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1.  Severe Microcytic Anemia but Increased Erythropoiesis in Mice Lacking Hfe or Tfr2 and Tmprss6 
Blood cells, molecules & diseases  2012;48(3):173-178.
Cell surface proteins Hfe, Tfr2, hemojuvelin and Tmprss6 play key roles in iron homeostasis. Hfe and Tfr2 induce transcription of hepcidin, a small peptide that promotes the degradation of the iron transporter ferroportin. Hemojuvelin, a co-receptor for bone morphogenic proteins, induces hepcidin transcription through a Smad signaling pathway. Tmprss6 (also known as matriptase-2), a membrane serine protease that has been found to bind and degrade hemojuvelin in vitro, is a potent suppressor of hepcidin expression. In order to examine if Hfe and Tfr2 are substrates for Tmprss6, we generated mice lacking functional Hfe or Tfr2 and Tmprss6. We found that double mutant mice lacking functional Hfe or Tfr2 and Tmprss6 exhibited a severe iron deficiency microcytic anemia phenotype mimicking the phenotype of single mutant mice lacking functional Tmprss6 (Tmprss6 msk/msk mutant) demonstrating that Hfe and Tfr2 are not substrates for Tmprss6. Nevertheless, the phenotype of the mice lacking Hfe or Tfr2 and Tmprss6 differed from Tmprss6 deficient mice alone, in that the double mutant mice exhibited much greater erythropoiesis. Hfe and Tfr2 have been shown to play important roles in the erythron, independent of their role in regulating liver hepcidin transcription. We demonstrate that lack of functional Tfr2 and Hfe allow for increased erythropoiesis even in the presence of high hepcidin expression, but the high levels of hepcidin levels significantly limit the availability of iron to the erythron, resulting in ineffective erythropoiesis. Furthermore, repression of hepcidin expression was unaffected by loss of functional Hfe, Tfr2 and Tmprss6.
doi:10.1016/j.bcmd.2011.12.005
PMCID: PMC3294186  PMID: 22244935
hepcidin; iron; TMPRSS6; hemochromatosis; anemia; HFE; TFR2; matriptase
2.  Iron-deficiency anemia from matriptase-2 inactivation is dependent on the presence of functional Bmp6 
Blood  2010;117(2):647-650.
Hepcidin is the master regulator of iron homeostasis. In the liver, iron-dependent hepcidin activation is regulated through Bmp6 and its membrane receptor hemojuvelin (Hjv) whereas, in response to iron deficiency, hepcidin repression seems to be controlled by a pathway involving the serine protease matriptase-2 (encoded by Tmprss6). To determine the relationship between Bmp6 and matriptase-2 pathways, Tmprss6−/− mice (characterized by increased hepcidin levels and anemia) and Bmp6−/− mice (exhibiting severe iron overload due to hepcidin deficiency) were intercrossed. We showed that loss of Bmp6 decreased hepcidin levels, increased hepatic iron and, importantly, corrected hematological abnormalities in Tmprss6−/− mice. This suggests that elevated hepcidin levels in patients with familial iron-refractory iron deficiency anemia are due to excess signaling through the Bmp6/Hjv pathway.
doi:10.1182/blood-2010-07-295147
PMCID: PMC3302207  PMID: 20940420
Anemia, Iron-Deficiency; metabolism; Animals; Antimicrobial Cationic Peptides; metabolism; Bone Morphogenetic Protein 6; metabolism; Female; Iron; metabolism; Iron, Dietary; metabolism; Liver; metabolism; Membrane Proteins; metabolism; Mice; Mice, Knockout; Serine Endopeptidases; metabolism; Signal Transduction; physiology; hepcidin; hemojuvelin; bmp6; matriptase2; tmprss6
3.  Iron-Refractory Iron Deficiency Anemia: New Molecular Mechanisms 
Kidney international  2009;76(11):1137-1141.
Iron deficiency is a common cause of anemia. In end-stage renal disease (ESRD), iron deficiency impairs the therapeutic efficacy of recombinant erythropoietin. Oral or parental iron supplements usually are effective in treating iron deficiency anemia (IDA). Some patients, however, respond poorly to iron supplements and are diagnosed as having iron-refractory iron deficiency anemia (IRIDA). The disease represents a medical challenge but its underlying mechanism was unclear. Hepcidin is a central player in iron homeostasis. It down-regulates the iron exporter ferroportin, thereby inhibiting iron absorption, release and recycling. In ESRD, plasma hepcidin levels are elevated, which contributes to iron deficiency in patients. Matriptase-2, a liver transmembrane serine protease, has been found to have a major role in controlling hepcidin gene expression. In mice, defects in the Tmprss6 gene encoding matriptase-2 result in high hepcidin expression and cause severe microcytic anemia. Similarly, mutations in the human TMPRSS6 gene have been identified in patients with IRIDA. Thus, matriptase-2 is critical for iron homeostasis and may play a role in renal disease.
doi:10.1038/ki.2009.357
PMCID: PMC2869468  PMID: 19776721
matriptase-2; TMPRSS6; hepcidin; end-stage renal disease; EPO resistance
4.  Modulation of bone morphogenetic protein signaling in vivo regulates systemic iron balance 
Journal of Clinical Investigation  2007;117(7):1933-1939.
