Duodenal expression of iron transport molecules in patients with hereditary hemochromatosis or iron deficiency

Disturbances of iron metabolism are observed in chronic liver diseases. In the present study, we examined gene expression of duodenal iron transport molecules and hepcidin in patients with hereditary hemochromatosis (HHC) (treated and untreated), involving various genotypes (genotypes which represent risk for HHC were examined), and in patients with iron deficiency anaemia (IDA). Gene expressions of DMT1, ferroportin, Dcytb, hephaestin, HFE and TFR1 were measured in duodenal biopsies using real-time PCR and Western blot. Serum hepcidin levels were measured using ELISA. DMT1, ferroportin and TFR1 mRNA levels were significantly increased in post-phlebotomized hemochromatics relative to controls. mRNAs of all tested molecules were significantly increased in patients with IDA compared to controls. The protein expression of ferroportin was increased in both groups of patients but not significantly. Spearman rank correlations showed that DMT1 versus ferroportin, Dcytb versus hephaestin and DMT1 versus TFR1 mRNAs were positively correlated regardless of the underlying cause, similarly to protein levels of ferroportin versus Dcytb and ferroportin versus hephaestin. Serum ferritin was negatively correlated with DMT1 mRNA in investigated groups of patients, except for HHC group. A decrease of serum hepcidin was observed in IDA patients, but this was not statistically significant. Our data showed that although untreated HHC patients do not have increased mRNA levels of iron transport molecules when compared to normal subjects, the expression is relatively increased in relation to body iron stores. On the other hand, post-phlebotomized HHC patients had increased DMT1 and ferroportin mRNA levels possibly due to stimulated erythropoiesis after phlebotomy.     

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.     

Regulatory networks for the control of body iron homeostasis and their dysregulation in HFE mediated hemochromatosis

Although the recent identification of several genes has extended our knowledge on the maintenance of body iron homeostasis, their tissue specific expression patterns and the underlying regulatory networks are poorly understood. We studied C57black/Sv129 mice and HFE knockout (HFE -/-) variants thereof as a model for hemochromatosis, and investigated the expression of iron metabolism genes in the duodenum, liver, and kidney as a function of dietary iron challenge. In HFE +/+ mice dietary iron supplementation increased hepatic expression of hepcidin which was paralleled by decreased iron regulatory protein (IRP) activity, and reduced expression of divalent metal transporter-1 (DMT-1) and duodenal cytochrome b (Dcytb) in the enterocyte. In HFE -/- mice hepcidin formation was diminished upon iron challenge which was associated with decreased hepatic transferrin receptor (TfR)-2 levels. Accordingly, HFE -/- mice presented with high duodenal Dcytb and DMT-1 levels, and increased IRP and TfR expression, suggesting iron deficiency in the enterocyte and increased iron absorption. In parallel, HFE -/- resulted in reduced renal expression of Dcytb and DMT-1. Our data suggest that the feed back regulation of duodenal iron absorption by hepcidin is impaired in HFE -/- mice, a model for genetic hemochromatosis. This change may be linked to inappropriate iron sensing by the liver based on decreased TfR-2 expression, resulting in reduced circulating hepcidin levels and an inappropriate up-regulation of Dcytb and DMT-1 driven iron absorption. In addition, iron excretion/reabsorption by the kidneys may be altered, which may aggravate progressive iron overload.     

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.     

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.

     

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.     

