hepcidin, a 25-amino acid peptide hormone, is the principal regulator of plasma iron concentrations. Hepcidin binding to its receptor, the iron exporter ferroportin, induces ferroportin internalization and degradation, thus blocking iron efflux from cells into plasma. The aim of this study was to characterize the fate of hepcidin after binding to ferroportin. We show that hepcidin is taken up by ferroportin-expressing cells in a temperature- and pH-dependent manner, and degraded together with its receptor. When Texas red-labeled hepcidin (TR-Hep) was added to ferroportin-GFP (Fpn-GFP) expressing cells, confocal microscopy showed co-localization of TR-Hep with Fpn-GFP. Using flow cytometry, we showed that the peptide was almost completely degraded by 24 h after its addition, but that lysosomal inhibitors completely prevented degradation of both ferroportin and hepcidin. In addition, using radio-labeled hepcidin and HPLC analysis we show that hepcidin is not recycled, and that only degradation products are released from the cells. Together these results show that the hormone hepcidin and its receptor ferroportin are internalized together and trafficked to lysosomes where both are degraded.     

Efficient oxidative folding and site-specific labeling of human hepcidin to study its interaction with receptor ferroportin

Hepcidin is a small disulfide-rich peptide hormone that plays a key role in the regulation of iron homeostasis by binding and mediating the degradation of the cell membrane iron efflux transporter, ferroportin. Since it is a small peptide, chemical synthesis is a suitable approach for the preparation of mature human hepcidin. However, oxidative folding of synthetic hepcidin is extremely difficult due to its high cysteine content and high aggregation propensity. To improve its oxidative folding efficiency, we propose a reversible S-modification approach. Introduction of eight negatively charged sulfonate moieties into synthetic hepcidin significantly decreased its aggregation propensity and, under optimized conditions, dramatically increased the refolding yield. The folded hepcidin displayed a typical disulfide-constrained β-sheet structure and could induce internalization of enhanced green fluorescent protein (EGFP) tagged ferroportin in transfected HEK293 cells. In order to study interactions between hepcidin and its receptor ferroportin, we propose a general approach for site-specific labeling of synthetic hepcidin analogues by incorporation of an l-propargylglycine during chemical synthesis. Following efficient oxidative refolding, a hepcidin analogue with Met20 replaced by l-propargylglycine was efficiently mono-labeled by a red fluorescent dye through click chemistry. The labeled hepcidin was internalized into the transfected cells together with the EGFP-tagged ferroportin, suggesting direct binding between hepcidin and ferroportin. The labeled hepcidin was also a suitable tool to visualize internalization of overexpressed or even endogenously expressed ferroportin without tags. We anticipate that the present refolding and labeling approaches could also be used for other synthetic peptides.     

The C19S Substitution Enhances the Stability of Hepcidin While Conserving Its Biological Activity

Hepcidin, the key hormone of iron homeostasis is responsible for lowering the serum iron level through its interaction with iron exporter ferroportin. Thus, hepcidin agonists provide a promising opportunity in the treatment of iron disorders caused by lacking or decreased hepcidin expression. We investigated the importance of each of the eight highly conserved cysteines for the biological activity of hepcidin. Eight cysteine mutants were created with site directed mutagenesis. The binding ability of these hepcidin mutants to the hepcidin receptor ferroportin was determined using bacterial two-hybrid system and WRL68 human hepatic cells. The biological activity of hepcidin mutants was determined by western blot analysis of ferroportin internalization and ferroportin ubiquitination. To investigate the effect of mutant hepcidins on the iron metabolism of the WRL68 cells, total intracellular iron content was measured with a colorimetric assay. The stability of M6 hepcidin mutant was determined using ELISA technique. Our data revealed that serine substitution of the sixth cysteine (M6) yielded a biologically active but significantly more stable peptide than the original hormone. This result may provide a promising hepcidin agonist worth testing in animal models.     

The hepcidin-binding site on ferroportin is evolutionarily conserved

Mammalian iron homeostasis is regulated by the interaction of the liver-produced peptide hepcidin and its receptor, the iron transporter ferroportin. Hepcidin binds to ferroportin resulting in degradation of ferroportin and decreased cellular iron export. We identify the hepcidin-binding domain (HBD) on ferroportin and show that a synthetic 19 amino acid peptide corresponding to the HBD recapitulates the characteristics and specificity of hepcidin binding to cell-surface ferroportin. The binding of mammalian hepcidin to ferroportin or the HBD shows an unusual temperature dependency with an increased rate of dissociation at temperatures below 15°C. The increased rate of dissociation is due to temperature- dependent changes in hepcidin structure. In contrast, hepcidin from poikilothermic vertebrates, such as fish or frogs, binds the HBD in a temperature-independent fashion. The affinity of hepcidin for the HBD permits a rapid, sensitive assay of hepcidin from all species and yields insights into the evolution of hepcidin.     

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.     

In-depth review: is hepcidin a marker for the heart and the kidney?