Systemic iron balance is regulated by hepcidin, a peptide hormone secreted by the liver. By decreasing cell surface expression of the iron exporter ferroportin, hepcidin decreases iron absorption from the intestine and iron release from reticuloendothelial stores. Hepcidin excess has been implicated in the pathogenesis of anemia of chronic disease, while hepcidin deficiency has a key role in the pathogenesis of the iron overload disorder hemochromatosis. We have recently shown that hemojuvelin is a coreceptor for bone morphogenetic protein (BMP) signaling and that BMP signaling positively regulates hepcidin expression in liver cells in vitro. Here we show that BMP-2 administration increases hepcidin expression and decreases serum iron levels in vivo. We also show that soluble hemojuvelin (HJV.Fc) selectively inhibits BMP induction of hepcidin expression in vitro and that administration of HJV.Fc decreases hepcidin expression, increases ferroportin expression, mobilizes splenic iron stores, and increases serum iron levels in vivo. These data support a role for modulators of the BMP signaling pathway in treating diseases of iron overload and anemia of chronic disease.
doi:10.1172/JCI31342
PMCID: PMC1904317  PMID: 17607365
5.  THE SERINE PROTEASE MATRIPTASE-2 (TMPRSS6) INHIBITS HEPCIDIN ACTIVATION BY CLEAVING MEMBRANE HEMOJUVELIN 
Cell metabolism  2008;8(6):502-511.
Summary
The liver peptide hepcidin regulates body iron, is upregulated in iron overload and inflammation and downregulated in iron deficiency/hypoxia. The transmembrane serine protease matriptase-2 (TMPRSS6) inhibits the hepcidin response and its mutational inactivation causes iron-deficient anemia in mice and humans. Here we confirm the inhibitory effect of matriptase-2 on hepcidin promoter; we show that matriptase-2 lacking the serine protease domain, identified in the anemic Mask mouse (matriptase-2MASK), is fully inactive and that mutant R774C found in patients with genetic iron deficiency has decreased inhibitory activity. Matriptase-2 cleaves hemojuvelin (HJV), a regulator of hepcidin, on plasma membrane; matriptase-2MASK shows no and the human mutant only partial cleavage capacity. Matriptase-2 interacts with HJV through the ectodomain since the interaction is conserved in matriptase-2MASK. The expression of matriptase-2 mutants in zebrafish results in anemia, confirming the matriptase-2 role in iron metabolism and its interaction with HJV.
doi:10.1016/j.cmet.2008.09.012
PMCID: PMC2648389  PMID: 18976966
6.  Control of Systemic Iron Homeostasis by the Hemojuvelin-Hepcidin Axis12 
Advances in Nutrition  2010;1(1):38-45.
Systemic iron homeostasis is maintained by the coordinate regulation of iron absorption in the duodenum, iron recycling of senescent erythrocytes in macrophages, and mobilization of storage iron in the liver. These processes are controlled by hepcidin, a key iron regulatory hormone. Hepcidin is a 25-amino acid peptide secreted predominantly from hepatocytes. It downregulates ferroportin, the only known iron exporter, and therefore inhibits iron efflux from duodenal enterocytes, macrophages, and hepatocytes into the circulation. Hepcidin expression is regulated positively by body iron load. Although the underlying mechanism of iron-regulated hepcidin expression has not been fully elucidated, several proteins have been identified that participate in this process. Among them, hemojuvelin (HJV) plays a particularly important role. HJV undergoes complicated post-translational processing in an iron-dependent manner, and it interacts with multiple proteins that are essential for iron homeostasis. In this review, I focus on the recent findings that elucidate the role of HJV and its interacting partners in the modulation of hepatic hepcidin expression.
doi:10.3945/an.110.1009
PMCID: PMC3042782  PMID: 22043450
7.  Iron regulation by hepcidin 
The Journal of Clinical Investigation  2013;123(6):2337-2343.