Hepcidin mRNA levels in mouse liver respond to inhibition of erythropoiesis

Hepcidin, a key regulator of iron metabolism, decreases intestinal absorption of iron and its release from macrophages. Iron, anemia, hypoxia, and inflammation were reported to influence hepcidin expression. To investigate regulation of the expression of hepcidin and other iron-related genes, we manipulated erythropoietic activity in mice. Erythropoiesis was inhibited by irradiation or posttransfusion polycythemia and stimulated by phenylhydrazine administration and erythropoietin. Gene expression of hepcidin and other iron-related genes (hemojuvelin, DMT1, ferroportin, transferrin receptors, ferritin) in the liver was measured by the real-time polymerase chain reaction. Hepcidin expression increased despite severe anemia when hematopoiesis was inhibited by irradiation. Suppression of erythropoiesis by posttransfusion polycythemia or irradiation also increased hepcidin mRNA levels. Compensated hemolysis induced by repeated phenylhydrazine administration did not change hepcidin expression. The decrease caused by exogenous erythropoeitin was blocked by postirradiation bone marrow suppression. The hemolysis and anemia decrease hepcidin expression only when erythropoiesis is functional; on the other hand, if erythropoiesis is blocked, even severe anemia does not lead to a decrease of hepcidin expression, which is indeed increased. We propose that hepcidin is exclusively sensitive to iron utilization for erythropoiesis and hepatocyte iron balance, and these changes are not sensed by other genes involved in the control of iron metabolism in the liver.     

Systemic regulation of intestinal iron absorption

The intestinal absorption of the essential trace element iron and its mobilization from storage sites in the body are controlled by systemic signals that reflect tissue iron requirements. Recent advances have indicated that the liver-derived peptide hepcidin plays a central role in this process by repressing iron release from intestinal enterocytes, macrophages and other body cells. When iron requirements are increased, hepcidin levels decline and more iron enters the plasma. It has been proposed that the level of circulating diferric transferrin, which reflects tissue iron levels, acts as a signal to alter hepcidin expression. In the liver, the proteins HFE, transferrin receptor 2 and hemojuvelin may be involved in mediating this signal as disruption of each of these molecules decreases hepcidin expression. Patients carrying mutations in these molecules or in hepcidin itself develop systemic iron loading (or hemochromatosis) due to their inability to down regulate iron absorption. Hepcidin is also responsible for the decreased plasma iron or hypoferremia that accompanies inflammation and various chronic diseases as its expression is stimulated by pro-inflammatory cytokines such as interleukin 6. The mechanisms underlying the regulation of hepcidin expression and how it acts on cells to control iron release are key areas of ongoing research.     

Intestinal iron absorption

Intestinal iron absorption is a critical process for maintaining body iron levels within the optimal physiological range. Iron in the diet is found in a wide variety of forms, but the absorption of non-heme iron is best understood. Most of this iron is moved across the enterocyte brush border membrane by the iron transporter divalent metal-ion transporter 1, a process enhanced by the prior reduction of the iron by duodenal cytochrome B and possibly other reductases. Enterocyte iron is exported to the blood via ferroportin 1 on the basolateral membrane. This transporter acts in partnership with the ferroxidase hephaestin that oxidizes exported ferrous iron to facilitate its binding to plasma transferrin. Iron absorption is controlled by a complex network of systemic and local influences. The liver-derived peptide hepcidin binds to ferroportin, leading to its internalization and a reduction in absorption. Hepcidin expression in turn responds to body iron demands and the BMP-SMAD signaling pathway plays a key role in this process. The levels of iron and oxygen in the enterocyte also exert important influences on iron absorption. Disturbances in the regulation of iron absorption are responsible for both iron loading and iron deficiency disorders in humans.     

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。     

Iron disorders of genetic origin: a changing world

Iron disorders of genetic origin are mainly composed of iron overload diseases, the most frequent being HFE-related hemochromatosis. Hepcidin deficiency underlies iron overload in HFE-hemochromatosis as well as in several other genetic iron excess disorders, such as hemojuvelin or hepcidin-related hemochromatosis and transferrin receptor 2-related hemochromatosis. Deficiency of ferroportin, the only known cellular protein iron exporter, produces iron overload in the typical form of ferroportin disease. By contrast, genetically enhanced hepcidin production, as observed in matriptase-2 deficiency, generates iron-refractory iron deficiency anemia. Diagnosis of these iron storage disorders is usually established noninvasively through combined biochemical, imaging and genetic approaches. Moreover, improved knowledge of the molecular mechanisms accounting for the variations of iron stores opens the way of novel therapeutic approaches aiming to restore normal iron homeostasis. In this review, we will summarize recent findings about these various genetic entities that have been identified owing to an exemplary interplay between clinicians and basic scientists.     