Iron is an essential trace element involved in oxidation–reduction reactions, oxygen transport and storage, and energy metabolism. Iron in excess can be toxic for cells, since iron produces reactive oxygen species and is important for survival of pathogenic microbes. There is a fine-tuning in the regulation of serum iron levels, determined by intestinal absorption, macrophage iron recycling, and mobilization of hepatocyte stores versus iron utilization, primarily by erythroid cells in the bone marrow. Hepcidin is the major regulatory hormone of systemic iron homeostasis and is upregulated during inflammation. Hepcidin metabolism is altered in chronic kidney disease. Ferroportin is an iron export protein and mediates iron release into the circulation from duodenal enterocytes, splenic reticuloendothelial macrophages, and hepatocytes. Systemic iron homeostasis is controlled by the hepcidin–ferroportin axis at the sites of iron entry into the circulation. Hepcidin binds to ferroportin, induces its internalization and intracellular degradation, and thus inhibits iron absorption from enterocytes, and iron release from macrophages and hepatocytes. Recent data suggest that hepcidin, by slowing or preventing the mobilization of iron from macrophages, may promote atherosclerosis and may be associated with increased cardiovascular disease risk. This article reviews the current data regarding the molecular and cellular pathways of systemic and autocrine hepcidin production and seeks the answer to the question whether changes in hepcidin translate into clinical outcomes of all-cause and cardiovascular mortality, and cardiovascular and renal end-points.

     

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.     

Production of biologically active forms of recombinant hepcidin, the iron-regulatory hormone

Hepcidin is a liver produced cysteine-rich peptide hormone that acts as the central regulator of body iron metabolism. Hepcidin is synthesized under the form of a precursor, prohepcidin, which is processed to produce the biologically active mature 25 amino acid peptide. This peptide is secreted and acts by controlling the concentration of the membrane iron exporter ferroportin on intestinal enterocytes and macrophages. Hepcidin binds to ferroportin, inducing its internalization and degradation, thus regulating the export of iron from cells to plasma. The aim of the present study was to develop a novel method to produce human and mouse recombinant hepcidins, and to compare their biological activity towards their natural receptor ferroportin. Hepcidins were expressed in Escherichia coli as thioredoxin fusion proteins. The corresponding peptides, purified after cleavage from thioredoxin, were properly folded and contained the expected four-disulfide bridges without the need of any renaturation or oxidation steps. Human and mouse hepcidins were found to be biologically active, promoting ferroportin degradation in macrophages. Importantly, biologically inactive aggregated forms of hepcidin were observed depending on purification and storage conditions, but such forms were unrelated to disulfide bridge formation.     

Evidence for two different broad-specificity oligopeptide transporters in intestinal cell line Caco-2 and colonic cell line CCD841

Recently the existence of two different Na(+)-coupled oligopeptide transport systems has been described in mammalian cells. These transport systems are distinct from the previously known H(+)/peptide cotransporters PEPT1 and PEPT2, which transport only dipeptides and tripeptides. To date, the only peptide transport system known to exist in the intestine is PEPT1. Here we investigated the expression of the Na(+)-coupled oligopeptide transporters in intestinal cell lines, using the hydrolysis-resistant synthetic oligopeptides deltorphin II and [d-Ala(2),d-Leu(5)]enkephalin (DADLE) as model substrates. Caco-2 cells and CCD841 cells, both representing epithelial cells from human intestinal tract, were able to take up these oligopeptides. Uptake of deltorphin II was mostly Na(+) dependent, with more than 2 Na(+) involved in the uptake process. In contrast, DADLE uptake was only partially Na(+) dependent. The uptake of both peptides was also influenced by H(+) and Cl(-), although to a varying degree. The processes responsible for the uptake of deltorphin II and DADLE could be differentiated not only by their Na(+) dependence but also by their modulation by small peptides. Several dipeptides and tripeptides stimulated deltorphin II uptake but inhibited DADLE uptake. These modulating small peptides were, however, not transportable substrates for the transport systems that mediate deltorphin II or DADLE uptake. These two oligopeptide transport systems were also able to take up several nonopioid oligopeptides, consisting of 9-17 amino acids. This represents the first report on the existence of transport systems in intestinal cells that are distinct from PEPT1 and capable of transporting oligopeptides consisting of five or more amino acids.     

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。     

Regulation of iron acquisition and iron distribution in mammals

Both cellular iron deficiency and excess have adverse consequences. To maintain iron homeostasis, complex mechanisms have evolved to regulate cellular and extracellular iron concentrations. Extracellular iron concentrations are controlled by a peptide hormone hepcidin, which inhibits the supply of iron into plasma. Hepcidin acts by binding to and inducing the degradation of the cellular iron exporter, ferroportin, found in sites of major iron flows: duodenal enterocytes involved in iron absorption, macrophages that recycle iron from senescent erythrocytes, and hepatocytes that store iron. Hepcidin synthesis is in turn controlled by iron concentrations, hypoxia, anemia and inflammatory cytokines. The molecular mechanisms that regulate hepcidin production are only beginning to be understood, but its dysregulation is involved in the pathogenesis of a spectrum of iron disorders. Deficiency of hepcidin is the unifying cause of hereditary hemochromatoses, and excessive cytokine-stimulated hepcidin production causes hypoferremia and contributes to anemia of inflammation.     