Hepcidin is a key hormone that is involved in the control of iron homeostasis in the body. Physiologically, hepcidin is controlled by iron stores, inflammation, hypoxia, and erythropoiesis. The regulation of hepcidin expression by iron is a complex process that requires the coordination of multiple proteins, including hemojuvelin, bone morphogenetic protein 6 (BMP6), hereditary hemochromatosis protein, transferrin receptor 2, matriptase-2, neogenin, BMP receptors, and transferrin. Misregulation of hepcidin is found in many disease states, such as the anemia of chronic disease, iron refractory iron deficiency anemia, cancer, hereditary hemochromatosis, and ineffective erythropoiesis, such as β-thalassemia. Thus, the regulation of hepcidin is the subject of interest for the amelioration of the detrimental effects of either iron deficiency or overload.
doi:10.1172/JCI67225
PMCID: PMC3668831  PMID: 23722909
8.  Hepcidin Regulation by BMP Signaling in Macrophages Is Lipopolysaccharide Dependent 
PLoS ONE  2012;7(9):e44622.
Hepcidin is an antimicrobial peptide, which also negatively regulates iron in circulation by controlling iron absorption from dietary sources and iron release from macrophages. Hepcidin is synthesized mainly in the liver, where hepcidin is regulated by iron loading, inflammation and hypoxia. Recently, we have demonstrated that bone morphogenetic protein (BMP)-hemojuvelin (HJV)-SMAD signaling is central for hepcidin regulation in hepatocytes. Hepcidin is also expressed by macrophages. Studies have shown that hepcidin expression by macrophages increases following bacterial infection, and that hepcidin decreases iron release from macrophages in an autocrine and/or paracrine manner. Although previous studies have shown that lipopolysaccharide (LPS) can induce hepcidin expression in macrophages, whether hepcidin is also regulated by BMPs in macrophages is still unknown. Therefore, we examined the effects of BMP signaling on hepcidin expression in RAW 264.7 and J774 macrophage cell lines, and in primary peritoneal macrophages. We found that BMP4 or BMP6 alone did not have any effect on hepcidin expression in macrophages although they stimulated Smad1/5/8 phosphorylation and Id1 expression. In the presence of LPS, however, BMP4 and BMP6 were able to stimulate hepcidin expression in macrophages, and this stimulation was abolished by the NF-κB inhibitor Ro1069920. These results suggest that hepcidin expression is regulated differently in macrophages than in hepatocytes, and that BMPs regulate hepcidin expression in macrophages in a LPS-NF-κB dependent manner.
doi:10.1371/journal.pone.0044622
PMCID: PMC3441567  PMID: 23028567
9.  The serine protease TMPRSS6 is required to sense iron deficiency 
Science (New York, N.Y.)  2008;320(5879):1088-1092.
Hepcidin, a liver-derived protein that restricts enteric iron absorption, is the key regulator of body iron content. Several proteins induce expression of the hepcidin-encoding gene Hamp in response to infection or high levels of iron. However, mechanism(s) of Hamp suppression during iron depletion are poorly understood. Here we describe mask, a recessive, chemically induced mutant mouse phenotype, characterized by progressive loss of body but not facial hair and microcytic anemia. The mask phenotype results from reduced absorption of dietary iron caused by high levels of hepcidin, and is due to a splicing defect in the transmembrane serine protease 6 gene Tmprss6. Overexpression of normal TMPRSS6 protein suppresses activation of the Hamp promoter, and the TMPRSS6 cytoplasmic domain mediates Hamp suppression via proximal promoter element(s). TMPRSS6 is an essential component of a pathway that detects iron deficiency and blocks Hamp transcription, permitting enhanced dietary iron absorption.
doi:10.1126/science.1157121
PMCID: PMC2430097  PMID: 18451267
10.  Hemojuvelin is essential for dietary iron sensing, and its mutation leads to severe iron overload 
Journal of Clinical Investigation  2005;115(8):2180-2186.
Iron homeostasis plays a critical role in many physiological processes, notably synthesis of heme proteins. Dietary iron sensing and inflammation converge in the control of iron absorption and retention by regulating the expression of hepcidin, a regulator of the iron exporter ferroportin. Human mutations in the glycosylphosphatidylinositol-anchored protein hemojuvelin (HJV; also known as RGMc and HFE2) cause juvenile hemochromatosis, a severe iron overload disease, but the way in which HJV intersects with the iron regulatory network has been unclear. Here we show that, within the liver, mouse Hjv is selectively expressed by periportal hepatocytes and also that Hjv-mutant mice exhibit iron overload as well as a dramatic decrease in hepcidin expression. Our findings define a key role for Hjv in dietary iron sensing and also reveal that cytokine-induced inflammation regulates hepcidin expression through an Hjv-independent pathway.
doi:10.1172/JCI25683
PMCID: PMC1180556  PMID: 16075058
11.  Iron metabolism: current facts and future directions 
Biochemia Medica  2012;22(3):311-328.
