The flatiron mutation in mouse ferroportin acts as a dominant negative to cause ferroportin disease

Abstract

IntroductionHereditary hemochromatosis is a common disorder in humans, characterized by iron overload resulting in tissue injury and ultimately organ failure. Typically, hemochromatosis exhibits an autosomal-recessive pattern of inheritance and is associated with mutations in HFE, hemojuvelin, hepcidin, or transferrin receptor 2. 1,2 Targeted deletion of these genes in the mouse results in hemochromatosis, providing mouse models for most forms of the disease. Hemochromatosis type IV, also referred to as ferroportin (Fpn) disease, results from mutations in the iron transporter ferroportin. Fpn is the only known iron exporter in mammalian cells and is present on the surface of macrophages, intestinal enterocytes, hepatocytes, and placental cells. [3][4][5] The level of cell surface Fpn is regulated by its interaction with hepcidin, a peptide secreted by the liver in response to iron stores and inflammation. Hepcidin binds to Fpn, inducing its internalization and degradation, thus regulating the export of iron from cells to plasma. 6 Mutations in Fpn lead to iron-overload disease but, in contrast to other forms of hemochromatasis, ferroportin disease exhibits an autosomal-dominant pattern of inheritance. 7 The disorder has different presentations depending on the Fpn mutation. Mutations leading to Fpn that is not internalized by hepcidin result in iron accumulation in hepatocytes and high transferrin saturation. 8,9 Mutations leading to Fpn that is not appropriately targeted to the cell surface result in iron accumulation in Kupffer cells and low transferrin saturation. [9][10][11] The mechanism by which the disease mutations exert a dominant effect is unclear. Some groups that study the disease suggest that it results from haploinsufficiency, 10,12 whereas others suggest that the disorder results from a dominantnegative effect of the mutant allele. 9,13 Importantly, all human mutations are missense mutations and mice that are heterozygous for a targeted deletion of Fpn do not show the disease. 14 Treatment for hemochromatosis aims to decrease iron load by repeated phlebotomy and this treatment works well for most patients. Many patients with ferroportin disease, however, become anemic with phlebotomy, highlighting the need for a mouse model to develop better treatments.We report here on a missense mutation in mouse Fpn that results in a disorder that is identical to classic human ferroportin disease. We show that macrophages isolated from mutant mice have no Fpn on their cell surface and that expression of Fpn constructs containing the missense mutation (H32R) affects the behavior of wild-type Fpn. These results show that Fpn disease is due to a dominant-negative effect of the mutant allele and provide the first mouse model for this disorder. Materials and methods Generation of mutant mice and identification of Fpn mutationThe ffe mouse line was identified in a screen for recessive ethylnitrosourea (ENU)-induced mutations that cause morphologic abnormalities at embryonic day (E) 12.5. [15][16][17] The ffe mutation...

Ferroportin deficiency impairs manganese metabolism in flatiron mice

“…Our characterization of these mice show properties reported in previous studies, with heterozygous ffe/+ mice having features of both Fe deficiency and loading. These characteristics recapitulate features of ferroportin disease (29). Importantly, our study is to our knowledge the first to directly demonstrate that intestinal Fe absorption is impaired in flatiron mice, consistent with deficiency of Fe export function across the enterocyte.…”

“…Fpn knockout mice are available, but homozygotes display embryonic lethality, and heterozygotes do not reproduce features of human Feloading disease caused by loss of Fpn function (28). Ffe mice heterozygous for a H32R missense mutation in Fpn are viable and show Fe loading in macrophages of reticuloendothelial system, with reduced hematocrit, high serum ferritin, and low transferrin saturation (29). These are known features of human ferroportin disease, a form of hereditary hemochromatosis (30).…”

“…These are known features of human ferroportin disease, a form of hereditary hemochromatosis (30). It has been shown that this phenotype arises because the Fpn mutant H32R acts as a dominant negative to mislocalize wild-type Fpn away from the cell surface (29). To resolve the outstanding questions about the role of Fpn in Mn export, we characterized Mn metabolism in flatiron mice.…”

Perturbation of hepcidin expression by BMP type I receptor deletion induces iron overload in mice

“…There were no hematologic abnormalities suggestive of the presence of a secondary, anemia-induced iron overload phenotype in mice with liverspecific Alk2 or Alk3 deficiency (supplemental Figure 3). Moreover, hepatic expression of hereditary hemochromatosis diseasecausing genes, including transferrin receptor-2 (Tfr2), 30 Hfe, 31,32 hemojuvelin (Hjv), 9,13,14,33 and ferroportin 1 (Fpn1), 34 did not differ between genotypes (supplemental Figure 4). These results demonstrate a mild iron overload phenotype in Alk2 fl/fl ; Alb-Cre mice and a severe iron overload phenotype in Alk3 fl/fl ; Alb-Cre mice and indicate that both Alk2 and Alk3 have roles in the regulation of systemic iron homeostasis.…”

Nramp1 and Other Transporters Involved in Metal Withholding during Infection

“…In vitro studies show that overexpression of ferroportin disrupts M. tuberculosis growth (77), diminishes growth of Salmonella (78), and reduces HIV replication (79). Conversely, studies of flatiron mice, which have ferroportin deficiency (70,76,80), demonstrate that loss of its export activity confers greater susceptibility to intracellular pathogens (81). Macrophages from these mice support greater growth of Chlamydophila psittaci, and this effect was lost upon iron chelation (81).…”