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.     

Hepcidin与疾病关系和其临床意义的研究进展

铁是人体必需的微量元素,是血红蛋白、肌红蛋白及多种酶的重要组成成分,广泛地参与氧气输运、氧化还原反应、细胞增殖与分化、基因表达调控等基本生命过程。机体铁稳态对生命体新陈代谢的平衡起着至关重要的作用。铁稳态依赖铁吸收、转运和储存、再循环利用等代谢过程共同调节。铁调素（Hepcidin）是铁代谢调节中最关键的调节分子,成熟的铁调素是一个由25个氨基酸组成的功能性小肽类激素,可以通过调节小肠上皮细胞和巨噬细胞表面的相关铁转运蛋白来调控机体内铁的储存和利用。铁调素同时受到机体铁水平的反馈,免疫应答和红细胞生成等因素的共同调节。许多铁代谢疾病、炎症和各种原因引起的贫血与铁调素的异常表达相关。因此,对于铁调素的检测不但可以反映机体的铁代谢状况,结合其他临床指标还能够辅助诊断和有针对性地检测相关疾病的治疗效果。     

Haemochromatosis protein is expressed on the terminal web of enterocytes in proximal small intestine of the rat

The haemochromatosis protein (HFE) is an important regulator of body iron stores. In the liver, HFE is required for appropriate expression of hepcidin, a humoral mediator of iron absorption. HFE is also present in enterocytes, though its function in the intestine is unknown; it is not intrinsically required for iron absorption, but can augment iron absorption when over-expressed—independent of hepcidin regulation by the liver. In this study, an antibody was raised against rat HFE and validated by enzyme-linked immunosorbent assay, Western blot and quenching of antibody function by the immunising peptide. The sub-cellular location of HFE in enterocytes of iron-deficient and control rats was determined by double-labelling experiments with markers for the microvillus membrane, terminal web, early endosomes, lysosomes and the transferrin receptor. Parallel studies were performed for the primary iron absorption protein, divalent metal transporter 1 (DMT1). HFE co-localised exclusively with the terminal web of intestinal enterocytes. HFE expression was increased in iron deficiency, consistent with a second regulatory role for HFE in iron absorption, independent of hepcidin from the liver. DMT1 was localised primarily on the microvillus membrane, but did partially co-localise with HFE raising the possibility that the two proteins may interact to regulate iron absorption.     

Hepcidin induction limits mobilisation of splenic iron in a mouse model of secondary iron overload

Venesection has been proposed as a treatment for hepatic iron overload in a number of chronic liver disorders that are not primarily linked to mutations in iron metabolism genes. Our aim was to analyse the impact of venesection on iron mobilisation in a mouse model of secondary iron overload. C57Bl/6 mice were given oral iron supplementation with or without phlebotomy between day 0 (D0) and D22, and the results were compared to controls without iron overload. We studied serum and tissue iron parameters, mRNA levels of hepcidin1, ferroportin, and transferrin receptor 1, and protein levels of ferroportin in the liver and spleen. On D0, animals with iron overload displayed elevations in iron parameters and hepatic hepcidin1 mRNA. By D22, in the absence of phlebotomies, splenic iron had increased, but transferrin saturation had decreased. This was associated with high hepatic hepcidin1 mRNA, suggesting that iron bioavailability decreased due to splenic iron sequestration through ferroportin protein downregulation. After 22 days with phlebotomy treatments, control mice displayed splenic iron mobilisation that compensated for the iron lost due to phlebotomy. In contrast, phlebotomy treatments in mice with iron overload caused anaemia due to inadequate iron mobilisation. In conclusion, our model of secondary iron overload led to decreased plasma iron associated with an increase in hepcidin expression and subsequent restriction of iron export from the spleen. Our data support the importance of managing hepcidin levels before starting venesection therapy in patients with secondary iron overload that are eligible for phlebotomy.