Iron metabolism has been intensively examined over the last decade and there are many new players in this field which are worth to be introduced. Since its discovery many studies confirmed role of liver hormone hepcidin as key regulator of iron metabolism and pointed out liver as the central organ of system iron homeostasis. Liver cells receive multiple signals related to iron balance and respond by transcriptional regulation of hepcidin expression. This liver hormone is negative regulator of iron metabolism that represses iron efflux from macrophages, hepatocytes and enterocytes by its binding to iron export protein ferroportin. Ferroportin degradation leads to cellular iron retention and decreased iron availability. At level of a cell IRE/IRP (iron responsive elements/iron responsive proteins) system allows tight regulation of iron assimilation that prevents an excess of free intracellular iron which could lead to oxidative stress and damage of DNA, proteins and lipid membranes by ROS (reactive oxygen species). At the same time IRE/IRP system provides sufficient iron in order to meet the metabolic needs.
Recently a significant progress in understanding of iron metabolism has been made and new molecular participants have been characterized. Article gives an overview of the current understanding of iron metabolism: absorption, distribution, cellular uptake, release, and storage. We also discuss mechanisms underlying systemic and cellular iron regulation with emphasis on central regulatory hormone hepcidin.
PMCID: PMC3900049  PMID: 23092063
hepcidin; hemojuvelin; iron metabolism
12.  Hepcidin expression in mouse retina and its regulation via lipopolysaccharide/Toll-like receptor-4 pathway independent of Hfe 
The Biochemical journal  2008;411(1):79-88.
Summary
Hepcidin is a hormone central to the regulation of iron homeostasis in the body. It is believed to be produced exclusively by the liver. Ferroportin, an iron exporter, is the receptor for hepcidin. This transporter/receptor is expressed in Müller cells, photoreceptor cells, and retinal pigment epithelium (RPE) within the retina. Since the retina is protected by the retinal-blood barriers, we asked whether ferroportin in the retina is regulated by hepcidin in the circulation or whether the retina produces hepcidin for regulation of its own iron homeostasis. Here we show that hepcidin is expressed robustly in Müller cells, photoreceptor cells, and RPE, closely resembling the expression pattern of ferroportin. We also show that bacterial lipopolysaccharide (LPS) is a regulator of hepcidin expression in Müller cells and RPE, both in vitro and in vivo, and that the regulation occurs at the transcriptional level. The action of LPS on hepcidin expression is mediated by the Toll-like receptor-4. The upregulation of hepcidin by LPS occurs independent of Hfe (Human leukocyte antigen-like protein involved in Fe homeostasis). The increase in hepcidin levels in retinal cells in response to LPS treatment is associated with a decrease in ferroportin levels. The LPS-induced upregulation of hepcidin and consequent downregulation of ferroportin is associated with increased oxidative stress and apoptosis within the retina in vivo. We conclude that retinal iron homeostasis may be regulated in an autonomous manner by hepcidin generated within the retina and that chronic bacterial infection/inflammation of the retina may disrupt iron homeostasis and retinal function.
doi:10.1042/BJ20071377
PMCID: PMC3731152  PMID: 18042040
hepcidin; retina; hemochromatosis; lipopolysaccharide; ferroportin; retinal pigment epithelium
13.  Hepcidin Regulation of Iron Transport123 
The Journal of nutrition  2008;138(11):2284-2288.
The discovery of hepcidin as a key regulator of iron homeostasis has advanced our current knowledge of this field. Liver-derived hepcidin peptide is secreted in response to iron and inflammation and interacts with the iron export protein ferroportin. This review summarizes recent advances discussed at the Symposium. A particular focus is on molecular interactions between hepcidin and ferroportin, the regulation of hepcidin expression by iron and inflammation, and emerging methods to measure serum hepcidin in human populations.
doi:10.3945/jn.108.096347
PMCID: PMC2764359  PMID: 18936232
14.  A novel TMPRSS6 mutation that prevents protease auto-activation causes IRIDA 
Biochemical Journal  2010;431(Pt 3):363-371.
IRIDA (iron-refractory iron-deficiency anaemia) is a rare autosomal-recessive disorder hallmarked by hypochromic microcytic anaemia, low transferrin saturation and high levels of the iron-regulated hormone hepcidin. The disease is caused by mutations in the transmembrane serine protease TMPRSS6 (transmembrane protease serine 6) that prevent inactivation of HJV (haemojuvelin), an activator of hepcidin transcription. In the present paper, we describe a patient with IRIDA who carries a novel mutation (Y141C) in the SEA domain of the TMPRSS6 gene. Functional characterization of the TMPRSS6(Y141C) mutant protein in cultured cells showed that it localizes to similar subcellular compartments as wild-type TMPRSS6 and binds HJV, but fails to auto-catalytically activate itself. As a consequence, hepcidin mRNA expression is increased, causing the clinical symptoms observed in this IRIDA patient. The present study provides important mechanistic insight into how TMPRSS6 is activated.
