Article

Ferroportin1 is required for normal iron cycling in zebrafish

Division of Hematology/Oncology, Children's Hospital, Karp Research Laboratories, Boston, Massachusetts 02115, USA.
Journal of Clinical Investigation (Impact Factor: 13.77). 07/2005; 115(6):1532-41. DOI: 10.1172/JCI23780
Source: PubMed

ABSTRACT Missense mutations in ferroportin1 (fpn1), an intestinal and macrophage iron exporter, have been identified between transmembrane helices 3 and 4 in the zebrafish anemia mutant weissherbst (weh(Tp85c-/-)) and in patients with type 4 hemochromatosis. To explore the effects of fpn1 mutation on blood development and iron homeostasis in the adult zebrafish, weh(Tp85c-/-) zebrafish were rescued by injection with iron dextran and studied in comparison with injected and uninjected WT zebrafish and heterozygotes. Although iron deposition was observed in all iron-injected fish, only weh(Tp85c-/-) zebrafish exhibited iron accumulation in the intestinal epithelium compatible with a block in iron export. Iron injections initially reversed the anemia. However, 8 months after iron injections were discontinued, weh(Tp85c-/-) zebrafish developed hypochromic anemia and impaired erythroid maturation despite the persistence of iron-loaded macrophages and elevated hepatic nonheme iron stores. Quantitative real-time RT-PCR revealed a significant decrease in mean hepatic transcript levels of the secreted iron-regulator hepcidin and increased intestinal expression of fpn1 in anemic weh(Tp85c-/-) adults. Injection of iron dextran into WT or mutant zebrafish embryos, however, resulted in significant increases in hepcidin expression 18 hours after injection, demonstrating that hepcidin expression in zebrafish is iron responsive and independent of fpn1's function as an iron exporter.

