Transferrin-a modulates hepcidin expression in zebrafish embryos

Abstract

The iron regulatory hormone hepcidin is transcriptionally up-regulated in response to iron loading, but the mechanisms by which iron levels are sensed are not well understood. Large-scale genetic screens in the zebrafish have resulted in the identification of hypochromic anemia mutants with a range of mutations affecting conserved pathways in iron metabolism and heme synthesis. We hypothesized that transferrin plays a critical role both in iron transport and in regulating hepcidin expression in zebrafish embryos. Here we report the identification and characterization of the zebrafish hypochromic anemia mutant, gavi, which exhibits transferrin deficiency due to mutations in transferrin-a. Morpholino knockdown of transferrin-a in wild-type embryos reproduced the anemia phenotype and decreased somite and terminal gut iron staining, while coinjection of transferrin-a cRNA partially restored these defects. Embryos with transferrin-a or transferrin receptor 2 (TfR2) deficiency exhibited low levels of hepcidin expression, however anemia, in the absence of a defect in the transferrin pathway, failed to impair hepcidin expression. These data indicate that transferrin-a transports iron and that hepcidin expression is regulated by a transferrin-a-dependent pathway in the zebrafish embryo. IntroductionTransferrin, a serum protein with 2 iron binding sites, is the major carrier of ferric iron in human plasma and is required to deliver iron to developing erythroid and other cells. Iron-bound holotransferrin binds transferrin receptor 1 (TfR1) on the surface of the cell, resulting in endocytosis of an endosome containing both transferrin and TfR1 (reviewed in Aisen 1 ). After acidification of the endosome, ferrous iron exits the endosome via the divalent metal transporter (DMT1). 2,3 Transferrin receptor 2 (TfR2), a homolog of TfR1, with restricted expression to hepatocytes and erythroid cells, 4 binds transferrin at lower affinity than TfR1 5,6 and is involved in the regulation of iron homeostasis.As humans have no active means of iron excretion, maintaining iron homeostasis requires adjustment of iron absorption to reflect the body's iron stores. Hepcidin is a peptide hormone, primarily expressed in the liver, 7 which modulates iron absorption and iron delivery to erythrocytes by binding the iron transporter ferroportin (fpn), resulting in internalization and degradation of fpn. 8 In mammalian models, hepcidin is transcriptionally up-regulated in response to inflammation 9,10 or iron overload 11 and down-regulated in response to anemia, iron deficiency, or hypoxia. 9 Although mutations in HFE, 12 transferrin receptor 2 (TFR2), 13 hepcidin (HAMP), 14 or hemojuvelin (HJV or HFE2) 15 have each been associated with hemochromatosis and impaired hepcidin expression, 16 the mechanisms by which iron overload is sensed and hepcidin is regulated are not completely understood.The zebrafish, Danio rerio, provides an excellent system for the identification and analysis of genes involved in iron metabolism and erythroid development. La...

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