doi:10.1042/BJ20100668
PMCID: PMC2958558  PMID: 20704562
haemojuvelin; hepcidin; iron-refractory irondeficiency anaemia (IRIDA); matriptase-2; transmembrane protease serine 6 (TMPRSS6); ALAS2, aminolevulinate δ synthase 2; BMP, bone morphogenetic protein; CUB domain, complement factor C1s/C1r, urchin embryonic growth factor and BMP domain; CMV, cytomegalovirus; DMEM, Dulbecco's modified Eagle's medium; EGFP, enhanced green fluorescent protein; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HJV, haemojuvelin; IRIDA, iron-refractory iron-deficiency anaemia; LDLR domain, low-density-lipoprotein receptor class A domain; ORF, open reading frame; PIC, phosphoinositidase C; qRT-PCR, quantitative real-time PCR; SEA domain, sea urchin sperm protein, enteropeptidase and agrin domain; SELDI–TOF, surface-enhanced laser-desorption ionization–time-of-flight; SLC11A2, solute carrier family 11, member 2; SLC25A28, solute carrier family 25, member 28; TMPRSS6, transmembrane protease serine 6; cdTMPRSS6, catalytic domain of TMPRSS6; TTSP, type II transmembrane serine protease
15.  Hepcidin mediates transcriptional changes that modulate acute cytokine-induced inflammatory responses in mice 
The Journal of Clinical Investigation  2010;120(7):2395-2405.
Hepcidin is a peptide hormone that regulates iron homeostasis and acts as an antimicrobial peptide. It is expressed and secreted by a variety of cell types in response to iron loading and inflammation. Hepcidin mediates iron homeostasis by binding to the iron exporter ferroportin, inducing its internalization and degradation via activation of the protein kinase Jak2 and the subsequent phosphorylation of ferroportin. Here we have shown that hepcidin-activated Jak2 also phosphorylates the transcription factor Stat3, resulting in a transcriptional response. Hepcidin treatment of ferroportin-expressing mouse macrophages showed changes in mRNA expression levels of a wide variety of genes. The changes in transcript levels for half of these genes were a direct effect of hepcidin, as shown by cycloheximide insensitivity, and dependent on the presence of Stat3. Hepcidin-mediated transcriptional changes modulated LPS-induced transcription in both cultured macrophages and in vivo mouse models, as demonstrated by suppression of IL-6 and TNF-α transcript and secreted protein. Hepcidin-mediated transcription in mice also suppressed toxicity and morbidity due to single doses of LPS, poly(I:C), and turpentine, which is used to model chronic inflammatory disease. Most notably, we demonstrated that hepcidin pretreatment protected mice from a lethal dose of LPS and that hepcidin-knockout mice could be rescued from LPS toxicity by injection of hepcidin. The results of our study suggest a new function for hepcidin in modulating acute inflammatory responses.
doi:10.1172/JCI42011
PMCID: PMC2898601  PMID: 20530874
16.  The Hepcidin Circuits Act: Balancing Iron and Inflammation 
Hepatology (Baltimore, Md.)  2011;53(5):1764-1766.
Hepcidin is a peptide hormone that regulates iron homeostasis and acts as an antimicrobial peptide. It is expressed and secreted by a variety of cell types in response to iron loading and inflammation. Hepcidin mediates iron homeostasis by binding to the iron exporter ferroportin, inducing its internalization and degradation via activation of the protein kinase Jak2 and the subsequent phosphorylation of ferroportin. Here we have shown that hepcidin-activated Jak2 also phosphorylates the transcription factor Stat3, resulting in a transcriptional response. Hepcidin treatment of ferroportin-expressing mouse macrophages showed changes in mRNA expression levels of a wide variety of genes. The changes in transcript levels for half of these genes were a direct effect of hepcidin, as shown by cycloheximide insensitivity, and dependent on the presence of Stat3. Hepcidin-mediated transcriptional changes modulated LPS-induced transcription in both cultured macrophages and in vivo mouse models, as demonstrated by suppression of IL-6 and TNF-α transcript and secreted protein. Hepcidin-mediated transcription in mice also suppressed toxicity and morbidity due to single doses of LPS, poly(I:C), and turpentine, which is used to model chronic inflammatory disease. Most notably, we demonstrated that hepcidin pretreatment protected mice from a lethal dose of LPS and that hepcidin-knockout mice could be rescued from LPS toxicity by injection of hepcidin. The results of our study suggest a new function for hepcidin in modulating acute inflammatory responses.
doi:10.1002/hep.24267
PMCID: PMC3095435  PMID: 21520181
17.  Leishmania-Mediated Inhibition of Iron Export Promotes Parasite Replication in Macrophages 
PLoS Pathogens  2014;10(1):e1003901.
