Hepcidin as a therapeutic tool to limit iron overload and improve anemia in β-thalassemic mice.

Excessive iron absorption is one of the main features of β-thalassemia and can lead to severe morbidity and mortality. Serial analyses of β-thalassemic mice indicate that while hemoglobin levels decrease over time, the concentration of iron in the liver, spleen, and kidneys markedly increases. Iron overload is associated with low levels of hepcidin, a peptide that regulates iron metabolism by triggering degradation of ferroportin, an iron-transport protein localized on absorptive enterocytes as well as hepatocytes and macrophages. Patients with β-thalassemia also have low hepcidin levels. These observations led us to hypothesize that more iron is absorbed in β-thalassemia than is required for erythropoiesis and that increasing the concentration of hepcidin in the body of such patients might be therapeutic, limiting iron overload. Here we demonstrate that a moderate increase in expression of hepcidin in β-thalassemic mice limits iron overload, decreases formation of insoluble membrane-bound globins and reactive oxygen species, and improves anemia. Mice with increased hepcidin expression also demonstrated an increase in the lifespan of their red cells, reversal of ineffective erythropoiesis and splenomegaly, and an increase in total hemoglobin levels. These data led us to suggest that therapeutics that could increase hepcidin levels or act as hepcidin agonists might help treat the abnormal iron absorption in individuals with β-thalassemia and related disorders.

The functional link between HFE and hepcidin- its contribution to iron metabolism regulation

Tese de doutoramento, Biologia (Biologia Molecular), Universidade de Lisboa, Faculdade de Ciências, 2013

Régulation de l'hepcidine et le rôle de la lipocaline 2 dans l'homéostasie du fer / Novel insights into the regulation of hepcidin and the role of lipocalin 2 in iron homeostasis

Le fer, un métal de transition, est requis pour la survie de presque tout les organismes vivant à cause de son habilité à accepter ou donner un électron et donc à catalyser plusieurs réactions biochimique fondamentales. Cependant, la même propriété permet aussi au fer ionique d’accélérer la formation de radicaux libres et donc le fer peut potentiellement avoir des effets néfastes. Conséquemment, l’homéostasie du fer doit être étroitement régulé, tant au niveau cellulaire que systémique. Notre étude met l’emphase sur deux molécules importante pour régulation du métabolisme du fer : la lipocaline 2 (Lcn2) et l’hepcidine. Lcn2, une protéine de phase aiguë, est impliquée dans le transport du fer par les sidérophores. Lcn2 est un candidat potentiel comme transporteur du fer qui pourrait être responsable de l’accumulation excessive du fer non lié à la transferrine dans le foie des patients atteints d’hémochromatose héréditaire (HH). Nous avons généré des souris double-déficiente HfeLcn2 pour évaluer l’importance de Lcn2 dans la pathogenèse de surcharge en fer hépatique dans les souris knock-out Hfe (Hfe -/-). Notre étude révèle que la délétion de Lcn2 dans les souris Hfe-/- n’influence pas leur accumulation de fer hépatique ou leur réponse à une surcharge en fer. Le phénotype des souries HfeLcn2-/- demeure indiscernable de celui des souris Hfe-/-. Nos données impliquent que Lcn2 n’est pas essentiel pour la livraison du fer aux hépatocytes dans l’HH. L’hepcidine, un régulateur clé du métabolisme du fer, est un petit peptide antimicrobien produit par le foie et qui régule l’absorption intestinale du fer et son recyclage par les macrophages. L’expression de l’hepcidine est induite par la surcharge en fer et l’inflammation, tandis que, à l'inverse, elle est inhibée par l'anémie et l'hypoxie. Dans certaine situations pathologique, l’hepcidine est régulée dans des directions opposées par plus d’un régulateur. Nous avons, en outre, analysé comment les différents facteurs influencent l’expression de l’hepcidine in vivo en utilisant un modèle de souris avec un métabolisme du fer altéré. Nous avons examiné la régulation de l’hepcidine en présence de stimuli opposés, ainsi que la contribution des médiateurs et des voix de signalisation en aval de l’expression de l’hepcidine. Nous avons démontré que l'érythropoïèse, lorsque stimulé par l’érythropoïétine, mais pas par l’hypoxie, diminue l’expression de l’hepcidine d’une façon dépendante de la dose, même en présence de lipopolysaccharides ou de surcharge de fer alimentaire, qui peuvent agir de manière additive. De plus, l’entraînement érythropoïétique inhibe tant la voix inflammatoire que celle de détection du fer, du moins en partie, par la suppression du signal IL-6/STAT3 et BMP/SMAD4 in vivo. Au total, nos données suggèrent que le niveau d’expression de l’hepcidine en présence de signaux opposés est déterminé par la force du stimulus individuel plutôt que par une hiérarchie absolue. Ces découvertes sont pertinentes pour le traitement de l’anémie des maladies chronique et les désordres de surcharge en fer.