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    • "Hepcidin binds ferroportin1, the only known vertebrate iron exporter, resulting in internalization and degradation of both proteins [6]. Degradation of ferroportin1 decreases intestinal iron absorption [6] and prevents the release of iron from macrophage iron stores to developing erythrocytes in the bone marrow [7]. "
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    ABSTRACT: Hepcidin, a peptide hormone produced in the liver, decreases intestinal iron absorption and macrophage iron release via effects on ferroportin. Bone morphogenic protein and Stat3 signaling regulate Hepcidin's transcription. Hepcidin is a potential drug target for patients with iron overload syndromes because its levels are inappropriately low in these individuals. To generate a tool for identifying small molecules that modulate Hepcidin expression, we stably transfected human hepatocytes (HepG2) cells with a reporter construct containing 2.7 kb of the human Hepcidin promoter upstream of a firefly reporter gene. We used high throughput methods to screen 10,169 chemicals in duplicate for their effect on Hepcidin expression and cell viability. Regulators were identified as chemicals that caused a change > 3 standard deviations above or > 1 standard deviation below the mean of the other chemicals (z-score > 3 or < 1), while not adversely affecting cell viability, quantified by fluorescence assay. Following validation assays, we identified 16 chemicals in a broad range of functional classes that promote Hepcidin expression. All of the chemicals identified increased expression of bone morphogenic protein-dependent and/or Stat3-dependent genes, however none of them strongly increased phosphorylation of Smad1,5,8 or Stat3.
    Blood Cells Molecules and Diseases 12/2014; 53(4). DOI:10.1016/j.bcmd.2014.06.002 · 2.33 Impact Factor
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    • "The negatively charged N-terminal region (usually 5 aa prior to the first conserved Cys) is known to be essential for its interaction with ferroportin (Nemeth et al., 2006). The QSHLS motif of the zebrafish (Danio rerio) HAMP1 is known to internalize ferroportin molecules, similar to the mammalian motif DTHEP (Fraenkel et al., 2005). The OJHAMP1, described in this study, represents a clearly conserved signature in the N-terminal region, and it shared an identical anionic pI value with all other fish HAMP1 orthologs, suggesting that the dual function of mammalian HAMP1s should be conserved in the broad teleostean taxa. "
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    ABSTRACT: Two hepcidin paralogs (ojhamp1 and ojhamp2) were isolated and characterized from a euryhaline Javanese ricefish (Oryzias javanicus: Beloniformes). The ojhamp1 cDNA encoded 90 or 91 amino acids (aa) of a typical HAMP1 preproprotein. This preproprotein is believed to cleave and yield the 66 or 67 aa-proprotein, followed by the 26 aa-mature peptide, composed of 8 conserved cysteine residues and the QSHL amino terminal motif. The ojhamp2 cDNA encoded 89 aa of HAMP2 preproprotein, cleaved to yield a 65 aa proprotein, and subsequently the 25 aa-mature peptide. The mature OJHAMP1 possessed a cationic isoelectric point (pI), whereas OJHAMP2 had an anionic charge. At the genomic level, both ojhamp1 and ojhamp2 share a conserved tripartite structure (three exons interrupted by two introns) with other vertebrate hepcidin genes. However, the ojhamp1 was shown to exist as two distinct mRNA species, encoding 90 or 91 aa, due to alternative splicing at the junction site between intron I and exon II. Both ojhamp1 and ojhamp2 transcripts were detected in a wide range of tissue types with varying levels of basal expression, although the highest expression was observed in the liver for both isoforms. Transcriptional response to bacterial challenge using Edwardsiella tarda showed that ojhamp1 was moderately upregulated in the liver but remained unchanged in the kidney. However, the ojhamp2 was significantly suppressed in both the kidney and liver, suggesting a potential diversification between the two paralogs.
    06/2011; 14(2). DOI:10.5657/FAS.2011.0093
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    • "With respect to iron homeostasis, HAMPs promote macrophage iron retention and diminish intestinal iron absorption. To exert this function, HAMP transcript levels increase in iron-overloaded fish [8] [10] [11] and reduce under severe anemia [12]. This regulation of HAMP gene expression has been associated to a defensive mechanism to limit the iron available for bacterial growth [13]. "
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    ABSTRACT: Hepcidin antimicrobial peptides (HAMPs) are key molecules of the innate immune system against bacterial infections and in iron metabolism. In this study we report the molecular cloning and genomic characterization of four HAMP genes (referred to as HAMP1, HAMP2, HAMP3 and HAMP4) in the redbanded seabream (Pagrus auriga). All these genes possessed the eight characteristic cysteine residues involved in protein folding. No canonical sequence for convertase-mediated processing of the HAMP3 propeptide was identified. At the genomic level, all four HAMP genes consisted of two introns and three exons. Phylogenetic analysis revealed that HAMPs could group in two main clusters with HAMP2, HAMP3 and HAMP4 belonging to the more complex and diversified HAMP2-like group of acanthopterygians. Quantitation of mRNA levels in adult tissues showed that HAMP1 was ubiquitously expressed, HAMP2 mainly in kidney, spleen and intestine, whereas HAMP3 and HAMP4 in liver. During development, HAMP2 and HAMP3 were expressed at a high level in embryos. Moreover, the expression levels of the four HAMP genes increased between 5 and 15 days after hatching when larvae started external feeding. Induction experiments with lipopolysaccharide revealed significant changes in gene expression of the four HAMP genes in kidney, liver and spleen. However, expression profiles differed in magnitude and time course response. HAMP1 mRNAs increased rapidly in kidney at 1 h p.i. whereas HAMP2 did later at 24 h. Moreover, HAMP4 transcripts increased more than 5000-fold in liver whereas HAMP2 mRNAs dropped significantly in spleen at 3 h p.i. All these data suggest that HAMPs are involved in the response against bacterial infections although additional functions in iron regulation and embryogenesis in fish should be considered.
    Fish &amp Shellfish Immunology 04/2009; 26(3):483-91. DOI:10.1016/j.fsi.2009.01.012 · 3.03 Impact Factor
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