Leishmania parasites infect macrophages, cells that play an important role in organismal iron homeostasis. By expressing ferroportin, a membrane protein specialized in iron export, macrophages release iron stored intracellularly into the circulation. Iron is essential for the intracellular replication of Leishmania, but how the parasites compete with the iron export function of their host cell is unknown. Here, we show that infection with Leishmania amazonensis inhibits ferroportin expression in macrophages. In a TLR4-dependent manner, infected macrophages upregulated transcription of hepcidin, a peptide hormone that triggers ferroportin degradation. Parasite replication was inhibited in hepcidin-deficient macrophages and in wild type macrophages overexpressing mutant ferroportin that is resistant to hepcidin-induced degradation. Conversely, intracellular growth was enhanced by exogenously added hepcidin, or by expression of dominant-negative ferroportin. Importantly, dominant-negative ferroportin and macrophages from flatiron mice, a mouse model for human type IV hereditary hemochromatosis, restored the infectivity of mutant parasite strains defective in iron acquisition. Thus, inhibition of ferroportin expression is a specific strategy used by L. amazonensis to inhibit iron export and promote their own intracellular growth.
Author Summary
Infection with the protozoan parasite Leishmania causes significant human disease in many parts of the world, particularly in the Middle East, India and South America. The parasite is transmitted by sand flies, which are difficult to control and are becoming increasingly common in urban areas. With domestic dogs serving as reservoirs of the disease and global travel increasing the population of infected human patients, the overall burden of leishmaniasis is on the rise. In mammals these parasites replicate inside macrophages, and therefore need strategies to survive within a cell that is specialized in killing pathogens. Earlier work demonstrated that iron is one of the essential nutrients that Leishmania must acquire from host cells to survive. Acquiring iron is particularly challenging inside macrophages, which play an important role in host iron homeostasis and export iron extracellularly through the membrane transporter ferroportin. We found that Leishmania amazonesis induces their host macrophages to produce hepcidin, a peptide that triggers internalization and degradation of ferroportin. This strategy increases the macrophage intracellular iron pool, and stimulates Leishmania replication. These results suggest that defects in iron homeostasis, which occur frequently in the human population, can have an important role in susceptibility to Leishmania infections.
doi:10.1371/journal.ppat.1003901
PMCID: PMC3907422  PMID: 24497831
18.  Estrogen Regulates Hepcidin Expression via GPR30-BMP6-Dependent Signaling in Hepatocytes 
PLoS ONE  2012;7(7):e40465.
Hepcidin, a liver-derived iron regulatory protein, plays a crucial role in iron metabolism. It is known that gender differences exist with respect to iron storage in the body; however, the effects of sex steroid hormones on iron metabolism are not completely understood. We focused on the effects of the female sex hormone estrogen on hepcidin expression. First, ovariectomized (OVX) and sham-operated mice were employed to investigate the effects of estrogen on hepcidin expression in an in vivo study. Hepcidin expression was decreased in the livers of OVX mice compared to the sham-operated mice. In OVX mice, bone morphologic protein-6 (BMP6), a regulator of hepcidin, was also found to be downregulated in the liver, whereas ferroportin (FPN), an iron export protein, was upregulated in the duodenum. Both serum and liver iron concentrations were elevated in OVX mice relative to their concentrations in sham-operated mice. In in vitro studies, 17β-estradiol (E2) increased the mRNA expression of hepcidin in HepG2 cells in a concentration-dependent manner. E2-induced hepatic hepcidin upregulation was not inhibited by ICI 182720, an inhibitor of the estrogen receptor; instead, hepcidin expression was increased by ICI 182720. E2 and ICI 182720 exhibit agonist actions with G-protein coupled receptor 30 (GPR30), the 7-transmembrane estrogen receptor. G1, a GPR30 agonist, upregulated hepcidin expression, and GPR30 siRNA treatment abolished E2-induced hepcidin expression. BMP6 expression induced by E2 was abolished by GPR30 silencing. Finally, both E2 and G1 supplementation restored reduced hepatic hepcidin and BMP6 expression and reversed the augmentation of duodenal FPN expression in the OVX mice. In contrast, serum hepcidin was elevated in OVX mice, which was reversed in these mice with E2 and G1. Thus, estrogen is involved in hepcidin expression via a GPR30-BMP6-dependent mechanism, providing new insight into the role of estrogen in iron metabolism.
doi:10.1371/journal.pone.0040465
PMCID: PMC3394730  PMID: 22792339
19.  Hepcidin regulation: ironing out the details 
Journal of Clinical Investigation  2007;117(7):1755-1758.