A Novel Isoform of the Hepatic Antimicrobial Peptide, Hepcidin (Hepc-CB1), from a Deep-Sea Fish, the Spinyjaw Greeneye Chlorophthalmus bicornis (Norman, 1939): Molecular Characterisation and Phylogeny

Hepcidin is cysteine-rich short peptide of innate immune system of fishes, equipped to perform prevention and proliferation of invading pathogens like bacteria and viruses by limiting iron availability and activating intracellular cascades. Hepcidins are diverse in teleost fishes, due to the varied aquatic environments including exposure to pathogens, oxygenation and iron concentration. In the present study, we report a 87-amino acid (aa) preprohepcidin (Hepc-CB1) with a signal peptide of 24 aa, a prodomain of 39 aa and a bioactive mature peptide of 24 aa from the gill mRNA transcripts of the deep-sea fish spinyjaw greeneye, Chlorophthalmus bicornis. Molecular characterisation and phylogenetic analysis categorised the peptide to HAMP2-like group with a mature peptide of 2.53 kDa; a net positive charge (?3) and capacity to form b-hairpin-like structure configured by 8 conserved cysteines. The present work provides new insight into the mass gene duplication events and adaptive evolution of hepcidin isoforms with respect to environmental influences and positive Darwinian selection. This work reports a novel hepcidin isoform under the group HAMP2 from a nonacanthopterygian deep-sea fish, C. bicornis

Molecular Characterisation and Phylogenetic Analysis of a Novel Isoform of Hepatic Antimicrobial Peptide, Hepcidin (Zc-hepc1), from the Coral Fish Moorish idol, Zanclus cornutus (Linnaeus,1758)

Hepcidin is a family of short cysteine-rich antimicrobial peptides (AMPs) participating in various physiological functions with inevitable role in host immune responses. Present study deals with identification and characterisation of a novel hepcidin isoform from coral fish Zanclus cornutus. The 81 amino acid (aa) preprohepcidin obtained from Z. cornutus consists of a hydrophobic aa rich 22 mer signal peptide, a highly variable proregion of 35 aa and a bioactive mature peptide with 8 conserved cysteine residues which contribute to the disulphide back bone. The mature hepcidin, Zc-hepc1 has a theoretical isoelectric point of 7.46, a predicted molecular weight of 2.43 kDa and a net positive charge of ?1. Phylogenetic analysis grouped Z. cornutus hepcidin with HAMP2 group hepcidins confirming the divergent evolution of hepcidin-like peptide in fishes. Zc-hepc1 can attain a b-hairpin-like structure with two antiparallel b-sheets. This is the first report of an AMP from the coral fish Z. cornutus.

Sequencing and expression analysis of hepcidin mRNA in donkey (Equus asinus) liver

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Identification, characterization and expression analysis of hepcidin gene in sheep

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Cloning, sequencing and expression analysis of the equine hepcidin gene by real-time PCR

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Lipopolysaccharide infusion up-regulates hepcidin mRNA expression in equine liver

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Influence of experimental inflammatory response on hepatic hepcidin gene expression and plasma iron concentration in sheep

Hepcidin is a highly conserved disulfide-bonded peptide that plays a central role in iron homeostasis. During systemic inflammation, hepcidin up-regulation is responsible for hypoferremia. This study aimed to analyze the influence of the inflammatory process induced by complete Freund's adjuvant (CFA) or lipopolysaccharide (LPS) on the liver expression of hepcidin mRNA transcripts and plasma iron concentration of sheep. The expression levels of hepcidin transcripts were up-regulated after CFA or LPS. Hypoferremic response was observed at 12 h (15.46 +/- 6.05 mu mol/L) or 6 h (14.59 +/- 4.38 mu mol/L) and iron reached its lowest level at 96 h (3.08 +/- 1.18 mu mol/L) or 16 h (4.06 +/- 1.58 mu mol/L) after CFA administration or LPS infusion, respectively. This study demonstrated that the iron regulatory hormone hepcidin was up-regulated in sheep liver in response to systemic inflammation. These findings extend our knowledge on the relationship between the systemic inflammatory response, hepcidin and iron, and provide a starting point for additional studies on iron metabolism and the inflammatory process in sheep. (C) 2011 Elsevier B.V. All rights reserved.

Freund's adjuvant-induced inflammation: clinical findings and its effect on hepcidin mRNA expression in horses

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Increased hepcidin expression in multibacillary leprosy

Iron is essential for all organisms and its availability can control the growth of microorganisms; therefore, we examined the role of iron metabolism in multibacillary (MB) leprosy, focusing on the involvement of hepcidin. Erythrograms, iron metabolism parameters, pro-inflammatory cytokines and urinary hepcidin levels were evaluated in patients with MB and matched control subjects. Hepcidin expression in MB lesions was evaluated by quantitative polymerase chain reaction. The expression of ferroportin and hepcidin was evaluated by immunofluorescence in paucibacillary and MB lesions. Analysis of hepcidin protein levels in urine and of hepcidin mRNA and protein levels in leprosy lesions and skin biopsies from healthy control subjects showed elevated hepcidin levels in MB patients. Decreases in haematologic parameters and total iron binding capacity were observed in patients with MB leprosy. Moreover, interleukin-1 beta, ferritin, soluble transferrin receptor and soluble transferrin receptor/log ferritin index values were increased in leprosy patients. Hepcidin was elevated in lepromatous lesions, whereas ferroportin was more abundant in tuberculoid lesions. In addition, hepcidin and ferroportin were not colocalised in the biopsies from leprosy lesions. Anaemia was not commonly observed in patients with MB; however, the observed changes in haematologic parameters indicating altered iron metabolism appeared to result from a mixture of anaemia of inflammation and iron deficiency. Thus, iron sequestration inside host cells might play a role in leprosy by providing an optimal environment for the bacillus.