Hepcidin: regulation of the master iron regulator

Iron, an essential nutrient, is required for many diverse biological processes. The absence of a defined pathway to excrete excess iron makes it essential for the body to regulate the amount of iron absorbed; a deficiency could lead to iron deficiency and an excess to iron overload and associated disorders such as anaemia and haemochromatosis respectively. This regulation is mediated by the iron-regulatory hormone hepcidin. Hepcidin binds to the only known iron export protein, ferroportin (FPN), inducing its internalization and degradation, thus limiting the amount of iron released into the blood. The major factors that are implicated in hepcidin regulation include iron stores, hypoxia, inflammation and erythropoiesis. The present review summarizes our present knowledge about the molecular mechanisms and signalling pathways contributing to hepcidin regulation by these factors.     

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.     

Impact of D181V and A69T on the function of ferroportin as an iron export pump and hepcidin receptor

Mutations in the only known mammalian iron exporter ferroportin cause a rare iron overload disorder termed ferroportin disease. Two distinct clinical phenotypes are caused by different disease mechanisms: mutations in ferroportin either cause loss of iron export function or gain of function due to resistance to hepcidin, the peptide hormone that normally downregulates ferroportin. The aim of the present study was to examine the disease mechanisms of the thus far unclassified A69T and D181V ferroportin mutations. We overexpressed wild-type and mutant ferroportin fused to green fluorescent protein in human embryonic kidney cells and used a ⁵⁹Fe-assay, intracellular ferritin concentrations, confocal microscopy and flow cytometry to study iron export function, subcellular localization and the responsiveness to hepcidin. While the A69T ferroportin mutation seems not to affect the iron export function it causes dose-dependent hepcidin resistance. We further found that D181V mutated ferroportin is iron export defective and hepcidin resistant, similar to the loss of function mutations A77D and C367X. This indicates that intact iron export might be necessary for hepcidin-induced downregulation of ferroportin. This hypothesis was investigated by studying the hepcidin response under modulation of iron availability. Incubation of wild-type ferroportin overexpressing cells with holo-transferrin increases the hepcidin effect whereas chelating extracellular ferrous iron causes hepcidin resistance. In this study we present data that postulates to classify the D181V ferroportin mutation as loss of function and the A69T mutation as dose-dependent hepcidin resistant and outline a possible causal link between iron export function and the hepcidin effect.     

Serum hepcidin / ferroportin levels in bipolar disorder and schizophrenia

BackgroundDespite several alternatives for cellular iron influx, the only mechanism for cellular iron efflux is ferroportin mediated active transport. In cases of ferroportin dysfunction, iron accumulates in the cell and causes ferroptosis. Hepcidin suppresses ferroportin levels and inflammatory activation increases hepcidin production. Mild inflammation in schizophrenia and bipolar disorder may alter hepcidin and ferroportin.MethodsThe study included a total of 137 patients aged 18–65 years, 57 diagnosed with schizophrenia and 80 with bipolar disorder, according to the DSM-IV diagnostic criteria, and a control group (HC) of 42 healthy individuals. Biochemical analyses, thyroid function tests, hemogram, serum iron level, iron-binding capacity, and ferritin levels were examined. Serum levels of hepcidin and ferroportin were measured with enzyme-linked immunosorbent assay (ELISA) method.ResultsA statistically significant difference was determined between the groups in terms of the serum ferroportin levels (F = 15.69, p < 0.001). Post-hoc analyses showed that the schizophrenia group had higher ferroportin levels than in the bipolar group (p < 0.001) and HCs (p < 0.001). Hepcidin levels did not differ between the groups. Chlorpromazine equivalent doses of antipsychotics correlated with ferroportin levels (p = 0.024).ConclusionFerroportin levels were increased in the schizophrenia group, although iron and hepcidin levels were within normal ranges. Antipsychotics may alter the mechanisms which control ferroportin levels. Further studies are needed to examine the relationships between antipsychotics and iron metabolism for determination of causal relationship.     

Effect of hepcidin on intracellular calcium in human osteoblasts

Hepcidin is known to increase intracellular iron through binding to and degrading ferroportin, which is a transmembrane protein that transports iron from the intracellular to the outside. However, it is not clear whether hepcidin has a similar effect on intracellular calcium. Here, we investigated the influence of hepcidin on intracellular calcium in human osteoblasts, with or without high environmental iron concentrations. Our data showed that hepcidin (<100 nmol/L) could increase intracellular calcium, and this effect was more significant when cells were exposed to high environmental iron concentrations. To further explore its underlying mechanisms, we pretreated human osteoblasts with Nimodipine, a L-type calcium channel blocker, and Dantrolene, a ryanodine receptor antagonist to inhibit abnormal calcium release from the sarco-endoplasmic reticulum. These treatments had not resulted in any alteration of intracellular calcium in human osteoblasts. Thus, these findings indicate that the increase of intracellular calcium induced by hepcidin is probably due to calcium release from endoplasmic reticulum, which is triggered by calcium influx.