Hepcidin is a peptide hormone secreted by the liver that plays a central role in the regulation of iron homeostasis. Increased hepcidin levels result in anemia while decreased expression is the causative feature in most primary iron overload diseases. Mutations in hemochromatosis type 2 (HFE2), which encodes the protein hemojuvelin (HJV), result in the absence of hepcidin and an early-onset form of iron overload disease. HJV is a bone morphogenetic protein (BMP) coreceptor and HJV mutants have impaired BMP signaling. In this issue of the JCI, Babitt and colleagues show that BMPs are autocrine hormones that induce hepcidin expression (see the related article beginning on page 1933). Administration of a recombinant, soluble form of HJV decreased hepcidin expression and increased serum iron levels by mobilizing iron from splenic stores. These results demonstrate that recombinant HJV may be a useful therapeutic agent for treatment of the anemia of chronic disease, a disorder resulting from high levels of hepcidin expression.
doi:10.1172/JCI32701
PMCID: PMC1904333  PMID: 17607352
20.  Hepcidin and Hfe in iron overload in β-thalassemia 
Hepcidin (HAMP) negatively regulates iron absorption, degrading the iron exporter ferroportin at the level of enterocytes and macrophages. We showed that mice with β-thalassemia intermedia (th3/+) have increased anemia and iron overload. However, their hepcidin expression is relatively low compared to their iron burden. We also showed that the iron metabolism gene Hfe is down-regulated in concert with hepcidin in th3/+ mice. These observations suggest that low hepcidin levels are responsible for abnormal iron absorption in thalassemic mice and that down-regulation of Hfe might be involved in the pathway that controls hepcidin synthesis in β-thalassemia. Therefore, these studies suggest that increasing hepcidin and/or Hfe expression could be a strategy to reduces iron overload in these animals. The goal of this paper is to review recent findings that correlate hepcidin, Hfe, and iron metabolism in β-thalassemia and to discuss potential novel therapeutic approaches based on these recent discoveries.
doi:10.1111/j.1749-6632.2010.05595.x
PMCID: PMC3652388  PMID: 20712796
β-thalassemia; iron overload; hepcidin; Hfe; lentiviral vectors
21.  In Vivo Imaging of Hepcidin Promoter Stimulation by Iron and Inflammation 
Blood cells, molecules & diseases  2007;38(3):253-257.
Hepcidin is an acute-phase response antimicrobial peptide that has emerged as a central regulator of iron absorption. Circulating hepcidin levels have been shown to affect iron uptake, release and storage. Hepcidin is mainly liver-derived and regulated, at least in part, transcriptionally. Hypoxia, erythroid demand, iron content and inflammation each have been shown to influence hepcidin mRNA expression in intact animals. In vitro, regulation of hepcidin by cytokines and by hypoxia is readily demonstrated in primary hepatocytes or in hepatocyte lines, but incubating the same cell lines with iron does not increase transcription of hepcidin. Thus, how iron excess stimulates hepcidin production in hepatocytes remains unknown. In addition, there is no current technique available that can investigate how iron induces hepcidin expression. To provide a better understanding of hepcidin gene expression in response to these regulatory stimuli, we have established a whole animal in vivo bioluminescence imaging assay to measure the activity of hepcidin promoter constructs in the animals liver after hydrodynamic transfection of hepcidin promoter/luciferase constructs into mice. Transfected hepcidin promoter constructs were shown to respond to both inflammatory and increasing iron stimuli in vivo. This work highlights the ability of this new imaging technique to investigate the key regions of the hepcidin promoter involved in iron induction of hepcidin expression.
doi:10.1016/j.bcmd.2007.01.004
PMCID: PMC1924465  PMID: 17331760
Hepcidin; IVIS; Luciferase; Iron Stimulation
22.  Serum and liver iron differently regulate the bone morphogenetic protein 6 (BMP6)-SMAD signaling pathway in mice 
Hepatology (Baltimore, Md.)  2011;54(1):273-284.
The bone morphogenetic protein 6 (BMP6)-SMAD signaling pathway is a central regulator of hepcidin expression and systemic iron balance. However, the molecular mechanisms by which iron is sensed to regulate BMP6-SMAD signaling and hepcidin expression are unknown. Here, we examined the effects of circulating and tissue iron on Bmp6-Smad pathway activation and hepcidin expression in vivo after acute and chronic enteral iron administration in mice. We demonstrated that both transferrin saturation and liver iron content independently influence hepcidin expression. While liver iron content is independently positively correlated with hepatic Bmp6 mRNA expression and overall activation of the Smad1/5/8 signaling pathway, transferrin saturation activates the downstream Smad1/5/8 signaling cascade, but does not induce Bmp6 mRNA expression in the liver. Hepatic inhibitory Smad7 mRNA expression is increased by both acute and chronic iron administration and mirrors overall activation of the Smad1/5/8 signaling cascade. In contrast to the Smad pathway, the extracellular signal-regulated kinase 1 and 2 (Erk1/2) mitogen-activated protein kinase (Mapk) signaling pathway in the liver is not activated by acute or chronic iron administration in mice.
Conclusion
Our data demonstrate that the hepatic Bmp6-Smad signaling pathway is differentially activated by circulating and tissue iron to induce hepcidin expression, while the hepatic Erk1/2 signaling pathway is not activated by iron in vivo.
doi:10.1002/hep.24359
PMCID: PMC3277401  PMID: 21488083
hepcidin; hemochromatosis; HFE; transferrin receptor 2; mitogen activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK1/2)
23.  Intracellular Iron Transport and Storage: From Molecular Mechanisms to Health Implications 
Antioxidants & Redox Signaling  2008;10(6):997-1030.
Abstract
Maintenance of proper “labile iron” levels is a critical component in preserving homeostasis. Iron is a vital element that is a constituent of a number of important macromolecules, including those involved in energy production, respiration, DNA synthesis, and metabolism; however, excess “labile iron” is potentially detrimental to the cell or organism or both because of its propensity to participate in oxidation–reduction reactions that generate harmful free radicals. Because of this dual nature, elaborate systems tightly control the concentration of available iron. Perturbation of normal physiologic iron concentrations may be both a cause and a consequence of cellular damage and disease states. This review highlights the molecular mechanisms responsible for regulation of iron absorption, transport, and storage through the roles of key regulatory proteins, including ferroportin, hepcidin, ferritin, and frataxin. In addition, we present an overview of the relation between iron regulation and oxidative stress and we discuss the role of functional iron overload in the pathogenesis of hemochromatosis, neurodegeneration, and inflammation. Antioxid. Redox Signal. 10, 997–1030.
Introduction
Iron Transport
Nonintestinal iron transport by transferring
Iron-bound transferrin binds the transferrin receptor for cellular iron uptake
Regulation of transferrin receptor 1 by iron regulatory element–iron regulatory protein system
Transcriptional regulation of transferrin receptor 1
Differential regulation of transferrin receptor 1 and transferrin receptor 2
Transferrin receptor 1 is regulated by hereditary hemochromatosis protein
Transferrin-independent cellular iron uptake
Intestinal iron absorption
Regulation of divalent metal transporter 1
Ferroportin is responsible for cellular iron efflux
Ferroportin associates and cooperates with ceruloplasmin
Ferroportin and hephaestin cooperate in iron efflux from intestinal cells
Hepcidin
Iron Storage and Ferritin
Structure, tissue distribution, and importance of cytoplasmic ferritin
Iron efflux and ferritin degradation
Serum ferritin and ferritin receptor
Mitochondrial ferritin
Nuclear ferritin
Regulation of Ferritin
Iron-mediated ferritin regulation
Ferritin regulation by reactive oxygen species
Ferritin transcriptional regulation by cytokines
Ferritin regulation in erythroleukemic cells
Frataxin and Iron Homeostasis
Frataxin and Friedreich ataxia
Frataxin and mitochondrial iron traffic
Frataxin, heme synthesis, and iron–sulfur cluster biogenesis
Frataxin gene regulation
Treatments
Functional Iron Overload and Human Health
Hereditary hemochromatosis
Mutant iron-responsive element-mediated iron overload
Iron regulation and neurodegeneration
Conclusions and Future Directions
doi:10.1089/ars.2007.1893
PMCID: PMC2932529  PMID: 18327971
24.  The Role of Ubiquitination in Hepcidin-Independent and Hepcidin-Dependent Degradation of Ferroportin 
Cell metabolism  2011;14(5):635-646.
SUMMARY
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.
doi:10.1016/j.cmet.2011.09.008
PMCID: PMC3229915  PMID: 22019085
25.  Role of Hepcidin in the Setting of Hypoferremia during Acute Inflammation 
PLoS ONE  2013;8(4):e61050.
The anemia of chronic disease (also called anemia of inflammation) is an acquired disorder of iron homeostasis associated with infection, malignancy, organ failure, trauma, or other causes of inflammation. It is now widely accepted that induction of hepcidin expression in response to inflammation might explain the characteristic hypoferremia associated with this condition. To determine the role of hepcidin in acute inflammation and the regulation of its receptor, the iron exporter, ferroportin, wild-type, heterozygote and hepcidin knockout mice (Hepc−/−) were challenged with sublethal doses of lipopolysaccharide (LPS). Six hours after injection, ferroportin mRNA and protein levels were assessed in the duodenum and the spleen and plasma iron was determined. Our results demonstrate that hepcidin is crucial, though not the sole mediator of LPS-mediated acute hypoferremia, and also that hepcidin major contribution relies on decreased ferroportin protein levels found in the spleen. Furthermore, we establish that LPS-mediated repression of the membrane iron transporter DMT1 and oxidoreductase Dcytb in the duodenum is independent of hepcidin. Finally, our results in the hepc+/− mice indicate that elevated hepcidin gene expression is not a prerequisite for the setting of hypoferremia during early inflammatory response, and they highlight the intimate crosstalk between inflammatory and iron-responsive pathways for the control of hepcidin.
doi:10.1371/journal.pone.0061050
PMCID: PMC3634066  PMID: 23637785

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